© 2002 Blackwell Science Ltd

Analysis of the Pseudomonas aeruginosa oprD genefrom clinical and environmental isolates

Introduction

Fluorescent pseudomonads are known for their strikingability to adapt to various ecological niches (Rainey andTravisano, 1998; Spiers et al., 2000). Pseudomonasaeruginosa, a ubiquitous pseudomonad, is also known asa human pathogen causing a variety of severe infectionsof nosocomial nature (Emori and Gaynes, 1993; Vincentet al., 1995). A classical example of the interplay of P.aeruginosa with its environment is antibiotic resistance.The introduction of antibiotic agents has led to the emer-gence of P. aeruginosa as one of the most problematicopportunistic pathogens in humans. Resistance againstthe limited number of antimicrobial agents displaying reliable antipseudomonal activity (e.g. carbapenems andfluoroquinolones) has been encountered with increasingfrequency (Quin et al., 1986; Masuda and Ohya, 1992).Carbapenems are not prone to inactivation by extended-spectrum b-lactamases and cross the outer membrane ofP. aeruginosa through a specialized pore protein, OprD,which allows selective permeation of basic amino acidsas well as small peptides containing these amino acidsand their structural analogues (Trias and Nikaido, 1990;Huang and Hancock, 1993). As such, carbapenems havea major role as initial empirical therapy as well as definitive therapy in severe P. aeruginosa infections(Bradley et al., 1999). However, prolonged treatment of P. aeruginosa-infected patients with imipenem, a carbapenem antibiotic, has often allowed for the emer-gence of imipenem-resistant mutants. These resistantstrains have either lost OprD (Quin et al., 1986; Lynchet al., 1987) or have strongly reduced OprD levels due to an nfxC-type of quinolone-resistant mutation, whichrepresses oprD and activates the mexEF–oprN multidrugefflux operon (Fukuda et al., 1995; Köhler et al., 1997). Itis unlikely that decreased penetration through the outermembrane, through mutational loss of OprD, alone canresult in high carbapenem resistance. The presence of aninducible or derepressed chromosomal b-lactamase,capable of hydrolysis at very low substrate concentra-tions, is also necessary (Satake et al., 1991; Livermore,1992). Recently Köhler et al. (1999) demonstrated thatmeropenem, but not imipenem, was a substrate of theconstitutively expressed MexAB–OprM efflux pump.

Nine years ago, Yoneyama and Nakae (1993) pub-lished the results of a study in which they analysed theelimination of OprD in 23 independent imipenem-resistant

Environmental Microbiology (2002) 4(12), 872–882

Jean-Paul Pirnay,1,2 Daniel De Vos,1,3 DimitrisMossialos,1† Alain Vanderkelen,2 Pierre Cornelis1

and Martin Zizi2,4*1Laboratory of Microbial Interactions, Department ofImmunology, Parasitology, and Ultrastructure, FlandersInteruniversity Institute of Biotechnology, Brussels FreeUniversity, B-1640 Sint-Genesius-Rode, Brussels,Belgium.2Science Department, Belgian Military Medical Service,Queen Astrid Military Hospital, B-1120 Neder-Over-Heembeek, Belgium.3Department of Infectious Diseases, Innogenetics NV,B-9052 Ghent, Belgium.4Department of Physiology, Faculty of Medicine andPharmacy, Brussels Free University, B-1090 Brussels,Belgium.

Summary

Genomes are constantly evolving. Our report high-lights the wide mutational diversity of clinical as wellas environmental isolates, compared with the labora-tory strain(s), through the systematic genetic analy-sis of a chromosomal porin gene (oprD) in relation toa specific antibiotic resistance. Mutational inactiva-tion of the oprD gene is associated with carbapenemresistance in Pseudomonas aeruginosa. Thesequence of the oprD gene of 55 Pseudomonasaeruginosa natural isolates obtained from across theworld – from sources as diverse as patients and rhizospheres – was analysed. A microscale mosaicstructure for this gene – resulting from multiple intra-and possibly interspecies recombinational events – isreported. An array of independent and seemingly fast-occurring defective oprD mutations were found,none of which had been described before. A burnwound isolate demonstrated unusually high overallsequence variability typical of mutator strains. Wealso present evidence for the existence of OprDhomologues in other fluorescent pseudomonads.

Received 20 November, 2001; accepted 25 January, 2002. *For cor-respondence. E-mail martin.zizi@vub.ac.be; Tel. (32) 2 477 4434;Fax (32) 2 477 4568. †Present address: Department of Biology, Imperial College of Science, Technology and Medicine, Sir AlexanderFleming Building, Imperial College Road, London SW7 2AZ, UK.Parts of this paper were presented at the Pseudomonas 2001 conference, Brussels, Belgium, 17–21 September.

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© 2002 Blackwell Science Ltd, Environmental Microbiology, 4, 872–882

mutants from TNP038, a laboratory strain harbouring aplasmid carrying the oprD gene and having a mutation inchromosomal oprD. The cloned oprD gene was derivedfrom strain PAO1 (Holloway, 1955; Stover et al., 2000),the prototype strain for sequencing, because it is the mostwidely used P. aeruginosa laboratory strain. They foundthat all 23 laboratory mutants could be divided into twogroups, both groups carrying a specific deletion in theoprD gene. One group had an 11 bp deletion in the codingregion, generating a frameshift resulting in a prematuretermination; the other group had a 1204 bp deletion covering the initiation codon and the promoter region.Although only plasmid-borne PAO1 oprD was analysed, itwas suggested that elimination of OprD from mostimipenem-resistant P. aeruginosa isolates is due to efficient selection of oprD deletion mutants. Since then,nobody verified that chromosomal mutations in imipenem-resistant clinical isolates are caused by the same mechanism. We sequenced the oprD gene from 37imipenem-sensitive and 18 imipenem-resistant P. aerug-inosa isolates, collected over a period of 15 years fromvarious, spatially separated, clinical and environmentalhabitats (Table 1), in order to assess the sequence diver-sity and the extent of naturally occurring chromosomalmutations. We also present evidence for the existence ofoprD-like genes in other fluorescent pseudomonads.

Results and discussion

Mosaic structure of the oprD gene

We found that all oprD sequences from P. aeruginosa iso-lates fell into three distinct groups (arbitrarily designatedA, B and C), with identities ranging from 91% to 93% forDNA and from 88% to 93% for amino acids. Visual inspec-tion of an alignment of oprD sequences showed clear evidence of a history of intraspecies recombinationalexchanges of DNA blocks of 100–300 bp, leading to a

mosaic gene structure and causing a further divergenceinto subgroups (Fig. 1). Such small-scale mosaics werealso suggested at the protein level for the VDAC chan-nels (or mitochondrial porins) (Song and Colombini,1996). The exchange of DNA blocks ranging in size from1 kb to 214 kb by mechanisms of horizontal transfer hasbeen described in P. aeruginosa (Römling et al., 1997),and more recently Kiewitz and Tümmler (2000) observedhypervariable regions in the flagellin and pillin genes of P.aeruginosa, indicating intragenic recombination events.All these findings add to the growing evidence that bac-teria have obtained a significant proportion of their geneticdiversity through the acquisition of sequences from rela-tively closely related bacteria (Smith et al., 1991; Ochmanet al., 2000), and that the uptake and chromosomal incorporation of DNA is not confined to some well-known naturally transformable genera such as Haemophilus(Kroll et al., 1994) and Neisseria (Halter et al., 1989).Recently, Wilderman et al. (2001) even suggested that P. aeruginosa had horizontally acquired a eukaryotic-like phospholipase D gene. The large variety of OprDhomologues in P. aeruginosa (http://cmdr.ubc.ca/bobh/OprDfamily.html) and in other bacteria presents opportu-nities for intrachromosomal exchange and horizontalgene transfer. To date, putative substrates have beenidentified for 8 of the 19 specific porins of the P. aerugi-nosa OprD family (Tamber et al., 2001), but so far onlyOprD appears to play a role in antibiotic uptake. A com-parison of P. aeruginosa OprD against the unfinishedgenomic sequences of P. fluorescens (strain Pf0-1), P. syringae (pv. Tomato) and P. putida (strain KT2440) in the NCBI database (http://www.ncbi.nlm.nih.gov/cgi-bin/Entrez/genom_table_cgi) using TBLASTN softwarerevealed the existence of such ‘OprD’ porin families in P.fluorescens (10 genes), P. syringae (11 genes) and P.putida (19 genes). By analogy with the P. aeruginosaOprD family, all members showed particularly highsequence similarity in regions that may constitute trans-

Isolation site

Geographical origin Wound Sputum Environment Others

Australia 1Belgium 4 2 2Bulgaria 2Czech Republic 1England 2Germany 3 5 5Hungary 1 1Pakistan 2Philippines 1Portugal 4The Netherlands 2 2Tunisia 1 2Turkey 3USA 5 4

Table 1. Sources of the 55 P. aeruginosaisolates used in this study.

© 2002 Blackwell Science Ltd, Environmental Microbiology, 4, 872–882

874 J.-P. Pirnay et al.

membrane domains and least similarity in the sequencescoding for the external loops and signal peptides. In each ‘OprD’ family we observed one member with ahigher similarity (51–65% amino acid homology) to P.aeruginosa OprD than the other members of the family(30–40%). An alignment of the amino acid sequences ofthese ‘true’ OprD homologues with representatives of thethree P. aeruginosa OprD groups we observed in thisstudy (A, B and C) revealed similarity, in the highly vari-able region of external loop 7, between the OprD homo-logues of P. syringae, P. fluorescens and representativesof the OprD groups A and B, but not C, of P. aeruginosa(Fig. 2). Strain PAO1, with which all substrate selectivityexperiments were carried out (Huang and Hancock,1993), belongs to subgroup ‘C1’ (Fig. 1). We were notable to determine whether this anomaly is the result of ahighly localized interspecies recombinational exchange orvertical evolution. As this analysis was based on contigsequences (P. syringae, August 2001; and P. putida,

September 2001) or a complete genomic sequence, but prior to final DNA assembly and annotation (P. fluorescens, June 2001), we are aware that the results ofour query are probably incomplete.

We found no correlation between the oprD groups andhabitats or geographic origin. The oprD sequence of aburn wound isolate obtained from London, for example,could be identical to that of a river water isolate from Pak-istan, or two strains isolated from the same patient inBrussels could be positioned very far apart. An OprDtopology model predicted eight external loops, and it wassuggested that loops 2 and 3 have a role in imipenembinding to the OprD channel, whereas loops 5, 7 and 8serve to restrict the OprD channel at its entrance, ham-pering non-specific passage of antibiotics (Huang et al.,1995; Huang and Hancock, 1996; Ochs et al., 2000). Aswe observed that amino acid variations between oprDgroups were particularly high at the external loops, includ-ing the loops putatively involved in antibiotic passage

Fig. 1. Graphical representation of the mosaic structure of the oprD gene in 55 unbiased P. aeruginosa strains. Although all sequences fellinto three main groups (A, B and C), which differ by 7–9% of nucleotides, several distant sequences contained nearly identical regions of afew hundred basepairs, witnesses of local exchanges of sequences between the groups. These recombinational events caused a furtherdivergence into subgroups. The members of each subgroup differed by <0.5% of nucleotides among themselves. Laboratory strain PAO1differed by about 1.4% of nucleotides from subgroup C1, mainly because of the exchange of a 250 bp sequence with group B. Regionsrepresented by the same colour are similar in sequence. *Including 10 isolates of a predominant clone, called clone C, derived fromenvironment and disease habitats (Römling et al., 1994). n, number of isolates belonging to a subgroup.

Fig. 2. Multiple amino acid alignment (UPGMA) of presumed loop 7 of the ‘true’ OprD homologues of P. syringae (pv. Tomato), P. fluorescens(Pf0-1), P. putida (KT2440) and representatives of P. aeruginosa OprD groups (A, B and C). Highly conserved residues are boxed in grey andgaps are represented by dots. Notice the relatively high similarity in the highly variable region of external loop 7 between OprD homologues ofP. syringae, P. fluorescens and P. aeruginosa OprD groups A and B.

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Fig. 3. Multiple amino acid alignment(UPGMA) of the external loops (L2, 3, 5, 7 and8), relevant for antibiotic passage, of the OprDgroups (A, B, C1 and C2), the ‘mutator’ strain,and laboratory strain PAO1. Highly conservedresidues are boxed in grey and gaps arerepresented by dots. Notice the mosaicstructure and the important sequence diversity,especially in loop 7, and the duplication ofsequence TACGGC in loop 7 of strain PAO1and subgroups C1 and C2. Numbering isbased on strain PAO1.

(Fig. 3), differences in substrate specificity between theoprD groups cannot be excluded. Although we did notobserve a direct correlation between the oprD groups andcarbapenem resistance, it is interesting that, recently, Eppet al. (2001) demonstrated that the C-terminal region, andmore specifically loop 7, of OprD modulates susceptibil-ity to meropenem. It is also true that high levels of recom-bination can be due to a ‘hitch-hiking’ effect caused bypositive selection for variation at a neighbouring gene,and Zhou et al. (1997) suggested that interspecies recom-binational exchanges could be observed even when thereappears to be no obvious selection for the recombinantphenotypes.

Mutator strain?

One burn wound isolate (BR680) demonstrated unusuallyhigh sequence variability in the oprD gene (Figs 1 and 3)as well as in other genes (oprL and oprI, data not shown).Similar aberrant sequence variability in different loci wasrecently detected in a P. aeruginosa ear infection isolate(Kiewitz and Tümmler, 2000). An augmented rate of variation, caused by spontaneous mutations, is typical ofhypermutable (mutator) strains, which allow bacterial populations to adapt to stressful environments (Leclercet al., 1996; Miller, 1996; Matic et al., 1997). An increasedrate of polymerase errors and/or defects in the proof-reading and mismatch repair mechanisms is believed tocause this state of hypermutation. In bacteria the initialerrors are probably polymerase errors, caused by spon-taneous mutations or triggered by damage to the templatestrand (Bridges, 2001). Recently, high frequencies of

hypermutable P. aeruginosa strains were detected incystic fibrosis lung infection (Oliver et al., 2000). Thesemutator strains persisted for years in most patients. Totest the hypothesis of the mutator phenotype, we esti-mated the frequency of mutation resulting into rifampicinresistance of strains BR680 and PAO1 as reported byOliver et al. (2000). According to their study, a strain wasconsidered a mutator when the mutation frequency wasat least 20-fold higher than that observed for PAO1. In ourcase, strains BR680 and PAO1 showed similar mutationfrequencies (~10-8). The combination of a mutator geno-type with a non-mutator phenotype can be explained asfollows: strain BR680 probably exhibited a mutator phe-notype, but once adapted, it reverted to the non-mutatorstate (Taddei et al., 1997). This is to be expected, as theaccumulation of deleterious mutations could decrease thefitness of the mutator population in the long term. StrainBR680 displayed a moderate imipenem resistance combined with a relatively high sensibility to meropenem,ceftazidime and aztreonam (see Table 3). An altered conformation of the OprD channel, which is likely, takinginto account the important aberrations in the amino acidsequence of BR680 OprD, could account for this unusualantibiotic pattern.

Mutational inactivation of oprD

In 12 imipenem-resistant isolates, defective chromosomaloprD mutations were observed (Table 2). Yet, all muta-tions were unique, and different from the two deletionspreviously described for laboratory strain TNP038(Yoneyama and Nakae, 1993). Different classes of muta-

© 2002 Blackwell Science Ltd, Environmental Microbiology, 4, 872–882

876 J.-P. Pirnay et al.

tions were found. The majority (7) were distinct base substitutions, causing a premature termination (5) or thereplacement of leucine by proline in the signal peptide (1)or external loop 7 (1). Two single-base deletions and onesingle-base duplication occurred at monotonic repeats(CCCC and GGGGG), causing a frameshift, resulting inthe creation of a translational stop codon in the regiondownstream of the mutation. A 44-base duplication wasobserved at a direct repeat of a 6 bp DNA motif(GCGCGG), separated by 44 intervening nucleotides(Fig. 4). These deletions and duplications could becaused by a mechanism first described by Streisinger

et al. (1966), which involves the slipped misalignment ofthe repeated sequences during DNA replication with theformation of a looped misaligned intermediate, leadingeither to nucleotide deletion (if the dislocated loop is onthe template strand) or to duplication (if the loop is on theprimer strand). Monotonic repeats and direct repeats aredosDNA (defined order sequence DNA) sequence ele-ments, unusual symmetry elements that can form alter-native DNA structures and are responsible – together withenzymes associated with DNA replication, recombinationand repair – for instability in chromosomes of prokaryoticand eukaryotic cells. These dosDNA sequence elementsfurther include inverted repeats, quasipalindromes andmirror repeats (Sinden et al., 1999). The slippage mis-alignment model has been used frequently to account forthe gain or loss of small or large sequences in bacteria(Streisinger et al., 1966; Farabaugh et al., 1978; Albertiniet al., 1982). Misalignment deletion and duplication arealso responsible for several human diseases (Krawczakand Cooper, 1991; Hu and Worton, 1992). In the p53tumour-suppressor gene in human cancer, all classes ofmutations were found, but the majority were base substi-tutions, and 10–15% of mutations were small intragenicdeletions or insertions caused by slippage misalignment(Greenblatt et al., 1996). Recently, a 23-base misalign-ment duplication mutation was detected in the TSC1 exon15 in an infant with cardiac rhabdomyomas (Smith andSperling, 1999). We also observed a case of slipped misalignment duplication in the oprL gene, coding for anouter membrane peptidoglycan-associated lipoprotein(Lim et al., 1997), in a P. pseudoalcaligenes strain. Theduplication resulted in the insertion of six additional aminoacids into the lipoprotein, with no apparent changes inphenotype (data not shown). The YG repeat in loop 7 ofgroup C is another probable case of replication misalign-ment duplication (Fig. 3). The fact that we were able towitness this type of mutation several times in a fewhundred sequences could mean that replication misalign-ment duplication plays a major role in the emergence ofnew alleles, or even new genes, in pseudomonads. Our

Table 2. Defective oprD mutations in P. aeruginosa clinical isolates.

Strain Mutation

BE128 Large deletion starting from nt 874 and covering the termination codonIS573 CÆT base substitution at nt 1018 fi premature terminationAA245 CÆT base substitution at nt 1243 fipremature terminationBU007 GÆT base substitution at nt 511 fi premature terminationHAC19 GÆA base substitution at nt 413 fi premature terminationLI010 GÆA base substitution at nt 831 fi premature terminationAA249 TÆC base substitution at nt 32 fi leucine replaced by proline in signal peptideBR670 TÆC base substitution at nt 1076 fi leucine replaced by proline in external loop 7BR718 Duplication between GCGCGG repeats (nt 573–8 and 617–22) fi frameshift fi stop codon at nt 730–2PR317 1-base duplication at monotonic repeat CCCC (nt 346–9) fi frameshift fi stop codon at nt 352–4MI162 1-base deletion at monotonic repeat CCCC (nt 346–9) fi frameshift fi stop codon at nt 379–81BO548 1-base deletion at monotonic repeat GGGGG (nt 631–5) fi frameshift fi stop codon at nt 713–5

Fig. 4. Possible mechanism for the 44-base duplication in the oprD gene of isolate BR718 involving slippage between two directrepeats (bold italic). The newly replicated single strand of DNA isdisplaced from the template and subsequently reannealed to it at the upstream copy of the direct repeat, forming a loopedmisaligned intermediate. When replication resumes, a 44-bpsequence (boxed) is duplicated, causing a frameshift, leading to apremature termination.

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© 2002 Blackwell Science Ltd, Environmental Microbiology, 4, 872–882

results indicate that defective chromosomal oprD muta-tions emerging in vivo in clinical isolates (at the site ofinfection) are far more versatile than those selected invitro from a laboratory strain carrying the plasmid-bornePAO1 oprD gene. It is well known that a complex networkof factors influence the rate and type of mutants that can be selected under antibiotic pressure (Martinez andBaquero, 2000). The important nucleotide diversityobserved in the oprD gene is clearly one of the factorsresponsible for the large variation of mutations. The pre-sence of dosDNA and the local sequence environment(e.g. the primary and secondary DNA structure betweendirect repeats) are major factors affecting the likelihood ofmisalignment mutations (Krawczak and Cooper, 1991;Trinh and Sinden, 1993). Furthermore, differences in thestructure and sequence of the surrounding DNA – aplasmid in the laboratory strain compared with a chromo-some in the clinical isolates – are also likely to contribute.

Transcription of oprD

All imipenem-resistant isolates, except BO548, showeddecreased transcription of oprD, compared withimipenem-sensitive isolates, while harbouring an oprDmutation (Table 3). Imipenem-sensitive isolate BR642, anearlier isolate of the imipenem-resistant strain BR718, asdetermined by amplified fragment length polymorphism(AFLP), serotyping and pyoverdine typing, alreadyshowed suppressed transcription of oprD (Table 3). Assuppression of oprD transcription also occurred inciprofloxacin-sensitive strains (Table 3), we suspect theexistence of a an OprD repression mechanism, acting atthe transcriptional level, but other than the nfxC-typemutation observed in quinolone-resistant isolates andpreceding the selection of oprD mutants in most P. aeruginosa clinical isolates.

Expression of OprD

Our results confirm that loss of OprD and the presence of a potent b-lactamase – illustrated by high minimuminhibitory concentrations (MICs) for aztreonam and ceftazidime in Table 3 – are the main requisites forimipenem resistance in P. aeruginosa. All isolates with aMIC for imipenem higher than 8 mg ml-1, except isolateBR670, completely lacked OprD, as observed in sodiumdodecyl sulphate polyacrylamide gel electrophoresis(SDS-PAGE) gels of outer membrane proteins andWestern blot analysis using YD10 anti-OprD antibody(Table 3). Isolate BR670 showed a 46 kDa protein, likelyto be OprD, in SDS-PAGE, but not in Western blot. Whenusing a mixture of anti-OprD antibodies, a fuzzy band wasobserved (Fig. 5). We suspect that the substitution of Profor Leu in external loop 7 of BR670 OprD (Table 2) caused

a conformational change in the loop, hampering thepassage of carbapenems and the binding of anti-OprDantibody YD10. This demonstrates the need for cautionwhen using monoclonal antibodies, which are – by defin-ition – directed against one epitope of one (laboratory)strain. Isolates BR776 and LO053 completely lackedOprD while showing no defective oprD mutation and atrue, albeit low, oprD transcription (Table 3). We thereforesuspect that, in these two isolates, loss of OprD wascaused by an OprD repression mechanism acting at thepost-transcriptional level. Recently, Köhler et al. (1999)showed that co-regulation of OprD and the MexEF–OprNefflux system occurred at the transcriptional but also at the post-transcriptional level. Ochs et al. (1999) alsosuggested that OprD is influenced by more than onemechanism of repression.

Conclusion

Carbapenem resistance in P. aeruginosa is mediated by chromosomal b-lactamases, drug efflux pumps andmostly by diminished expression or loss of OprD. It hasbeen suggested that these mechanisms work in concertin clinical isolates (Pai et al., 2001). Expression of oprDis hampered by a seemingly unlimited number of de-fective oprD mutations and several mechanisms acting on the transcriptional and post-transcriptional level, the multiplicity and complexity of which are likely to complicate DNA-based assays for the detection of carbapenem resistance in P. aeruginosa. Jenks et al.(1999) also suggested that, although metronidazole resis-tance in Helicobacter pylori is frequently associated withmutational inactivation of the rdxA gene, other mecha-nisms of resistance are likely to exist in H. pylori. The high levels of sequence variation and intra- and possibly inter-species recombinations within oprD are not surprising.The main function of OprD is the uptake of basic amino

Fig. 5. Western blot analysis of the outer membrane proteins of P. aeruginosa strains BR670 and ATCC 27853 and E. coli strainsC644 (expressing oprD) and DH5a with a mixture of anti-OprDMAbs (A) or anti-OprD MAb YD10 (B). BR670 OprD is detectedwith the mixture of MAbs, but not with YD10. PPS, precisionprotein standards.

© 2002 Blackwell Science Ltd, Environmental Microbiology, 4, 872–882

878 J.-P. Pirnay et al.

Tab

le3.

opr

Dm

utat

ions

, le

vels

of

tran

scrip

tion

and

tran

slat

ion

of o

prD

, an

d an

tibio

tic s

usce

ptib

ilitie

s of

clin

ical

P.

aeru

gino

sais

olat

es.

MIC

d(m

gm

l–1)

ofG

eogr

aphi

cal

Sam

plin

gop

rDop

rDTr

ansc

riptio

nbP

rese

nce

Isol

ate

orig

insi

tegr

oupa

mut

atio

nof

opr

Dof

Opr

Dc

IPM

ME

MC

AZ

AT

MC

IP

AA

249

Aac

hen

(Ger

man

y)B

urn

wou

ndB

Yes

Low

No

6432

832

2M

I162

Mic

higa

n (U

SA

)B

urn

wou

ndC

2Ye

sV

ery

low

No

6416

128

128

<0.

5B

R77

6B

russ

els

(Bel

gium

)T

hroa

tB

No

Low

No

3232

128

128

4B

E12

8B

ever

wijk

(T

he N

ethe

rland

s)B

ronc

hus

C2

Yes

Low

No

328

216

<0.

5B

R71

8B

russ

els

(Bel

gium

)F

loor

AYe

sV

ery

low

No

328

416

<0.

5P

R31

7P

ragu

e (C

zech

Rep

ublic

)B

urn

wou

ndC

2Ye

sLo

wN

o16

322

3232

BU

007

Bud

apes

t (H

unga

ry)

Bur

n w

ound

C2

Yes

Low

No

1616

256

6416

BR

670

Bru

ssel

s (B

elgi

um)

Bro

nchu

sB

Yes

Low

Yese

1616

864

1A

A24

5A

ache

n (G

erm

any)

Bur

n w

ound

C2

Yes

Low

No

168

256

>25

632

LI01

0Li

sboa

(P

ortu

gal)

CF

-pat

ient

BYe

sLo

wN

o16

825

612

8<

0.5

HA

C19

Han

nove

r (G

erm

any)

CF

-pat

ient

BYe

sLo

wN

o16

832

32<

0.5

LO05

3Lo

ndon

(E

ngla

nd)

Bur

n w

ound

C2

No

Low

No

168

432

1B

O54

8B

osto

n (U

SA

)B

urn

wou

ndC

1Ye

sN

orm

alN

o16

464

32<

0.5

IS57

3Is

tanb

ul (

Turk

ey)

Bur

n w

ound

C2

Yes

Low

No

816

432

64M

I159

Mic

higa

n (U

SA

)P

ress

ure

sore

BN

oV

ery

low

Yes

84

6464

<0.

5LO

049

Lond

on (

Eng

land

)B

urn

wou

ndA

No

Ver

y lo

wYe

s8

42

82

BO

546

Bos

ton

(US

A)

Bur

n w

ound

BN

oLo

wYe

s8

2>

256

256

<0.

5B

E13

6B

ever

wijk

(T

he N

ethe

rland

s)B

ronc

hus

AN

oV

ery

low

Yes

8<

0.25

216

<0.

5B

R68

0B

russ

els

(Bel

gium

)B

urn

wou

nd‘M

utat

or’

No

Nor

mal

Yes

4<

0.25

11

<0.

5B

R64

2B

russ

els

(Bel

gium

)B

ed p

anA

No

Ver

y lo

wYe

s2

<0.

251

16<

0.5

RO

124

Rot

terd

am (

The

Net

herla

nds)

Bur

n w

ound

AN

oN

orm

alYe

s2

42

32<

0.5

BU

004

Bud

apes

t (H

unga

ry)

Thr

oat

C1

No

Nor

mal

Yes

22

14

<0.

5A

TC

C 2

7853

Bos

ton

(US

A)

Blo

odB

No

Nor

mal

Yes

21

14

<0.

5B

R25

7B

russ

els

(Bel

gium

)R

hizo

sphe

reB

No

Nor

mal

Yes

20.

51

8<

0.5

PA

O1

Mel

bour

ne (

Aus

tral

ia)

Wou

ndC

1N

oN

orm

alYe

s1

0.5

21

<0.

5

a.B

ased

on

oprD

sequ

ence

alig

nmen

t an

d cl

uste

ring.

b.

Leve

l of

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Pseudomonas oprD gene analysis 879

acids. This means that OprD is important for bacterialadaptability to changing environments, and it is knownthat such so-called contingency genes are highly mutablecompared with housekeeping genes, which are relevantfor basic bacterial metabolism and structure and whichmutate at an expected low frequency (Moxon et al., 1994).Our results highlight the need for very careful interpreta-tion of results obtained with laboratory strains. Boucheret al. (2001) recently demonstrated that strains of thesame species can differ in gene content by as much as20%. Now that the genomes of P. aeruginosa strain PAO1(Stover et al., 2000) and more than 40 other bacteria are completely sequenced (http://www.ncbi.nlm.nih.gov/PMGifs/Genomes/micr.html for an updated list), analysisof various clinical and environmental isolates should be encouraged. The use of large batches of clinical andenvironmental isolates, instead of laboratory strains, inmodels to predict the frequency and type of resistantmutants that may arise during therapy, will generate morereliable data.

Experimental procedures

Bacterial strains and growth conditions

The isolates used in this study were kindly provided by Dr A. T. McManus, US Army Institute of Surgical Research,Texas, USA; Dr L. Ménesi, General Hospital St. Istvan,Budapest, Hungary; Dr A. Vanderkelen, Queen Astrid MilitaryHospital, Neder-Over-Heembeek, Belgium; Dr J. A. Clark,Queen Mary’s University Hospital, London, England; Dr A. F.Vloemans, Rode Kruis Ziekenhuis, Beverwijk, The Nether-lands; Dr T. Taddonio, University of Michigan, MI, USA; Dr A. Radke, Klinik für Verbrennungs- und Plastische Wiederherstellungschirurgie, Aachen, Germany; ProfessorR. Konigova, Charles University Hospital, Prague, CzechRepublic; Dr R. G. Tompkins, Burns Institute, Shriners Hospital for Children, Boston, MA, USA; Dr B. Tümmler,Medizinische Hochschule, Hannover, Germany; Dr M.Caneira, Hospital de Santa Maria, Lisboa, Portugal; Profes-sor A. Boudabous, Science Faculty, Tunis, Tunisia; Dr M.Mergeay, Environmental Technology Expertise Centre, Mol,Belgium; Dr A. E. Lim Jr., St. Scholastica’s College of HealthSciences, Tacloban City, Philippines; Professor O. Hadjiiski,Scientific Institute of Emergency Medicine Pirogov, Sofia,Bulgaria; Professor K. Taviloglu, University of Istanbul, Istanbul, Turkey; and Dr W. D. H. Hendriks, Zuiderziekenhuis,Rotterdam, The Netherlands. E. coli strain C644 was kindlyprovided by Dr R. E. W. Hancock (University of British Colum-bia, Vancouver, Canada) and P. aeruginosa strain PAO1 byDr C. K. Stover (PathoGenesis Corporation, Seattle, WA,USA). P. aeruginosa strain ATCC 27853 was purchased from Gibson Laboratories. All isolates, except 10 isolatesbelonging to clone C (Römling et al., 1994), were non-clonalas determined by the AFLP technique. Unless otherwise indicated, strains were grown on Luria–Bertani broth medium(Gibco-BRL-Life Technologies) at 37°C on a rotary shaker(150 r.p.m.).

AFLP

AFLP utilized an ABI 377 automated fluorescence sequencer(PE Applied Biosystems) and the AFLP™ Microbial Fingerprinting Kit (PE Applied Biosystems) as detailed in the manufacturers’ protocols. The enzymes used were T4DNA ligase, EcoRI and Tru9I (all purchased from RocheDiagnostics). The primer pair used was EcoRI-0/MseI-C.Band patterns were analysed using BioNumerics software(Applied Maths).

Susceptibility tests

The MIC was determined in Mueller–Hinton broth (Gibco–BRL–Life Technologies) by the broth microdilution method(Amsterdam, 1991). Microtitre plates were inoculated with afinal concentration of 2 ¥ 104 to 105 CFU ml-1 and incubatedfor 18 h at 37°C. The antibiotics and their sources were asfollows: aztreonam (ATM), S.A. Bristol-Myers Squibb; cef-tazidime (CAZ), GlaxoWellcome; ciprofloxacin (CIP), Bayer;imipenem (IPM), Merck Sharp and Dome; and meropenem(MEM), Zeneca.

Mutation frequency measurement

Mutation frequencies to rifampicin were determined forstrains BR680 and PAO1 as described by Oliver et al. (2000).In short, one bacterial colony was suspended in 20 ml ofMueller–Hinton broth (MHB) and incubated overnight at37°C. Bacterial cells were collected at 3000 r.p.m. for 5 minand resuspended in 1 ml of MHB. One hundred microlitresamples of 10-fold dilutions were plated onto Mueller–Hintonagar (Gibco-BRL-Life Technologies) plates, with and withoutrifampicin (300 mg ml-1). Colony counting was performed after36 h. Experiments were performed in triplicate.

PCR and sequencing of the oprD gene

DNA was extracted from overnight P. aeruginosa culturesusing the High Pure™ PCR Template Preparation Kit (RocheDiagnostics) according to the manufacturer’s guidelines.

The oprD gene was amplified by polymerase chain reaction(PCR), using primers pDF1 (5¢-ATGAAAGTGATGAAGTGGAGC-3¢) and pDR1 (5¢-CAGGATCGACAGCGGATAGT-3¢), derived from the published oprD nucleotide sequence(Yoneyama et al., 1992). PCR was performed in 200 ml microcentrifuge tubes. The PCR mixture (50 ml final volume)contained the following: 25.5 ml of sterile distilled water, 5 ml of 10¥ PCR buffer (500 mmol l-1 KCl and 100 mmol l-1 Tris-HCl, pH 8.3), 4 ml of a deoxynucleotide mixture (dGTP, dTTP,dATP and dCTP; 2 mmol l-1 each), 5 ml of MgCl2 (2.5 mmol l-1),2.5 ml of primer pDF1 (10 mmol l-1), 2.5 ml of primer pDR1 (2.5 mmol l-1), 5 ml of template DNA and 0.5 ml of AmpliTaq DNA polymerase (5 U ml-1). All PCR reagents and primerswere ordered from PE-Applied Biosystems. The amplificationwas performed in a GeneAmp® PCR system 2400 (PE Applied Biosystems). The amplification programme was set at 50 cycles of denaturation at 94°C for 30 s, annealing at50°C for 30 s and elongation at 72°C for 1 min The reactionmixture was put on an agarose gel of 1.5% (w/v) for elec-

© 2002 Blackwell Science Ltd, Environmental Microbiology, 4, 872–882

trophoresis and visualization of the PCR product after stain-ing with ethidium bromide on a transilluminator. The DNAband corresponding to the amplified oprD gene was excisedfrom the agarose gel with a clean scalpel. DNA was extractedfrom the gel slice using the QIAEX II Gel Extraction Kit (Westburg) according to the manufacturer’s recommenda-tions. Five microlitres of the purified PCR fragment was usedas a template in the sequencing reaction. Sequencing of thecoding and anticoding strand of the oprD PCR productsnecessitated two additional internal primers, pDF2 (5¢-AACCTCAGCGCCTCCCT-3¢) and pDR2 (5¢-AGGGAGGCGCTGAGGTT-3¢). DNA sequencing utilized an ABI 377 automatedfluorescence sequencer (PE Applied Biosystems) and the ABI Prism® BigDye™ Terminator Cycle Sequencing ReadyReaction Kit (PE Applied Biosystems) as detailed in the manufacturers’ protocols. The oprD gene of isolate BE128was sequenced directly from genomic DNA. PCR and se-quencing were performed in duplicate in order to be able todetect eventual PCR mistakes. Sequences were aligned andclustered using the unweighted pair group method using arithmetic averages (UPGMA) and BioNumerics software(Applied Maths).

Reverse transcriptase PCR of oprD mRNA

RNA was extracted from overnight cultures using the HighPure™ RNA Isolation Kit (Roche Diagnostics) according tothe manufacturer’s guidelines. Extracted RNA was quantifiedspectrophotometrically at 260 nm. One microgram of RNAwas used as template for the reverse transcriptase (RT)-PCRreaction. The Titan™ One Tube RT-PCR System (RocheDiagnostics) was used. RT-PCR was performed in 200 mlmicrocentrifuge tubes. The RT-PCR mixture (50 ml finalvolume) contained the following: 23.25 µl of sterile distilledwater, 10 ml of 5¥ RT-PCR buffer, 4 ml of a deoxynucleotidemixture (dGTP, dTTP, dATP and dCTP; 2 mmol l-1 each), 5 mlof MgCl2 (2.5 mmol l-1), 2 ml of primer pDF1 (10 mmol ml-1), 2 ml of primer pDR1 (pDR2 for strain BE128) (10 mmol l-1), 5ml of template RNA, 2.5 ml of dithiothreitol (100 mM), 0.25 mlof RNase inhibitor (40 U ml-1) and 1 ml of Enzyme Mix. All RT-PCR reagents were ordered from Roche Diagnostics.Reverse transcription and amplification were performed in aGeneAmp® PCR system 2400 (PE Applied Biosystems).Reverse transcription was performed at 50°C for 30 min. The amplification programme was set at 35 cycles of denaturation at 94°C for 30 s, annealing at 49°C for 30 s andelongation at 68°C for 1 min (after 10 cycles an additionalelongation of 5 s was added to each cycle). Absence of con-taminating DNA was examined by PCR, without precedentRT reaction. No amplification product was obtained. Labora-tory strain ATCC 27853 and imipenem-sensitive isolatesBU004, RO124, and BR257, 642 and 680 were included asreferences.

Outer membrane protein preparation, SDS-PAGE andWestern blot analysis

Outer membranes were prepared by the Sarkosyl solubiliza-tion method (Filip et al., 1973) as previously described (Cornelis et al., 1989). The proteins were solubilized in

sample buffer, heated at 99°C for 5 min and separated bySDS-PAGE (12% precast criterion gels, Bio-Rad). The gelswere stained with Coomassie blue (Bio-Rad). Bio-Rad preci-sion protein standards were used as molecular weightmarkers. For the Western blot analysis, electrophoresedouter membrane proteins were electrophoretically transferredto a nitrocellulose membrane (Amersham–Pharmacia–Biotech). Anti-OprD antibody YD10 (Ochs et al., 1999) wasused as primary antibody, except for strain BR670, in whicha mixture of YD03, 11, 12, and17 antibodies was used. Alkaline phosphatase-conjugated goat antibody to mouseimmunoglobulin G (Sigma) was used as the secondary antibody. For colour development, NBT/BCP (Innogenetics)was used. Escherichia coli strains C644, expressing oprD,and DH5a were used as a control.

Acknowledgements

We are grateful to N. Gotoh (Department of Microbiology,Kyoto Pharmaceutical University, Kyoto, Japan) for providinganti-OprD antibodies and to all the collectors and providersof the bacterial isolates used in this study. We also thank M. Denoyette and A. Kholti for assistance with Western blotanalysis. This work was supported by the ‘Alphonse and JeanForton Fund’ and grants JSM-R and T G98/02 from theBelgian Department of Defense.

References

Albertini, A.M., Hofer, M., Calos, M.P., and Miller, J.H. (1982)On the formation of spontaneous mutations: the impor-tance of short sequence homologies in generation of largedeletion. Cell 29: 319–328.

Amsterdam, D. (1991) Susceptibility testing of antimicrobialsin liquid media. In: Antibiotics in Laboratory Medicine.Lorian, V. (ed.). Baltimore: Williams & Wilkins, pp. 72–78.

Boucher, Y., Nesbo, C.L., and Doolittle, W.F. (2001) Microbialgenomes: dealing with diversity. Curr Opin Microbiol 4:285–289.

Bradley, J.S., Gareau, J., Lode, H., Rolston, K.V.I., Wilson,S.E., and Quin, J.P. (1999) Carbapenems in clinical practice: a guide to their use in serious infection. Int JAntimicrob Agents 11: 93–100.

Bridges, B.A. (2001) Hypermutation in bacteria and other cellular systems. Phil Trans R Soc Lond B Biol Sci356: 29–39.

Cornelis, P., Bouia, A., Belarbi, A., Guyonvarch, A., Kam-merer, B., Hannaert, V., and Hubert, J.C. (1989) Cloningand analysis of the gene for the major outer membranelipoprotein from Pseudomonas aeruginosa. Mol Microbiol3: 421–428.

Emori, T.G., and Gaynes, R.P. (1993) An overview of nosocomial infections, including the role of the micro-biology laboratory. Clin Microbiol Rev 6: 428–442.

Epp, S.F., Köhler, T., Plésiat, P., Michéa-Hamzehpour, M.,Frey, J., and Pechère, J.C. (2001) C-terminal region ofPseudomonas aeruginosa outer membrane porin OprDmodulates susceptibility to meropenem. Antimicrob AgentsChemother 45: 1780–1787.

Farabaugh, P.J., Schmeissner, U., Hofer, M., and Miller, J.H.(1978) Genetic studies of the Lac repressor on the molec-

© 2002 Blackwell Science Ltd, Environmental Microbiology, 4, 872–882

880 J.-P. Pirnay et al.

Pseudomonas oprD gene analysis 881

ular nature of spontaneous hotspots in the LacI gene ofEscherichia coli. J Mol Biol 126: 847–857.

Filip, C., Fletcher, G., Wulff, J.L., and Earhart, C.F. (1973)Solubilization of the cytoplasmic membrane of Escherichiacoli by the ionic detergent sodium lauryl sarcosinate. JBacteriol 115: 717–722.

Fukuda, H., Hosaka, M., Iyobe, S., Gotoh, N., Nishino, T., andHirai, K. (1995) nfxC-type quinolone resistance in a clini-cal isolate of Pseudomonas aeruginosa. Antimicrob AgentsChemother 39: 790–792.

Greenblatt, M.S., Grollman, A.P., and Harris, C.C. (1996)Deletions and insertions in the p53 tumor suppressor genein human cancers: confirmation of the DNA polymeraseslippage/misalignment model. Cancer Res 56: 2130–2136.

Halter, R., Pohlner, J., and Meyer, T.F. (1989) Mosaic-likeorganization of IgA protease genes in Neisseria gonor-rhoeae generated by horizontal genetic exchange in vivo.EMBO J 8: 2737–2744.

Holloway, B.W. (1955) Genetic recombination inPseudomonas aeruginosa. J General Microbiol 13:572–581.

Hu, X., and Worton, R.G. (1992) Partial gene duplication asa cause of human disease. Hum Mutat 1: 3–12.

Huang, H., and Hancock, R.E.W. (1993) Genetic definition ofthe substrate selectivity of outer membrane porin proteinOprD of Pseudomonas aeruginosa. J Bacteriol 175: 7793–7800.

Huang, H., and Hancock, R.E.W. (1996) The role of specificsurface loop regions in determining the function of theimipenem-specific pore protein OprD of Pseudomonasaeruginosa. J Bacteriol 178: 3085–3090.

Huang, H., Jeanteur, D., Pattus, F., and Hancock, R.E.W.(1995) Membrane topology and site-specific mutagenesisof Pseudomonas aeruginosa porin OprD. Mol Microbiol16: 931–941.

Jenks, P.J., Ferrero, R.L., and Labigne, A. (1999) The role ofthe rdxA gene in the evolution of metronidazole resistancein Helicobacter pylori. J Antimicrob Chemother 43: 753–758.

Kiewitz, C., and Tümmler, B. (2000) Sequence diversity ofPseudomonas aeruginosa: impact on population structureand genome evolution. J Bacteriol 182: 3125–3135.

Köhler, T., Epp, S.F., Kocjancic Curty, L., and Pechère, J.C. (1999) Characterization of MexT, the regulator of the MexE–MexF–OprN multidrug efflux system ofPseudomonas aeruginosa. J Bacteriol 181: 6300–6305.

Köhler, T., Michéa-Hamzehpour, M., Henze, U., Gotoh, N.,Curty, L.K., and Pechère, J.C. (1997) Characterization ofMexE-MexF-OprN, a positively regulated multidrug effluxsystem of Pseudomonas aeruginosa. Mol Microbiol 23:345–354.

Krawczak, M., and Cooper, D.N. (1991) Gene deletionscausing human genetic disease: mechanisms of mutage-nesis and the role of local DNA sequence environments.Hum Genet 86: 425–441.

Kroll, J.S., Moxon, E.R., and Loynds, B.M. (1994) Naturalgenetic transfer of a putative virulence-enhancing mutationto Haemophilus influenzae type a. J Infect Dis 169: 676–679.

Leclerc, J.E., Li, B., Payne, W.L., and Cebula, T.A. (1996)

High mutation frequencies among Escherichia coli andSalmonella pathogens. Science 274: 1208–1211.

Lim, A. Jr, De Vos, D., Brauns, M., Mossialos, D., Gaballa,A., Qing, D., and Cornelis, P. (1997) Molecular andimmunological characterization of OprL, the 18-kDa outermembrane peptidoglycan-associated (PAL) lipoprotein of Pseudomonas aeruginosa. Microbiology 143: 1709–1716.

Livermore, D.M. (1992) Interplay of impermeability and chro-mosomal b-lactamase activity in imipenem-resistantPseudomonas aeruginosa. Antimicrob Agents Chemother36: 2046–2048.

Lynch, M.J., Drusano, G.L., and Mobley, H.L.T. (1987) Emergence of resistance to imipenem in Pseudomonasaeruginosa. Antimicrob Agents Chemother 31: 1892–1896.

Martinez, J.L., and Baquero, F. (2000) Mutation frequenciesand antibiotic resistance. Antimicrob Agents Chemother44: 1771–1777.

Masuda, N., and Ohya, S. (1992) Cross-resistance tomeropenem, cephems, and quinolones in Pseudomonasaeruginosa. Antimicrob Agents Chemother 36: 1847–1851.

Matic, I., Radman, M., Taddei, F., Picard, B., Doit, C., Bingen,E. et al. (1997) Highly variable mutation rates in commen-sal and pathogenic Escherichia coli. Science 277: 1833–1834.

Miller, J.H. (1996) Spontaneous mutators in bacteria, insightsinto pathways of mutagenesis and repair. Annu Rev Microbiol 50: 625–643.

Moxon, E.R., Rainey, P.B., Nowak, M.A., and Lenski, R.E.(1994) Adaptive evolution of highly mutable loci in patho-genic bacteria. Curr Biol 4: 24–33.

Ochman, H., Lawrence, J.G., and Groisman, E.A. (2000)Lateral gene transfer and the nature of bacterial innova-tion. Nature 405: 299–304.

Ochs, M.M., McCusker, M., Bains, M., and Hancock, R.E.W.(1999) Negative regulation of the Pseudomonas aerugi-nosa outer membrane porin OprD selective for imipenemand basic amino acids. Antimicrob Agents Chemother43: 1085–1090.

Ochs, M.M., Bains, M., and Hancock, R.E.W. (2000) Role ofputative loops 2 and 3 in imipenem passage through thespecific porin OprD of Pseudomonas aeruginosa. Anti-microb Agents Chemother 44: 1983–1985.

Oliver, A., Canton, R., Campo, P., Baquero, F., and Blazquez,J. (2000) High frequency of hypermutable Pseudomonasaeruginosa in cystic fibrosis lung infection. Science 288:1251–1254.

Pai, H., Kim, J.W., Kim, J., Lee, J.H., Choe, K.W., and Gotoh,N. (2001) Carbapenem resistance in Pseudomonas aerug-inosa clinical isolates. Antimicrob Agents Chemother45: 480–484.

Quin, J.P., Dudek, E.J., DiVincenzo, C.A., Lucks, D.A., andLerner, S.A. (1986) Emergence of resistance to imipenemduring therapy for Pseudomonas aeruginosa infections. J Infect Dis 154: 289–294.

Rainey, P.B., and Travisano, M. (1998) Adaptive radiation ina heterogeneous environment. Nature 394: 69–72.

Römling, U., Wingender, J., Müller, H., and Tümmler, B.(1994) A major Pseudomonas aeruginosa clone commonto patients and aquatic habitats. Appl Environ Microbiol60: 1734–1738.

© 2002 Blackwell Science Ltd, Environmental Microbiology, 4, 872–882

© 2002 Blackwell Science Ltd, Environmental Microbiology, 4, 872–882

882 J.-P. Pirnay et al.

Römling, U., Schmidt, K.D., and Tümmler, B. (1997) Largegenome rearrangements discovered by the detailed analy-sis of 21 clone C isolates found in environment and diseasehabitats. J Mol Biol 271: 386–404.

Satake, S., Yoneyama, H., and Nakae, T. (1991) Role ofOmpD2 and chromosomal b-lactamase in carbapenemresistance in clinical isolates of Pseudomonas aeruginosa.J Antimicrob Chemother 28: 199–207.

Sinden, R.R., Hashem, V.I., and Rosche, W.A. (1999) DNA-directed mutations. Ann N Y Acad Sci 870: 173–189.

Smith, J.M., Dowson, C.G., and Spratt, B.G. (1991) Local-ized sex in bacteria. Nature 349: 29–31.

Smith, M., and Sperling, D. (1999) Novel 23-base-pair dupli-cation in TSC1 exon 15 in an infant presenting with cardiacrhabdomyomas. Am J Med Genet 84: 346–349.

Song, J., and Colombini, M. (1996) Indications of a commonfolding pattern for VDAC channels from all sources. JBioenerg Biomembr 28: 153–161.

Spiers, A.J., Buckling, A., and Rainey, P.B. (2000) The causesof Pseudomonas diversity. Microbiology 146: 2345–2350.

Stover, C.K., Pham, X.-Q., Erwin, A.L., Mizoguchi, S.D., Warrener, P., Hickey, M.J. et al. (2000) Complete genomesequence of Pseudomonas aeruginosa, an opportunisticpathogen. Nature 406: 959–964.

Streisinger, G., Okada, Y., Emrich, J., Newton, J., Tsugita, A.,Terzaghi, E., and Inouye, M. (1966) Frameshift mutationsand the genetic code. Cold Spring Harbor Symp QuantBiol 31: 77–84.

Taddei, F., Radman, M., Maynard-Smith, J., Toupance, B.,Gouyon, P.H., and Godelle, B. (1997) Role of mutatoralleles in adaptive evolution. Nature 387: 700–702.

Tamber, S., Warrener, P., and Hancock, R.E.W. (2001) Roleof OprD and OprD homologues in nutrient uptake inPseudomonas aeruginosa. Poster presentation (abstract

PS204) at the Pseudomonas 2001 conference, Brussels,Belgium, 17–21September.

Trias, J., and Nikaido, H. (1990) Outer membrane protein D2catalyzes facilitated diffusion of carbapenems and penemsthrough the outer membrane of Pseudomonas aeruginosa.Antimicrob Agents Chemother 34: 52–57.

Trinh, T.Q., and Sinden, R.R. (1993) The influence of primaryand secondary DNA structure in deletion and duplicationbetween direct repeats in Escherichia coli. Genetics 134:409–422.

Vincent, J.L., Bihari, D.J., Suter, P.M., Bruining, H.A., White,J., Nicolas-Chanoin, M.H. et al. (1995) The prevalence ofnosocomial infection in intensive care units in Europe.Results of the European Prevalence of Infection in Inten-sive Care (EPIC) Study. EPIC International Advisory Com-mittee. J Am Med Assoc 274: 639–644.

Wilderman, P.J., Vasil, A.I., Johnson, Z., and Vasil, M.L.(2001) Genetic and biochemical analyses of a eukaryotic-like phospholipase D of Pseudomonas aeruginosa suggesthorizontal acquisition and a role for persistence in a chronicpulmonary infection model. Mol Microbiol 39: 291–303.

Yoneyama, H., and Nakae, T. (1993) Mechanism of efficientelimination of protein D2 in outer membrane of imipenem-resistant Pseudomonas aeruginosa. Antimicrob AgentsChemother 37: 2385–2390.

Yoneyama, H., Yoshihara, E., and Nakae, T. (1992)Nucleotide sequence of the protein D2 gene ofPseudomonas aeruginosa. Antimicrob Agents Chemother36: 1791–1793.

Zhou, J., Bowler, L.D., and Spratt, B.G. (1997) Interspeciesrecombination, and phylogenetic distortions, within the glu-tamine synthetase and shikimate dehydrogenase genes ofNeisseria meningitidis and commensal Neisseria species.Mol Microbiol 23: 799–812.