Identification of Francisella tularensis in natural water samples by PCR

ELSEVIER FEMS Microbiology Ecology 16 (1995) 83-92

Identification of Francisella tularensis in natural water samples by PCR

Mats Forsman *+‘, Anna NY&n a, Anders Sjiistedt aTb, Lilian Sjiikvist a, Gunnar Sandstriim aTb

a Department of Microbiology, National Defense Research Establishment, S-901 82 Umeit, Sweden ’ Department of Infectious Diseases, University of Umeii, S-901 87, Umeii, Sweden

Received 18 May 1994; revision received and accepted 6 September 1994

Abstract

Francisella tulurensis, the causative agent of the epizootic disease tularemia in mammals, can be isolated from mud and water. To study the spread and persistence of Francisella tulurensis in water, different strategies for pre-treatment of natural water samples prior to identification of the bacterium by polymerase chain reaction (PCR) were evaluated. A method for handling of samples taken from natural waters was developed. Applied on natural water samples amended with F. tularensis, the method rendered identification by PCR reproducible and it resulted in an amplified Francisella-specific product in all samples from natural waters tested. In addition, by employing primers targeting conserved regions of the 16s rDNA the presence of bacteria was demonstrated in all samples investigated. The results presented will, in combination with other techniques that allow identification, improve studies on the epizootiology and epidemiology of the genus Francisella.

Keywords: Polymerase chain reaction; Water; Identification; Francisella tularensis; Purification

1. Introduction

Francisella tularensis is the causative agent of

tularemia. The manifestation of the infection differs

depending on the route of transmission. Ulceroglan-

dular or glandular tularaemia is seen after infection

by arthropods or after direct contact with infected

animals. When the portal of entry of the bacterium is

the conjunctiva of the eye, oculoglandular tularemia

occurs. Respiratory tularemia results from inhalation

of contaminated dust, whereas after ingestion of

contaminated food or water oropharyngeal tularemia

* Corresponding author. Tel: ( f 46)90-106669; Fax: ( + 46)90- 106800.

is seen. Finally, a typhoid form of tularemia is

known of which the mechanism of infection is un- clear.

The bacterium is a small Gram-negative rod which occurs as two main types [ll. Type A also designated F. tularensis var. tularensis is predominately found in North America where it is frequently transmitted to humans by ticks [2,3]. The other main type is type B, F. tularensis var. palaearctica, which can be found in all epizootic areas, including Sweden, where tularemia occurs [2-41. This type is often seen as a disease in man attributed to contact with muskrats and/or beavers. Accordingly, this type of the bacterium has a close connection to water and it has been reported that it can be isolated from ponds and streams [5]. The bacterium is highly infectious and as

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84 M. Forsman et al. / FEMS Microbiology Ecology 16 (1995) 83-92

few as ten bacteria are needed to cause infection [4]. The infection dose might be somewhat higher by ingestion of contaminated water compared to transmission by vectors or by inhalation of contaminated aerosols [3,5].

In several countries bacteria of the type B variant of F. tularensis have been isolated from water samples as the cause of outbreaks of tularemia [3,5-g]. Isolation of the bacterium has been performed from

water samples used for domestic purposes [3,6-8] as well as from natural water systems [3,5]. However, in Sweden, isolates of F. tularensis from water samples have never been reported, although from the Northern part of the country, north of limes norrlandicus, there are annual reports of sporadic cases or minor epidemics of tularemia [9,10]. In this area tularemia in man is generally connected to transmission of the bacterium by mosquitoes [lo], even though the largest outbreak reported of the disease was due to inhalation of dust from hay contaminated with vole faeces [9].

One emerging method for identification of infectious agents is the polymerase chain reaction (PCR). This method has been applied for detection and identification of microorganisms in different samples taken from humans, animals, and the environment. Although the method is powerful, each application has to be evaluated because different samples may contain organic and/or inorganic material which interfere with the reaction.

In the present paper the PCR with primers specific for identification of the genus Francisella, was utilised to identify the bacterium in different water samples taken in the Northern part of Sweden. PCR was also used to amplify the total pool of 16s rRNA genes in the samples, by the aid of primers comple- mentary to conserved regions of genes encoding 16s rRNA. Moreover, to evaluate the PCR for identification of the bacteria with special references to F. tularensis, investigations in order to optimise methods for pre-treatment of samples were carried out.

F. tularensis is a fastidious bacterium which re- quires special supplementation of media for its growth, which makes isolation of the bacterium com- plicated since other bacteria may be favoured on most media utilizable for growth of F. tularensis [ll]. There are, however, other methods for identification of F. tularensis in water samples based on the use of antibodies to trap the bacterium [12]. Such methods have the drawbacks of low sensitivity and they are also time and labour consuming. F. tularensis has been applied to identification methods aimed at the genetic information hosted by the bacterium [13]. With such methods it has been possible to identify bacteria in samples taken directly from a

patient [141.

2. Material and methods

2.1. Bacteria

Francisella tularensis LVS, live vaccine strain, obtained from the US Army Medical Research Insti- tute of Infectious Diseases, Fort Detrick, Frederick, MD, USA, was used throughout this study.

2.2. Primers

The primers used in this study are listed in Table

1.

Table 1 Sequence and positions of the primers used in the polymerase chain reaction

Desig- Gene Position a Annealing Sequence

nation temperature

Fl

R13 F2

RI4 F5

Fll

16%rRNA 9-27 68°C 16S-rRNA 1544-1525 68°C

16S-rRNA 31-51 68°C 16%rRNA 985-964 68°C 16S-rRNA 1290-1272 62°C

16S-rRNA 149-168 62°C

5’-GAG’I-ITGATCCI’GGCTCAG-3’ Universal region 1251 S-AGAAAGGAGGTGATCCAGCC-3’ Universal region [251 5’-GAACGCTGGCGGCAGGCCAA-3’ Universal region This study 5’-GGTAAGGTTCTTCGCG’lTCXAT-3’ Universal region This study

5’-CC’ITI-ITGAGTITCGCTCC-3’ Francise~~a specific region [191

5’-TACCAGTTGGAAACGACTGT-3’ Francisella specific region [ 191

16s rDNA Target Ref.

a Positions of the primers are numbered according to the nomenclature of E. coli.

M. Forsman et al. / FEMS Microbiology Ecology 16 (1995) 83-92 85

2.3. Water sample collection

Water samples was collected in sterile lOO-ml bottles at an average depth of 1 m during July and August 1993 from lakes and rivers in Sweden north of limes norrlandicus. The bottles were immersed at a depth of 1 m and the caps were opened to allow inflow of water. One water sample was taken from a well on a farm in the county of JHmtland where, in 1973, 13 cases of tularemia occurred. Although not disclosed, the well was suspected to be the source of transmission of F. tularensis.

2.4. Enumeration of bacteria

Ten ml of each water sample were filtered onto 47-mm nitro-cellulose filters with a pore size of 0.45 pm. Each sample was collected onto two filters. One filter of each sample was incubated on LA [15] plates and the other one on modified Thayer-Martin agar plates containing Gc medium base (36 g/l; Difco Laboratories, Detroit, MI), haemoglobin (10 g/l; Difco), and IsoVitaleX (10 mg/l; BBL Micro- biology Systems, Cockeysville, MD), and incubated for three days at 37°C in 5% CO,. Viable counts after each incubation were determined. Duplicates of the plates were incubated under aerobic conditions at 37°C.

extraction prior to PCR (B). One ml of the water samples were treated by the alkaline method as described [17], and the resulting material was used in the PCR (C). Chromosomal DNA from 1 ml of the water samples were prepared as described [18] and subjected to PCR (D). The final method used (Table 3, E) for pre-treatment utilised concentration of the bacteria on sterile filters. 445 ~1 of each water sample with added bacteria was filtered through a Ultrafree-MC filter (low-binding Durapore, 0.22 pm pore size, Millipore Inc.) by centrifugation at 12000 X g for 2 min. Bacteria on the filter was resuspended in 95 ~1 of TE-buffer. To lyse bacteria 5 ~1 of 10% (wt/vol) sodium dodecyl sulfate (SDS) and 0.5 ~1 of (10 mg/ml) Proteinase K (Sigma Chemi- cal Co., St. Louis, MO) were added to the samples and incubated at 37°C for 10 min. Next, 17 ~1 of 5 M NaCl and 13 ~1 of CTAB/NaCl solution (10% hexadecyltrimethyl ammonium bromide in 0.7 M NaCl) was added, followed by incubation at 65°C for 10 min. The DNA was further purified by adding 125 ~1 of phenol-chloroform-isoamyl alcohol (24 : 24 : 2). The total liquid phase was then passed through the filter by centrifugation 12000 X g for 5 min. The aqueous eluate was collected and further purified on a microspin column (SR-400 HR, Phar- macia Biotech) according to the instructions given by the manufacturer. Ten ~1 of the final eluate was used in the PCR.

2.5. Release and purification of DNA of filter-bound bacteria prior to PCR 2.6. PCR amplification

Several methods for pre-treatment of the water PCR was performed in a total volume of 25 ~1. samples were evaluated (see Table 3). In all experi- The final reaction mixture contained primers at a ments the waters were amended with F. tularensis to concentration of 0.4 PM, dATP, dCTP, dGTP, and a final concentration of 8 X 103/ml. The water sam- d’ITP, all at a concentration of 200 PM, 20 mM ples were pre-treated according to Bej et al. [16]. (NH&SO,, 75 mM Tris-HCl (pH 9.0 at 25”C), One ml of water was filtrated through FGLP filters. 0.01% Tween 80, 4.5 mM MgCl,, and 1 U of The DNA was released by repetitive freeze-thawing Thermus aquaticus enzyme (Taq DNA polymerase; cycles, and the PCR were carried out in the presence Advanced Biotechnologies Inc.). Samples were sub- of the filter (A). Bacterial cells in 1 ml of water were jected to amplification in a DNA thermal cycler concentrated by centrifugation at 12000 X g for 1 9600 (Perkin Elmer Cetus, Norwalk, CT). An ampli- min. The pellets were resuspended in 200 ~1 Insta- fication cycle consisted of denaturation for 15 s at Gene purification matrix (Bio-Rad Laboratories, 94°C primer annealing to the template at a tempera- Hercules, CA) and then treated according to the ture unique to each primer pair for 15 s, and primer instructions given by the manufacturer, with the extension at 72°C for 22 s. Different annealing tem- exception that the DNA was further purified by peratures were evaluated for each primer pair. Under using phenol-chloroform-isoamyl alcohol (24 : 24 : 2) the condition used, the temperatures given in Table 1

86 M. Forsman et al. /FEMS Microbiology Ecology 16 (1995) 83-92

resulted in an amplified product of the expected size. In all experiments, 35 cycles were performed. For nested PCR two sets of primers (Fl-R13 and F2-R14) targeting conserved regions of prokaryotic 16s rRNA genes were used. First, 20 cycles of amplification with the Fl-R13 primer pair (Table 1) was performed using the same conditions as described above. This resulted in an amplified product of 1550 bp. One ~1 of this PCR mixture was transferred to new tubes and subjected to a nested PCR using the F2-R14 primer pair (Table l), resulting in a 950-bp DNA fragment as expected. The nested PCR’s were performed for 30 cycles. Ten ~1 of the PCR products were electrophoresed in a 1.0% agarose gel, stained with ethidium bromide, and photographed.

3. Results

3.1. Sensitivity of the PCR

Primers have previously been designed to be used in PCR for specific detection of the genus Fran- cisella [19]. The sensitivity of the assay in non-contaminated water samples was investigated by use of these primers after addition of F. tularensis to deionized water and tap water. Bacteria were suspended in the water samples and subjected to amplification by PCR using the genus-specific primers F5-Fll (Table 1). Samples were analyzed after 35 cycles of amplification. The detection limit of the assay was approx. 10 bacteria/sample in deionized water or tap-water (Fig. 1).

3.2. Collection and characterisation of lake waters

Tularemia occurs as sporadic cases or minor outbreaks in the Northern part of Sweden. Water samples were collected from lakes and rivers north of limes norrlandicus (Fig. 2). Viable counts of bacteria in water samples were determined by plating. LA medium and an enriched medium, modified Thayer- Martin agar. suitable for growth of F. tularensis were used. Viable counts were similar on the two types of media. The water samples contained lo-lo3 bacteria/ml (Table 2). Most cultures contained more than one colony type, but usually less than five. Colonies that showed macroscopic resemblance to

12 34 56 78 910

4,0-

2,0-

l.O-

Fig. 1. Sensitivity of PCR in deionized- and tap water samples

using Francisella genus-specific primers. Lane 1: Positive con-

trol, 8 X lo3 F. tularensis/ml resuspended in PCR buffer. Lanes

2-5: F. hdarensis resuspended in deionized water, numbers of

bacteria respectively 0.8, 8, 8 X lo’, 8 X 103/ml. Lanes 6-9: F.

tularensis resuspended in tap water, numbers of bacteria respec-

tively 0.8, 8, 8 X lo’, 8 X 103/ml. Lane 10: negative control, PCR

buffer without added bacteria.

F. tularensis were further analysed by microscopy and slide agglutination. No isolates were found to have the properties of Francisella. Similar results of viable microorganisms were obtained both after incubation in 5% CO, at 37°C and ambient atmo- sphere at 37°C. The relative content of humic acids in the water samples was estimated by measurement of the absorbance at 260 nm. The obtained values varied between 0.06 to 0.64 OD units (Table 2). It should be noted that the presence of free nucleic acids in the samples could not be excluded.

3.3. Detection of native bacterial DNA in natural water samples by PCR

First it was investigated whether it was possible to directly amplify native bacterial DNA from the water samples. Primers were utilised that target conserved regions of genes encoding 16s rRNA. No amplified DNA products could be detected in agarose gels from any of the 21 different water samples, although they obviously contained bacteria (Table 2). There- fore, a nested PCR was tried. Using two sets of primers specific to conserved regions of prokaryotic 16s rRNA genes, a 950-bp DNA fragment was expected to be amplified from the original 1550-bp


PCR product. First, 20 cycles of amplification with the Fl-R13 primer pair (Table 1) was performed, whereupon 1 ~1 of this PCR mixture was transferred to new tubes and subjected to a nested PCR using the F2-R14 primer pair (Table 1). This strategy enabled detection of PCR amplified products in ap- proximately 70% of the samples (Table 2).

It was then determined whether filtration of the water samples, followed by nested PCR, would be a more advantageous system for detection of the native bacteria contained in the waters. Ten ml of the different water samples were filtered onto different types of filters. The following filters were investigated: FHLP (hydrophobic), FGLP (hydrophobic), HVHP (hydrophilic), GVHP (hydrophilic), GSWP (hydrophobic) with a pore size of 0.2 pm (Millipore Inc.). Filters were then put into microcentrifuge tubes.

Bacteria were released either by boiling or by sub- jecting the tubes to repeated cycles of freeze-thawing. Freeze-thawing appeared to yield somewhat better results than boiling for 5 min. Before PCR amplification, the filters were removed from the tubes. However, no additional water sample became positive after more than two cycles of freeze-thawing. Virtually no difference in amplification was demonstrated irrespective of the type of filter used (data not shown).

To judge the potential advantage combining filtration and nested PCR, bacteria from ten ml of sample were filtrated through FGLP filters. Bacteria were lysed by two cycles of freeze-thawing. Thereafter, released DNA was subjected to nested PCR. The system did not result in any higher sensitivity than did the direct nested amplification without filtration

Table 2

Characteristics of the waters and results of PCR using universal 16S-rRNA primers

No. Water source Number of Absorbance Direct

bacteria at 260 nm PCR a

x lo3

Direct

nested

PCR b

Filtration

prior nested PCR b,c

1 NIstansjijln 1.00 0.17 - + -

2 ;isele 0.50 0.21 _ - _

3 Gortistjlm 0.80 0.19 - + +

4 ~gennanalven 0.50 0.14 _ + +

5 Volgsjlin 1.00 0.06 + +

6 T%sjo 1.00 0.21 - - -

7 Braxele 0.50 0.64 - _

8 Skogsbacken 0.20 0.32 -

9 Miellejohka 0.10 0.02 - + +

10 Umellven 0.11 0.23 _ +

11 Stalonbkken 0.09 0.11 + +

12 Skarvsjon 0.07 0.12 + +

13 Vistansjdn 0.08 0.06 + +

14 Ratan 0.12 0.10 - + + 15 Knyttabacken 0.03 0.17 - - _

16 Atjikb?icken 0.01 0.10 + +

17 Bullerforsen 0.13 0.08 + +

18 V%jm%n 0.09 0.06 - + + 19 TaIlsjo 0.20 0.30 - _

20 Abisko 0.10 0.07 + +

21 Uman 0.10 0.07 + +

1 The direct PCR were carried out directly on 10 11 unpurified water from each sample using the Fl-R13 primer pair. The nested PCR were carried out with two sets of primers specific to conserved regions of prokaryotic 16s rRNA genes. First, 20 cycles

of amplification with Fl-R13 primer pair (Table 1) was performed, where upon nested PCR was done using the F2-R14 primer pair

(Table 1).

’ 10 ml of each water were filtrated through FGLP filters. Bacteria were lysed by two cycles of freeze-thawing. Thereafter, released DNA was subjected to the nested PCR.

M. Forsman et al. /FEMS Microbiology Ecology 16 (1995) 83-92

polar circle .________

Fig. 2. Map of Sweden. The black line indicate limes norrlandicus. This line separates the northern boreal (or taiga) zone from

the southern borea-nemoral zone. Sporadic cases and endemics of

tularemia occur above limes norrlandicus. The water samples

were taken mostly from the counties of Vasterbotten and Ilimt-

land.

(Table 2). Thus, with two exceptions, no amplification was obtained from the water samples irrespective of the method used. Viable counts in these negative PCR samples were, however, not lower than in any other samples, but showed a higher average absorbance at 260 nm. Hence, either nested PCR or a combination of nested PCR with filtration was sufficient to obtain DNA in all samples that were amenable for amplification.

3.4. Identification of F. tularensis in natural water samples

With the aim of developing a method to detect the possible presence of F. tularensis in natural water samples, further work was initiated to determine the cause of the negative PCR reactions. To accomplish this, F. tularensis was added to the samples from natural water sources and several different proce- dures to pretreat the samples prior to PCR were evaluated (Table 3, column A-E). Throughout this series of experiments the genus-specific Francisella primer pair (F5Fll) was used in the PCR. The method described by Bej et al. [16], which involves filtration of the bacteria on FGLP filters and subse- quent release of DNA by repeated freeze-thawing, showed low sensitivity (Table 3, column A). Further- more, it was found that the samples in which no natural bacteria could be detected also inhibited the

Table 3

Evaluation of different methods for pre-treatment of the waters

prior to PCR

No. Water source Method

A B C D E

1 Nastansjiiin

2 Lele

3 Gortistjlm

4 &rgermanllven

5 Volgsjiin

6 Tisjd

7 Braxele

8 Skogsbacken 9 Miellejohka

10 Umeiilven 11 Stalonbacken

12 Skarvsjon

13 Viistansjon

14 Ratan

15 Knyttabacken

16 Atjikbacken 17 Bullerforsen

18 V%jm%n

19 Tallsjii

20 Abisko 21 Uman

+ - _ _ + + + - + - _ + _ + _ + + + _ + _ + + + + + + + + + + + + - + -

_ + + + -

_ + + + +

+ - + _ + +

+ + +

+ + + - + + + + + + - + - f + + + + + + + + + + + + + + + + + + + + - + + + + + f + + + +

In all experiments the waters were amended with F. tularensis to

a final concentration of 8 X 103/ml. PCR were carried out with the genus-specific primer pair F5-Fll. For further details, see Materials and Methods,


12 14 16 18 20 B

Al23 45 67 89108 A II 13 15 17 19 21

Fig. 3. Identification of F. rulurensis by PCR using the Francisellu genus-specific primer pair (F5-Fll) in the different water samples

indicated in Table 3. Lane A: Positive control, 8 X lo3 F. tuZurensis/ml resuspended in PCR buffer. Lanes 1-21: 8 X lo3 F. tdarensis ml

resuspended in the different waters (corresponding numbers) indicated in Table 3. Lanes B: negative control, PCR buffer without added

bacteria.

amplification of added bacteria when this method was used. Four other methods were also evaluated and the outcome of this was that three of the four methods showed a similar lack of sensitivity and reproducibility (Table 3). Therefore, methodological considerations had to be taken, and one improved method based on the following preparative procedure resulted in reproducible identification of F. tularensis by PCR. First, a given amount of water amended with F. thrensis was filtered through a sterile filter (Ultrafree-MC, Millipore Inc.). Bacteria retained on the filter were lysed by treatment with SDS and Proteinase K. Proteins and carbohydrates were pre- cipitated by the addition of CTAB in high salt concentration. The DNA was further purified by the addition of phenol-chloroform-isoamyl alcohol. The liquid phase containing the DNA was driven through the filter by centrifugation. After collection the obtained material was further purified on a microspin column (SR-400 HR, Pharmacia Biotech). Finally, an aliquot of the recovered eluate was used directly . _ --_-_ .- in the PCK. T‘he whole procedure is completed in less than 1 h.

A representative result of the assay, including the 21 different water samples specified in Table 3 amended with F. tularensis, is shown in Fig. 3. Also, it was possible to amplify 16s rRNA genes from 100% of the water samples with this assay (Fig. 4, I>, in contrast to the use of nested PCR or

filtration combined with nested PCR (Table 2). How- ever, it was not possible to detect any amplified product in non-amended water samples with the Francisella-specific primer pair (Fig. 4, II). In addition, another 53 waters from different locations in the Northern part of Sweden has been tested for the

Fig. 4. Result of PCR-assisted amplification in natural water

samples (specified in Table 3) by the Fl-R13 primer pair comple-

mentary to conserved regions in all eubacterial 16s rRNA se-

quences (I) and the Francisella genus-specific F5-Fll primer pair (II). Lane A: Positive control, F. tdarensis resuspended in PCR

buffer. Lanes 1-21: corresponding to the different waters indi-

cated in Table 3. Lane B: negative control, PCR buffer without

added bacteria. The same DNA molecular weieht marker was used in both experiments.


presence of F. tularensis. In PCR using the Fran- cisellu genus-specific primers, all these water samples showed an amplified product when amended with F. tularensis. However, none of these water samples seemed, a priori, to be contaminated with F. tularensis as determined by PCR.

3.5. Sensitivity of the assay in natural waters

The sensitivity of the assay in natural water samples was investigated by addition of F. tularensis to the 21 different water samples specified in Table 3. Bacteria were suspended and diluted to different concentrations (loo-104), based on viable counts, in the waters and thereafter subjected to amplification by PCR using the genus-specific primers F5-Fll. Samples were then analysed directly after 35 cycles of amplification. The same sensitivity in natural water samples as in distilled water samples was obtained. In all cases tested the sensitivity was lower than 10 bacteria/ml which corresponded to 0.4 bacteria in the PCR tube. Thus, less than 10 F. tulurensis could be detected using this method which is in agreement with the results of identification of F. tularensis in distilled water samples.

4. Discussion

In the present study, results are presented on the highly sensitive PCR for detection of F. tularensis in water samples taken in Northern Sweden where tularemia occurs as sporadic cases or minor epidemic outbreaks. Although tularemia can be transmitted to man by ingestion of drinking water or by direct contact with rodents operating in the surroundings of water [3,.5-81, no human case of tularemia in Swe- den has been documented where water-borne transmission has been proven. The reason for this is unknown, but it might be speculated that the bacterium occurs in low numbers in water and, due to its requirements for growth, over-growth of contaminat- ing bacteria in a water sample may occur [ll], thus resulting in lack of identification of F. tularensis. Moreover, since all documented cases of tularemia in Sweden have transmission routes other than water [9,10], the possibility of water contamination may be overlooked. Recently it has been shown that atypical

F. tularensis strains exist, and accordingly such strains may be classified as non-typable Gram-negative bacteria [20]. It is, however, not known if such F. tularensis strains are present in Sweden.

Usually tularemia in mammals is diagnosed by immunological methods using samples taken from subjects with symptoms of the disease [21]. Further- more, F. tularensis bacteria may be identified as a cause of illness, by the use of media enriched to support growth [11,22] or by immunoassays with specific antibodies against the bacterium [12]. Akin to the methods for identification of the bacterium are methods that target the harboured genetic information of F. tularensis. Such methods have been used to identify and discriminate strains of F. tularensis [13], identify bacteria from pus from a patient [14], and to study the phylogeny of the genus to which F. tularensis belongs [ 191.

PCR is a powerful tool for both rapid and specific identification of microorganisms in different samples [23]. Naturally, the method is easier to judge and is more reproducible the less contaminated a sample is with organic and/or inorganic material. To over- come the drawbacks of such materials present in samples obtained from the environment, many different methods have been reported [16]. These include use of filtration, treatment with phenol-chloroform, freeze-thawing cycles, different types of chelators, purification by gel filtration etc. In the report by Bej et al. [16] it was found that microorganisms trapped on hydrophobic filters followed by extraction of bacterial DNA by repetitive freeze-thawing resulted in reproducible detection. However, in this paper it was found that filtration with different filters and freeze-thawing did not result in improved possibility to identify microorganisms including F. tularensis. The reason for the failure described here of filtration through hydrophobic filters followed by identification was not further explored. Instead, filtration in combination with chemical extraction of nucleic acids followed by gel filtration rendered F. tularensis assessable for detection in all water samples tested.

Advantages of the method described are its rapid- ity and simple concept which should make it amenable to the development of an automatic pro- cessing protocol. It should be emphasized that identification with this method relies on material trapped on the filter. This means that non-cultivable viable

M. Forsman et al. / FEMS Microbiology Ecology 16 (I 995) 83-92 91

microorganisms would be detected. Although it was possible to detect F. tularensis in all the water samples included in this study, it cannot be excluded that other water samples may contain other types of inhibitors to the PCR than those eliminated here. Accordingly, to further evaluate the method for treatment of water samples prior to PCR, more water samples have to be tested.

Examination of the species or taxonomic compo- sition of a total bacterial assemblage in a pond, stream or lake is of general interest. To this end, the study of 16s rRNA sequences from nucleic acids extracted from such environmental samples is of vital importance, since only a fraction of the ob- served bacteria in nature is readily cultivable [24 and references therein]. In this context, it is noteworthy that the methodological concept presented is not just limited to identification of the genus Francisella and species therein. With the described method it was possible to extract and purify DNA accessible to amplification by PCR, from native contained bacteria in all waters, as demonstrated by the successful amplification with the prokaryotic universal primer- pair Fl-R13. Any of the Fl-R13 amplified products obtained in this study could be cloned and sequenced in order to obtain a better knowledge of the total bacterial assemblages in these water samples. How- ever, it should be taken into consideration that a sequence originating from a PCR-product itself may not disclose identification of a microorganism, since at least for cultivable infectious microorganisms it is important to fulfil Koch postulate. Nevertheless, PCR can give rapid and valuable information about a causative agent and the emerging possibilities of molecular description might render such techniques sufficient for identification.

One of the waters (TLsjii) tested here was obtained in the neighbourhood of a previous minor outbreak of tularemia which was thought to be due to contamination of F. tularensis in a well located on a farm in the county of JEmtland in Northern Sweden. Neither in this water nor any of the other waters tested from the Northern part of Sweden resulted in a positive PCR, indicating that the water samples lack the genetic material from F. tularensis

searched for. Nevertheless, the presented method for pre-treatment of water samples followed by PCR gives rise to new possibilities to detect and identify

the causative agent of tularemia in water. Moreover, this improved method for detection of F. tularensis

opens up new ways of studying the epizootology and epidemiology of F. tularensis.

Acknowledgements

This work was in part supported by the UMI- NOVA foundation, P.O. Box 1450, S-901 29 UmeH, Sweden. We thank Thorsten Johansson for collecting the water samples.

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Identification of Francisella tularensis in natural water samples by PCR

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