Elisenda Ballesté

Multiplex PCR with 16S rRNA Gene-Targeted Primers of Bifidobacterium spp. To Identify Sources of Fecal Pollution.

ABSTRACT Bifidobacteria are one of the most common bacterial types found in the intestines of humans and other animals and may be used as indicators of human fecal pollution. The presence of nine human-related Bifidobacterium species was analyzed in human and animal wastewater samples of different origins by using species-specific primers based on 16S rRNA sequences. Only B. adolescentis and B. dentium were found exclusively in human sewage. A multiplex PCR approach with strain-specific primers was developed. The method showed a sensitivity threshold of 10 cells/ml. This new molecular method could provide useful information for the characterization of fecal pollution sources.

Virulence and antimicrobial resistance profiles among Escherichia coli strains isolated from human and animal wastewater

To gain insight into whether Escherichia coli isolated from humans and resistant to some common antimicrobial agents are derived from animals, 85 E. coli strains were selected by ERIC-PCR from human and animal wastewater samples. Phylogroup, pathogenicity islands (PAIs), resistance to quinolones, fluoroquinolones and presence of extended-spectrum beta-lactamases (ESBLs) were analyzed. Among the total, 55% were resistant to nalidixic acid and 38% to ciprofloxacin; 12% produced ESBLs. Chicken-derived strains were associated with quinolone and fluoroquinolone resistance and presence of ESBLs, while human strains were associated with susceptibility. Group B2 E. coli strains were associated with human origin, susceptibility to fluoroquinolones and presence of PAIs, whereas groups A, B1 and D showed a low virulence profile and a high level of antimicrobial resistance. In both human and animal wastewater, E. coli A, B1 and D were prevalent, and strains from both origins showed a similar virulence profile in each phylogroup. These findings led us to hypothesize that abusive antibiotic use in food animal production may promote the development of resistance among these intestinal E. coli phylogroups, which could later be transmitted to humans through the food supply. The low prevalence of E. coli group B2 in the animal gut may explain, at least in part, the absence of emergence of resistant B2 isolates.

Persistence of Bacteroides species populations in a river as measured by molecular and culture techniques.

ABSTRACT Given the interest in Bacteroides species as microbial source tracking (MST) markers, and the limited knowledge of the survival of Bacteroides species in the environment, here we examine the survival of Bacteroides fragilis, B. thetaiotaomicron, and environmental species of Bacteroides by use of culture techniques and molecular tools. Two kinds of experiments were performed: (i) on-site experiments, in which bacteria were exposed to changes in the levels of several environmental parameters in a river, and (ii) microcosm assays in the laboratory, with controlled temperatures. On-site experiments showed different survival patterns for the cultivable Bacteroides strains. B. fragilis die-off rate was strongly affected by the combined effect of high temperatures and grazing predators, which were more active under warmer conditions. However, the survival rates of cultivable B. thetaiotaomicron and environmental Bacteroides spp. were more affected by dissolved oxygen (DO) concentration in water. Environmental Bacteroides strains survived longer than either type strain, due to better adaptation to environmental conditions. However, the period of their survival was shorter than that observed for fecal coliforms and enterococci, suggesting Bacteroides species as markers of recent fecal pollution. The total Bacteroides species were detected by molecular techniques throughout the experiment in winter, but they were detected on only two or three days in the summer. This indicates that temperature is the main factor affecting DNA degradation, regardless of species. The use of microcosms in the laboratory also pointed to temperature as the main factor affecting Bacteroides survival, regardless of species. However, the conditions in the laboratory may mask the effects of the environmental factors and their interactions. The observed variability in die-off rate as a function of the species analyzed, the experimental conditions, and the methodology used should be taken into consideration in future persistence studies.

Molecular Indicators Used in the Development of Predictive Models for Microbial Source Tracking

ABSTRACT A number of chemical, microbial, and eukaryotic indicators have been proposed as indicators of fecal pollution sources in water bodies. No single one of the indicators tested to date has been able to determine the source of fecal pollution in water. However, the combined use of different indicators has been demonstrated to be the best way of defining predictive models suitable for determining fecal pollution sources. Molecular methods are promising tools that could complement standard microbiological water analysis. In this study, the feasibility of some proposed molecular indicators for microbial source tracking (MST) was compared (names of markers are in parentheses): host-specific Bacteroidetes (HF134, HF183, CF128, and CF193), Bifidobacterium adolescentis (ADO), Bifidobacterium dentium (DEN), the gene esp of Enterococcus faecium, and host-specific mitochondrial DNA associated with humans, cattle, and pigs (Humito, Bomito, and Pomito, respectively). None of the individual molecular markers tested enabled 100% source identification. They should be combined with other markers to raise sensitivity and specificity and increase the number of sources that are identified. MST predictive models using only these molecular markers were developed. The models were evaluated by considering the lowest number of molecular indicators needed to obtain the highest rate of identification of fecal sources. The combined use of three molecular markers (ADO, Bomito, and Pomito) enabled correct identification of 75.7% of the samples, with differentiation between human, swine, bovine, and poultry sources. Discrimination between human and nonhuman fecal pollution was possible using two markers: ADO and Pomito (84.6% correct identification). The percentage of correct identification increased with the number of markers analyzed. The best predictive model for distinguishing human from nonhuman fecal sources was based on 5 molecular markers (HF134, ADO, DEN, Bomito, and Pomito) and provided 90.1% correct classification.

Quantification of Shiga Toxin-Converting Bacteriophages in Wastewater and in Fecal Samples by Real-Time Quantitative PCR

ABSTRACT Shiga toxin-converting bacteriophages (Stx phages) are involved in the pathogenicity of some enteric bacteria, such as Escherichia coli O157:H7. Stx phages are released from their bacterial hosts after lytic induction and remain free in the environment. Samples were analyzed for the presence of free Stx phages by an experimental approach based on the use of real-time quantitative PCR (qPCR), which enables stx to be detected in the DNA from the viral fraction of each sample. A total of 150 samples, including urban raw sewage samples, wastewater samples with fecal contamination from cattle, pigs, and poultry, and fecal samples from humans and diverse animals, were used in this study. Stx phages were detected in 70.0% of urban sewage samples (10 to 103 gene copies [GC] per ml) and in 94.0% of animal wastewater samples of several origins (10 to 1010 GC per ml). Eighty-nine percent of cattle fecal samples were positive for Stx phages (10 to 105 GC per g of sample), as were 31.8% of other fecal samples of various origins (10 to 104 GC per g of sample). The stx2 genes and stx2 variants were detected in the viral fraction of some of the samples after sequencing of stx2 fragments amplified by conventional PCR. The occurrence and abundance of Stx phages in the extraintestinal environment confirm the role of Stx phages as a reservoir of stx in the environment.

New Molecular Quantitative PCR Assay for Detection of Host-Specific Bifidobacteriaceae Suitable for Microbial Source Tracking

ABSTRACT Bifidobacterium spp. belong to the commensal intestinal microbiota of warm-blooded animals. Some strains of Bifidobacterium show host specificity and have thus been proposed as host-specific targets to determine the origin of fecal pollution. Most strains have been used in microbial-source-tracking (MST) studies based on culture-dependent methods. Although some of these approaches have proved very useful, the low prevalence of culturable Bifidobacterium strains in the environment means that molecular culture-independent procedures could provide practical applications for MST. Reported here is a set of common primers and four Bifidobacterium sp. host-associated (human, cattle, pig, and poultry) probes for quantitative-PCR (qPCR) assessment of fecal source tracking. This set was tested using 25 water samples of diverse origin: urban sewage samples, wastewater from four abattoirs (porcine, bovine, and poultry), and water from a river with a low pollution load. The selected sequences showed a high degree of host specificity. There were no cross-reactions between the qPCR assays specific for each origin and samples from different fecal origins. On the basis of the findings, it was concluded that the host-specific qPCRs are sufficiently robust to be applied in environmental MST studies.

Bifidobacterial diversity and the development of new microbial source tracking indicators.

ABSTRACT Many studies suggest a close relationship between species of Bifidobacterium and their hosts. Thus, species such as B. adolescentis and B. thermacidophilum subsp. porcinum have been proposed as potential indicators of human and porcine fecal pollution. The diversity of bifidobacteria in wastewaters (human and animal) and slurries is analyzed using nested PCR followed by denaturant gradient gel electrophoresis (DGGE). The sewage samples showed similar DGGE patterns. The predominant bands were recognized as B. adolescentis, B. longum, and two unidentified species related to B. adolescentis. A single band detected in poultry samples was identified as B. saeculare. Bifidobacterial diversity was higher within porcine and bovine samples. The main bands in porcine samples were identified as B. minimum, an unknown species, and B. thermophilum/B. thermacidophilum subsp. porcinum. The latter species was also identified among the main bands in bovine samples together with B. pseudolongum and B. ruminantium. We then attempted to isolate the host-specific strains. DGGE bands were examined to develop specific probes to screen environmental samples by colony hybridization and further isolation of strains from positively hybridized colonies. Bifidobacterial strains that are host associated by DGGE bands to human and pig were successfully isolated from the environment: B. adolescentis from human sewage samples and the unidentified species related to pig from slurries and slaughterhouse wastewater. Neither the poultry-associated B. saeculare nor the ruminant-associated B. pseudolongum could be isolated with the current methodology, suggesting either a low prevalence in the samples or failure of the culture to grow in the media used.

Neoscardovia arbecensis gen. nov., sp. nov., isolated from porcine slurries

Three Gram-positive, anaerobic, pleomorphic strains (PG10(T), PG18 and PG22), were selected among five strains isolated from pig slurries while searching for host specific bifidobacteria to track the source of fecal pollution in water. Analysis of the 16S rRNA gene sequence showed a maximum identity of 94% to various species of the family Bifidobacteriaceae. However, phylogenetic analyses of 16S rRNA and HSP60 gene sequences revealed a closer relationship of these strains to members of the recently described Aeriscardovia, Parascardovia and Scardovia genera, than to other Bifidobacterium species. The names Neoscardovia gen. nov. and Neoscardovia arbecensis sp. nov. are proposed for a new genus and for the first species belonging to this genus, respectively, and for which PG10(T) (CECT 8111(T), DSM 25737(T)) was designated as the type strain. This new species should be placed in the Bifidobacteriaceae family within the class Actinobacteria, with Aeriscardovia aeriphila being the closest relative. The prevailing cellular fatty acids were C(16:0) and C(18:1)ω9c, and the major polar lipids consisted of a variety of glycolipids, diphosphatidyl glycerol, two unidentified phospholipids, and phosphatidyl glycerol. The peptidoglycan structure was A1γmeso-Dpm-direct. The GenBank accession numbers for the 16S rRNA gene and HSP60 gene sequences of strains PG10(T), PG18 and PG22 are JF519691, JF519693, JQ767128 and JQ767130, JQ767131, JQ767133, respectively.

Beyond the canonical strategies of horizontal gene transfer in prokaryotes

Efforts to identify and characterize strategies for horizontal gene transfer (HGT) in prokaryotes could have overlooked some mechanisms that do not entirely fit in with the canonical ones most often described (conjugation of plasmids, phage transduction and transformation). The difficulty in distinguishing the different HGT strategies could have contributed to underestimate their real extent. Here we review non classical HGT strategies: some that require mobile genetic elements (MGEs) and others independent of MGE. Among those strategies that require MGEs, there is a range of newly reported, hybrid and intermediate MGEs mobilizing only their own DNA, others that mobilize preferentially bacterial DNA, or both. Considering HGT strategies independent of MGE, a few are even not restricted to DNA transfer, but can also mobilize other molecules. This review considers those HGT strategies that are less commonly dealt with in the literature. The real impact of these elements could, in some conditions, be more relevant than previously thought.

Determination of crAssphage in water samples and applicability for tracking human faecal pollution

In recent decades, considerable effort has been devoted to finding microbial source‐tracking (MST) markers that are suitable to assess the health risks of faecally polluted waters, with no universal marker reported so far. In this study, the abundance and prevalence of a crAssphage‐derived DNA marker in wastewaters of human and animal origins were studied by a new qPCR assay with the ultimate aim of assessing its potential as an MST marker. crAssphage showed up to 106 GC/ml in the sewage samples of human origin, in both the total DNA and the viral DNA fraction. In wastewaters containing animal faecal remains, 39% of the samples were negative for the presence of the crAssphage sequence, while those showing positive results (41% of the samples) were at least 1 log10 unit lower than the samples of human origin. Noteworthy, the log10 values of the ratio (R) crAssphage (GC/ml)/Escherichia coli (CFU/ml) varied significantly depending on the human or animal origin (R > 1.5 for human samples and R < −1.5 for animal wastewater samples. This study opens the way for further research to explore if different specific animal variants of crAssphage exist and whether other zones of the crAssphage genome are better suited to source discrimination.