Use of Ribotyping in Epidemiological Surveillance of Nosocomial Outbreaks

CLINICAL MICROBIOLOGY REVIEWS, JUlY 1994, P. 311-327 Vol0893-8512/94/$04.00+0Copyright © 1994, American Society for Microbiology

Use of Ribotyping in Epidemiological Surveillance ofNosocomial Outbreaks

EDOUARD H. BINGEN,1* ERICK DENAMUR,2'3 AND JACQUES ELION2'3Laboratoire de Microbiologie,' Laboratoire de Biochimie Genetique,2 and Institut National de la

Sante et de la Recherche Medicale U120,3 Hopital Robert Debre, 75019 Paris, France

INTRODUCTION .............................................................. 311DNA POLYMORPHISM .............................................................. 312rRNA OPERONS .............................................................. 312RIBOTYPING METHOD............................................................... 313APPLICATIONS OF RIBOTYPING .............................................................. 314

Enterobacter cloacae ............................................................... 314Enterobacter sakazakii............................................................... 315Providencia stuartii............................................................... 315Klebsiela pneumoniae ............................................................... 316Acinetobacter calcoaceticus-Acinetobacter baumanii............................................................... 316Xanthomonas maltophilia ............................................................... 316Pseudomonas aeruginosa............................................................... 316Pseudomonas cepacia ............................................................... 317Legionela pneumophila ............................................................... 318Moraxela (Branhamela) catarrhalis ............................................................... 319Rhodococcus spp...............................................................................319Tsukamurella paurometabolum ............................................................... 319Enterococcus spp...............................................................319Streptococcus pyogenes............................................................... 320Staphylococcus aureus ............................................................... 320Coagulase-Negative Staphylococcus spp.............................................................. 320Other Gram-Positive Bacteria .............................................................. 320Miscellaneous .............................................................. 320

WHAT TO EXPECT FROM RIBOTYPING IN NOSOCOMIAL OUTBREAKS ..............................................321FUTURE TRENDS............................................................... 321CONCLUSIONS............................................................... 322ACKNOWLEDGMENTS ............................................................... 322REFERENCES............................................................... 322

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INTRODUCTION

Determination of the relatedness of microbial isolates hasbecome increasingly important as the number and spectrum ofnosocomial pathogens continue to expand. Indeed, this ap-proach is critical to identifying outbreaks, to determining themode of acquisition of pathogens, and, accordingly, to definingeffective preventive and therapeutic measures.A variety of phenotypic techniques have been employed for

strain differentiation. Bacterial pathogens have been typed byusing biochemical profiles, antisera, bacteriophage susceptibil-ity, bacteriocin production and susceptibility, outer membraneprotein profiles, and antibiotic susceptibility profiles. Althoughvery useful, there are some problems associated with thesetechniques. For example, phage and bacteriocin typing systemsare not available for all bacterial species. Serotyping requires abattery of antisera, which may be difficult and expensive toprepare and standardize, and a collection of well-characterizedtype strains. Moreover, the utility of these methods is notablylimited because phenotypic markers may not be stably ex-pressed under certain environmental or culture conditions and

* Corresponding author. Mailing address: Laboratoire de Microbi-ologie, H6pital Robert Debre, 48 Boulevard Serurier, 75019 Paris,France. Phone: 33 (1) 40 03 23 40. Fax: 33 (1) 40 03 24 50.

may vary under the pressure of antibiotic usage in a hospitalsetting. Antigenic traits, for example, are unstable and prone tovary under immune pressure. Moreover, some of the pheno-typic methods require reagents that are not commerciallyavailable; this is a major obstacle to comparative investigationsby other investigators.

Analysis of DNA offers several advantages (74, 106, 123,144, 148). First, DNA can always be extracted from bacteria sothat the notion of untypeable strains disappears; second, theanalytical strategy is always the same and can be applied to anyDNA, whatever its source; and third, many of the proceduresused do not require species-specific reagents. Another advan-tage is that it is possible to study neutral markers, which, atleast on chromosomal DNA, tend to be rather stable.Although plasmid analysis has provided important insights,

this approach is limited. For instance, analysis of plasmid DNAis obviously limited to plasmid-containing strains (95). Inaddition, plasmid profiles are not always discriminating enoughfor an adequate strain characterization since different strainsmay carry nonidentical plasmids of the same size. To improveselectivity, plasmids can be subjected to restriction enzymedigestion before comparison by electrophoresis. Similar diges-tion patterns virtually assure plasmid identity and strengthenthe case for strain clonality. However, even this refinementmay be inadequate for some epidemic studies since plasmids

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are by nature unstable and mobile. Similar plasmids may befound in different strains from the same bacterial species oreven in strains from different bacterial species (103). Indeed,many plasmids, particularly R plasmids, are readily lost oracquired, as they are capable of promiscuous interstrain,interspecies, and intergeneric transfer.

Analysis of chromosomal DNA is a useful alternative toplasmid analysis. It is based on the study of polymorphism ofeither total DNA or selected regions of the bacterial chromo-some. In the latter case, the choice of chromosomal markers isof utmost importance since DNA that encodes a highly select-able phenotype may be just as unstable as the phenotype itself.On the contrary, DNA encoding a stringently designed struc-ture will be much more stable. Analysis of DNA polymorphismin the chromosomal regions containing the highly conservedrRNA genes (rrn), and referred to as ribotyping, has beenwidely used as a molecular approach to the surveillance ofnosocomial outbreaks. One should mention, however, that it isstill unclear what portion of the studied polymorphisms iswithin the rn operons and what portion is in the flankingDNA, i.e., in regions that are not expected to be as stringentlyconserved.The purpose of this review is to describe the principles of

ribotyping and to illustrate its advantages and limitations instudying nosocomial outbreaks.

DNA POLYMORPHISM

DNA polymorphism corresponds to variations of the nucle-otide sequence between individuals of the same species. Tech-nically, the simplest way to explore DNA polymorphism is touse restriction enzyme analysis. These enzymes are endonucle-ases that cleave double-stranded DNA at specific recognitionsequences usually composed of 4 to 6 bp and referred to asrestriction sites. Once DNA has been cleaved with a restrictionenzyme, the size of the generated fragments is easy to analyze,most commonly, by electrophoresis on agarose gels. Interindi-vidual and, for our purpose, strain-to-strain variations infragment sizes are referred to as restriction fragment lengthpolymorphisms (RFLPs). Most commonly, RFLPs result fromsingle-base-pair variations that account for the presence orabsence of a restriction site at given positions in the genome.Credit for the discovery of RFLPs is due to Kan and Dozy (75),who, in 1978, first reported the existence of a polymorphic sitefor the enzyme HpaI in the human P-globin gene. Because thisvariation was neutral, it had no functional consequence andwas not by itself selected for. In that sense, such polymor-phisms constitute ideal genetic markers. But RFLP can alsoresult from insertions, deletions, or rearrangement of DNA inthe region spanning two constant restriction sites. AlthoughRFLP analysis explores only a small part of the polymorphicvariations, i.e., those affecting restriction sites, it represents ahighly powerful method for bacterial strain differentiation.

Restriction fragments resulting from digestion with enzymesrecognizing 6-bp sites commonly vary in size from 1,000 to20,000 bp. They can be easily visualized under UV light bystaining the DNA in agarose gels with ethidium bromide.However, a very large number of fragments is usually pro-duced, and because of their number, the fragments are oftenpoorly resolved (Fig. 1). Thus, strain comparison may requireseveral electrophoretic runs in which strains generating closelyrelated patterns in preliminary experiments are analyzed incontiguous lanes. Systematic inclusion of DNA size standardsin the analyzed samples has been proposed to improve reli-ability, but comparison remains difficult (49). One way tocircumvent this problem is to analyze patterns composed of a

1 2 3 4 5 6 7 8 9 Kb

- 48.5

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FIG. 1. RFLP patterns of total DNA of X maltophilia obtainedafter BclI digestion and ethidium bromide staining. Patterns of lanes 3to 9 are identical. Numbers on the right are DNA size standards.

limited number of well-separated bands. Enzymes that arespecific for longer recognition sequences will generate asmaller number of large DNA fragments. Pulsed-field gelelectrophoresis (PFGE) resolves large chromosomal frag-ments that are not separated by conventional agarose gelelectrophoresis (135). This technique requires long and deli-cate procedures for isolating and restricting high-molecular-weight genomic DNA, sophisticated equipment, extended elec-trophoresis times, and considerable standardization. However,new developments in instrumentation, as seen in the second-generation PFGE systems, and simplification of protocols nowrender PFGE easier to perform.

Reducing the number of bands to be analyzed can also beachieved by using labelled DNA probes to highlight only someof the restriction fragments. Bacteriophage M13 DNA hasbeen used as a nonspecific probe for this purpose (69, 152).Most often, however, the probe is targeted to a small numberof specific loci. These probes may include cloned randomchromosomal sequences of the bacterial species to be studied,virulence factor or antibiotic resistance genes, and the trans-posable repetitive insertion sequences. The potential of suchprobes for distinguishing individual strains of bacteria has beenclearly shown (144). However, these probes are species specificor even specific for only some strains within a particularspecies. In addition, some of these probes are of limitedavailability. From 1970 onwards, polymorphisms within andaround the Escherichia coli rRNA genes (nrn) were identifiedand characterized (7, 47, 67, 73, 102). Demonstration that theubiquitous and polymorphic rm loci are highly conserved in theeubacterial kingdom (108) led to the brilliant idea of Grimontand Grimont (58) to use E. coli rRNA as a universal probe tostudy restriction patterns for taxonomic purposes. Later, Stullet al. (143) independently developed this concept of universalprobe, demonstrated its utility for epidemiological studies, andcoined the name ribotyping. Typical results of this approachare shown in Fig. 2.

rRNA OPERONS

The three species of rRNA (23S, 16S, and 5S) are present inequimolar amounts in 70S ribosome particles. They are co-

transcribed as a large 30S precursor RNA, which undergoes a

maturation process to yield the three different mature speciesof rRNA. The corresponding genes are thus organized into a

polycistronic transcription unit, the mn operon. Starting fromthe promoter, the individual genes are arranged in the follow-ing order: 5'-16S-23S-5S-3'. A typical E. coli n-n operon spans6,000 to 7,000 bp of DNA.While most genes in bacteria are present in one copy, mr

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FIG. 2. RFLP patterns of the rDNA region ofX maltophilia afterBamHI digestion. Lane 1, ATCC 13637. Patterns of lanes 4 to 10 areidentical. Numbers on the right are DNA size standards.

operons are unique in that they are present in multiple copies.Depending on the species, the number of copies varies from 2to 11 per haploid genome, i.e., per bacterial cell. The E. colichromosome, for instance, contains seven mn operons locatedin a region that spans half of the genome. Rare exceptions havebeen reported for some strains of Mycoplasma and Mycobac-terium spp. and for Borrelia burgdorfen strains, which carry onlyone rn operon (124). The possibility that the number of nmoperons is proportional to the size of the genome (5) and tothe rate of growth (14) has been suggested. B. burgdorferi is aslow-growing organism, and its genome is approximately 106bp long (13, 48); for comparison, the size of the E. coli genomeis 5 x 106 bp. The number of rn operons is important toconsider in the context of ribotyping, because the higher thenumber, the higher the potential discriminative power of thetechnique.Between the genes for 16S and 23S rRNAs is a spacer region

that contains genes encoding several tRNAs and several directrepeat sequences (11). These regions are heterogeneous. Forinstance, the seven rm operons of E. coli are divided into twoclasses depending on the nature of the spacer region (32). Oneof these contains the tRNA gene for glutamic acid, while theother contains tRNA genes for alanine and isoleucine (98).Other heterogeneities have been reported in the spacer re-gions of Bacillus (54) and Aspergillus (141) species. In additionto having tRNA genes in the 16S-23S intergenic spacer, somern operons also have tRNA genes in their 3'-flanking regions(97). Depending on the operon, these genes vary in nature andcopy number.The rrn nucleotide sequences encoding 16S and 23S rRNAs

appear to have changed little during evolution. A high degreeof homology for these sequences exists among various bacteria,despite the genetic diversity of the remainder of the chromo-some. In their broad-ranging study, Grimont and Grimont (58)used 32P-labelled E. coli 16S+23S rRNA to probe rrn loci in 40different species of gram-positive and gram-negative bacteria.They found that the probe reacted with the DNA of speciesthat were phylogenetically remote from E. coli and that a widevariety of restriction fragment patterns was observed amongthe strains studied. The explanation is that the highly con-served coding sequences in each rrn operon are surrounded bythe heterogeneous spacer and flanking sequences, describedabove, which vary from operon to operon in a given speciesand from one species to another. In addition, as might beexpected from the sequence redundancy, reciprocal recombi-nation has occurred frequently during evolution, thus contrib-uting to the observed high level of diversity of the nm loci.Therefore, the peculiar structure of rrn operons allows both abroad-spectrum hybridization of the E. coli RNA probe and a

A B C

FIG. 3. RFLP patterns of the rDNA regions of P. aeruginosa afterdigestion with HindIII (A), EcoRI (B), and BclI (C). Isolates wererecovered after 0 (lanes 1, 5, and 9), 10 (lanes 2, 6, and 10), 20 (lanes3, 7, and 11), and 30 (lanes 4, 8, and 12) passages in vitro. Numbers onthe right are DNA size standards.

high level of polymorphism. RFLP patterns actually reveal twolevels of genetic diversity. Some of the observed fragments arespecies specific and some, within a given species, are strainspecific. Accordingly, ribotyping can be considered a tool forboth taxonomic and epidemiological studies. Several studieshave shown that these polymorphic markers are stable in vitro(Fig. 3) and in vivo during an outbreak, and the value ofprobing rm loci with E. coli rRNA has now been clearlydemonstrated for the epidemiological typing of any species ofbacteria (58, 59, 70, 143).

RIBOTYPING METHOD

Restriction enzyme analysis requires pure DNA prepara-tions devoid of protein contamination. Most researchers use 3to 20 ml of broth from an overnight culture as the startingmaterial. Lysis of the bacterial cell wall is generally achievedwith ionic or nonionic detergents, after a pretreatment withlysozyme. The procedure may vary for capsulated or noncap-sulated bacteria and for gram-positive or gram-negative organ-isms. Proteins are eliminated by a proteinase K treatment inthe presence of sodium dodecyl sulfate and/or by a phenol-chloroform extraction. Finally, DNA is precipitated with eth-anol or isopropanol, dried, and resuspended in a low-ionic-strength buffer. DNA is then digested with restriction enzymes,and the generated fragments are size separated by electro-phoresis on a submarine agarose gel. According to the classicprocedure of Southern (139), DNA is transferred to a nylonmembrane and then hybridized with the radioactive probe.DNA fragments that react with the probe on the membraneare then revealed by autoradiography (Fig. 4).Most often, ribotyping uses labelled probes containing E.

coli 23S, 16S, and SS rRNA sequences (58, 143), but both thenature of the probe and the mode of labelling vary. Initially,Grimont and Grimont used commercially available E. coli16S+23S rRNA (58), a material known to be heavily contam-inated with SS rRNA. Similarly, Stull et al. used total rRNA(143). Now the probe most often used is DNA. It can be cDNAsynthesized directly from rRNA by reverse transcription, arecombinant plasmid in which the nm DNA has been cloned, or

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Enzyme cleavage DNA

Agarose gelelectrophoresis

Transfer on a /

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Membrane

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FIG. 4.printing.

Schematic representation of Southern hybridization finger-

a synthetic oligonucleotide constructed from the 16S or16S+23S rRNA gene sequences. E. coli rRNA is not the onlyheterologous probe that has been used for the study of otherspecies; for example, De Buyser et al. (39) used Bacillus subtilisrRNA to probe Staphylococcus DNA. Rather than using asingle heterologous probe, some workers prefer to use homol-ogous probes derived from the specific rRNA sequences of theorganism under study, as has been done in studies withMycoplasma sp. (159), Providencia stuartii (107, 119), Pseudo-monas cepacia and Haemophilus influenzae (143), Legionellasp. (133), Campylobacter sp. (111, 130), Corynebacterium jeikei-um (118), and Bacillus species (54). In addition, investiga-tors do not always use sequences of the same DNA regionsas probes. For instance, whereas as mentioned above, se-quences most often are 16S+23S (+5S) rRNA, Altwegg et al.(3) and others used a recombinant pBR322-derived plasmid(pKK3535) (35) containing the 16S, 23S, and 5S genes, thespacer region, two open reading frames in the sequencesflanking the rm operon, a portion of phage K DNA, and theother components of the vector, including the amp resistancegene. In contrast, Patton et al. used a probe to the 16S rRNAgene only (111), and in other studies, synthetic oligonucleo-tides were constructed from sequences of the conserved regionof 16S rRNA (99, 130) or of the variable region of the 16S or

23S rRNA genes (52). Since the hybridization bands obviouslydepend on the region of DNA that is probed, the results fromseparate studies are quite often not directly comparable.

Probes were initially labelled with 32p. For RNA probes andfor oligonucleotides, end labelling is used, and nick translationor random oligopriming is used for DNA probes (132). Oneadvantage of isotopic probes is that relatively few steps are

required for hybridization and subsequent washing. But theeffective nonisotopic cold-labelling systems that have now been

rRNA

hexanucleotide primers

Reverse transcriptase+ dCTP+ dATP+ dGTP+Dig-dUTP 4

rRNA

Digoxigenin-labelled rDNA

FIG. 5. Method for digoxigenin (Dig) labelling of rDNA.

developed have many advantages over the radioactive systems,including safety, easy disposal, and stability of the probes (4).Several nonradioactive probe labelling kits are now commer-cially available, and these represent an important step towardthe use of ribotyping in the clinical microbiology laboratory.Nonisotopic labelling may be done by incorporating biotiny-lated nucleotides in the DNA probes during cDNA synthesisor by nick translation or random oligopriming of plasmidscontaining rm sequences. After hybridization to the bacterialDNA fragments on the membrane, the biotin-labelled probecan be detected by using a streptavidin-alkaline phosphataseconjugate. Other labelling molecules have been used. In ourstudies, 16S+23S rRNA from E. coli was cold labelled byrandom oligopriming, using a mixture of hexanucleotide prim-ers and cloned mouse mammary leukemia virus reverse tran-scriptase (115) in the presence of deoxynucleotide triphos-phates, including digoxigenin-11-dUTP (18) (Fig. 5). Analkaline phosphatase-conjugated antidigoxigenin antibody isthen added, and the enzymatic activity is revealed by using thechemiluminescent substrate adamantyl-1,2-dioxetane phos-phate. As is the case when radioactive probes are used,hybridized DNA bands on the membrane are detected bycontact with an X-ray film (Fig. 6). We found this probe to bestable for several months and simple to synthesize. It providedresults as good as or superior to those obtained with isotopiclabelling. rRNA has also been labelled dlrectly withacetylaminofluorene, and the hybridized probe has been de-tected by an antibody-alkaline phosphatase conjugate (57). Inyet another procedure, a horseradish peroxidase-polyethyl-eneimine complex is covalently coupled to rRNA, followed byenhanced chemiluminescence detection (63).

APPLICATIONS OF RIBOTYPING

Enterobacter cloacae

Ribotyping may be an extremely powerful epidemiologicaltool when the classical phenotyping methods available in theclinical microbiology laboratory fail to discriminate betweenstrains. E. cloacae is a good example because of the high levelof diversity of the rrn loci among strains of the species (Fig. 7).Indeed, in this species, ribotyping greatly improves discrimina-tion obtained with biochemical patterns and antibiotic suscep-tibility profiles. For instance, we used ribotyping for the



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/ Filter-bound digestedchromosomal DNA

Digoxigenin-labelledrDNA

Hybridization

Anti digoxigeninantibody alkalinephosphatase conjugate

Chemiluminescentsignal

Exposure to X-ray film

FIG. 6. Schematic representation of chemiluminescent ribotyping.AMPPD, adamantyl-1,2-dioxetane phosphate.

epidemiological evaluation of 10E. cloacae nosocomial iso-lates obtained from nine patients in our hospital (21). Five ofthese patients had been hospitalized during overlapping peri-ods of time, thus raising the question of cross-contamination.Indeed, a single biochemical pattern and a single antibioticsusceptibility profile were observed for all but one isolate.However, ribotypes established after digestion of DNA withEcoRI and BamHI demonstrated the genetic unrelatedness ofthe isolates and thus excluded a common source of contami-nation or patient-to-patient transfer. These results suggestedthe possibility of an endogenous origin of infection, whichcould be demonstrated in at least one case of neonatalmeningitis (82). In these two studies, the discriminative power

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FIG. 7. RFLP patterns of the rDNA regions of Enterobacter cloa-

cae strains after EcoRl digestion. Each pattern is different. Numbers

on the right are DNA size standards.

of ribotyping was similar to that of total DNA RFLP analysisby conventional agarose gel electrophoresis.

Validation of this approach forE. cloacae typing comes fromthe work of Garaizar et al. (50). For E. cloacae strains, theseauthors compared ribotyping with more sophisticated butclassical phenotypic methods, including 0serotyping, phagesusceptibility, and biotype pattern. Forty-five isolates of E.cloacae from 36 patients in nine hospitals were examined. Byconventional typing, only 26 isolates (57.8%) could be assignedto a specific serotype, and 6 isolates (13.3%) were autoagglu-tinable owing to rough lipopolysaccharide antigens. All isolateswere assigned to one of the three biotypes of the species butcould be distinguished by their different phage susceptibilitypatterns. By combining the results of the three phenotypingapproaches, 29 different strains were identified. With threerestriction enzymes, EcoRI,BamnHI, and HindIII, 30 ribotypeswere identified. EcoRI patterns alone were sufficient to distin-guish between the great majority of strains, illustrating theimportance of the choice of restriction enzymes. EcoRI gen-erated the largest number of fragments over the widest sizerange. Overall, the agreement between ribotyping and thecombination of conventional typing methods was good(84.4%). The authors conclude that ribotyping is a highlydiscriminatory and reproducible method for typing E. cloacaebut that in most outbreaks it offers little in increased discrim-ination over traditional methods. One should mention, how-ever, that (i) the phenotyping techniques used in the study are

not widely available in clinical microbiology laboratories, (ii)all strains generated a clear ribotype, and (iii) discrepanciesbetween the two approaches were generally confined to sero-

logically untypeable strains or resulted from variability inbiotype codes.

Enterobacter sakazakiiThe study of Clark et al. (37) on Enterobacter sakazakii is

interesting to relate because ribotyping is compared with a

highly discriminative molecular phenotyping approach, mul-tilocus enzyme analysis. In addition, and in contrast to thework described above, it shows how ribotyping can identify an

exogenous source of infection. Two unrelated hospital out-breaks involving meningitis, bacteremia, and colonization ofneonates were investigated. In each of them, E. sakazakii was

isolated both from the patients and from dried infant formula.This extensive study combined antibiograms, plasmid analysis,total DNA RFLP analysis, ribotyping, and multilocus enzyme

electrophoresis to evaluate strain relatedness in each outbreak.Antibiograms determined by the disk agar diffusion methodoften varied among colonies selected from each isolate. Plas-mid analysis, total DNA RFLP, ribotyping, and multilocusenzyme electrophoresis all were effective for typing E. sakaza-kii strains, especially when used in combination. Multilocusenzyme analysis alone was more discriminative than any of theother methods. The typing results differed between the two

outbreaks, but in each of them, patients' isolates and isolatesfrom the formula shared the same typing pattern. Thus, E.sakazakii from contaminated dried infant formula had beenthe source of the neonatal infections (37).

Providencia stuartiiRadioactive labelling of the probes initially used for ribotyp-

ing represented an obvious limitation to the use of ribotypingin clinical microbiology laboratories. While studying the epi-

demiology of hospital strains of P. stuartii, Owen et al. (107)demonstrated that biotin was an excellent probe-reportermolecule and a suitable alternative to the radiolabelled probes

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used for ribotyping in many other studies. Here, 16S+23SrRNA from P. stuartii was used as a homologous probe. The 11rm RFLP profiles obtained for the 26 P. stuartii strains studiedafter digestion of DNA with EcoRI and HindIlI were quitedistinct from those obtained with strains of other Providenciaand Proteus species, thus illustrating the taxonomic value ofribotyping, as originally reported (58).

KiebsieUa pneumoniaeInvestigations conducted on multiresistant K pneumoniae

are of interest because they illustrate the advantages of ri-botypes compared with plasmid profiles.

Analysis of plasmid profiles was the first broadly applicablegenotypic method. By 1980, it had been used to study severaloutbreaks of multiresistant K pneumoniae infections in aneonatal intensive care unit in Virginia (93). In this study, thepresence of a single plasmid of about 106 kb, found in all of theisolates, was taken as evidence for the spread of a single strain.More recently, K pneumoniae strains which are simultaneouslyresistant to broad-spectrum cephalosporins and aminoglyco-sides because they produce the novel plasmid-mediated P-lac-tamases CTX-1 (137), SHV-2 (78), and SHV-3 (72) thathydrolyze oxyimino-,-lactams have been isolated. Epidemio-logical investigations of outbreaks with these organisms showthat plasmid analysis and ribotyping are complementary meth-ods that provide different types of information. In our hospital,over a 12-month period, 43 children in eight different wardswere infected or colonized with K pneumoniae strains produc-ing extended broad-spectrum ,-lactamase (25). Using twoenzymes, EcoRI and HindlIl, we observed 15 ribotypes amongthe 43 clinical strains, but 60% of the isolates in six wardsbelonged to two ribotypes only, and nine ribotypes wereobserved only once. Twelve isolates from different wards,representative of the most common eight ribotypes, showedseven different plasmid profiles by direct analysis or afterEcoRI digestion but only four different ,-lactamase isoelectricfocusing patterns. Thus, at least two genetically unrelatedstrains in the same ward had the same plasmid content. Theseresults showed the complexity of an outbreak in which patient-to-patient cross-contamination was associated with severalepidemic strains with different plasmid contents interspersedwith sporadic cases with nonepidemic strains and indicated thepossible spread of a plasmid. They show that plasmid profileanalysis by itself is not sufficient to identify the relationshipbetween isolates. Only the combination of plasmid analysis andribotyping was capable of delineating the details of this out-break, because the two approaches provided different butcomplementary information. Conclusions of previous studiesbased on plasmid analysis alone and suggesting that a singlestrain was responsible for outbreaks of multiresistant K pneu-moniae must therefore be taken with caution (8, 93). Evidently,a given plasmid may disseminate in different strains of thesame species and even among different species (103).

Acinetobacter calcoaceticus-Acinetobacter baumaniiGerner-Smidt considered ribotyping a reference method for

taxonomic studies and strain identification in the A. calcoace-ticus-A. baumanii complex (51) but did not compare ribotypingwith another genotyping method. Dijkshoorn et al. have usedribotyping and electrophoresis of cell envelope proteins tostudy the epidemiology of Acinetobacter isolates from hospitaloutbreaks (44). They found the two methods to have a

comparable discriminative power, higher than those of biotyp-ing and comparison of antibiograms, but the highest discrimi-nation was obtained when the results of the two methods were

combined. The authors also concluded that several markersshould be sought before isolates that are indistinguishable by agiven typing method are designated as representing the sameepidemic strain.

Xanthomonas maltophiliaWe used the analysis of total DNA RFLP and ribotyping to

study the relationship among 11 isolates of X. maltophiliaobtained from seven patients with nosocomial bacteremia infour distinct wards of our hospital (20). In this rather smallseries of isolates, results of the two approaches were in perfectagreement. Whereas analysis of biochemical profiles and anti-biotic susceptibilities would have led to the conclusion of thespread of two epidemiological strains throughout the hospital,the genotypic data indicated that cross-infection among pa-tients had occurred in two wards only and that cases in the twoother wards were independent infectious episodes.

Pseudomonas aeruginosaP. aeruginosa is an important organism to consider in terms

of nosocomial outbreaks, most particularly in pediatric clinicsbecause of the high incidence of infections among identifiablegroups of high-risk patients. For instance, it is the mostcommon cause of devastating pulmonary infections in cysticfibrosis (CF) patients. Despite this fact, the epidemiology ofthis pathogen is not well understood. Conflicting reports on thesource and spread of P. aeruginosa in a patient population areprimarily due to inadequate strain identification techniques.A number of phenotyping systems have been used for the

epidemiological investigation of P. aeruginosa, including deter-mination of antimicrobial susceptibility profiles, pyocin suscep-tibility or pyocin production, susceptibility to bacteriophages,biotyping, and 0 serotyping (120). Both pyocin type andbiotype are subject to change during the course of an apparentoutbreak, and antibiograms may change in response to antimi-crobial agent use (34). Changes in 0 serotype have beenreported after lysogenization with a bacteriophage in vitro(90), and some believe that similar changes may also occur invivo (83). Serotyping is of limited use if the 0-antigenicdeterminants are lost (105). Up to 70% of the strains isolatedfrom CF patients may be autoagglutinable or nontypeable (40).These factors contribute to the inability of current typingschemes to reliably establish relationships between individualisolates of P. aeruginosa.

In this context, the genotyping approach is of great interest:first, because chromosomal markers are rather stable, andsecond, because DNA can be extracted from any strain. Ogleet al. used RFLPs in the exotoxin A gene, exoA, and surround-ing sequences as epidemiological markers for P. aeruginosaisolates from CF patients (104). Some isolates that varied incolonial morphology, serotype, and biotype produced identicalRFLP patterns, indicating that phenotypic variation in P.aeruginosa does not necessarily reflect genetic heterogeneity.On the other hand, isolates from two unrelated patients thatwere indistinguishable by serotyping, biotyping, and antibio-grams were easily distinguished by Southern blot analysis ofexoA. Unfortunately, approximately 5% of P. aeruginosa strainslack exoA and thus are not typeable by this approach (131). Asa potential alternative, Samadpour et al. (131) and Speert et al.(140) used a probe derived from the gene encoding theantigenically variable P. aeruginosa pilin polypeptide. Variabil-ity in the pilin coding region is reflected by variations in theRFLP patterns obtained when the gene-specific probe is used.Samadpour et al. found that use of both the exotoxin A and thepilin probes was necessary to obtain a discrimination level high



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enough to be useful in the epidemiological survey of P.aeruginosa outbreaks (131). As might be expected, whenprobes targeted to a single locus are used, the results are

unpredictable. For instance, probes derived from algD andfrom the elastase gene were not useful in discriminating P.aeruginosa strains (91). In most cases, the discrimination levelwill increase only by combining data obtained by probingseveral informative loci. Along the same line, multilocusenzyme electrophoresis has been used successfully for epide-miological studies of P. aeruginosa strains (36, 85). Finally, use

of probes directed to specific virulence genes has the obviousdisadvantage of being restricted to a single bacterial species.

Pitt et al. were the first to use ribotyping for the study ofmultiresistant serotype 012 P. aenrginosa strains from Europe(121). Results agreed with those of outer membrane proteinelectrophoresis, lipopolysaccharide analysis, and esterase typ-ing in showing uniform characteristics suggestive of a commonorigin of the strains. Similar results were found by Grattard etal. (55). Denamur et al. compared ribotyping and esteraseelectrophoretic typing for differentiating 102 P. aeruginosaclinical isolates from 23 CF patients with chronic lung infection(40). All strains were typeable with this molecular approach.Three restriction enzymes were used in the study (HindIII,EcoRI, and BclI), which generated 16 ribotypes, while esteraseelectrophoresis revealed 25 zymotypes. Combination of dataobtained with the two systems led to the identification of 30different types. Interestingly, the data highlight the pathologi-cal complexity of P. aeruginosa infection in patients with CF, as

in six cases several types were found among the isolates fromindividual patients. On the other hand, two unique types werefound in two and three patients, respectively, raising thepossibility of cross-infections (40). Nosocomial acquisition ofP. aeruginosa by CF patients has also been reported by others(150). Using the same ribotyping and esterase typing scheme,Bingen et al. compared 27 P. aeruginosa strains isolated beforeand after 2-week courses of antipseudomonal treatment inseven CF patients (24). A total of 12 courses of therapy were

studied. In 10 cases, at least one of the posttherapy isolates wasfound to be resistant to the antimicrobial agent, and in 5 casesat least one of the posttherapy isolates was still susceptible.These data indicate that treatment failure in a given patientmay result from emergence of resistance and/or failure toeradicate a susceptible strain. Again, ribotyping was less dis-criminative than esterase electrophoresis. Among the 27 iso-lates, eight zymotypes and five ribotypes were identified.Resistant posttherapy isolates were identical to pretherapyisolates in all cases but one. However, in one case an additionalresistant strain was isolated after therapy. In all five cases inwhich susceptibility was still observed after treatment, pre-

therapy and posttherapy isolates were indistinguishable (24).Similar results were obtained by Ogle et al., who comparedpre- and posttherapy isolates of P. aeruginosa, using the exoAprobe (104). In most cases, failure to eradicate P. aeruginosawas due to the development of resistance rather than toreinfection. Yet, using an exoA probe, Wolz et al. (156)presented evidence that (i) cross-infection may occur in CFclinics as well as between siblings at home, (ii) P. aeruginosa isnot eradicated but only suppressed after successful antibiotictherapy, and (iii) CF patients normally harbor only one P.aeruginosa strain at a time in their sputa, but after 6 months thegenotype of the strain in sputum changes in 26% of theinfected patients.PFGE, as a molecular approach to studying P. aeruginosa

epidemiology (1, 33, 61), also was found to be more discrimi-native than ribotyping. For instance, Poh et al. studied a

collection of P. aeruginosa serotype 011 strains isolated in

seven hospitals in Singapore (122). With two restriction en-zymes, EcoRI and Sacl, they identified 11 ribotypes, whereasusing either SpeI or DraI and PFGE, they differentiated 41different strain types among the 44 clinical isolates.The apparently low sensitivity of ribotyping for the study of

P. aeruginosa strains might be related to a lack of optimizationof the technique and particularly to an insufficient search forthe most appropriate restriction enzyme(s). Blanc et al. (28)studied 55 epidemiologically unrelated isolates from threegeographic areas of Switzerland and 11 isolates obtainedduring an outbreak of P. aeruginosa infections in a burn unit.With four selected restriction enzymes (BamHI, ClaI, EcoRI,and PstI), the 55 unrelated isolates could be classified into 33ribotypes. To assess the value of the method for epidemiolog-ical investigations, the authors calculated an index of discrim-ination which takes into consideration both the number oftypes defined by the typing method and their relative frequen-cies. Their ribotyping system obtained a high index of discrim-ination of 0.958, comparing well with different typing schemesfor which indices of discrimination could be calculated fromthe published data. All clinical isolates from the outbreak inthe burn unit belonged to the same ribotype. Gruner et al. usedeight restriction enzymes to ribotype P. aeruginosa strains.They found that PvuII provided the highest discriminationbecause it alone allowed the discrimination of 15 of 18 strains(62). Table 1 summarizes the different molecular methods usedfor epidemiological studies of P. aeruginosa infections.

Pseudomonas cepacia

P. cepacia is increasingly recognized as an important patho-gen (127). Again, the understanding of the epidemiology ofinfections with P. cepacia has been impaired mostly by the lackof a standardized typing method. Conventional methods (66,128) have relied on the analysis of phenotypic characteristicsthat may not be stably expressed. Rabkin et al. (127) usedribotyping, serologic reactions, biochemical tests, bacteriocinproduction and susceptibility, and antimicrobial susceptibilityto study the relatedness of isolates obtained during sevenoutbreaks. This is the only study which uses tightly definedepidemiology as a gold standard for comparison of typingsystems. Ribotyping was the most accurate (97%) in detectingoutbreak concordance (127).

P. cepacia also has special relevance in patients with CF (88).Pulmonary infection with this organism results in a rapidclinical deterioration and death in some patients (145). Sincepulmonary colonization is generally refractory to antibiotictreatment, prevention of P. cepacia acquisition has become alogical goal in the management of these patients. The clustersof colonized patients found at certain CF centers and the highrisk of colonization associated with a recent hospital admissionor with the colonization of a sibling (145) suggest nosocomialacquisition and person-to-person transmission (146). Differentauthors have used ribotyping for the study of P. cepaciaepidemiology in CF (27, 88). For instance, LiPuma et al.analyzed isolates from 68 patients attending three CF centers(88). A predominant ribotype accounted for 52 to 61% of theisolates in each center. The predominant ribotype in eachcenter was different from the predominant ribotype in theother two. None of the other ribotypes were found in morethan two strains (88). These data are consistent with theepidemiological observations suggesting nosocomial acquisi-tion of this pathogen. However, the finding of unique ribotypesat each of the three centers also suggests that acquisition of P.cepacia occurs from unrelated and therefore unidentifiedsources. Previous studies that failed to identify an environmen-

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TABLE 1. Molecular method used for the epidemiological study of P. aeruginosa nosocomial infections

Ribotyping Other RFLPs PFGE Electrophoretic Reference(s)[probe/enzyme(s)] [probe/enzyme(s)] enzyme enzyme typing

E. coli rRNA 16S+23S/EcoRI, HindIll, BclI Esterase Denamur et al. (40), Bingen et al. (24)P. aeruginosa 16S+23S rRNA/EcoRI, Sacl SpeI, Dral Poh et al. (122)P. aeruginosa rRNA/EcoRI, HindIll, SmaI Esterase Pitt et al. (121)pKK 3535 containing E. coli rRNA operonBamHI, Clal, EcoRI, PstI Blanc et al. (28)PstI, SmaI, EcoRI, BclI, HindIll, ClaI, Gruner et al. (62)

SphI, PvuIIEcoRI, BamHI, HindIll Grattard et al. (55)

Pilin/PstI, HindIl Speert et al. (140)Pilin/BamHI, EcoRV, exoAl Samadpour et al. (131)BamHI, Sall, BclII

e-xoA/BglII, Sall, XhoI Ogle et al. (104), Wolz (156)algD/SalI, SmaI Loutit and Tompkins (91)

Multilocus Charnock and Bergan (36), Levin et al.(85)

tal reservoir support the possibility that nosocomial transmis-sion occurs by direct contact between CF patients. LiPuma etal. investigated the acquisition of P. cepacia by a CF patient(86). Ribotype analysis of the isolates recovered from thisindex patient and his contacts showed person-to-person trans-mission of the organism. This type of information is essential todevise effective prevention strategies to limit the acquisition ofthe pathogen by CF patients.The studies of LiPuma et al. (87) on P. cepacia are important

because they demonstrate the stability of the genetic markersexplored by ribotyping. Ribotypes from isolates that had beensubcultured 100 times in vitro were stable, and so were theribotypes of isolates recovered from chronically colonizedmice. Additional evidence for the stability of ribotypes in vivocomes from the results obtained with isolates collected fromchronically colonized CF patients (87), which led to theconclusion that most patients harbor the same strain forprolonged periods.

Panlilio et al. used ribotyping to document the occurrence ofP. cepacia pseudoinfections due to the intrinsic contaminationof a povidone-iodine product (109). Blood cultures from sixpatients in a Texas pediatric facility yielded P. cepacia after thenurses had wiped the tops of blood culture bottles with thepovidone-iodine solution before inoculation. P. cepacia iso-lates from the blood cultures and from the povidone-iodinehad similar antibiograms and identical plasmid profiles andribotypes. Pegues et al. also used ribotyping to study anoutbreak of P. cepacia bacteremia among patients with long-term indwelling central venous catheters (112).

Ribotyping was used together with PFGE to distinguish P.cepacia isolates from a nosocomial outbreak (6). Routinecultures for management of febrile illnesses at the MinneapolisVA Medical Center identified 40 isolates of P. cepacia from 18patients. Both PFGE and ribotyping revealed a single patternfor all 36 isolates from 16 of the 18 patients (89%). A seconddistinct pattern distinguished the four isolates from the tworemaining patients. Similarly, ribotyping and PFGE wereequally effective in differentiating all 25 control isolates unre-lated to the outbreak. Ribotyping was also compared withPFGE and with the recently introduced genotyping strategybased on the arbitrarily primed PCR (153, 154) for theepidemiological investigation of 23 P. cepacia isolates from 11CF patients (27). In that study, PFGE was slightly morediscriminative than the other two methods, and ribotyping andarbitrarily primed PCR were identical in resolving power. In

vivo stability of the patterns obtained with the three methodswas observed over periods extending from 3 to 41 months (27).

Legionella pneumophila

Legionella pneumophila is an important cause of nosocomialpneumonia, particularly among patients with impaired hostdefenses. Potable water systems, cooling towers, and respira-tory devices are among the environmental sources implicatedin outbreaks. Because of the ubiquity of Legionella isolates inaquatic habitats, precise epidemiological evaluations are veryimportant for the effective control of nosocomial outbreaks.Grimont et al. were the first to use ribotyping for the

taxonomic study of Legionella spp. (60). They identified a totalof 28 species of Legionella after digestion with either EcoRV orHindIll. Van Ketel and De Wever combined plasmid analysis,total DNA RFLP, and ribotyping to compare L. pneumophilastrains from six patients with six strains isolated from theenvironment (151). Strains from the patients, the air-condi-tioning system, and the cooling tower contained plasmids ofthe same molecular weight, but strains from the hot watersupply were plasmidless. The results of the three genotypingmethods correlated completely with each other in showing thateither the cooling tower or the air-conditioning system was theenvironmental source for the examined cluster of cases ofLegionnaires' disease.

Saunders et al. compared different types of probes forribotyping L. pneumophilia (133). The probes included cDNAstranscribed from Legionella or E. coli rRNA and a cloned L.pneumophila rRNA gene. The cloned homologous rRNA geneprobe gave the best discrimination. In the same study, ribotyp-ing was compared with RFLP analysis with anonymous, ran-domly selected cloned fragments of the Legionella chromo-some, none of which contained mr sequences. The greatestdiscrimination was achieved with one of these random probes.Because there was little correlation between the results ob-tained with the rRNA and random probes, the overall discrim-ination of RFLP typing was enhanced by combining the twosets of data. Tram et al. also used E. coli rRNA and a 15-kbrandomly cloned L. pneumophila DNA probe to investigatetwo nosocomial outbreaks at the Necker and Pitie hospitals inParis (149). None of the isolates studied harbored plasmids.The authors used a single restriction enzyme, HindlIl. A singlepattern was obtained when the filters were hybridized with therRNA probe, whereas three patterns were obtained after



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hybridization with the random probe. Results of this molecularanalysis showed that these two outbreaks of legionellosis wereunrelated and linked the outbreak at the Necker hospital tocontaminated tap water. In these two studies, the randomlycloned probe discriminated strains that were indistinguishableby ribotyping (149).

In the study by Schoonmaker et al., ribotyping and PFGEwere used to compare isolates of L. pneumophila obtainedfrom patients and the environment during a nosocomial out-break in the renal transplant unit of an upstate New Yorkhospital (134). Both techniques were successful at decipheringthe details of this complex outbreak. Of the three geneticallyunrelated strains observed in the patients, only two could berelated to an identified environmental source, which was thewater supply system. Three additional strains were found in thehospital environment. However, PFGE was more efficient thanribotyping in differentiating the epidemiologically unrelatedstrains that were studied as controls.

Moraxella (Branhamella) catarrhalis

To our knowledge, only one nosocomial outbreak of M.(Branhamella) catarrhalis has been documented to date by agenotypic method (110). The study used RFLP of total DNAand showed that the strains from five patients were identical tostrains isolated from two staff members. Denamur et al. (41)compared total DNA RFLP and ribotyping for the epidemio-logical comparison of M. catarrhalis strains. The strains studiedhad been selected for their diversity by esterase electrophoreticanalysis and belonged to 20 distinct zymotypes. All strainscould be differentiated by either ribotyping or RFLP of totalDNA, but the use of five restriction enzymes was necessary toobtain such a level of discrimination with ribotyping.

Rhodococcus spp.

Rhodococcus species are ubiquitous in the environment, andseveral of them have been reported to be potentially patho-genic for humans. Rhodococcus equi, in particular, can causeinfections in patients with AIDS (84). Identification of Rhodo-coccus species by conventional biochemical tests is problem-atic, and there is no other simple and reproducible method fortheir rapid identification and differentiation. Lasker et al.found that ribotyping could clearly distinguish the type strainsof the 20 recognized species in the genus Rhodococcus (84). Intheir study, they used only two restriction enzymes, PvuI andPstl, and a digoxigenin-labelled E. coli rDNA probe. Analysisof four clinical or environmental isolates confirmed as Rhodo-coccus bronchialis showed no interstrain variation of ribotype,but analysis of 15 isolates confirmed as R. equi and isolatedfrom 13 patients showed 11 different ribotypes. No discernibledifferences were observed in the ribotype patterns between R.equi isolates from AIDS patients and those from patients whodid not have AIDS, and there was no evidence of geographicclustering of R. equi ribotype patterns. Therefore, ribotypeanalysis may provide an important adjunct to the identificationand epidemiological study of environmental and clinical iso-lates of Rhodococcus species (84). Richet et al. analyzedplasmid profiles, total DNA RFLP, and ribotypes to study ahospital outbreak of surgical would infections caused by R.bronchialis (129). The last two methods were equally discrim-inatory in demonstrating that a single genetically distinct strainwas involved in the outbreak and traced its source to the dogof a circulating operating room nurse.

Tsukamurella paurometabolum

From January 1988 to May 1989, 12 strains of T. pauro-metabolum were identified in samples from 10 patients with noidentifiable common risk factors at a hospital in South Caro-lina (10). Case control studies revealed that the positivespecimens had been tissue samples, all of which had beenprocessed in the tuberculosis-fungal room of the laboratoryand handled by the same technician. Total DNA RFLPanalysis and ribotyping were used in addition to biochemicaltests and antibiograms to study this apparent outbreak. Thediscriminative powers of the two genotypic methods wereidentical. The results showed that the patients were notinfected and that contamination of the samples had occurredduring handling in the laboratory. Apparently, T pauronietab-olumn is present both in the environment and as a culturecontaminant more often than previously recognized but israrely the true cause of infection in humans.

Enterococcus spp.

Until recently, epidemiological studies of enterococci havebeen limited by the lack of convenient and accessible methodsfor comparing strains. Vancomycin-resistant enterococci rep-resent a special problem in terms of nosocomial infections asthey are found with increasing frequency. We conducted anepidemiological investigation on 16 vancomycin-resistant En-terococcus faeciumn strains isolated from 15 patients in fourdifferent wards of our children's hospital over a period of 17months (22). The analysis combined RFLP of total DNA andribotyping. Each strain produced a different total DNA RFLPpattern after both HindllI and PvuII digestions except for twostrains that were isolated from a single patient. Ribotyping wasless discriminatory than RFLP of total DNA. This studyshowed the genetic unrelatedness of the nosocomial strainsstudied and excluded the possibility of patient-to-patient straintransmission either in the same ward or between wards.Woodford et al. used a probe specific for the glycopeptideresistance gene, vanA, in combination with ribotyping to studya cluster of vancomycin-resistant Enterococcus faecalis and E.faecium isolates from patients in the renal unit at a Londonhospital (157). As in our study, the 23 vancomycin-resistant E.faecium isolates showed a heterogeneity, evidenced by eightdifferent ribotypes, which suggested that multiple strains wereinvolved. But in addition, 21 of the 23 isolates harbored a24-MDa plasmid that hybridized with the vanA probe, implyingthat interstrain dissemination of a vancomycin resistance plas-mid may have occurred. In contrast, 12 of the 13 E. faecalisisolates studied exhibited a common ribotype, and the vanAprobe hybridized with chromosomal DNA in these 12 isolates.The other isolate had a different ribotype, and vanzA waslocated on plasmid DNA. These data suggest that cross-infection with a single strain of vancomycin-resistant E. faecalishad occurred in all cases but one. Because vanA can be locatedeither on chromosomal DNA or on a plasmid, details ofoutbreaks with vancomycin-resistant enterococci clearly can beobtained only from an approach that combines analysis ofchromosomal polymorphism and of plasmid DNA and prefer-ably includes the use of a vanA probe. The ability of ribotypingto distinguish strains of Enterococcus spp. at the subspecieslevel was compared with that of PFGE. Ribotyping with threeenzymes, EcoRI, HinidIII, and BscI, appears to be less discrim-inatory than PFGE for strain differentiation (53, 65).

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Streptococcus pyogenes

Together with RFLP of total DNA, ribotyping was used forstrain differentiation of S. pyogenes (19). This typing approachwas used to document vertical mother-to-infant transmissionof S. pyogenes and to investigate the spread of the organism inan obstetrics unit (19). Two isolates from a newborn, twoisolates from his mother, and two isolates from two othermothers were studied. RFLP of total DNA, after both Hindllland PvuII digestions, gave indistinguishable patterns for thestrains isolated from the neonate, his mother, and one of theother mothers. On the contrary, strains from the third motherand six unrelated strains studied for comparison showeddifferent patterns. In this system, ribotyping was less discrimi-native than total DNA RFLP analysis.

Staphylococcus aureusRibotyping was used to differentiate species of Staphylococ-

cus (39, 147). Hadorn et al. combined conventional bacterio-phage typing and ribotyping for the analysis of methicillin- andciprofloxacin-resistant S. aureus strains that had been isolatedwith increasing frequency since the introduction of the new4-quinolones at the Tel Aviv Medical Center (64). Ribotypeswere established after digestion of bacterial DNA with EcoRIand HindlIl. The patterns obtained were identical for all of thenosocomial strains but one and different for all unrelatedstrains studied as controls. Bacteriophage typing was in goodagreement with these genotypic results. Thus, the increasingoccurrence of resistant S. aureus was due to the spread of aresistant strain rather than to the development of resistance inmany strains (64). However, rather different results were foundby Blumberg et al. while investigating the emergence andspread of ciprofloxacin-resistant S. aureus at the AtlantaVeterans Administration Medical Center (30). These authorsalso used ribotyping and phage typing together with plasmidanalysis. For ribotyping, they first tested the DNA of selectedisolates with 20 different restriction enzymes and then theyused EcoRI only, because it gave the best discrimination. Bygenerating 15 different patterns among the 50S. aureus isolatesstudied, ribotyping was highly informative when comparedwith the other two techniques. Indeed, many of the isolateswere nontypeable by phages or harbored few plasmids thatwere useful as markers. The results demonstrated that, in thatinstitution, ciprofloxacin resistance had emerged in multiplestrains of methicillin-resistant S. aureus but from a single cloneof methicillin-susceptible S. aureus.The study of Preheim et al. is of interest because it relates

the investigation of 46 strains of methicillin-susceptible and-resistant S. aureus of various phage types and resistotypesfrom seven countries (125). In addition, the authors demon-strate that, in their series, ribotyping was not affected bychanges in plasmid, transposon, enterotoxin A, or phagecontent. S. aureus DNA was restricted with HindIll or EcoRIand probed with a homologous rm cDNA probe. Analysis ofthe results included calculation of the percentage of similarity,using the Dice coefficient (43), and clustering was based on theunweighted-pair-group arithmetic average algorithm. Epi-demic methicillin-resistant isolates from Australia and fromthe United Kingdom showed a high degree of similarity, butthe pattern was not unique and was also found in methicillin-susceptible and other methicillin-resistant strains. Again, all ofthe strains could be typed, including the non-phage-typeablestrains. Thus, ribotyping may provide new clues to the clonalevolution of the S. aureus population.

Prevost et al. (126) compared PFGE and ribotyping for thedifferentiation of methicillin-resistant S. aureus isolates. After

restriction of chromosomal DNA from 239 strains from 142patients, PFGE produced 26 different fingerprints. The de-duced size of the bacterial chromosome ranged from 2,200 to3,100 kb (±100 kb). A total of 81 isolates taken from 65patients were then typed by PFGE and ribotyping with ClaI,EcoRI, and HindIII. Ribotypes were less discriminating thanPFGE.

Coagulase-Negative Staphylococcus spp.

Bialkowska-Hobrzanska et al. first used ribotyping andRFLP of total DNA to type 53 clinical isolates of coagulase-negative staphylococci and 25 reference type strains from theAmerican Type Culture Collection (15). Both methods clearlydistinguished all 15 species of coagulase-negative staphylococciand most individual strains within each species. Eight restric-tion enzymes were tested for ribotyping, and the most discrim-inating results were obtained with ClaI. An exception wasStaphylococcus wameri, for which HpaI and AvaI were morespecific than ClaI for strain differentiation. The patternsproduced by ribotyping were much simpler and thus easier tointerpret in this rather large series than were correspondingpatterns of total DNA RFLP. However, ribotyping was slightlyless discriminating.

Since then, ribotyping has been applied to other epidemio-logical studies of coagulase-negative staphylococci (38, 71,155). Eighty-six S. epidermidis strains were ribotyped intraspe-cifically by Izard et al. (71). Eleven ribotypes were found afterdigestion with EcoRI, and 10 ribotypes were found withHindIII. Use of a combination of the two restriction enzymesincreased the discriminatory power from 14.3 to 31.6%. Com-bining ribotyping with biotyping raised the discriminatorypower to 48.6%. Ribotypes were stable after more than 400generations of growth. Among the tested S. epidermidis strains,no correlation was observed between methicillin resistance andcertain ribotypes. Wilton et al. evaluated several techniques forthe epidemiological typing of S. epidermidis isolates frompatients in an intensive care unit and from neonates (155).RFLPs studied with the enzyme BclI were effective epidemio-logical markers. total DNA RFLP profiles were highly discrim-inative but difficult to interpret. Profiles obtained with a clonedE. coli rm probe were less discriminative and thus less suitablefor intraspecies typing than those generated with a panel ofrandomly cloned genomic fragments of S. epidermidis DNA.The three approaches were reproducible and superior totyping on the bases of determination of antigen and plasmidprofiles, antibiotic susceptibility patterns, and biotypes.

Other Gram-Positive BacteriaUsing ribotyping and the analysis of plasmid content and

antibiotic susceptibility, Pitcher et al. demonstrated that strainsof C. jeikeium may be transferred between hospital patients(118). Patterns of antibiotic susceptibility appeared not to beplasmid encoded and correlated broadly with the ribotypes.

MiscellaneousRibotyping has also been applied to other bacterial species,

among which some might be involved in nosocomial infections.These include Serratia marcescens (26), E. coli (2, 9, 17, 23, 89,114), Proteus mirabilis (16), Salmonella spp. (3, 94, 101, 117),Shigella spp. (46, 68, 142), Yersinia enterocolitica (29, 116),Aeromonas sp. (100), Campylobacter spp. (77), Neisseria men-ingitidis (158), Vibrio cholerae (79), Haemophilus spp. (70, 143),Leptospira spp. (113), and Listeria monocytogenes (12, 56). ForSalmonella typhi, ribotyping may be the only subtyping method



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that offers sufficient discrimination for use in epidemiologicalinvestigations (3, 45, 101). Interestingly, ribotypes have alsobeen related to virulence in E. coli (114) and H. influenzae (70).

WHAT TO EXPECT FROM RIBOTYPING INNOSOCOMIAL OUTBREAKS

Obviously, our goal when investigating a nosocomial out-break is to determine if two isolates are identical. But do wereally know what two identical strains are? Stricto sensu, theanswer should be: two strains for which the complete nucle-otide sequences of both chromosomal and extrachromosomalDNA are identical. In that case, molecular epidemiologyshould provide the ultimate answer. But in the context ofnosocomial infection, this is not necessarily so and not onlybecause of technological limitations. The same strain may havebeen transmitted from one patient to another but nucleotidesubstitutions may have occurred on the chromosome, at firstsilent and neutral, but then with phenotypic consequences, ora plasmid may have been acquired or lost. Because we do notyet have the tools to determine the complete nucleotidesequence of bacterial DNA in a few hours, nor do we knowexactly what constitutes two identical strains in a nosocomialinfection, we have no way to identify the gold standard withwhich the results of a new typing procedure can be comparedto appreciate its exact value for investigating nosocomialinfections.

Ribotyping is relatively easy to implement in the clinicalmicrobiology laboratory, and ribotyping always provides re-sults. This method is advantageous over the classical pheno-typic methods for some strains that are untypeable, such asmucoid P. aeruginosa strains in patients with CF, or strains thatare extremely difficult to type, such as Legionella or Rhodococ-cus spp. In many other instances, ribotyping easily demon-strates a genetic diversity that is not reflected by classicalphenotyping (Acinetobacter and Pseudomonas species). But dothe results of ribotyping always lead to the correct answer? Iftwo strains exhibit different ribotypes, they most probably aredifferent. This has been confirmed by the good correlationbetween the results of ribotyping and phenotypic methods forspecies for which the latter techniques are highly discrimina-tive. But as already mentioned, the discriminatory thresholdthat makes differences relevant in the context of nosocomialoutbreaks is not known. In all cases, good judgment must beused before a conclusion is reached, especially when identicalribotypes suggest that two strains are identical. Understandingthe limitations of ribotyping, confirming its results with thoseof the other typing procedures, and, most importantly, corre-lating results with clinical epidemiological data are essential.The limitations of ribotyping for the investigation of noso-

comial outbreaks must be considered at different levels. Thefirst one concerns the general limitations of typing in nosoco-mial infections. When the studied species is present in alimited number of clones worldwide, as has been suggested forgroup B streptococci (42), methicillin-resistant S. aureus (81),or multiresistant serotype 012 P. aeruginosa (55, 121), forexample, then neither a phenotypic nor a genotypic techniqueis likely to be useful and the investigation will have to rely onclassical epidemiological studies. This is even more true when,among the various clones of a species, only one is virulent, e.g.,H. influenzae biogroup aegyptus for Brazilian purpuric fever(70) or the E. coli strain responsible for hemolytic uremicsyndrome (92).The second consideration is in cases in which ribotyping is

less discriminatory than analysis of total DNA RFLP byconventional electrophoresis or by PFGE or when randomly

cloned chromosomal probes are used. In these instances, twoexplanations should be considered. Some of these cases mayrepresent a real limitation if the extent of rm RFLP is smallerthan that of total DNA. After all, rm operons cover only about0.1% of chromosomal DNA. This is likely to happen forspecies in which the number of rn operons is small. It is alsopossible that, in some species, because of the clustering of rnloci in a very small region of the chromosome, ribotypes wouldnot reflect the overall extent of RFLP. In E. coli, four of theseven nn operons span 10% of the genome only. However, inmany instances, ribotypes are established from patterns ob-tained with only one, two, or three restriction enzymes so thatthe maximal level of sensitivity of the method has not beenreached. Table 2 lists the different restriction endonucleasesused to study the organisms discussed in this review. Increasingthe number of enzymes increases the labor intensity but alsoincreases discriminatory power (31, 41, 123, 158, 159).The most discriminative typing schemes may be those that

analyze several loci evenly scattered on the chromosomebecause they are most likely to reflect the overall genomicDNA polymorphism. Phenotypic analysis by multilocus en-zyme electrophoresis (136), and probably esterase electro-phoresis (116), meet these criteria. Accordingly, they arehighly discriminative and correlate directly with the genotype.PFGE or use of cloned random probes provides similar results,but these methods require specific equipment and long andcomplex experimental protocols or probes specific for onespecies only. However, it is fair to state that laboratoriesperforming PFGE on a routine basis are quite comfortablewith this approach and may consider the blotting proceduresand labelling techniques required for ribotyping to be difficult.

In many of the examples of application given above, ribotyp-ing was a good compromise because its discrimination wasappropriate for the species under investigation. In all cases, itwas superior to gene-specific probes because they are targetedto one locus only, most often a virulence gene which may notbe present in all strains of a species (92, 131), and which alsomay be horizontally transferred (9). Clearly, ribotyping is not a"miracle" method, but neither are any of the other typingmethods. Combining all available stable markers, both geno-typic and phenotypic, is probably the best way to appreciateoverall genomic DNA polymorphism (76) and thus to obtainhighly discriminatory data which, properly combined withtightly defined epidemiological parameters, are likely to pro-vide the most relevant answers to nosocomial outbreaks.

FUTURE TRENDS

Ribotyping has provided invaluable information during theinvestigation of many nosocomial infections. Since the devel-opment of cold-labelling systems, radioactive isotopes are nolonger an obstacle to its implementation in the clinical micro-biology laboratory. Computer-assisted image analysis and sta-tistical treatment of the data (107, 138) will probably speed upand improve the reliability of RFLP pattern comparison (144).A few machines for automated DNA purification are commer-cially available, and some have been developed for automatedSouthern blotting during the conduction of the Human Ge-nome Project. At present, they are highly sophisticated andexpensive pieces of equipment, and it is unlikely that thenumber of samples to be treated will justify their installation ina clinical microbiology laboratory, except perhaps in somereference centers.Over the past few years, PCR has revolutionized the ap-

proach to studying DNA by the gain of time it provides andalso because only minute quantities of DNA are necessary. In

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TABLE 2. Summary of nosocomial microbial species investigated by ribotyping with the different restriction endonucleases useda

Organisms Restriction endonuclease(s) used Reference

Enterobacter cloacae EcoRI, BamHI Bingen et al. (21)EcoRI, BamHI, HindlIl Garaizar et al. (50)

Enterobacter sakazakii HindIII, BamHI Clark et al. (37)Providencia stuartii EcoRI, HindIII Owen et al. (107)Klebsiella pneumoniae EcoRI, HindIll Bingen et al. (25)Acinetobacter spp. EcoRI, Clal, Sall Gerner-Smidt (51)

EcoRI, HindIII Dijkshoorn et al. (44)Xanthomonas maltophilia EcoRI, Clal, SalI Bingen et al. (20)Pseudomonas cepacia EcoRI LiPuma et al. (88)

EcoRI Panlilio et al. (109)EcoRI Bingen et al. (27)EcoRI Anderson et al. (6)EcoRI, Clal Pegues et al. (112)

Legionella spp. EcoRV, HindlIl Grimont et al. (60)EcoRI, HindIII Van Ketel and De Wever (151)ClaI, NciI Saunders et al. (133)HindIII Tram et al. (149)EcoRI, HpaI Schoonmaker et al. (134)

Moraxella catarrhalis PstI, TaqI, EcoRV, HaeIII, Hinfl Denamur et al. (41)Rhodococcus spp. PvuII, PstI Lasker et al. (84)Rhodococcus bronchialis PvuII Richet et al. (129)Tsukamurella paurometabolum PstI Auerbach et al. (10)Enterococcus spp. HindIll, PvuII Bingen et al. (22)

BamHI Woodford, et al. (157)EcoRI, HindIll, Bscl Hall et al. (65)EcoRI, Hindlll Gordillo et al. (53)

Streptococcus pyogenes HindIll, PvuII Bingen et al. (19)Staphylococcus aureus EcoRI, HindlIl Hadorn et al. (64)

EcoRI Blumberg et al. (30)HindIII, EcoRI Preheim et al. (125)HindIll, EcoRI, ClaI Prevost et al. (126)

Coagulase-negative staphylococci Clal Bialkowska-Hobrzanska et al. (15)Staphylococcus warnei HpaI, AvaI Bialkowska-Hobrzanska et al. (15)Staphylococcus epidermidis EcoRl, HindIII Izard et al. (71)

Bcll Wilton et al. (155)Corynebacterium jeikeium Hindlll, Pvull Pitcher et al. (118)" Data for P. aeruginosa are given in Table 1.

addition, DNA purity is no longer a crucial factor so that wholecells (at least for gram-positive bacteria) can be used directly inthe PCR tube and DNA purification is no longer necessary(96). As an alternative to classical ribotyping, length polymor-phisms have been sought in the 16S-23S spacer regions ofbacterial rRNA genes by the use of PCR with oligonucleotideprimers complementary to the highly conserved sequencesflanking the spacer regions (80). By this method, variation inlength of the amplified DNA fragments (PCR ribotyping)distinguished unrelated isolates of P. cepacia, whereas isolatesimplicated in person-to-person transmission produced identi-cal amplification patterns. The full potential of this newapproach to ribotyping remains to be appreciated.

CONCLUSIONS

Ribotyping has now been used for the study of all bacterialspecies most frequently responsible for nosocomial infections.In vitro and in vivo stability of the studied markers has beenshown. Although there are limitations to the technique, inmany cases it compares well with other genotypic methods andis generally easier to implement in the clinical microbiologylaboratory. It was found to be particularly discriminative formembers of the Enterobacteriaceae, P. cepacia, and X malto-philia. In many of the reported examples, it greatly improvedthe understanding of the mechanism of nosocomial acquisitionof organisms by allowing distinction between endogenous and

exogenous infections and, in the latter case, between individualand epidemic strains, thus allowing differentiation of cross-infection from independent acquisition. It is possible, however,that this method will be superseded in the future by still easierto use PCR-based techniques.

ACKNOWLEDGMENTS

We are extremely thankful to Rajagopal Krishnamoorthy andBertrand Picard for enlightening nightly discussions on ribotyping andto Michelle Fageon for typing the manuscript. E.D. is particularlygrateful to Francine and Patrick A. D. Grimont for teaching him thebases of molecular epidemiology.Our work is supported by institutional funding of the Unite 120 by

the Institut National de la Sante et de la Recherche Medicale(INSERM), by a Contrat de Recherche Externe (INSERM-CRE92-0602), and by grants from the 1993 cooperative research programbetween INSERM and the Caisse Nationale d'Assurance Maladie desTravailleurs Salaries (CNAMTS), from the Delegation a la RechercheClinique de l'Assistance Publique-H6pitaux de Paris, and from theUniversite Denis Diderot-Paris 7.

REFERENCES1. Allardet-Servent, A., N. Bouzigues, M. J. Carles-Nurit, G. Bourg,

A. Gouby, and M. Ramuz. 1989. Use of low-frequency-cleavagerestriction endonucleases for DNA analysis in epidemiologicalinvestigations of nosocomial bacterial infections. J. Clin. Micro-biol. 27:2057-2061.

2. Alos, J. I., T. Lambert, and P. Courvalin. 1993. Comparison of



R6

http://cmr.asm

.org/D

ownloaded from

RIBOTYPING 323

two molecular methods for tracing nosocomial transmission ofEscherichia coli KI in a neonatal unit. J. Clin. Microbiol. 31:1704-1709.

3. Altwegg, M., F. W. Hickman-Brenner, and J. J. Farmer III. 1989.Ribosomal RNA gene restriction patterns provide increasedsensitivity for typing Salmonella typhi strains. J. Infect. Dis.160:145-149.

4. Altwegg, M., and L. W. Mayer. 1989. Bacterial molecular epide-miology based on a nonradioactive probe complementary toribosomal RNA. Res. Microbiol. 140:325-333.

5. Amikam, D., G. Glaser, and S. Razin. 1984. Mycoplasmas(Mollicutes) have a low number of rRNA genes. J. Bacteriol.158:376-378.

6. Anderson, D. J., J. S. Kuhns, M. L. Vasil, D. N. Gerding, andE. N. Janoff. 1991. DNA fingerprinting by pulsed-field gelelectrophoresis and ribotyping to distinguish Pseudomonas cepa-

cia isolates from a nosocomial outbreak. J. Clin. Microbiol.29:648-649.

7. Anderson, R. P., and J. R. Roth. 1977. Tandem genetic duplica-tions in phage and bacteria. Annu. Rev. Microbiol. 31:473-505.

8. Arlet, G., M. J. Sanson-le Pors, M. Rouveau, G. Fournier, 0.

Marie, B. Schlemmer, and A. Philippon. 1990. Outbreak ofnosocomial infections due to Klebsiella pneumoniae producingSHV-4 ,B-lactamase. Eur. J. Clin. Microbiol. Infect. Dis. 9:797-803.

9. Arthur, M., R. D. Arbeit, C. Kim, P. Beltran, H. Crowe, S.Steinbach, C. Campanelli, R. A. Wilson, R. K. Selander, and R.Goldstein. 1990. Restriction fragment length polymorphismamong uropathogenic Escherichia coli isolates: pap-related se-

quences compared with rn- operons. Infect. Immun. 58:471-479.10. Auerbach, S. B., M. M. McNeil, J. M. Brown, B. A. Lasker, and

W. R. Jarvis. 1992. Outbreak of pseudoinfection with Tsuka-murella paurometabolum traced to laboratory contamination:efficacy of joint epidemiological and laboratory investigation.Clin. Infect. Dis. 14:1015-1022.

11. Bacot, C. M., and R. H. Reeves. 1991. Novel tRNA gene

organization in the 16S-23S intergenic spacer of the Streptococcuspneumoniae rRNA gene cluster. J. Bacteriol. 173:4234-4236.

12. Baloga, A. O., and S. K. Harlander. 1991. Comparison ofmethods for discrimination between strains of Listena monocy-

togenes from epidemiological surveys. AppI. Environ. Microbiol.57:2324-2331.

13. Baril, C., C. Richaud, G. Baranton, and I. Saint-Girons. 1989.Linear chromosome of Borrelia burgdorfen. Res. Microbiol. 140:507-516.

14. Bercovier, H., 0. Kafri, and S. Sela. 1986. Mycobacteria possess

a surprisingly small number of ribosomal RNA genes in relationto the size of their genome. Biochem. Biophys. Res. Commun.136:1136-1141.

15. Bialkowska-Hobrzanska, H., V. Harry, D. Jaskot, and 0. Ham-merberg. 1990. Typing of coagulase-negative staphylococci bySouthern hybridization of chromosomal DNA fingerprints usinga ribosomal RNA probe. Eur. J. Clin. Microbiol. Infect. Dis.9:588-594.

16. Bingen, E., C. Boissinot, P. Desjardins, H. Cave, N. Brahimi, N.Lambert-Zechovsky, E. Denamur, P. Blot, and J. Elion. 1993.Arbitrarily primed polymerase chain reaction provides rapiddifferentiation of Proteus mirabilis isolates from a pediatrichospital. J. Clin. Microbiol. 31:1055-1059.

17. Bingen, E., H. Cave, Y. Aujard, N. Lambert-Zechovsky P. Des-jardins, J. Elion, and E. Denamur. 1993. Molecular analysis ofmultiply recurrent meningitis due to Escherichia coli KI in an

infant. Clin. Infect. Dis. 16:82-85.18. Bingen, E., E. Denamur, N. Lambert-Zechovsky, Y. Aujard, N.

Brahimi, P. Geslin, and J. Elion. 1992. Analysis of DNA restric-tion fragment length polymorphism extends the evidence forbreast milk transmission in Streptococcus agalactiae late-onsetneonatal infection. J. Infect. Dis. 165:569-573.

19. Bingen, E., E. Denamur, N. Lambert-Zechovsky, C. Boissinot, N.Brahimi, Y. Aujard, P. Blot, and J. Elion. 1992. Mother-to-infantvertical transmission and cross-colonization of Streptococcus pyo-genes confirmed by DNA restriction fragment length polymor-phism analysis. J. Infect. Dis. 165:147-149.

20. Bingen, E., E. Denamur, N. Lambert-Zechovsky, A. Bourdois, P.Mariani-Kurkdjian, J. P. Cezard, J. Navarro, and J. Elion. 1991.DNA restriction fragment length polymorphism differentiatescrossed from independent infections in nosocomial Xanthomonasmaltophilia bacteremia. J. Clin. Microbiol. 29:1348-1350.

21. Bingen, E., E. Denamur, N. Lambert-Zechovsky, N. Brahimi, M.El Lakany, and J. Elion. 1992. Rapid genotyping shows theabsence of cross-contamination in Enterobacter cloacae nosoco-mial infections. J. Hosp. Infect. 21:95-101.

22. Bingen, E., E. Denamur, N. Lambert-Zechovsky, and J. Elion.1991. Evidence for the genetic unrelatedness of nosocomialvancomycin-resistant Enterococcus faecium strains in a pediatrichospital. J. Clin. Microbiol. 29:1888-1892.

23. Bingen, E., E. Denamur, B. Picard, P. Goullet, N. Lambert-Zechovsky, N. Brahimi, J. C. Mercier, F. Beaufils, and J. Elion.1992. Molecular epidemiology unravels the complexity of neona-tal Escherichia coli acquisition in twins. J. Clin. Microbiol.30:1896-1898.

24. Bingen, E., E. Denamur, B. Picard, P. Goullet, N. Lambert-Zechovsky, P. Foucaud, J. Navarro, and J. Elion. 1992. Molecularepidemiological analysis of Pseudomonas aeruginosa strains caus-ing failure of antibiotic therapy in cystic fibrosis patients. Eur. J.Clin. Microbiol. Infect. Dis. 11:432-437.

25. Bingen, E., P. Desjardins, G. Arlet, F. Bourgeois, P. Mariani-Kurkdjian, N. Lambert-Zechovsky, E. Denamur, A. Philippon,and J. Elion. 1993. Molecular epidemiology of plasmid spreadamong extended broad-spectrum 3-lactamase-producing Kleb-siella pneumoniae in a pediatric hospital. J. Clin. Microbiol.31:179-184.

26. Bingen, E., P. Mariani-Kurkdjian, N. Lambert-Zechovsky, P.Desjardins, E. Denamur, Y. Aujard, E. Vilmer, and J. Elion.1992. Ribotyping provides efficient differentiation of nosocomialSerratia marcescens isolates in a pediatric hospital. J. Clin.Microbiol. 30:2088-2091.

27. Bingen, E., M. Weber, J. Derelle, N. Brahimi, N. Lambert-Zechovsky, M. Vidailhet, J. Navarro, and J. Elion. 1993. Arbi-trarily-primed polymerase chain reaction as a rapid method todifferentiate crossed from independent Pseudomonas cepaciainfections in cystic fibrosis patients. J. Clin. Microbiol. 31:2589-2593.

28. Blanc, D. S., H. H. Siegrist, R. Sahli, and P. Francioli. 1993.Ribotyping of Pseudomonas aeruginosa: discriminatory powerand usefulness as a tool for epidemiological studies. J. Clin.Microbiol. 31:71-77.

29. Blumberg, H. M., J. A. Kiehibauch, and I. K. Wachsmuth. 1991.Molecular epidemiology of Yersinia enterocolitica 0:3 infections:use of chromosomal DNA restriction fragment length polymor-phism of rRNA genes. J. Clin. Microbiol. 29:2368-2374.

30. Blumberg, H. M., D. Rimland, J. A. Kiehlbauch, P. M. Terry, andI. K. Wachsmuth. 1992. Epidemiologic typing of Staphylococcusaureus by DNA restriction fragment length polymorphisms ofrRNA genes: elucidation of the clonal nature of a group ofbacteriophage-nontypeable, ciprofloxacin-resistant, methicillin-susceptible S. aureus isolates. J. Clin. Microbiol. 30:362-369.

31. Blumberg, H. M., D. S. Stephens, C. Licitra, N. Pigott, R.Facklam, B. Swaminathan, and I. K. Wachsmuth. 1992. Molec-ular epidemiology of group B streptococcal infections: use ofrestriction endonuclease analysis of chromosomal DNA andDNA restriction fragment length polymorphisms of ribosomalRNA genes (ribotyping). J. Infect. Dis. 166:574-579.

32. Boros, I., A. Kiss, and P. Venetianer. 1979. Physical map of theseven ribosomal RNA genes of Escherichia coli. Nucleic AcidsRes. 6:1817-1830.

33. Boukadida, J., M. de Montalembert, G. Lenoir, P. Scheinmann,M. Veron, and P. Berche. 1993. Molecular epidemiology ofchronic pulmonary colonisation by Pseudomonas aeruginosa incystic fibrosis. J. Med. Microbiol. 38:29-33.

34. Brokopp, C. D., and J. J. Farmer. 1979. Typing methods forPseudomonas aeruginosa, p. 89-133. In R. G. Doggett (ed.),Pseudonmonas aeruginosa: clinical manifestations of infections andcurrent therapy. Academic Press, Inc., New York.

35. Brosius, J., A. Ullrich, M. A. Raker, A. Gray, T. J. Dull, R. R.Gutell, and H. F. Noller. 1981. Construction and fine mapping of

VOL. 7, 1994


R6

http://cmr.asm

.org/D

ownloaded from

324 BINGEN ET AL.

recombinant plasmids containing the rnB ribosomal RNAoperon of E. coli. Plasmid 6:112-118.

36. Charnock, C., and T. Bergan. 1992. Multilocus enzyme electro-phoresis of major 0-antigen reference strains of Pseudomonasaeruginosa. Eur. J. Clin. Microbiol. Infect. Dis. 11:810-816.

37. Clark, N. C., B. C. Hill, C. M. O'Hara, 0. Steingrimsson, andR. C. Cooksey. 1990. Epidemiologic typing of Enterobacter saka-zakii in two neonatal nosocomial outbreaks. Diagn. Microbiol.Infect. Dis. 13:467-472.

38. Cookson, B. D., P. Stapleton, and H. Ludlam. 1992. Ribotyping ofcoagulase-negative staphylococci. J. Med. Microbiol. 36:414-419.

39. De Buyser, M. L., A. Morvan, F. Grimont, and N. El Solh. 1989.Characterization of Staphylococcus species by ribosomal RNAgene restriction patterns. J. Gen. Microbiol. 135:989-999.

40. Denamur, E., B. Picard, P. Goullet, E. Bingen, N. Lambert, andJ. Elion. 1991. Complexity of Pseudomonas aeruginosa infectionin cystic fibrosis: combined results from esterase electrophoresisand rDNA restriction fragment length polymorphism analysis.Epidemiol. Infect. 106:531-539.

41. Denamur, E., N. Picard-Pasquier, C. Mura, B. Picard, J. Orfila,and R. Krishnamoorthy. 1991. Comparison of molecular epide-miological tools for Branhamella catarrhalis typing. Res. Micro-biol. 142:585-589.

42. Denning, D. W., C. J. Baker, N. J. Troup, and L. S. Tompkins.1989. Restriction endonuclease analysis of human and bovinegroup B streptococci for epidemiologic study. J. Clin. Microbiol.27:1352-1356.

43. Dice, L. R. 1945. Measures of the amount of ecologic associationbetween species. Ecology 26:297-302.

44. Dijkshoorn, L., H. Aucken, P. Gerner-Smidt, M. E. Kaufmann, J.Ursing, and T. L. Pitt. 1993. Correlation of typing methods forAcinetobacter isolates from hospital outbreak. J. Clin. Microbiol.31:702-705.

45. Esteban, E., K. Snipes, D. Hird, R. Kasten, and H. Kinde. 1993.Use of ribotyping for characterization of Salmonella serotypes. J.Clin. Microbiol. 31:233-237.

46. Faruque, S. M., K. Haider, M. M. Rahman, A. R. M. A. Alim,Q. S. Ahmad, M. J. Albert, and R. B. Saclk 1992. Differentiationof Shigella flexneri strains by rRNA gene restriction patterns. J.Clin. Microbiol. 30:2996-2999.

47. Fellner, P., and J. P. Ebel. 1970. Observations on the primarystructure of the 23S ribosomal RNA from E. coli. Nature(London) 225:1131-1132.

48. Ferdows, M. S., and A. G. Barbour. 1989. Megabase-sized linearDNA in the bacterium Borrelia burgdorferi, the Lyme diseaseagent. Proc. Natl. Acad. Sci. USA 86:5969-5973.

49. Forbes, K. J., D. Bruce, J. Z. Jordens, A. Ball, and T. H.Pennington. 1991. Rapid methods in bacterial DNA fingerprint-ing. J. Gen. Microbiol. 137:2051-2058.

50. Garaizar, J., M. E. Kaufmann, and T. L. Pitt. 1991. Comparisonof ribotyping with conventional methods for the type identifica-tion of Enterobacter cloacae. J. Clin. Microbiol. 29:1303-1307.

51. Gerner-Smidt, P. 1992. Ribotyping of the Acinetobacter calcoace-ticus-Acinetobacter baumanii complex. J. Clin. Microbiol. 30:2680-2685.

52. Gobel, U. B., A. Geiser, and E. J. Stranbridge. 1987. Oligonucle-otide probes complementary to variable regions of ribosomalRNA discriminate between Mycoplasma species. J. Gen. Micro-biol. 133:1969-1974.

53. Gordillo, M., K. Singh, and B. Murray. 1993. Comparison ofribotyping and pulsed-field gel electrophoresis for subspeciesdifferentiation of strains of Enterococcus faecalis. J. Clin. Micro-biol. 31:1570-1574.

54. Gottlieb, P., and R. Rudner. 1985. Restriction site polymorphismof ribosomal ribonucleic acid gene sets in members of the genusBacillus. Int. J. Syst. Bacteriol. 35:244-252.

55. Grattard, F., 0. G. Gaudin, B. Pozzetto, A. Ros, and A. D. Mbida.1993. Genotypic homogeneity of nosocomial Pseudomonasaeruginosa 012 strains demonstrated by analysis of proteinprofiles, DNA fingerprints and rRNA gene restriction patterns.Eur. J. Clin. Microbiol. Infect. Dis. 12:57-61.

56. Graves, L. M., B. Swaminathan, M. W. Reeves, and J. Wenger.1991. Ribosomal DNA fingerprinting of Listeria monocytogenes

using a digoxigenin-labeled probe. Eur. J. Epidemiol. 7:77-82.57. Grimont, F., D. Chevrier, P. A. D. Grimont, M. Lefevre, and J. L.

Guesdon. 1989. Acetylaminofluorene-labelled ribosomal RNAfor use in molecular epidemiology and taxonomy. Res. Microbiol.140:447-454.

58. Grimont, F., and P. A. D. Grimont. 1986. Ribosomal ribonucleicacid gene restriction patterns as potential taxonomic tools. Ann.Inst. Pasteur Microbiol. 137B:165-175.

59. Grimont, F., and P. A. D. Grimont. 1991. DNA fingerprinting, p.249-280. In E. Stackebrandt and M. Goodfellow (ed.), Nucleicacid techniques in bacterial systematics: modern microbiologicalmethods. John Wiley & Sons, Chichester, England.

60. Grimont, F., M. Lefevre, E. Ageron, and P. A. D. Grimont. 1989.rRNA gene restriction patterns of Legionella species: a molecularidentification system. Res. Microbiol. 140:615-626.

61. Grothues, D., U. Koopmann, H. Von Der Hardt, and B. Tummler.1988. Genome fingerprinting of Pseudomonas aeruginosa indi-cates colonization of cystic fibrosis siblings with closely relatedstrains. J. Clin. Microbiol. 26:1973-1977.

62. Gruner, E., A. Kropec, J. Huebner, M. Altwegg, and F. Daschner.1993. Ribotyping of Pseudomonas aeruginosa strains isolatedfrom surgical intensive care patients. J. Infect. Dis. 167:1216-1220.

63. Gustaferro, C. A., and D. H. Persing. 1992. Chemiluminescentuniversal probe for bacterial ribotyping. J. Clin. Microbiol.30:1039-1041.

64. Hadorn, K., W. Lenz, F. H. Kayser, I. Shalit, and C. Krasemann.1990. Use of a ribosomal RNA gene probe for the epidemiolog-ical study of methicillin and ciprofloxacin resistant Staphylococ-cus aureus. Eur. J. Clin. Microbiol. Infect. Dis. 9:649-653.

65. Hall, L. M., B. Duke, M. Guiney, and R. Williams. 1992. Typingof Enterococcus species by DNA restriction fragment analysis. J.Clin. Microbiol. 30:915-919.

66. Heidt, A., H. Monteil, and C. Richard. 1983. 0 and H serotypingof Pseudomonas cepacia. J. Clin. Microbiol. 18:738-740.

67. Hill, C. W., and B. W. Harnish. 1981. Inversions betweenribosomal RNA genes of Escherichia coli. Proc. Natl. Acad. Sci.USA 78:7069-7072.

68. Hinojosa-Ahumada, M., B. Swaminathan, S. B. Hunter, D. N.Cameron, J. A. Kiehlbauch, I. K. Wachsmuth, and N. A. Strock-bine. 1991. Restriction fragment length polymorphisms in rRNAoperons for subtyping Shigella sonnei. J. Clin. Microbiol. 29:2380-2384.

69. Huey, B., and J. Hall. 1989. Hypervariable DNA fingerprinting inEscherichia coli: minisatellite probe from bacteriophage M13. J.Bacteriol. 171:2528-2532.

70. Irino, K., F. Grimont, I. Casin, P. A. D. Grimont, and theBrazilian Purpuric Fever Study Group. 1988. rRNA gene restric-tion patterns of Haemophilus influenzae biogroup aegyptiusstrains associated with Brazilian purpuric fever. J. Clin. Micro-biol. 26:1535-1538.

71. Izard, N. C., H. Hachler, M. Grehn, and F. H. Kayser. 1992.Ribotyping of coagulase-negative staphylococci with special em-phasis on intraspecific typing of Staphylococcus epidermidis. J.Clin. Microbiol. 30:817-823.

72. Jarlier, V., M. H. Nicolas, G. Fournier, and A. Philippon. 1988.Extended broad-spectrum f3-lactamases conferring transferableresistance to newer P-lactam agents in Enterobacteniaceae: hos-pital prevalence and susceptibility patterns. Rev. Infect. Dis.10:867-878.

73. Jarry, B., and R. Rosset. 1973. Localization of some 5S RNAcistrons on Escherichia coli chromosome. Mol. Gen. Genet.121:151-162.

74. John, J. F., Jr. 1989. Molecular analysis of nosocomial epidemics.Infect. Dis. Clin. North Am. 3:683-700.

75. Kan, Y. W., and A. Dozy. 1978. Polymorphism of DNA sequenceadjacent to human 1-globin structural gene: relationship to sicklemutation. Proc. Natl. Acad. Sci. USA 75:5631-5635.

76. Karl, S. A., and J. C. Avise. 1992. Balancing selection at allozymeloci in oysters: implications from nuclear restriction fragmentlength polymorphisms. Science 256:100-102.

77. Kiehlbauch, J. A., B. D. Plikaytis, B. Swaminathan, D. N.Cameron, and I. K. Wachsmuth. 1991. Restriction fragment



R6

http://cmr.asm

.org/D

ownloaded from

RIBOTYPING 325

length polymorphism in the ribosomal genes for species identifi-cation and subtyping of aerotolerant Campylobacter species. J.Clin. Microbiol. 29:1670-1676.

78. Kliebe, C., B. A. Nies, J. F. Meyer, R. M. Tolxdorff-Neutzling, andB. Wiedemann. 1985. Evolution of plasmid-coded resistance tobroad-spectrum cephalosporins. Antimicrob. Agents Chemother.28:302-307.

79. Koblavi, S., F. Grimont, and P. A. D. Grimont. 1990. Clonaldiversity of Vibrio cholerae 01 evidenced by rRNA gene restric-tion patterns. Res. Microbiol. 141:645-657.

80. Kostman, J. R., T. D. Edlind, J. J. LiPuma, and T. L. Stull. 1992.Molecular epidemiology of Pseudomonas cepacia determined bypolymerase chain reaction ribotyping. J. Clin. Microbiol. 30:2084-2087.

81. Kreiswirth, B., J. Kornblum, R. D. Arbeit, W. Eisner, J. N.Maslow, A. McGeer, D. E. Low, and R. P. Noviclk 1993. Evidencefor a clonal origin of methicillin resistance in Staphylococcusaureus. Science 259:227-230.

82. Lambert-Zechovsky, N., E. Bingen, E. Denamur, N. Brahimi, P.Brun, H. Mathieu, and J. Elion. 1992. Molecular analysis pro-vides evidence for the endogenous origin for Enterobacter cloacaebacteremia and meningitidis in an infant. Clin. Infect. Dis.15:30-32.

83. Lanyi, B., and J. Lantos. 1976. Antigenic changes in Pseudomo-nas aeruginosa in vivo and after lysogenization in vitro. ActaMicrobiol. Hung. 23:337-351.

84. Lasker, B. A., J. M. Brown, and M. M. McNeil. 1992. Identifica-tion and epidemiological typing of clinical and environmentalisolates of the genus Rhodococcus with use of a digoxigenin-labeled rDNA gene probe. Clin. Infect. Dis. 15:223-233.

85. Levin, M. H., R. A. Weinstein, C. Nathan, R. K. Selander, H.Ochman, and S. A. Kabins. 1984. Association of infection causedby Pseudomonas aeruginosa serotype 011 with intravenous abuseof pentazocine mixed with tripelennamine. J. Clin. Microbiol.20:758-762.

86. LiPuma, J. J., S. E. Dasen, D. W. Nielson, R. C. Stern, and T. L.Stull. 1990. Person-to-person transmission of Pseudomonas cepa-cia between patients with cystic fibrosis. Lancet 3:1094-1096.

87. LiPuma, J. J., M. C. Fisher, S. E. Dasen, J. E. Mortensen, andT. L. Stull. 1991. Ribotype stability of serial pulmonary isolates ofPseudomonas cepacia. J. Infect. Dis. 164:133-136.

88. LiPuma, J. J., J. E. Mortensen, S. E. Dasen, T. D. Edlind, D. V.Schidlow, J. L. Burns, and T. L. Stull. 1988. Ribotype analysis ofPseudomonas cepacia from cystic fibrosis treatment centers. J.Pediatr. 113:859-862.

89. LiPuma, J. J., T. L. Stull, S. E. Dasen, K. A. Pidcock, D. Kaye,and 0. M. Korzeniowski. 1989. DNA polymorphism amongEscherichia coli isolated from bacteriuric women. J. Infect. Dis.159:526-532.

90. Liu, P. V. 1969. Changes in somatic antigens of Pseudomonasaeruginosa induced by bacteriophages. J. Infect. Dis. 119:237-246.

91. Loutit, J. S., and L. S. Tompkins. 1991. Restriction enzyme andSouthern hybridization analyses of Pseudomonas aeruginosastrains from patients with cystic fibrosis. J. Clin. Microbiol.29:2897-2900.

92. Mariani-Kurkdjian, P., E. Denamur, A. Milon, B. Picard, H.Cave, N. Lambert-Zechovsky, C. Loirat, P. Goullet, P. J. San-sonetti, and J. Elion. 1993. Identification of a clone of Escherichiacoli 0103:H2 as a potential agent of hemolytic-uremic syndromein France. J. Clin. Microbiol. 31:296-301.

93. Markowitz, S. M., J. M. Veazy, F. L. Macrima, C. G. Mayhall,and V. A. Lamb. 1980. Sequential outbreaks of infection dueto Klebsiella pneumoniae in a neonatal intensive care unit:implication of a conjugative R plasmid. J. Infect. Dis. 142:106-112.

94. Martinetti, G., and M. Altweeg. 1990. rRNA gene restrictionpatterns and plasmid analysis as a tool for typing Salmonellaenteritidis. Res. Microbiol. 141:1151-1162.

95. Mayer, L. W. 1988. Use of plasmid profiles in epidemiologicsurveillance of disease outbreaks and in tracing the transmissionof antibiotic resistance. Clin. Microbiol. Rev. 1:228-243.

96. Mazurier, S. I., and K. Wernars. 1992. Typing of Listeria strains

by random amplification of polymorphic DNA. Res. Microbiol.143:499-505.

97. Morgan, E. A., T. Ikemura, L. Lindahl, A. M. Fallon, and M.Nomura. 1978. Some rRNA operons in E. coli have tRNA genesat their distal ends. Cell 13:335-344.

98. Morgan, E. A., T. Ikemura, and M. Nomura. 1977. Identificationof spacer tRNA genes in individual ribosomal RNA transcriptionunits of Escherichia coli. Proc. Natl. Acad. Sci. USA 74:2710-2714.

99. Moureau, P., I. Derclaye, D. Gregoire, M. Janssen, and G. R.Cornelis. 1989. Campylobacter species identification based onpolymorphism of DNA encoding rRNA. J. Clin. Microbiol.27:1514-1517.

100. Moyer, N. P., G. M. Luccini, L. A. Holcomb, N. H. Hall, and M.Altwegg. 1992. Application of ribotyping for differentiating aero-monads isolated from clinical and environmental sources. Appl.Environ. Microbiol. 58:1940-1944.

101. Nastasi, A., C. Mammina, and M. R. Villafrate. 1991. rDNAfingerprinting as a tool in epidemiological analysis of Salmonellatyphi infections. Epidemiol. Infect. 107:565-576.

102. Nomura, M. 1973. Assembly of bacterial ribosomes. Science179:864-873.

103. O'Brien, T. F., D. G. Ross, M. A. Guzman, A. A. Medeiros, R. W.Hedges, and D. Bostein. 1980. Dissemination of an antibioticresistance plasmid in hospital patient flora. Antimicrob. AgentsChemother. 17:537-543.

104. Ogle, J. W., J. M. Janda, D. E. Woods, and M. L. Vasil. 1987.Characterization and use of a DNA probe as an epidemiologicalmarker for Pseudomonas aeruginosa. J. Infect. Dis. 155:119-126.

105. Ojeniyi, B., L. Baek, and N. Hoiby. 1985. Polyagglutinability dueto loss of 0-antigenic determinants in Pseudomonas aeruginosastrains isolated from cystic fibrosis patients. Acta Pathol. Micro-biol. Scand. Sect. B 93:7-13.

106. Owen, R. J. 1989. Chromosomal DNA fingerprinting: a newmethod of species and strain identification applicable to micro-bial pathogens. J. Med. Microbiol. 30:89-99.

107. Owen, R. J., A. Beck, P. A. Dayal, and C. Dawson. 1988. Detectionof genomic variation in Providencia stuartii clinical isolates byanalysis of DNA restriction fragment length polymorphismscontaining rRNA cistrons. J. Clin. Microbiol. 26:2161-2166.

108. Pace, N. R., G. J. Olsen, and C. R. Woese. 1986. Ribosomal RNAphylogeny and the primary lines of evolutionary descent. Cell45:325-326.

109. Panlilio, A. L., C. M. Beck-Sague, J. D. Siegel, R. L. Anderson,S. Y. Yetts, K. A. Thomassen, R. W. Vess, B. C. Hill, 0. C. Tablan,and W. R. Jarvis. 1992. Infections and pseudoinfections due topovidone-iodine solution contaminated with Pseudomonas cepa-cia. Clin. Infect. Dis. 14:1078-1083.

110. Patterson, T. F., J. E. Patterson, B. L. Masecar, G. E. Barden,W. J. Hierholzer, and M. J. Zervos. 1988. A nosocomial outbreakof Branhamella catarrhalis confirmed by restriction endonucleaseanalysis. J. Infect. Dis. 157:996-1001.

111. Patton, C. M., I. K. Wachsmuth, G. M. Evins, J. A. Kiehlbauch,B. D. Plikaytis, N. Troup, L. Tompkins, and H. Lior. 1991.Evaluation of 10 methods to distinguish epidemic-associatedCampylobacter strains. J. Clin. Microbiol. 29:680-688.

112. Pegues, D. A., L. A. Carson, L. R. Anderson, M. J. Norgard, T. A.Argent, W. R. Jarvis, and C. H. Woernle. 1993. Outbreak ofPseudomonas cepacia bacteremia in oncology patients. J. Infect.Dis. 16:407-411.

113. Perolat, P., F. Grimont, B. Regnault, P. A. D. Grimont, E.Fournie, H. Thevenet, and G. Baranton. 1990. rRNA generestriction patterns of Leptospira: a molecular typing system. Res.Microbiol. 141:159-171.

114. Picard, B., N. Picard-Pasquier, R. Krishnamoorthy, and P.Goullet. 1991. Characterization of highly virulent Escherichia colistrains by ribosomal DNA restriction fragment length polymor-phism. FEMS Microbiol. Lett. 82:183-188.

115. Picard-Pasquier, N., M. Ouagued, B. Picard, P. Goulet, and R.Krishnamoorthy. 1989. A simple, sensitive method for analyzingbacterial ribosomal DNA polymorphism. Electrophoresis 10:186-189.

116. Picard-Pasquier, N., B. Picard, S. Heeralal, R. Krishnamoorthy,

VOL. 7, 1994


R6

http://cmr.asm

.org/D

ownloaded from

326 BINGEN ET AL.

and P. Goullet. 1990. Correlation between ribosomal DNApolymorphism and electrophoretic enzyme polymorphism inYersinia. J. Gen. Microbiol. 136:1655-1666.

117. Pignato, S., G. Gimmanco, F. Grimont, and P. A. D. Grimont.1992. Molecular typing of Salmonella enterica subsp. entericaserovar Wien by rRNA gene restriction patterns. Res. Microbiol.143:703-709.

118. Pitcher, D., A. Johnson, F. Allerberger, N. Woodford, and R.George. 1990. An investigation of nosocomial infection withCorynebacterium jeikeium in surgical patients using a ribosomalRNA gene probe. Eur. J. Clin. Microbiol. Infect. Dis. 9:643-648.

119. Pitcher, D. G., R. J. Owen, P. Dyal, and A. Beck. 1987. Synthesisof a biotinylated DNA probe to detect ribosomal RNA cistrons inProvidencia stuartii. FEMS Microbiol. Lett. 48:283-287.

120. Pitt, T. L. 1988. Epidemiological typing of Pseudomonas aerugi-nosa. Eur. J. Clin. Microbiol. Infect. Dis. 7:238-247.

121. Pitt, T. L., D. M. Livermore, D. Pitcher, A. C. Vatopoulos, andN. J. Legakis. 1989. Multiresistant serotype 012 Pseudomonasaeruginosa: evidence for a common strain in Europe. Epidemiol.Infect. 103:565-576.

122. Poh, C. L., C. C. Yeo, and L. Tay. 1992. Genome fingerprinting bypulsed-field gel electrophoresis and ribotyping to differentiatePseudomonas aeruginosa serotype O11 strains. Eur. J. Clin.Microbiol. Infect. Dis. 11:817-822.

123. Popovic, T., C. A. Bopp, 0. Olsvik, and J. A. Kiehlbauch. 1993.Ribotyping in molecular epidemiology, p. 573-583. In D. H.Persing, T. F. Smith, F. C. Tenover, and T. J. White (ed.),Diagnostic molecular microbiology: principles and applications.American Society for Microbiology, Washington, D.C.

124. Postic, D., C. Edlinger, C. Richaud, F. Grimont, Y. Dufresne, P.Perolat, G. Baranton, and P. A. D. Grimont. 1990. Two genomicspecies in Borrelia burgdorferi. Res. Microbiol. 141:465-475.

125. Preheim, L., D. Pitcher, R. Owen, and B. Cookson. 1991. Typingof methicillin resistant and susceptible Staphylococcus aureusstrains by ribosomal RNA gene restriction patterns using abiotinylated probe. Eur. J. Clin. Microbiol. Infect. Dis. 10:428-436.

126. Prevost, G., B. Jaulhac, and Y. Piemont. 1992. DNA fingerprint-ing by pulsed-field gel electrophoresis is more effective thanribotyping in distinguishing among methicillin-resistant Staphylo-coccus aureus isolates. J. Clin. Microbiol. 30:967-973.

127. Rabkin, C. S., W. R. Jarvis, R. L. Anderson, J. Govan, J. Klinger,J. J. LiPuma, W. J. Martone, H. Monteil, C. Richard, S. Shigeta,A. Sosa, T. L. Stull, J. Swenson, and D. Woods. 1989. Pseudomo-nas cepacia typing systems: collaborative study to assess theirpotential in epidemiologic investigations. Rev. Infect. Dis. 11:600-607.

128. Richard, C., H. Monteil, F. Megraud, R. Chatelain, and B.Laurent. 1981. Caracteres phenotypiques de 100 souches dePseudomonas cepacia. Proposition d'un schema de biovars. Ann.Biol. Clin. 39:9-15.

129. Richet, H. M., P. C. Craven, J. M. Brown, B. A. Lasker, C. D. Cox,M. M. McNeil, A. D. Tice, W. R. Jarvis, and 0. C. Tablan. 1991.A cluster of Rhodococcus (Gordona) bronchialis sternal-woundinfections after coronary-artery bypass surgery. N. Engl. J. Med.324:104-109.

130. Romaniuk, P. J., and T. J. Trust. 1987. Identification of Campy-lobacter species by Southern hybridization of genomic DNA usingan oligonucleotide probe for 16S rRNA genes. FEMS Microbiol.Lett. 43:331-335.

131. Samadpour, M., S. L. Moseley, and S. Lory. 1988. BiotinylatedDNA probes for exotoxin A and pilin genes in the differentiationof Pseudomonas aeruginosa strains. J. Clin. Microbiol. 26:2319-2323.

132. Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecularcloning: a laboratory manual, 2nd ed. Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y.

133. Saunders, N. A., T. G. Harrison, N. Kachwalla, and A. G. Taylor.1988. Identification of species of the genus Legionella using acloned rRNA gene from Legionella pneumophila. J. Gen. Micro-biol. 134:2363-2374.

134. Schoonmaker, D., T. Heimberger, and G. Birkhead. 1992. Com-parison of ribotyping and restriction enzyme analysis using

pulsed-field gel electrophoresis for distinguishing Legionellapneumophila isolates obtained during a nosocomial outbreak. J.Clin. Microbiol. 30:1491-1498.

135. Schwartz, D. C., and C. R. Cantor. 1984. Separation of yeastchromosome-sized DNAs by pulsed field gradient gel electro-phoresis. Cell 37:67-75.

136. Selander, R. K., D. A. Caugant, H. Ochman, J. M. Musser, M. N.Gilmour, and T. S. Whittam. 1986. Methods of multilocusenzyme electrophoresis for bacterial population genetics andsystematics. Appl. Environ. Microbiol. 51:873-884.

137. Sirot, D., J. Sirot, R. Labia, A. Morand, P. Courvalin, A.Darfeuille-Michaud, R. Perroux, and R. Cluzel. 1987. Transfer-able resistance to third-generation cephalosporins in clinicalisolates of Klebsiella pneumoniae. Identification of CTX-1, anovel 3-lactamase. J. Antimicrob. Chemother. 20:323-334.

138. Soto, A., 0. G. Pitcher, and F. Soriano. 1991. A numericalanalysis of ribosomal RNA gene patterns for typing clinicalisolates of Corynebacterium group D2. Epidemiol. Infect. 107:263-272.

139. Southern, E. M. 1975. Detection of specific sequences amongDNA fragments separated by gel electrophoresis. J. Mol. Biol.98:503-517.

140. Speert, D. P., M. E. Campbell, S. W. Farmer, K. Volpel, A. M.Joffe, and W. Paranchych. 1989. Use of a pilin gene probe tostudy molecular epidemiology of Pseudomonas aeruginosa. J.Clin. Microbiol. 27:2589-2593.

141. Spreadbury, C. L., B. W. Bainbridge, and J. Cohen. 1990.Restriction fragment length polymorphisms in isolates of As-pergillus fumigatus probed with part of the intergenic spacerregion from the ribosomal RNA gene complex of Aspergillusnidulans. J. Gen. Microbiol. 136:1991-1994.

142. Strockbine, N. A., J. Parsonnet, K. Greene, J. A. Kiehlbauch, andI. K. Wachsmuth. 1991. Molecular epidemiologic techniques inanalysis of epidemic and endemic Shigella dysenteriae type 1strains. J. Infect. Dis. 163:406-409.

143. Stull, T. L., J. J. LiPuma, and T. D. Edlind. 1988. A broad-spectrum probe for molecular epidemiology of bacteria: ribo-somal RNA. J. Infect. Dis. 157:280-285.

144. Swaminathan, B., and G. M. Matar. 1993. Molecular typingmethods, p. 26-50. In D. H. Persing, T. H. Smith, F. C. Tenover,and T. J. White (ed.), Diagnostic molecular microbiology: prin-ciples and applications. American Society for Microbiology,Washington, D.C.

145. Tablan, 0. C., T. L. Chorba, D. V. Schidlow, J. W. White, K. A.Hardy, P. H. Gilligan, W. M. Morgan, L. A. Carson, W. J.Martone, J. M. Jason, and W. R. Jarvis. 1985. Pseudomonascepacia colonization in patients with cystic fibrosis: risk factorsand clinical outcome. J. Pediatr. 107:382-387.

146. Taylor, R. F. H., L. Dalla Costa, M. E. Kaufmann, T. L. Pitt, andM. E. Hodson. 1992. Pseudomonas cepacia pulmonary infectionin adults with cystic fibrosis: is nosocomial acquisition occurring?J. Hosp. Infect. 21:199-204.

147. Thomson-Carter, F. M., P. E. Carter, and T. H. Pennington.1989. Differentiation of staphylococcal species and strains byribosomal RNA gene restriction patterns. J. Gen. Microbiol.135:2093-2097.

148. Tompkins, L. S. 1992. The use of molecular methods in infectiousdiseases. N. Engl. J. Med. 18:1290-1297.

149. Tram, C., M. Simonet, M. H. Nicolas, C. Offredo, F. Grimont, M.Lefevre, E. Ageron, A. Debure, and P. A. D Grimont. 1990.Molecular typing of nosocomial isolates of Legionella pneumo-phila serogroup 3. J. Clin. Microbiol. 28:242-245.

150. Tummler, B., U. Koopmann, D. Grothues, H. Weissbrodt, G.Steinkamp, and H. Von der Hardt. 1991. Nosocomial acquisitionof Pseudomonas aeruginosa by cystic fibrosis patients. J. Clin.Microbiol. 29:1265-1267.

151. Van Ketel, R. J., and B. De Wever. 1989. Genetic typing in a

cluster of Legionella pneumophila infections. J. Clin. Microbiol.27:1105-1107.

152. Wei, M. Q., D. M. Groth, A. H. Mendis, J. Sampson, J. D.Wetherall, and W. B. Grubb. 1992. Typing of methicillin-resistantStaphylococcus aureus with an M13 repeat probe. J. Hosp. Infect.20:233-245.



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RIBOTYPING 327

153. Welsh, J., and M. McClelland. 1990. Fingerprinting genomes

using PCR with arbitrary primers. Nucleic Acids Res. 18:7213-7218.

154. Williams, J. G., A. R. Kubelik, K. J. Livak, J. A. Rafalski, andS. V. Tingey. 1990. DNA polymorphisms amplified by arbitraryprimers are useful as genetic markers. Nucleic Acids Res.18:6531-6535.

155. Wilton, J., K. Jung, I. Vedin, B. Aronsson, and J. I. Flock. 1992.Comparative evaluation of a new molecular method for typingStaphylococcus epidermidis. Eur. J. Clin. Microbiol. Infect. Dis.11:515-521.

156. Wolz, C., G. Kiosz, J. W. Ogle, M. L. Vasil, U. Schaad, K.Botzenhartand, and G. Dbring. 1989. Pseudomonas aeruginosacross-colonization and persistence in patients with cystic fibrosis.Use of a DNA probe. Epidemiol. Infect. 102:205-214.

157. Woodford, N., D. Morrison, A. P. Johnson, V. Briant, R. C.George, and B. Cookson. 1993. Application of DNA probes forrRNA and vanA genes to investigation of a nosocomial cluster ofvancomycin-resistant enterococci. J. Clin. Microbiol. 31:653-658.

158. Woods, T. C., L. 0. Helsel, B. Swaminathan, W. F. Bibb, R. W.Pinner, B. G. Gellin, S. F. Collin, S. H. Waterman, M. W. Reeves,D. J. Brenner, and C. V. Broome. 1992. Characterization ofNeisseria meningitidis serogroup C by multilocus enzyme electro-phoresis and ribosomal DNA restriction profiles (ribotyping). J.Clin. Microbiol. 30:132-137.

159. Yogev, D., D. Halachmi, G. E. Kenny, and S. Razin. 1988.Distinction of species and strains of mycoplasmas (Mollicutes) bygenomic DNA fingerprints with an rRNA gene probe. J. Clin.Microbiol. 26:1198-1201.

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