Bacteriophages may bias outcome of bacterial enrichment cultures

Abstract

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-microm 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.

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.

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.

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.