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Applied and Environmental Microbiology, August 2005, p. 4269-4275, Vol. 71, No. 8
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.8.4269-4275.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Bacteriophages May Bias Outcome of Bacterial Enrichment Cultures

Maite Muniesa, Anicet R. Blanch, Francisco Lucena, and Juan Jofre*

Department of Microbiology, Faculty of Biology, University of Barcelona, Diagonal 645, E-08028 Barcelona, Spain

Received 27 October 2004/ Accepted 7 March 2005


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Enrichment cultures are widely used for the isolation of bacteria in clinical, biotechnological, and environmental studies. However, competition, relative growth rates, or inhibitory effects may alter the outcome of enrichment cultures, causing the phenomenon known as enrichment bias. Bacteriophages are a major component in many microbial systems, and it abounds in natural settings. This abundance means that bacteriophages are likely to be present in many laboratory enrichment cultures. Our hypothesis was that bacteriophages present in the sample might bias the enriched subpopulation, since it can infect and lyse the target bacteria during the enrichment step once the bacteria reach a given density. Here we show that the presence of bacteriophages in Salmonella and Shigella enrichment cultures produced a significant reduction (more than 1 log unit) in the number of these bacteria compared with samples in which bacteriophages had been reduced by filtration through 0.45-µm non-protein-binding membranes. Furthermore, our data indicate that the Salmonella biotypes isolated after the enrichment culture change if bacteriophages are present, thus distorting the results of the analysis.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The awareness that bacteriophages are an abundant and ubiquitous component of many natural settings (9, 10, 46, 48) has stimulated a great deal of interest in its role in microbial systems, either in the regulation of the density or the diversity of bacterial populations (23, 27, 41, 47) or in horizontal gene transfer (13, 28, 42, 49). Additionally, bacteriophages interfere with many artificial processes, such as cheese making (12), malolactic fermentation in wine making (26), or activated sludge wastewater treatment (29, 30). This abundance and ubiquity means that bacteriophages are likely to be present in many laboratory cultures initiated from natural samples. The question then arises as to what extent individual members of the bacterial community in the culture can be affected by the presence of bacteriophages and, consequently, whether bacteriophages influence the outcome of laboratory experiments.

Many microbiological methods rely either totally or partially on enrichment cultures, and many of these cultures are initiated with natural samples. These techniques are based on the axiom that certain microorganisms from a mixture can be enriched with respect to other microorganisms by using batch liquid cultures that favor their growth. Such cultures are used either for the enrichment of a given group of bacteria, such as bacterial indicators (e.g., indicators of water quality), bacterial pathogens, and bacteria expressing specific phenotypes for the evaluation of metabolic pathways (nitrification-denitrification, oxidation and reduction of sulfur compounds, hydrocarbon degradation, certain fermentations, and reduction of mineral ions—Mn, Fe, and As, etc.) of naturally occurring microbial populations. This presence/absence determined by enrichment in batch liquid cultures can be applied in multiple tube assays to enumerate the bacteria by methods such as the most-probable number (MPN) method. Enrichment cultures are widely used for the detection and isolation of bacterial indicators and pathogens from food, water, and biosolids; bacterial pathogens from clinical samples; and bacteria with a given activity in both biotechnological and environmental microbiology studies. Many standardized methods in environmental and food quality control are based on enrichment cultures. In spite of the emergence of genomic methods that may replace enrichment cultures in certain cases, the latter are still extensively used in laboratory practices. Even new methodological approaches based on genomic techniques contemplate preliminary steps for the selective or nonselective enrichment of the target microorganisms (21, 22, 44). However, enrichment bias may alter the outcome of such cultures (17). The factors most frequently implicated in these effects are competition, relative growth rates, and growth inhibitors. Our hypothesis was that bacteriophages can contribute to the enrichment bias.

The densities of bacteria and bacteriophages needed for phage replication and consequent host cell destruction (31, 38, 45) are far below the numbers of bacteria attained in enrichment cultures. Moreover, the physiological status of bacteria being enriched, which are actively growing, is optimal to support the replication of bacteriophages (38). Thus, if specific phages are present in the sample, the densities of host bacteria reached during the enrichment period could be high enough to cause phage infection and replication, even with a low initial number of phages (31, 38, 45). Thus, phages may devastate the bacterial population and cause bias at the end of the enrichment process. Consequently, the presence in the initial sample of phages infecting a given bacterial subpopulation would bias the outcome of the enrichment.

On the other hand, filtration through 0.22-µm- or 0.45-µm-pore-size membranes reduces the numbers of free phage particles in a natural sample, since bacteria, but not phage, are retained by the membrane. This procedure does not remove prophages in lysogenic bacteria, which can be a significant portion of the bacteria in certain environments (40). Optimal reduction is provided by non-protein-binding membrane filters such as those made of polyvinylidene fluoride or polyether sulfone, which allow most phages to pass (36, 43). On the other hand, the membranes of cellulose esters habitually used to retain bacteria adsorb a high proportion of the phages present in the sample (35).

We studied the effect of removing phages in the sample on enrichment for Salmonella and Shigella. These pathogens are associated with water and food, and their detection by enrichment frequently gives spurious results, even when well-standardized methods based on enrichment procedures are used (4, 19).


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Strains and media.
Clinical isolates of Salmonella enterica serovar Typhimurium (Tcr and Ampr) and Salmonella enterica serovar Enteritidis (Nalr) were used as host strains to evaluate the effect of phages on Salmonella detection and as positive controls in the PCR assays. A clinical isolate of Shigella sonnei (Tcr and Ampr) was used in the experiments conducted with Shigella and as a host strain for phages infecting Shigella. Escherichia coli WG5 (6) was used for the determination of phages in the sample and to evaluate the reduction of phages in the phage-reduced sample.

Salmonella enterica serovar Brandenberg, Salmonella enterica serovar Wirchow, and Salmonella enterica serovar Rissen clinical isolates were additionally used as host strains to evaluate the number of bacteriophages present in sewage infecting different Salmonella species.

Selenite-F broth (Scharlau Microbiology, Barcelona, Spain) and bismuth sulfite (BS) agar (Oxoid, Madrid, Spain) were used for the enrichment and detection of Salmonella. Tryptic soy broth (TSB) and xylose lysine deoxycholate (XLD) agar (Oxoid, Madrid, Spain) were used for the enrichment and detection of Shigella. TSB and semisolid TSB (TSB containing 0.7% agar) were used for the enumeration of bacteriophages infecting Salmonella and Shigella and for the evaluation of aerobic bacteria. Modified Scholten's medium and agar (6) were used for the detection of phages infecting E. coli WG5.

Tetracycline (25 µg/ml), ampicillin (100 µg/ml), and nalidixic acid (200 µg/ml) were used for the selection of the strains used.

Samples.
Raw sewage samples were collected from inflowing raw sewage at three municipal wastewater treatment plants in the Barcelona area.

Bacteriophage enumeration.
Phages infecting E. coli were counted in sewage samples following the protocol described in ISO 10705-2 (6). Phages infecting several Salmonella species and serovars and S. sonnei were counted in the enrichment cultures by the double agar layer method (1) in TSB.

Aerobic bacteria determination.
Aerobic bacteria present in sewage or in the phage-reduced sample and grown in tryptic soy agar (TSA) were evaluated by performing decimal dilutions in phosphate-buffered saline (PBS), plating 0.1 ml of each dilution in TSA, and incubating the samples under aerobic conditions at 37°C for 18 h.

Phage reduction procedure.
Volumes of 1 or 10 ml of sewage were filtered through 0.45-µm polyvinylidene fluoride DURAPORE membranes (Millipore, Bedford, Massachusetts), described by the manufacturer as low-protein-binding membranes, which allowed the bacteriophages to pass through while the bacteria were retained. To further remove phages retained on the filters, 10 ml of PBS was added to the surface of the filter, gently agitated, and eliminated by filtration. Preliminary experiments assessed whether one or two washing steps were necessary to achieve 99% phage reduction. Finally, the experiments were performed with two consecutive washing steps, since the reduction of phage was improved without significant loss of bacteria, as evaluated in TSA and as described above, both before and after the phage reduction procedure (see Table 2). These analyses were only conducted in a first set of experiments. In subsequent experiments, the recovered bacteria were used for the enrichment procedures. For this, the bacteria retained by the filter were recovered in 1.2 ml of PBS. From this, 1 ml was used for the enrichment cultures and the remaining 0.2 ml was used to assess the phage reduction.


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  		TABLE 2. Reduction of naturally occurring somatic coliphages and total aerobic bacteria grown in TSA after filtration through non-protein-binding membrane filters

 
Isolation of Salmonella from sewage.
The presence of Salmonella was studied in 1 ml of sewage from an untreated sample or from a phage-reduced sample obtained as described above.

Salmonella was detected and isolated from the sample following previously described protocols (4) with few modifications. Briefly, the sample was preenriched with 4 ml of TSB for 4 h, enriched in 45 ml of selenite-F broth, and then incubated at 35 to 37°C for 18 h. Decimal dilutions of the enrichment culture were plated on BS agar and incubated at 37°C for 18 h, where colonies suspected to be Salmonella are black, with or without a metallic sheen with a "halo."

After each enrichment selection process, 10 suspect Salmonella colonies were isolated and identified by PCR with specific primers for Salmonella spp. (24). The primers used were 5'-GATCATCCATTCGGCATTAAACA-3' and 5'-TTCTCAGCGACGGAAGGGTAAATC-3'. PCR conditions were 2 min of initial denaturation at 94°C, followed by 35 cycles of 92°C for 1 min, 48°C for 1 min, and 72°C for 1 min. Amplimers were visualized in agarose gels stained with ethidium bromide.

Isolates identified as Salmonella by PCR were further confirmed and characterized with the commercial identification kit for enterobacteria API-20E (BioMérieux, La Balme, France) in accordance with the manufacturer's instructions.

Detection of seeded S. enterica serovar Typhimurium and S. enterica serovar Enteritidis.
Two clinical isolates of S. enterica (serovar Typhimurium [Tcr Ampr] and serovar Enteritidis [Nalr]) and one of Shigella (S. sonnei [Tcr Ampr]), chosen on the basis of the numbers of phages found in sewage that infected these strains, were seeded into raw sewage in independent experiments. S. enterica serovar Typhimurium grown on TSB was inoculated into 20 ml of a 50-fold-diluted sewage sample to a final concentration of 50 to 100 CFU/ml. Ten milliliters was directly inoculated into 40 ml of selenite-F broth. Ten milliliters was filtered as described above, washed twice in PBS, and finally eluted in 10 ml PBS. The filtered sample was inoculated into 40 ml of selenite-F broth.

The enrichment procedure and detection of seeded Salmonella were performed as described above, with BS containing tetracycline and ampicillin to select for the growth of S. enterica serovar Typhimurium.

The same procedure, using agar plates containing nalidixic acid, was used for the detection of S. enterica serovar Enteritidis.

Detection of S. sonnei.
One clinical isolate of Shigella (S. sonnei [Tcr Ampr]), selected on the basis of the numbers of phages from sewage which infected this strain, was seeded into raw sewage in independent experiments. In this case, the growth of Shigella was studied in two different liquid media: selenite-F broth and TSB broth supplemented with tetracycline and ampicillin. The use of both media was due to the poor growth of Shigella in selenite-F broth. Experiments were conducted in the same way as those described for Salmonella but using Tcr and Ampr in the XLD agar plates as selective media. Suspect Shigella colonies are black-centered red colonies on XLD.

Data computation and statistics.
An analysis of variance test was performed to determine the significance of the differences between the series of results. Similarity and clustering analyses between isolates were performed by nearest-neighbor and single-linkage distances. Both statistical analyses were performed using the Statistical Package for Social Science (7).


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Presence of bacteriophages infecting Salmonella and Shigella in sewage.
Experiments conducted to evaluate the presence of phages infecting Salmonella and Shigella (Table 1) showed the presence, in sewage, of phages infecting the different species and serovars tested. The occurrence and levels of phages were fairly consistent in samples of the different settings where sewage was obtained and also in the samples of the same setting taken at different days (Table 1).


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  		TABLE 1. Bacteriophages present in sewage detected with different Salmonella and Shigella strains

 
Evaluation of the phage reduction procedure.
In preliminary experiments to optimize the phage reduction procedure, phages infecting E. coli that occur naturally in sewage was enumerated to determine whether the filtration step followed by either one or two washing steps reduced the number of phages in a given sample without significant reduction in the number of bacteria. The sample obtained after one filtration followed by one washing step in PBS still presented a number of phages ranging between 6 to 47% (average of 20%) of the initial samples. After a second washing step, the filtered sample presented only a proportion ranging from 0.9 to 1.1% (average 1.0%) of free phage particles compared with the initial sample. To evaluate whether filters let phages pass through, phages eliminated from the sample were detected in preliminary experiments by analyzing the filtrate of the first and second steps for phage enumeration.

With this procedure (two washing steps), 10 samples were tested in independent experiments for the presence of phages of E. coli (Table 2) in the material retained by the membrane. A significant (Student's t test, P < 0.01) 99.3% average reduction in the number of phages retained was attained. Thus, when the bacteria retained by the filter were resuspended in water, the phage-reduced sample differed from the untreated sample in its bacteriophages content. No variation in the bacterial content was produced by the phage reduction procedure, since results of the first set of experiments evaluating the recovery of aerobic bacteria in TSA did not show significant reduction in the number of bacteria (Table 2).

The removal of phage was monitored in every experiment by counting the phage remaining in the sample after the phage reduction procedure.

Evaluation of seeded Salmonella and Shigella in the phage-reduced samples after enrichment.
The numbers of phages infecting bacteria ranged from 3 ± 1 PFU per ml for S. enterica serovar Typhimurium and 8 ± 2 PFU per ml for S. sonnei. With the 99.3% reduction reported above, the probability of finding phages infecting these clinical isolates in the diluted sewage was low. Such phages were not detected in 0.2 ml of the phage-reduced samples.

The antibiotic resistance of both strains of Salmonella (serovars Typhimurium and Enteritidis) was a suitable marker for monitoring the variation in numbers of the inoculated strains, since some background flora present in sewage grows in the selenite-F broth used as enrichment medium. Averaged results from five independent experiments are shown in Fig. 1. Panel A shows the numbers of both Salmonella serovars attained after 18 h of enrichment of the untreated sample and the phage-reduced sample. The differences were significant (Student's t test, P < 0.05 [serovar Enteritidis], P < 0.01 [serovar Typhimurium]). In parallel, the numbers of phages forming plaques on each of the inoculated bacteria after enrichment are shown in panel B. Whereas the numbers of phages in the enrichments corresponding to the untreated samples were 107 and 5 x 106 PFU/ml, respectively, those of the phage-reduced samples were undetectable (panel B).



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  		FIG. 1. Determination of S. enterica serovar Typhimurium and serovar Enteritidis. Panel A corresponds to bacterial determinations and shows the numbers of both Salmonella serovars attained after 18 h of enrichment of the untreated sample and the phage-reduced one; panel B corresponds to the numbers of phages plaguing each of the inoculated bacteria after enrichment. Grey bars indicate the values of the untreated sample, and white bars correspond to the values of the phage-reduced sample. Values shown are the averages of the results from five independent experiments. Standard deviations are represented by error bars.

 
Similar trends to those obtained with Salmonella were observed with enrichments of samples seeded with S. sonnei. As described above, the growth of Shigella was studied in selenite-F broth and TSB broth supplemented with tetracycline and ampicillin. In both approaches, the numbers of Shigella in the enrichments of the phage-reduced samples were significantly higher (Student's t test, P < 0.01) than in the enrichments of the untreated samples (Fig. 2A). In addition, the number of phages showed the same trend as with the Salmonella enrichments (Fig. 2B). This is an increase in the number of phages concurrent with a decrease in the number of bacteria in the untreated samples.



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  		FIG. 2. Determination of Shigella sonnei using selenite or TSB with nalidixic acid in the enrichment culture. Panel A corresponds to bacterial determinations, and panel B corresponds to bacteriophage enumeration. Grey bars indicate the values of the untreated sample, and white bars correspond to the values of the phage-reduced sample. Values shown are the averages of the results from five independent experiments. Standard deviations are represented by error bars.

 
These results clearly show that phages affect the outcome of these enrichment cultures as a consequence of infection and lysis of the host bacteria.

Evaluation of naturally occurring Salmonella in phage-reduced samples after enrichment. (i) Quantitative variation of Salmonella.
Analyses were performed to assess the influence of phages on the detection of naturally occurring Salmonella in sewage using a standardized method. From each of the enrichments, 10 Salmonella suspect colonies were isolated and confirmed by specific PCR amplification of a fragment of the 16S RNA gene and biochemical phenotyping (API-20E profiles). The results (Table 3) indicate significantly higher numbers (Student's t test, P < 0.01) of suspect and confirmed Salmonella in the phage-reduced cultures. There was also a difference in the percentage of samples in which Salmonella was detected; while Salmonella was detected in only three untreated samples, it was detected in seven phage-reduced samples.


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  		TABLE 3. Detection of naturally occurring Salmonella in sewage

 
(ii) Qualitative variation of Salmonella.
For each sample, isolates from the enrichment of the untreated sample showed different API-20E profiles (used to prepare dendrogram in Fig. 3) than those from the enrichment with the phage-reduced sample in a group of naturally occurring Salmonella isolated in samples with or without phage reduction. In addition, clustering analyses for enriched samples with phages (untreated samples) showed higher diversity in the API-20E profiles of Salmonella strains, whereas almost a single biochemical profile appeared after the phage reduction procedure (reduced samples). Figure 3 shows two main clusters, corresponding mostly to phage-reduced isolates from two different sewage samples (A and B). Inside these two clusters, the scale distance between isolates was lower than 1.0, indicating that all isolates had identical biochemical profiles. Differences between the two large clusters (grey boxes) were revealed by only two biochemical tests (lysine decarboxylase and melobiose). The similarity distances between the rest of the isolates (mostly corresponding to untreated samples) were greater, indicating their diversity. Although this experiment was performed only with two different samples in which Salmonella was isolated in the untreated and phage-reduced samples, results clearly show that the absence of phage allows detection of a preponderant strain or strains that were not detected when phages were present in the sample.



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  		FIG. 3. Nearest-neighbor and single-linkage dendrogram showing clustering of 23 Salmonella strains isolated from two sewage samples (A and B) after the phage reduction procedure (Red) or from untreated (Unt) samples. The dendrogram has been constructed using raw results from the tests included in the API-20E gallery. The scale along the x axis indicates similarities between different clusters. Cluster distances lower than 1.0 inside the same cluster indicate that isolates are identical or that differences in the biochemical profile are reported. Grey boxes show the two big clusters identified: one corresponded to reduced samples of sewage sample B and the second corresponded to reduced samples of sewage sample A.

 

   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Evaluation of phages infecting local clinical isolates of Salmonella and Shigella shows that phages infecting strains circulating among the human population was present in all sewage samples tested, with numbers ranging from about 103 to 105 PFU per 100 ml. Moreover, the numbers of phages were of the same order of magnitude at different time intervals. Similar numbers of phages infecting S. enterica serovar Typhimurium in raw sewage had been reported in the United States (15).

Taking into consideration these data and published data regarding phage die-off in the environment (15, 18, 34), the presence of phages infecting many Salmonella species and serovars is probable in different kinds of samples, for example, source drinking water (2) and shellfish (3), to which enrichment for Salmonella is routinely performed in public health laboratories.

However, phages can easily be reduced from aqueous natural samples. The reduction of the numbers of bacteriophages in the natural sample, applying the complete set of phage reduction methods described here, was significant, since data regarding somatic coliphages indicate an average reduction of 99.3%. The reduction of phages in the samples to be enriched can certainly be done more efficiently, but the aim of this study was merely to show the role of phages in enrichment cultures. Regarding our experiments, it is clear that we removed the phages infecting Salmonella in most of the samples spiked and those tested for naturally occurring Salmonella and that the Salmonella that may have been lost in the phage reduction process is compensated for by the beneficial effect of the process in the enrichment. Therefore, it seems clear that phages can be removed from natural samples without significantly impairing the numbers of bacteria in the sample to be enriched. In fact, a prefiltration step and incubation of the membrane filter and retained material is currently used to increase the detection of pathogens in water by enrichment (5, 14, 20). However, methods recommended do not detail the kind of membranes to be used and, consequently, they do not account for phage behavior. But taking into consideration that, depending on the chemical nature of the membranes, some membrane filters, such as those of cellulose esters, do adsorb phages (11, 35), we strongly recommend using non-protein-binding membranes and washing off the material retained by the membrane filters once or twice, since according to present knowledge on adsorption to and elution from surfaces (35), phages retained in the filters will be eluted by most enrichment media. The use of non-protein-binding membranes, such as those of polyvinylidene fluoride or polyether sulfone, and the introduction of a washing step will improve the performance of this prefiltration step. In the experiments reported here, 0.45-µm-pore-size non-protein-binding membranes were used. The use of 0.22-µm-pore-size non-protein-binding membranes should be explored when the bacteria to be enriched, such as Vibrio, may go through the 0.45-µm pores. Obviously, the procedure proposed here does not remove either all the free phage particles or the temperate phages in lysogenic bacteria, which had been reported to be quantitatively significant in some environments (40). At present, however, to our knowledge, treatments to reduce the number of lysogenic bacteria in a sample prior to enrichment are not feasible.

The results regarding the tests performed with natural samples indicate that the filtration step performed to remove phages influences both the quantitative results and the qualitative ones. Thus, the numbers of Salmonella grown in the enrichment cultures with the filtered sample were significantly higher than those in the untreated samples. The difference is large and, in many enrichment cultures with untreated samples, it renders Salmonella undetectable among the background flora also growing in the enrichment, thereby giving false-negative test results in many samples. Moreover the Salmonella isolates from phage-reduced samples were different to from those isolated in the non-phage-reduced samples. It can then be concluded that the enrichment may also bias the identity of bacterial isolates. Thus, if in a given sample, phages infecting the most abundant bacteria are present, the enrichment may lead to the failure to isolate the most abundant bacteria. Our results on the spiked samples indicate that bacteriophages play a major role in the enrichment bias of both Shigella and Salmonella enrichment cultures, since the reduction in the final number of bacteria in the untreated samples parallels the increase in the number of specific bacteriophage.

Enrichment bias in quantification of bacteria is frequently mentioned in the literature. A typical manifestation of this bias is the discrepancy between numbers detected by CFU and numbers detected by MPN of enrichment cultures in liquid media, with the surprising results that CFU give higher counts than MPN (32, 39). Another potential manifestation is the difficulty in isolating Shigella both in carriers and sewage-polluted waters (20, 44). Results here and elsewhere (37) indicate that the number of phages infecting Shigella in sewage is high, probably because Shigella shares many bacteriophages with E. coli (37) and, consequently, the phages in the sample may be partially responsible for the low success in the isolation of Shigella from many types of samples.

Also, the effect of enrichments in the evaluation of the diversity and type distribution of bacteria in natural samples has been described in different kinds of samples (17, 39). Furthermore, the apparent lack of coincidence of the pathogens (Salmonella and Yersinia) isolated in sewage or sewage-polluted water in a given area compared with the clinical isolates of the same area (8, 16, 22, 33) may be explained, at least partially, by the enrichment bias. A potential negative effect of reducing the number of phages from a sample will be a reduction in the diversity of the bacterial isolates after enrichment. However, the diversity and type distribution after enrichment cannot be the same as the diversity and type distribution in the environment from where the sample was taken. Consequently, enrichment is not a good procedure for studying the diversity and distribution of bacteria in natural samples, since factors such as growth rates or differential growth inhibitors may change the proportions of the various enriched bacteria and even mask the most abundant bacteria in the sample.

Thus, enrichment bias is manifested both in the numbers of bacteria detected and in the diversity and distribution of the bacterial isolates, leading to erroneous conclusions in many kinds of studies. This bias has been mostly imputed to differences in growth rate of the organisms obtained from the environment (25), but the present article provides data indicating that this bias is at least in part caused by bacteriophages present in the sample and that reducing the numbers of bacteriophages is feasible. We suggest that in many enrichment procedures, mostly in complex samples, it will be recommendable to introduce an additional step, such as filtration with non-protein-binding membranes. This would allow reduction of the number of phages in the sample if the objective is to detect the presence of the most abundant bacteria and to enumerate the sample accurately.


   ACKNOWLEDGMENTS
 
We thank Ruth Serra-Moreno, Cristina Valdivieso, and Susana Calle for technical assistance and Guillem Prats for providing the clinical isolates of Shigella and Salmonella.

This study was supported by the Generalitat de Catalunya (2001SGR00099) and the Centre de Referència en Biotecnologia (CeRBa). M.M. is a researcher of the "Ramon y Cajal" program of the Spanish Ministry of Science and Technology.


   FOOTNOTES
 
* Corresponding author. Mailing address: Department of Microbiology, Faculty of Biology, University of Barcelona, Diagonal 645, E-08028 Barcelona, Spain. Phone: 34 934 021487. Fax: 34 934 039047. E-mail: jjofre{at}ub.edu. Back


   REFERENCES
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Applied and Environmental Microbiology, August 2005, p. 4269-4275, Vol. 71, No. 8
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.8.4269-4275.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.




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