Enterococcus species distribution among human and animal hosts using multiplex PCR

B A Layton 1, S P Walters 1, L H Lam 1, A B Boehm 1

Affiliations

PMID: 20132375
DOI: 10.1111/j.1365-2672.2010.04675.x

Free article https://sfamjournals.onlinelibrary.wiley.com/doi/10.1111/j.1365-2672.2010.04675.x

Abstract

Aims: This study evaluated the use of Enterococcus species differentiation as a tool for microbial source tracking (MST) in recreational waters.

Methods and results: Avian, mammalian and human faecal samples were screened for the occurrence of Enterococcus avium, Enterococcus casseliflavus, Enterococcus durans, Enterococcus gallinarum, Enterococcus faecium, Enterococcus faecalis, Enterococcus hirae and Enterococcus saccharolyticus using multiplex PCR. Host-specific patterns of Enterococcus species presence were observed only when data for multiple Enterococcus species were considered in aggregate.

Conclusions: The results suggest that no single Enterococcus species is a reliable indicator of the host faecal source. However, Enterococcus species composite 'fingerprints' may offer auxiliary evidence for bacterial source identification.

Significance and impact of study: This study presents novel information on the enterococci species assemblages present in avian and mammalian hosts proximate to the nearshore ocean. These data will aid the development of appropriate MST strategies, and the approach used in this study could potentially assist in the identification of faecal pollution sources.

Discussion

To date, the Enterococcus species diversity in avian and mammalian hosts adjacent to coastal areas has not been fully characterized. This study provides a snapshot of the predominant Enterococcus species populations in gulls, dogs, horses, seals, sea lions and humans proximate to the Northern California coast. These data underscore the ubiquitous nature of enterococci in animal faeces and sewage, while revealing host-specific variations in the composition of species assemblages. Nearly every species of Enterococcus that was tested for was found in each host examined, which suggests that no single species of Enterococcus is a reliable indicator of human faeces.

Significantly different patterns of Enterococcus species presence between hosts were identified, although when individual ‘fingerprints’ were assembled into a dendrogram, horses were the only host group that formed an exclusive cluster. The observed groupings may be driven by similarities in the hosts’ diet and/or habitat. For example, high similarities were seen among the marine hosts as well as between humans and dogs. The observed variation between the sewage samples may be because of temporal separation: fewer species were detected in the winter than in summer. Repeated faecal sampling across seasonal timescales would be informative, as the diet and/or habitat (and therefore the intestinal microbiota) of the hosts may change with time. The groupings could also be verified by screening a broad range of host species in various locations.

The robustness of the fingerprints could be further optimized by evaluating the host distribution of other Enterococcus species beyond the eight considered here, as it was evident that additional Enterococcus species were likely present. For example, one avian (seagull) faecal sample did not produce any PCR products when screened with the species-specific primers, although it did generate amplicons with the Enterococcus genus primers. This suggests that this individual carried enterococci other than the species targeted in this study; possibilities include Enterococcus columbae or Enterococcus cecorum, which have been detected previously in avian hosts (Aarestrup et al. 2002). Inclusion of these or other Enterococcus species could potentially improve the observed fingerprint clustering among the gulls (Fig. 3).

The results from the mixed-template control experiment highlight a drawback of using enrichment cultures followed by PCR for Enterococcus speciation. Different species may have different growth or recovery rates on selective media and TSB (Lleo et al. 2001), leading to some species out-competing others in the enrichment. In addition, the various PCR assays may have different sensitivities in complex mixtures of target and nontarget DNA. However, the enrichment technique is consistent with the method being used for the Ent. faecium esp gene human faecal marker assay (Scott et al. 2005; Yamahara et al. 2007; Layton et al. 2009) and provides a straightforward way to rapidly and simultaneously detect multiple species in a sample. While the PCR multiplexes used in this study provide a reasonable estimate of the species present in a particular sample, the mixed-template control experiment indicated that false negative results for Ent. durans and Ent. saccharolyticus are possible.

Despite the drawbacks of the method, multiple unique enrichment cultures from each host individual produced consistent results in each species assay a great majority (84%) of the time. Given the expected heterogeneity among organisms initially present in replicates of membrane filtration, this level of agreement is quite satisfactory. The validity of the method was further confirmed by subculturing isolates from sewage membrane filters prior to enrichment. Each species that was isolated was also detected in the corresponding enrichment culture, and for more densely populated filters, additional species were detected in the enrichment culture that were not among the randomly selected isolates. These results suggest that despite the potential for enrichment bias, the method still provides a reasonable snapshot of the enterococci species present in a given sample.

The distribution of Enterococcus species presence among hosts observed in the present study differs from previous works. For example, Wheeler et al. (2002) reported very low frequencies of Ent. gallinarum in humans and dogs, whereas the PCR multiplexes used here detected Ent. gallinarum in 38% of dogs and 33% of humans (Table 2). Similarly, Wheeler et al. (2002) did not detect Ent. avium or Ent. durans in dogs or humans, while both of these Enterococcus species were observed in dogs and humans in this study. However, other studies of Enterococcus species differentiation (Wheeler et al. 2002; Harwood et al. 2004; Ferguson et al. 2005) utilized biochemical assays; thus, it may not be possible to directly compare those results to the data presented here. To date, this is the first study to use exclusively molecular methods to determine Enterococcus species distribution in human and nonhuman animal faeces.

It is notable that Ent. casseliflavus was detected in 21 (34%) of 62 host individuals, as well as both sewage samples, as this species is considered epiphytic (Mundt and Graham 1968; Müller et al. 2001). It is conceivable that this organism may be incorporated into the microbiota of the digestive tract after consumption. The present study supports the idea that the presence of Ent. casseliflavus in faeces could be attributable to host diet: Ent. casseliflavus was detected most frequently in horses, the only exclusive herbivore that was sampled. Nevertheless, these data illustrate that detection of Ent. casseliflavus in the environment cannot be attributed exclusively to nonfaecal sources.

The analysis shown here suggests that Enterococcus species assemblages may be conserved within host species, which supports previous findings (Kühn et al. 2003). Additional insight into host-specific Enterococcus assemblages – including relative abundances – could be obtained by extracting DNA directly from water or faeces, with no enrichment step. The method could then potentially be adapted for quantitative PCR or high-throughput sequencing. The host specificity of Enterococcus assemblages suggests possible MST applications for Enterococcus species fingerprints; however, this prospect is confounded by several factors. Both differential survival of Enterococcus species in the environment and variable recovery from the VBNC state have been observed (Lleo et al. 2001, 2005); thus, the ability to detect each species could vary depending on environmental conditions and the duration of environmental exposure. Additional work is needed to determine whether host-specific fingerprints could be discerned among various ratios of mixed faecal inputs and ‘background’ enterococci in the environment. The ability to parse mixed fingerprints in the environment could be further complicated by the presence of all eight species in sewage (Table 2); detection of all eight species in the environment could indicate either sewage or a mixture of animal inputs. This problem could potentially be rectified by utilizing a different combination of Enterococcus species in the fingerprints. Applying species fingerprinting as an MST method would likely require the creation of a fingerprint library for different geographical locations and thus may be prohibitively expensive. Lastly, species fingerprints could only offer information on the faecal source and not the risk to health.

The work presented here offers valuable information on faecal Enterococcus assemblages that is pertinent to recreational water quality monitoring. It is evident from this and other studies that no single species of Enterococcus is an accurate indicator of human faecal contamination and that detection of a given species can neither confirm nor rule out a specific source. Finally, these data illustrate the cosmopolitan nature of the genus in hosts proximate to the nearshore environment.

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Enterococcus species distribution among human and animal hosts using multiplex PCR

Abstract

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