Michael Elk

Development of Quantitative PCR Assays Targeting the 16S rRNA Genes of Enterococcus spp. and Their Application to the Identification of Enterococcus Species in Environmental Samples

ABSTRACT The detection of environmental enterococci has been determined primarily by using culture-based techniques that might exclude some enterococcal species as well as those that are nonculturable. To address this, the relative abundances of enterococci were examined by challenging fecal and water samples against a currently available genus-specific assay (Entero1). To determine the diversity of enterococcal species, 16S rRNA gene-based group-specific quantitative PCR (qPCR) assays were developed and evaluated against eight of the most common environmental enterococcal species. Partial 16S rRNA gene sequences of 439 presumptive environmental enterococcal strains were analyzed to study further the diversity of enterococci and to confirm the specificities of group-specific assays. The group-specific qPCR assays showed relatively high amplification rates with targeted species (>98%), although some assays cross-amplified with nontargeted species (1.3 to 6.5%). The results with the group-specific assays also showed that different enterococcal species co-occurred in most fecal samples. The most abundant enterococci in water and fecal samples were Enterococcus faecalis and Enterococcus faecium, although we identified more water isolates as Enterococcus casseliflavus than as any of the other species. The prevalence of the Entero1 marker was in agreement with the combined number of positive signals determined by the group-specific assays in most fecal samples, except in gull feces. On the other hand, the number of group-specific assay signals was lower in all water samples tested, suggesting that other enterococcal species are present in these samples. While the results highlight the value of genus- and group-specific assays for detecting the major enterococcal groups in environmental water samples, additional studies are needed to determine further the diversity, distributions, and relative abundances of all enterococcal species found in water.

Detection of fecal bacteria and source tracking identifiers in environmental waters using rRNA-based RT-qPCR and rDNA-based qPCR assays.

In this study, we evaluated the use of RT-qPCR assays targeting rRNA gene sequences for the detection of fecal bacteria in water samples. We challenged the RT-qPCR assays against RNA extracted from sewage effluent (n = 14), surface water (n = 30), and treated source water (n = 15) samples. Additionally, we applied the same assays using DNA as the qPCR template. The targeted fecal bacteria were present in most of the samples tested, although in several cases, the detection frequency increased when RNA was used as the template. For example, the majority of samples that tested positive for E. coli and Campylobacter spp. in surface waters, and for human-specific Bacteroidales, E. coli, and Enterococcus spp. in treated source waters were only detected when rRNA was used as the original template. The difference in detection frequency using rRNA or rDNA (rRNA gene) was sample- and assay-dependent, suggesting that the abundance of active and nonactive populations differed between samples. Statistical analyses for each population exhibiting multiple quantifiable results showed that the rRNA copy numbers were significantly higher than the rDNA counterparts (p < 0.05). Moreover, the detection frequency of rRNA-based assays were in better agreement with the culture-based results of E. coli, intestinal enterococci, and thermotolerant Campylobacter spp. in surface waters than that of rDNA-based assays, suggesting that rRNA signals were associated to active bacterial populations. Our data show that using rRNA-based approaches significantly increases detection sensitivity for common fecal bacteria in environmental waters. These findings have important implications for microbial water quality monitoring and public health risk assessments.

Biofilms on Hospital Shower Hoses: Characterization and Implications for Nosocomial Infections

ABSTRACT Although the source of drinking water (DW) used in hospitals is commonly disinfected, biofilms forming on water pipelines are a refuge for bacteria, including possible pathogens that survive different disinfection strategies. These biofilm communities are only beginning to be explored by culture-independent techniques that circumvent the limitations of conventional monitoring efforts. Hence, theories regarding the frequency of opportunistic pathogens in DW biofilms and how biofilm members withstand high doses of disinfectants and/or chlorine residuals in the water supply remain speculative. The aim of this study was to characterize the composition of microbial communities growing on five hospital shower hoses using both 16S rRNA gene sequencing of bacterial isolates and whole-genome shotgun metagenome sequencing. The resulting data revealed a Mycobacterium-like population, closely related to Mycobacterium rhodesiae and Mycobacterium tusciae, to be the predominant taxon in all five samples, and its nearly complete draft genome sequence was recovered. In contrast, the fraction recovered by culture was mostly affiliated with Proteobacteria, including members of the genera Sphingomonas, Blastomonas, and Porphyrobacter. The biofilm community harbored genes related to disinfectant tolerance (2.34% of the total annotated proteins) and a lower abundance of virulence determinants related to colonization and evasion of the host immune system. Additionally, genes potentially conferring resistance to β-lactam, aminoglycoside, amphenicol, and quinolone antibiotics were detected. Collectively, our results underscore the need to understand the microbiome of DW biofilms using metagenomic approaches. This information might lead to more robust management practices that minimize the risks associated with exposure to opportunistic pathogens in hospitals.

Intestinal Microbiota and Species Diversity of Campylobacter and Helicobacter spp. in Migrating Shorebirds in Delaware Bay

ABSTRACT Using 16S rRNA gene sequencing analysis, we examined the bacterial diversity and the presence of opportunistic bacterial pathogens (i.e., Campylobacter and Helicobacter) in red knot (Calidris canutus; n = 40), ruddy turnstone (Arenaria interpres; n = 35), and semipalmated sandpiper (Calidris pusilla; n = 22) fecal samples collected during a migratory stopover in Delaware Bay. Additionally, we studied the occurrence of Campylobacter spp., enterococci, and waterfowl fecal source markers using quantitative PCR (qPCR) assays. Of 3,889 16S rRNA clone sequences analyzed, the bacterial community was mostly composed of Bacilli (63.5%), Fusobacteria (12.7%), Epsilonproteobacteria (6.5%), and Clostridia (5.8%). When epsilonproteobacterium-specific 23S rRNA gene clone libraries (i.e., 1,414 sequences) were analyzed, the sequences were identified as Campylobacter (82.3%) or Helicobacter (17.7%) spp. Specifically, 38.4%, 10.1%, and 26.0% of clone sequences were identified as C. lari (>99% sequence identity) in ruddy turnstone, red knot, and semipalmated sandpiper clone libraries, respectively. Other pathogenic species of Campylobacter, such as C. jejuni and C. coli, were not detected in excreta of any of the three bird species. Most Helicobacter-like sequences identified were closely related to H. pametensis (>99% sequence identity) and H. anseris (92% sequence identity). qPCR results showed that the occurrence and abundance of Campylobacter spp. was relatively high compared to those of fecal indicator bacteria, such as Enterococcus spp., E. faecalis, and Catellicoccus marimammalium. Overall, the results provide insights into the complexity of the shorebird gut microbial community and suggest that these migratory birds are important reservoirs of pathogenic Campylobacter species.

Development and Evaluation of a Quantitative PCR Assay Targeting Sandhill Crane (Grus canadensis) Fecal Pollution

ABSTRACT While the microbial water quality in the Platte River is seasonally impacted by excreta from migrating cranes, there are no methods available to study crane fecal contamination. Here we characterized microbial populations in crane feces using phylogenetic analysis of 16S rRNA gene fecal clone libraries. Using these sequences, a novel crane quantitative PCR (Crane1) assay was developed, and its applicability as a microbial source tracking (MST) assay was evaluated by determining its host specificity and detection ability in environmental waters. Bacteria from crane excreta were dominated by bacilli and proteobacteria, with a notable paucity of sequences homologous to Bacteroidetes and Clostridia. The Crane1 marker targeted a dominant clade of unclassified Lactobacillales sequences closely related to Catellicoccus marimammalium. The host distribution of the Crane1 marker was relatively high, being positive for 69% (66/96) of the crane excreta samples tested. The assay also showed high host specificity, with 95% of the nontarget fecal samples (i.e., n = 553; 20 different free-range hosts) being negative. Of the presumed crane-impacted water samples (n = 16), 88% were positive for the Crane1 assay, whereas none of the water samples not impacted by cranes were positive (n = 165). Bayesian statistical models of the Crane1 MST marker demonstrated high confidence in detecting true-positive signals and a low probability of false-negative signals from environmental water samples. Altogether, these data suggest that the newly developed marker could be used in environmental monitoring studies to study crane fecal pollution dynamics.

Distribution of human-specific bacteroidales and fecal indicator bacteria in an urban watershed impacted by sewage pollution, determined using RNA- and DNA-based quantitative PCR assays.

ABSTRACT The identification of fecal pollution sources is commonly carried out using DNA-based methods. However, there is evidence that DNA can be associated with dead cells or present as “naked DNA” in the environment. Furthermore, it has been shown that rRNA-targeted reverse transcription-quantitative PCR (RT-qPCR) assays can be more sensitive than rRNA gene-based qPCR assays since metabolically active cells usually contain higher numbers of ribosomes than quiescent cells. To this end, we compared the detection frequency of host-specific markers and fecal bacteria using RNA-based RT-qPCR and DNA-based qPCR methods for water samples collected in sites impacted by combined sewer overflows. As a group, fecal bacteria were more frequently detected in most sites using RNA-based methods. Specifically, 8, 87, and 85% of the samples positive for general enterococci, Enterococcus faecalis, and Enterococcus faecium markers, respectively, were detected using RT-qPCR, but not with the qPCR assay counterpart. On average, two human-specific Bacteroidales markers were not detected when using DNA in 12% of the samples, while they were positive for all samples when using RNA (cDNA) as the template. Moreover, signal intensity was up to three orders of magnitude higher in RT-qPCR assays than in qPCR assays. The human-specific Bacteroidales markers exhibited moderate correlation with conventional fecal indicators using RT-qPCR results, suggesting the persistence of nonhuman sources of fecal pollution or the presence of false-positive signals. In general, the results from this study suggest that RNA-based assays can increase the detection sensitivity of fecal bacteria in urban watersheds impacted with human fecal sources.

Effects of Cr(III) and Cr(VI) on nitrification inhibition as determined by SOUR, function-specific gene expression and 16S rRNA sequence analysis of wastewater nitrifying enrichments.

The effect of Cr(III) and Cr(VI) on nitrification was examined with samples from nitrifying enrichment cultures using three different approaches: by measuring substrate (ammonia) specific oxygen uptake rates (SOUR), by using RT-qPCR to quantify the transcripts of functional genes involved in nitrification, and by analysis of 16S rRNA sequences to determine changes in structure and activity of the microbial communities. The nitrifying bioreactor was operated as a continuous reactor with a 24 h hydraulic retention time. The samples were exposed in batch vessels to Cr(III) (10-300 mg/L) and Cr(VI) (1-30 mg/L) for a period of 12 h. There was considerable decrease in SOUR with increasing dosages for both Cr(III) and Cr(VI), however Cr(VI) was more inhibitory than Cr(III). Based on the RT-qPCR data, there was reduction in the transcript levels of amoA and hao for increasing Cr(III) dosage, which corresponded well with the ammonia oxidation activity measured via SOUR. For Cr(VI) exposure, there was comparatively little reduction in amoA expression while hao expression decreased for 1-3 mg/L Cr(VI) and increased at 30 mg/L Cr(VI). While Nitrosomonas spp. were the dominant bacteria in the bioreactor, based on 16S rRNA sequencing, there was a considerable reduction in Nitrosomonas activity upon exposure to 300 mg/L Cr(III). In contrast, a relatively small reduction in activity was observed at 30 mg/L Cr(VI) loading. Our data that suggest that both Cr(III) and Cr(VI) were inhibitory to nitrification at concentrations near the high end of industrial effluent concentrations.

Comparison of two poultry litter qPCR assays targeting the 16S rRNA gene of Brevibacterium sp.

Chicken feces commonly contain human pathogens and are also important sources of fecal pollution in environmental waters. Consequently, methods that can detect chicken fecal pollution are needed in public health and environmental monitoring studies. In this study, we compared a previously developed SYBR green qPCR assay (LA35) to a novel TaqMan qPCR assay (CL) for the environmental detection of poultry-associated fecal pollution. We tested both assays against chicken litter (n = 40), chicken fecal samples (n = 186), non-chicken fecal sources (n = 484), and environmental water samples (n = 323). Most chicken litter samples (i.e., ≥ 98%) were positive for both assays with relatively high signal intensities, whereas only 23% and 12% of poultry fecal samples (n = 186) were positive with the LA35 and the CL assays, respectively. Data using fecal samples from non-target animal species showed that the assays are highly host-associated (≥ 95%). Bayesian statistical models showed that the two assays are associated with relatively low probability of false-positive and false-negative signals in water samples. The CL marker had a lower prevalence than the LA35 assay when tested against environmental water samples (i.e., 21% vs. 31% positive signals). However, by combining the results from the two assays the detection levels increased to 41%, suggesting that using multiple assays can improve the detection of chicken-fecal pollution in environmental waters.

Inhibitory effect of cyanide on wastewater nitrification determined using SOUR and RNA‐based gene‐specific assays

The effect of cyanide (CN−) on nitrification was examined with samples from nitrifying bacterial enrichments using two different approaches: by measuring substrate (ammonia) specific oxygen uptake rates (SOUR), and by using RT‐qPCR to quantify the transcripts of functional genes involved in nitrification. The nitrifying bioreactor was operated as a continuous reactor with a 24 h hydraulic retention time. The samples were exposed in batch vessels to cyanide for a period of 12 h. The concentrations of CN− used in the batch assays were 0·03, 0·06, 0·1 and 1·0 mg l−1. There was considerable decrease in SOUR with increasing dosages of CN−. A decrease of more than 50% in nitrification activity was observed at 0·1 mg l−1 CN−. Based on the RT‐qPCR data, there was notable reduction in the transcript levels of amoA and hao for increasing CN− dosage, which corresponded well with the ammonia oxidation activity measured via SOUR. The inhibitory effect of cyanide may be attributed to the affinity of cyanide to bind ferric haeme proteins, which disrupt protein structure and function. The correspondence between the relative expression of functional genes and SOUR shown in this study demonstrates the efficacy of RNA‐based function‐specific assays for better understanding of the effect of toxic compounds on nitrification activity in wastewater.

Bacterial community changes in copper and PEX drinking water pipeline biofilms under extra disinfection and magnetic water treatment

To study the stability of biofilms and water quality in pilot scale drinking water copper and PEX pipes in changing conditions (extra disinfection, magnetic water treatment, MWT).