Anicet R. Blanch

Comparison of enterococcal populations in animals, humans, and the environment - a European study

The objectives of the present study were to generate knowledge of enterococcal populations in the food chain, by studying the population structure (in measures of abundance and diversity) among enterococci in different geographical regions and in different parts of the food chain, as well as the similarities between different enterococcal populations. Altogether, 2868 samples were collected from humans (healthy and hospitalised individuals and clinical isolates), animals (slaughterhouse carcasses and farm animals), and the environment (pig farms, sewage, and surface water) in four European countries-Sweden, Denmark, UK, and Spain. The samples were characterised with regard to presence and numbers of enterococci, and eight (for faecal samples) or 24 (for environmental samples) isolates per sample were phenotyped and preliminarily identified with the PhP-RF system. In total, more than 20,000 isolates were typed. A majority of the samples (77%) showed the presence of presumed enterococci. The diversities of enterococci in environmental samples were generally high, and also faecal samples normally showed presence of more than one enterococcal strain. The most common species found were Enterococcus faecium (33%), E. faecalis (29%), and E. hirae (24%), but different enterococcal populations differed in their species distribution. Clinical isolates, hospitalised patients, and hospital sewage in Sweden showed a clear dominance of E. faecalis (80%, 57%, and 54%, respectively) whereas healthy individuals and urban sewage contained less E. faecalis (39% and 40%, respectively). The species distribution among isolates from slaughterhouses varied between animal species and also between countries, but E. faecalis seemed to be mainly associated with broiler, and E. hirae with cattle and pigs. The results from the study have indicated a simplified method to study the diversity of bacterial populations. Instead of collecting many samples and analysing one or a few isolates per sample, it is possible to collect fewer samples and analyse several isolates per sample. Both approaches yielded similar information on the diversity of the populations. Another useful information was that since samples from hospital sewage, urban sewage, and manure contained enterococcal populations that reflected those in faecal samples of hospitalised patients, healthy humans, and animals, respectively, such samples may be used as pooled faecal samples and may replace cumbersome samplings from many individuals.

Integrated Analysis of Established and Novel Microbial and Chemical Methods for Microbial Source Tracking

ABSTRACT Several microbes and chemicals have been considered as potential tracers to identify fecal sources in the environment. However, to date, no one approach has been shown to accurately identify the origins of fecal pollution in aquatic environments. In this multilaboratory study, different microbial and chemical indicators were analyzed in order to distinguish human fecal sources from nonhuman fecal sources using wastewaters and slurries from diverse geographical areas within Europe. Twenty-six parameters, which were later combined to form derived variables for statistical analyses, were obtained by performing methods that were achievable in all the participant laboratories: enumeration of fecal coliform bacteria, enterococci, clostridia, somatic coliphages, F-specific RNA phages, bacteriophages infecting Bacteroides fragilis RYC2056 and Bacteroides thetaiotaomicron GA17, and total and sorbitol-fermenting bifidobacteria; genotyping of F-specific RNA phages; biochemical phenotyping of fecal coliform bacteria and enterococci using miniaturized tests; specific detection of Bifidobacterium adolescentis and Bifidobacterium dentium; and measurement of four fecal sterols. A number of potentially useful source indicators were detected (bacteriophages infecting B. thetaiotaomicron, certain genotypes of F-specific bacteriophages, sorbitol-fermenting bifidobacteria, 24-ethylcoprostanol, and epycoprostanol), although no one source identifier alone provided 100% correct classification of the fecal source. Subsequently, 38 variables (both single and derived) were defined from the measured microbial and chemical parameters in order to find the best subset of variables to develop predictive models using the lowest possible number of measured parameters. To this end, several statistical or machine learning methods were evaluated and provided two successful predictive models based on just two variables, giving 100% correct classification: the ratio of the densities of somatic coliphages and phages infecting Bacteroides thetaiotaomicron to the density of somatic coliphages and the ratio of the densities of fecal coliform bacteria and phages infecting Bacteroides thetaiotaomicron to the density of fecal coliform bacteria. Other models with high rates of correct classification were developed, but in these cases, higher numbers of variables were required.

Occurrence and Relatedness of Vancomycin-Resistant Enterococci in Animals, Humans, and the Environment in Different European Regions

ABSTRACT Vancomycin-resistant enterococcci (VRE) in Europe are thought to have emerged partly due to the use of the glycopeptide avoparcin in animal husbandry. We compared the occurrence of VRE in geographical regions of Europe in which until 1997 large amounts of avoparcin were used (Spain, United Kingdom, and Denmark) with the occurrence of VRE in Sweden, where avoparcin was banned in 1986. We also studied the relatedness between VRE strains from different regions and habitats. In total, 2,580 samples were collected from humans, animals, and the environment (soil, sewage, recipient water). VRE resistant to 20 μg/ml vancomycin were identified in 8.2% of the samples and were found most frequently in raw and treated urban sewage samples (means, 71% and 36% of the samples, respectively), pig manure (17%), and hospital sewage (16%). The proportions of VRE-positive sewage samples were similar in Sweden, Spain, and the United Kingdom, whereas pig feces and manure were more often positive in Spain than in Sweden (30% versus 1%). Most VRE were Enterococcus faecium carrying vanA, and computerized biochemical phenotyping of the isolates of different ecological origins showed a high degree of polyclonality. In conclusion, it seems that animal-associated VRE probably reflect the former use of avoparcin in animal production, whereas VRE in human-associated samples may be a result of antibiotic use in hospitals. Since there seems to be a reservoir of the resistance genes in all countries studied, precautions must be taken to limit the use of antibiotics and antibiotic-like feed additives.

Identification of Vibrio spp. with a set of dichotomous keys

Aims:  To define a binary biochemical key for the identification of all recognized Vibrio spp.

Microbial source tracking : methods, applications, and case studies

Chapter 1: Overview.- Chapter 2: Performance Criteria.- Chapter 3: Library-dependent Source Tracking Methods.- Chapter 4: Library-Independent Source Tracking Methods.- Chapter 5: Viruses as Tracers of Fecal Contamination.- Chapter 6: Phage Methods.- Chapter 7: Pathogenic Protozoa.- Chapter 8: Chemical-Based Fecal Source Tracking Methods.- Chapter 9: Statistical Approaches for Modeling in Microbial Source Tracking.- Chapter 10: Mitochondrial DNA as Source Tracking Markers of Fecal Contamination.- Chapter 11: Community Analysis-Based Methods.- Chapter 12: Public Perception of and Public Participation in Microbial Source Tracking.- Chapter 13: Use of Microbial Source Tracking in the Legal Arena: Benefits and Challenges.- Chapter 14: Applications of Microbial Source Tracking in the TMDL Process.- Chapter 15: Relating MST Results to Fecal Indicator Bacteria, Pathogens, and Standards.- Chapter 16: Minimizing Microbial Source Tracking at All Costs.- Chapter 17: Environmental Persistence and Naturalization of Fecal Indicator Organisms.- Chapter 18: Agricultural and Rural Watersheds.- Chapter 19: Case Studies of Urban and Suburban Watersheds.- Chapter 20: Beaches and Coastal Environmenta.- Chapter 21: Source tracking in Australia and New Zealand: Case Studies.- Chapter 22: Microbial Source Tracking in China and Developing Nations.- Chapter 23: A National Security Perspective of Microbial Source Tracking.- Chapter 24: Applications of Quantitative Microbial Source Tracking (QMST) and Quantitative Microbial Risk Assessment (QMRA).- Chapter 25: Food Safety and Implications for Microbial Source Tracking.- Chapter 26: Training Future Scientists: Teaching Microbial Source Tracking (MST) to Undergraduates.- Index.

Description of Bacillus toyonensis sp. nov., a novel species of the Bacillus cereus group, and pairwise genome comparisons of the species of the group by means of ANI calculations

Strain BCT-7112(T) was isolated in 1966 in Japan from a survey designed to obtain naturally occurring microorganisms as pure cultures in the laboratory for use as probiotics in animal nutrition. This strain, which was primarily identified as Bacillus cereus var toyoi, has been in use for more than 30 years as the active ingredient of the preparation TOYOCERIN(®), an additive for use in animal nutrition (e.g. swine, poultry, cattle, rabbits and aquaculture). Despite the fact that the strain was initially classified as B. cereus, it showed significant genomic differences from the type strains of the B. cereus group that were large enough (ANI values below 92%) to allow it to be considered as a different species within the group. The polyphasic taxonomic study presented here provides sufficient discriminative parameters to classify BCT-7112(T) as a new species for which the name Bacillus toyonensis sp. nov. is proposed, with BCT-7112(T) (=CECT 876(T); =NCIMB 14858(T)) being designated as the type strain. In addition, a pairwise comparison between the available genomes of the whole B. cereus group by means of average nucleotide identity (ANI) calculations indicated that besides the eight classified species (including B. toyonensis), additional genomospecies could be detected, and most of them also had ANI values below 94%. ANI values were on the borderline of a species definition only in the cases of representatives of B. cereus versus B. thuringiensis, and B. mycoides and B. weihenstephanensis.

Comparison of enterococcal populations related to urban and hospital wastewater in various climatic and geographic European regions

Aims: Scarce knowledge about the distribution of enterococci species in wastewaters limits any statement on their reliability as faecal indicators or the implications of antibiotic resistance transmission by these organisms through the water cycle. Enterococci have been involved in nosocomial infections and the spreading of antibiotic resistance through the food chain. The species distribution of enterococci and the presence of resistant strains to vancomycin and erythromycin were analysed in more than 400 raw and treated urban wastewaters, surface waters receiving these treated wastewaters and hospital wastewaters from three European countries.

Identification and typing of Vibrio anguillarum - a comparison of different methods.

Summary The majority (91%) of 260 isolates initially identified as Vibrio anguillarum , that were obtained from a wide range of hosts, habitats and geographical locations, were recovered in a single cluster based on the ribotype and were pathogenic to Atlantic salmon. A significant proportion of isolates (78% of the total) were allocated to 15 serogroups (O1–O10 and five previously undescribed groups referred to as VaNT1, VaNT2, VaNT4, NaNT5 and VaNT7). A minority of isolates (6%) reacted with more than one antiserum or were self-agglutinating, and the remainder did not react with any of the antisera tested. Good correlation was noted between serogroups and lipopolysaccharide profiles, particularly with respect to isolates belonging to serogroups O1, 02 and 04ߝ010. Plasmids were recognized in some serogroups. especially O1, which contained the 67 kb plasmid associated with virulence. However, the 19 profiles based on outer membrane protein patterns did not correspond to the results obtained with the other typing methods. Generally, the isolates were heterogeneous in their biochemical characteristics; 117 profiles were obtained with the API 20E system, and 9 and 32 clusters recognised from the results of BIOLOG fingerprinting and Biotype-100 biotyping methods, respectively. Three dominant dusters were defined from fatty acid methyl esters profiles.

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.

Method for Isolation of Bacteroides Bacteriophage Host Strains Suitable for Tracking Sources of Fecal Pollution in Water

ABSTRACT Bacteriophages infecting Bacteroides are potentially a good tool for fecal source tracking, but different Bacteroides host strains are needed for different geographic areas. A feasible method for isolating Bacteroides host strains for phages present in human fecal material is described. Useful strains were identified for application in Spain and the United Kingdom. One strain, GA-17, identified as Bacteroides thetaiotaomicron, was tested in several locations in Europe with excellent performance in Southern Europe.