Occurrence of Diarrheagenic Virulence Genes and Genetic Diversity in Escherichia coli Isolates from Fecal Material of Various Avian Hosts in British Columbia,Canada

Contamination of surface water by fecal microorganisms originating from human and nonhuman sources is a public health concern. In the present study, Escherichia coli isolates (n = 412) from the feces of various avian host sources were screened for various virulence genes: stx₁ and stx₂ (Shiga toxin-producing E. coli [STEC]), eae (enteropathogenic E. coli [EPEC]), est-h, est-p, and elt (encoding heat-stable toxin [ST] variants STh and STp and heat-labile toxin [LT], respectively) (enterotoxigenic E. coli [ETEC]), and ipaH (enteroinvasive E. coli [EIEC]). None of the isolates were found to be positive for stx₁, while 23% (n = 93) were positive for only stx₂, representing STEC, and 15% (n = 63) were positive for only eae, representing EPEC. In addition, five strains obtained from pheasant were positive for both stx₂ and eae and were confirmed as non-O157 by using an E. coli O157 rfb (rfb_O157) TaqMan assay. Isolates positive for the virulence genes associated with ETEC and EIEC were not detected in any of the hosts. The repetitive element palindromic PCR (rep-PCR) fingerprint analysis identified 143 unique fingerprints, with an overall Shannon diversity index of 2.36. Multivariate analysis of variance (MANOVA) showed that the majority of the STEC and EPEC isolates were genotypically distinct from nonpathogenic E. coli and clustered independently. MANOVA analysis also revealed spatial variation among the E. coli isolates, since the majority of the isolates clustered according to the sampling locations. Although the presence of virulence genes alone cannot be used to determine the pathogenicity of strains, results from this study show that potentially pathogenic STEC and EPEC strains can be found in some of the avian hosts studied and may contaminate surface water and potentially impact human health.     

Comparative Analysis of Virulence Genes, Genetic Diversity, and Phylogeny of Commensal and Enterotoxigenic Escherichia coli Isolates from Weaned Pigs

If the acquisition of virulence genes (VGs) for pathogenicity were not solely acquired through horizontal gene transfers of pathogenicity islands, transposons, and phages, then clonal clusters of enterotoxigenic Escherichia coli (ETEC) would contain few or even none of the VGs found in strains responsible for extraintestinal infections. To evaluate this possibility, 47 postweaning diarrhea (PWD) ETEC strains from different geographical origins and 158 commensal E. coli isolates from the gastrointestinal tracts of eight group-housed healthy pigs were screened for 36 extraintestinal and 18 enteric VGs using multiplex PCR assays. Of 36 extraintestinal VGs, only 8 were detected (fimH, traT, fyuA, hlyA, kpsMtII, k5, iha, and ompT) in the ETEC collection. Among these, hlyA (α-hemolysin) and iha (nonhemagglutinating adhesin) occurred significantly more frequently among the ETEC isolates than in the commensal isolates. Clustering analysis based on the VG profiles separated commensal and ETEC isolates and even differentiated serogroup O141 from O149. On the other hand, pulsed-field gel electrophoresis (PFGE) successfully clustered ETEC isolates according to both serotype and geographical origin. In contrast, the commensal isolates were heterogeneous with respect to both serotype and DNA fingerprint. This study has validated the use of VG profiling to examine pathogenic relationships between porcine ETEC isolates. The clonal relationships of these isolates can be further clarified by PFGE fingerprinting. The presence of extraintestinal VGs in porcine ETEC confirmed the hypothesis that individual virulence gene acquisitions can occur concurrently against a background of horizontal gene transfers of pathogenicity islands. Over time, this could enable specific clonotypes to respond to host selection pressure and to evolve into new strains with increased virulence.     

Antimicrobial Resistance and Virulence Genes of Escherichia coli Isolates from Swine in Ontario

A total of 318 Escherichia coli isolates obtained from diarrheic and healthy pigs in Ontario from 2001 to 2003 were examined for their susceptibility to 19 antimicrobial agents. They were tested by PCR for the presence of resistance genes for tetracycline, streptomycin, sulfonamides, and apramycin and of 12 common virulence genes of porcine E. coli. Antimicrobial resistance frequency among E. coli isolates from swine in Ontario was moderate in comparison with other countries and was higher in isolates from pigs with diarrhea than in isolates from healthy finisher pigs. Resistance profiles suggest that cephamycinases may be produced by ≥8% of enterotoxigenic E. coli (ETEC). Resistance to quinolones was detected only in enterotoxigenic E. coli (≤3%). The presence of sul3 was demonstrated for the first time in Canada in porcine E. coli isolates. Associations were observed among tetA, sul1, aadA, and aac(3)IV and among tetB, sul2, and strA/strB, with a strong negative association between tetA and tetB. The paa and sepA genes were detected in 92% of porcine ETEC, and strong statistical associations due to colocation on a large plasmid were observed between tetA, estA, paa, and sepA. Due at least in part to gene linkages, the distribution of resistance genes was very different between ETEC isolates and other porcine E. coli isolates. This demonstrates that antimicrobial resistance epidemiology differs significantly between pathogenic and commensal E. coli isolates. These results may have important implications with regards to the spread and persistence of resistance and virulence genes in bacterial populations and to the prudent use of antimicrobial agents.     

Antimicrobial Resistance,Virulence Factors and Genetic Diversity of Escherichia coli Isolates from Household Water Supply in Dhaka,Bangladesh

Background

Unsafe water supplies continue to raise public health concerns, especially in urban areas in low resource countries. To understand the extent of public health risk attributed to supply water in Dhaka city, Bangladesh, Escherichia coli isolated from tap water samples collected from different locations of the city were characterized for their antibiotic resistance, pathogenic properties and genetic diversity.

Methodology/Principal Findings

A total of 233 E. coli isolates obtained from 175 tap water samples were analysed for susceptibility to 16 different antibiotics and for the presence of genes associated with virulence and antibiotic resistance. Nearly 36% (n = 84) of the isolates were multi-drug(≥3 classes of antibiotics) resistant (MDR) and 26% (n = 22) of these were positive for extended spectrum β-lactamase (ESBL). Of the 22 ESBL-producers, 20 were positive for bla _CTX-M-15, 7 for bla _OXA-1-group (all had bla _OXA-47) and 2 for bla _CMY-2. Quinolone resistance genes, qnrS and qnrB were detected in 6 and 2 isolates, respectively. Around 7% (n = 16) of the isolates carried virulence gene(s) characteristic of pathogenic E. coli; 11 of these contained lt and/or st and thus belonged to enterotoxigenic E. coli and 5 contained bfp and eae and thus belonged to enteropathogenic E. coli. All MDR isolates carried multiple plasmids (2 to 8) of varying sizes ranging from 1.2 to >120 MDa. Ampicillin and ceftriaxone resistance were co-transferred in conjugative plasmids of 70 to 100 MDa in size, while ampicillin, trimethoprim-sulfamethoxazole and tetracycline resistance were co-transferred in conjugative plasmids of 50 to 90 MDa. Pulsed-field gel electrophoresis analysis revealed diverse genetic fingerprints of pathogenic isolates.

Significance

Multi-drug resistant E. coli are wide spread in public water supply in Dhaka city, Bangladesh. Transmission of resistant bacteria and plasmids through supply water pose serious threats to public health in urban areas.     

Occurrence of Virulence Genes Associated with Diarrheagenic Pathotypes in Escherichia coli Isolates from Surface Water

Escherichia coli isolates (n = 300) collected from six sites in subtropical Brisbane, Australia, prior to and after storm events were tested for the presence of 11 virulence genes (VGs) specific to diarrheagenic pathotypes. The presence of eaeA, stx₁, stx₂, and ehxA genes specific for the enterohemorrhagic E. coli (EHEC) pathotype was detected in 56%, 6%, 10%, and 13% of isolates, respectively. The VGs astA (69%) and aggR (29%), carried by enteroaggregative (EAEC) pathotypes, were frequently detected in E. coli isolates. The enteropathogenic E. coli (EPEC) gene bfp was detected in 24% of isolates. In addition, enteroinvasive E. coli (EIEC) VG ipaH was also detected in 14% of isolates. During dry periods, isolates belonging to the EAEC pathotype were most commonly detected (23%), followed by EHEC (11%) and EPEC (11%). Conversely, a more uniform prevalence of pathotypes, EPEC (14%), EAEC (12%), EIEC (10%), EHEC (7%), and ETEC (7%), was observed after the storm events. The results of this study highlight the widespread occurrence of potentially diarrheagenic pathotypes in the urban aquatic ecosystems. While the presence of VGs in E. coli isolates alone is insufficient to determine pathogenicity, the presence of diarrheagenic E. coli pathotypes in high frequency after the storm events could lead to increased health risks if untreated storm water were to be used for nonpotable purposes and recreational activities.     

Serotypes,Virulence Factors,and Antimicrobial Susceptibilities of Vaginal and Fecal Isolates of Escherichia coli from Giant Pandas

Although Escherichia coli typically colonizes the intestinal tract and vagina of giant pandas, it has caused enteric and systemic disease in giant pandas and greatly impacts the health and survival of this endangered species. In order to understand the distribution and characteristics of E. coli from giant pandas, 67 fecal and 30 vaginal E. coli isolates from 21 giant pandas were characterized for O serogroups, phylogenetic groups, antimicrobial susceptibilities, and pulsed-field gel electrophoresis (PFGE) profiles. In addition, these isolates were tested for the presence of extraintestinal pathogenic E. coli (ExPEC) and diarrheagenic E. coli (DEC) by multiplex PCR detection of specific virulence genes. The most prevalent serogroups for all E. coli isolates were O88, O18, O167, O4, and O158. ExPEC isolates were detected mostly in vaginal samples, and DEC isolates were detected only in fecal samples. Phylogenetic group B1 predominated in fecal isolates, while groups B2 and D were frequently detected in vaginal isolates. Resistance to trimethoprim-sulfamethoxazole was most frequently observed, followed by resistance to nalidixic acid and tetracycline. All except five isolates were typeable by using XbaI and were categorized into 74 PFGE patterns. Our findings indicate that panda E. coli isolates exhibited antimicrobial resistance, and potentially pathogenic E. coli isolates were present in giant pandas. In addition, these E. coli isolates were genetically diverse. This study may provide helpful information for developing strategies in the future to control E. coli infections of giant pandas.     

Prevalence and Genetic Characterization of Shiga Toxin-Producing Escherichia coli Isolates from Slaughtered Animals in Bangladesh

To determine the prevalence of Shiga toxin (Stx)-producing Escherichia coli (STEC) in slaughter animals in Dhaka, Bangladesh, we collected rectal contents immediately after animals were slaughtered. Of the samples collected from buffalo (n = 174), cows (n = 139), and goats (n = 110), 82.2%, 72.7%, and 11.8% tested positive for stx₁ and/or stx₂, respectively. STEC could be isolated from 37.9%, 20.1%, and 10.0% of the buffalo, cows, and goats, respectively. STEC O157 samples were isolated from 14.4% of the buffalo, 7.2% of the cows, and 9.1% of the goats. More than 93% (n = 42) of the STEC O157 isolates were positive for the stx₂, eae, katP, etpD, and enterohemorrhagic E. coli hly (hly_EHEC) virulence genes. STEC O157 isolates were characterized by seven recognized phage types, of which types 14 (24.4%) and 31 (24.4%) were predominant. Subtyping of the 45 STEC O157 isolates by pulsed-field gel electrophoresis showed 37 distinct restriction patterns, suggesting a heterogeneous clonal diversity. In addition to STEC O157, 71 STEC non-O157 strains were isolated from 60 stx-positive samples from 23.6% of the buffalo, 12.9% of the cows, and 0.9% of the goats. The STEC non-O157 isolates belonged to 36 different O groups and 52 O:H serotypes. Unlike STEC O157, most of the STEC non-O157 isolates (78.9%) were positive for stx₁. Only 7.0% (n = 5) of the isolates were positive for hly_EHEC, and none was positive for eae, katP, and etpD. None of the isolates was positive for the iha, toxB, and efa1 putative adhesion genes. However, 35.2% (n = 25), 11.3% (n = 8), 12.7% (n = 9), and 12.7% (n = 9) of the isolates were positive for the lpf_O113, saa, lpfA_O157/01-141, and lpfA_O157/OI-154 genes, respectively. The results of this study provide the first evidence that slaughtered animals like buffalo, cows, and goats in Bangladesh are reservoirs for STEC, including the potentially virulent STEC strain O157.     

Prevalence of Antimicrobial Resistance and Transfer of Tetracycline Resistance Genes in Escherichia coli Isolates from Beef Cattle

The aim of this study was to investigate the prevalence and transferability of resistance in tetracycline-resistant Escherichia coli isolates recovered from beef cattle in South Korea. A total of 155 E. coli isolates were collected from feces in South Korea, and 146 were confirmed to be resistant to tetracycline. The tetracycline resistance gene tet(A) (46.5%) was the most prevalent, followed by tet(B) (45.1%) and tet(C) (5.8%). Strains carrying tet(A) plus tet(B) and tet(B) plus tet(C) were detected in two isolates each. In terms of phylogenetic grouping, 101 (65.2%) isolates were classified as phylogenetic group B1, followed in decreasing order by D (17.4%), A (14.2%), and B2 (3.2%). Ninety-one (62.3%) isolates were determined to be multidrug resistant by the disk diffusion method. MIC testing using the principal tetracyclines, namely, tetracycline, chlortetracycline, oxytetracycline, doxycycline, and minocycline, revealed that isolates carrying tet(B) had higher MIC values than isolates carrying tet(A). Conjugation assays showed that 121 (82.9%) isolates could transfer a tetracycline resistance gene to a recipient via the IncFIB replicon (65.1%). This study suggests that the high prevalence of tetracycline-resistant E. coli isolates in beef cattle is due to the transferability of tetracycline resistance genes between E. coli populations which have survived the selective pressure caused by the use of antimicrobial agents.     

A Virulence and Antimicrobial Resistance DNA Microarray Detects a High Frequency of Virulence Genes in Escherichia coli Isolates from Great Lakes Recreational Waters

Escherichia coli is generally described as a commensal species with occasional pathogenic strains. Due to technological limitations, there is currently little information concerning the prevalence of pathogenic E. coli strains in the environment. For the first time, using a DNA microarray capable of detecting all currently described virulence genes and commonly found antimicrobial resistance genes, a survey of environmental E. coli isolates from recreational waters was carried out. A high proportion (29%) of 308 isolates from a beach site in the Great Lakes carried a pathotype set of virulence-related genes, and 14% carried antimicrobial resistance genes, findings consistent with a potential risk for public health. The results also showed that another 8% of the isolates had unusual virulence gene combinations that would be missed by conventional screening. This new application of a DNA microarray to environmental waters will likely have an important impact on public health, epidemiology, and microbial ecology in the future.     

Putative Virulence Genes and Biofilm Production Among Typical Enteroaggregative Escherichia coli Isolates from Diarrhoeic Children in Kashmir and Andhra Pradesh

Fifty-eight typical EAEC isolates from children with diarrhoea were examined for HEp-2 cell adherence assay, presence of dispersin (aap), yersiniabactin (irp2), plasmid encoded toxins (pet), Shigella enterotoxin1 (set1A) and cryptic open reading frame (shf) putative virulence genes by polymerase chain reaction as well as for biofilm production. All the isolates showed aggregative adherence pattern on HEp-2 cells. All but five isolates (91.3 %) carried aap gene. While irp2, pet, set1A and shf genes were detected in 68.9, 5.1, 39.6, and 60.3 % isolates, respectively. Thirty-three (64.7 %) isolates out of 51 tested were found to produce biofilm which was found to be significantly associated only with set1A virulence gene (P = 0.025). Highest amount of biofilm was produced by a strain that possessed all the genes studied. Out of 14 isolates in which the most frequent gene combination (aap, irp2 and shf) was observed, only six produced biofilm. It is concluded that there is significant heterogeneity in putative virulence genes of EAEC isolates from diarrhoeic children and biofilm formation is associated with multiple genes.     

High Diversity among Environmental Escherichia coli Isolates from a Bovine Feedlot

Approximately 280 Escherichia coli isolates were isolated from a bovine feedlot at the University of Connecticut campus via enrichment in lauryl tryptose broth and random selection from MacConkey plates. The E. coli subspecies diversity was estimated by employing whole-cell BOX-PCR genomic fingerprints. A total of 89 distinct operational taxonomic units (OTUs) were identified by employing a criterion of 85% fingerprint similarity as a surrogate for an OTU, while the Chao1 index estimated the E. coli population richness at 128 OTUs. One genotype (at a similarity level of 60%) dominated the population at 66% regardless of sampling depth or location, while no significant vertical distribution pattern was observed in terms of genotype, mobility, antibiotic resistance profile, or biofilm-forming ability. Motility, measured by a soft agar assay, had a very broad range among the E. coli population and was positively correlated with biofilm-forming ability in minimal medium (Spearman's rank correlation coefficient r = 0.619, P < 10⁻⁴) but not in Luria broth. Only an estimated 48% of the population possessed gene agn43, which encodes Ag43, a phase-variable outer membrane protein that has been implicated in biofilm formation in minimal medium. We observed significantly more biofilm formation in both minimal medium and Luria broth for agn43⁺ strains, with a larger effect in minimal medium. This study represents an exhaustive inventory of extant E. coli population diversity at a bovine feedlot and reveals significant subspecies heterogeneity in interfacial behavior.     

Phylogenetic Groups and Pathogenicity Island Markers in Fecal Escherichia coli Isolates from Asymptomatic Humans in China

The study of phylogenetic groups and pathogenicity island (PAI) markers in commensal Escherichia coli strains from asymptomatic Chinese people showed that group A strains are the most common and that nearly half of all fecal strains which were randomly selected harbor PAIs.Escherichia coli is a well-diversified commensal species in the intestine of healthy humans but also includes intestinal or extraintestinal pathogens. It has been reported that pathogenic E. coli may be derived from fecal strains by acquisition of virulence determinants (11). The relationship between the E. coli genetic background and the acquisition of virulence factors is now better understood (1, 5). Extraintestinal E. coli strains may harbor several virulence factors, such as adhesins, fimbriae, and hemolysin, which can contribute to bacterial pathogenesis. These traits are usually encoded on pathogenicity islands (PAIs), which have been studied in pathogenic E. coli previously (15). The E. coli population includes 4 major phylogroups (A, B₁, B₂, and D) (2). Pathogenic strains belong mainly to groups B₂ and D, while most fecal isolates belong to groups A and B₁. Strains of groups B₂ and D often carry virulence factors that are lacking in group A and B₁ strains (3, 9, 13).In this study, we examined the distribution of phylogroups and the prevalence of PAIs in commensal E. coli strains isolated from asymptomatic persons in one region of China.     

Vaginal versus Obstetric Infection Escherichia coli Isolates among Pregnant Women: Antimicrobial Resistance and Genetic Virulence Profile

Vaginal Escherichia coli colonization is related to obstetric infections and the consequent development of infections in newborns. Ampicillin resistance among E. coli strains is increasing, which is the main choice for treating empirically many obstetric and neonatal infections. Vaginal E. coli strains are very similar to extraintestinal pathogenic E. coli with regards to the virulence factors and the belonging to phylogroup B2. We studied the antimicrobial resistance and the genetic virulence profile of 82 E. coli isolates from 638 vaginal samples and 63 isolated from endometrial aspirate, placental and amniotic fluid samples from pregnant women with obstetric infections. The prevalence of E. coli in the vaginal samples was 13%, which was significant among women with associated risk factors during pregnancy, especially premature preterm rupture of membranes (p<0.0001). Sixty-five percent of the strains were ampicillin-resistant. The E. coli isolates causing obstetric infections showed higher resistance levels than vaginal isolates, particularly for gentamicin (p = 0.001). The most prevalent virulence factor genes were those related to the iron uptake systems revealing clear targets for interventions. More than 50% of the isolates belonged to the virulent B2 group possessing the highest number of virulence factor genes. The ampicillin-resistant isolates had high number of virulence factors primarily related to pathogenicity islands, and the remarkable gentamicin resistance in E. coli isolates from women presenting obstetric infections, the choice of the most appropriate empiric treatment and clinical management of pregnant women and neonates should be carefully made. Taking into account host-susceptibility, the heterogeneity of E. coli due to evolution over time and the geographical area, characterization of E. coli isolates colonizing the vagina and causing obstetric infections in different regions may help to develop interventions and avoid the aetiological link between maternal carriage and obstetric and subsequent puerperal infections.     

Association between the Presence of Class 1 Integrons, Virulence Genes, and Phylogenetic Groups of Escherichia coli Isolates from River Water

Ninety-six class 1 integron-positive and 96 integron-negative Escherichia coli isolates cultured from the water of the Warta River, Poland, were characterized for their phylogenetic group affiliation and for the presence of genes associated with virulence. Most strains belonged to phylogenetic group A, but phylogenetic group affiliation was not related with the presence of integrons. The occurrence of heat-stable toxin gene of enterotoxigenic E. coli, S fimbriae subunit gene sfaS, and siderophore receptor genes, fyuA and iutA, was associated with the presence of class 1 integrons. Moreover, virulence factor score (the total number of virulence-associated genes) was associated with the presence of integrons in groups. The results bring new insight into relations between the presence of integrons in E. coli, virulence traits, as well as phylogenetic group affiliation.     

Extraintestinal Escherichia coli Carrying Virulence Genes in Coastal Marine Sediments

Despite the recognized potential of long-term survival or even growth of fecal indicators bacteria (FIB) in marine sediments, this compartment is largely ignored by health protection authorities. We conducted a large-scale study over approximately 50 km of the Marche coasts (Adriatic Sea) at depths ranging from 2 to 5 m. Total and fecal coliforms (FC) were counted by culture-based methods. Escherichia coli was also quantified using fluorescence in situ hybridization targeting specific 16S rRNA sequences, which yielded significantly higher abundances than culture-based methods, suggesting the potential importance of viable but nonculturable E. coli cells. Fecal coliforms displayed high abundances at most sites and showed a prevalence of E. coli. FC isolates (n = 113) were identified by API 20E, additional biochemical tests, and internal transcribed spacer-PCR. E. coli strains, representing 96% of isolates, were then characterized for genomic relatedness and phylogenetic group (A, B1, B2, and D) of origin by randomly amplified polymorphic DNA and multiplex-PCR. The results indicated that E. coli displayed a wide genotypic diversity, also among isolates from the same station, and that 44 of the 109 E. coli isolates belonged to groups B2 and D. Further characterization of B2 and D isolates for the presence of 11 virulence factor genes (pap, sfa/foc, afa, eaeA, ibeA, traT, hlyA, stx₁, stx₂, aer, and fyuA) showed that 90% of B2 and 65% of D isolates were positive for at least one of these. Most of the variance of both E. coli abundance and assemblage composition (>62%) was explained by a combination of physical-chemical and trophic variables. These findings indicate that coastal sediments could represent a potential reservoir for commensal and pathogenic E. coli and that E. coli distribution in marine coastal sediments largely depends upon the physical and trophic status of the sediment. We conclude that future sampling designs aimed at monitoring the microbiological quality of marine coastal areas should not further neglect the analysis of the sediment and that monitoring of these environments can be improved by including molecular methods as a complement of culture-based techniques.Marine environments contaminated by fecal material, derived from human or animal waste, may contain a large variety of pathogenic microorganisms. Health protection and monitoring programs analyze the contamination of aquatic ecosystems (20) but, due to technical and practical difficulties, the search of fecal indicator bacteria (FIB) is routinely preferred to the systematic search of all potential pathogens to assess the sanitary risk of a water body (17). Recreational seawaters are, for instance, classified on the basis of the concentration of Escherichia coli and Enterococcus spp. (21, 33, 40), assumed to be indicators of fecal contamination and of the presence of other pathogenic enteric bacteria. Exposure to waters contaminated with E. coli and Enterococcus spp. have been associated with an increased risk of contracting gastrointestinal and respiratory illnesses (10, 24, 31, 62, 64). Although most E. coli strains are harmless, some strains can cause a variety of intestinal and extraintestinal diseases (11, 57, 58, 62) such as diarrhea, urinary tract infections, bacteremia, sepsis, and meningitis (57). Phylogenetic analyses have shown that E. coli includes four main phylogroups (A, B1, B2, and D) and that most virulent extraintestinal strains belong to the groups B2 and D (11, 23, 46).The microbiological quality of marine waters is typically based exclusively on the water column, whereas sediments have received attention only recently (7, 14, 27, 45). Fecal coliforms (FC) and enterococci have been reported from marine sediments (5, 19, 41), and it has been also proposed that FIB accumulated in the sediments have the potential to contaminate the overlying waters by resuspension of sediment particles (35). There is evidence that FIB and pathogenic bacteria can survive longer in aquatic sediments than in the overlying water column (12, 34). However, the available knowledge on the environmental factors influencing the ecology of pathogenic bacteria in marine sediments is still extremely scant, and there are only few detailed studies on the pathogenic potential, genetic diversity, or population structure of FIB in sediments (1, 63).The development of molecular methods has permitted a range of new approaches to monitor the safety of recreational waters (2). Among the available molecular methods, the fluorescence in situ hybridization (FISH) based on probes specific to 16S or 23S rRNA can be utilized to detect and enumerate specific prokaryotic taxa (16, 59). Since the number of ribosomes varies, generally between 10³ and 10⁵ per cell, depending on the species and physiological state, FISH has also been used to provide evidence of an active metabolic state of the detected cells (2, 8). FISH can thus represent a good complement to culture-based methods, and provides reliable quantitative data in a short time (within 4 h). With regard to FIB, the use of FISH to detect total coliforms (TC) has proven to be difficult, due to their high phylogenetic heterogeneity (55). Conversely, the use of species-specific probes for the detection of single species, such as E. coli, is routinely used (22, 47, 53); however, it has been never tested on marine sediments.The objective of the present study was to investigate the microbiological quality of coastal marine sediments along a large area of the Adriatic Sea (Central Mediterranean Sea) and to evaluate the presence and distribution of specific bacterial genotypes associated with different marine areas. More specifically, it was our aim to evaluate whether marine sediments may be a potential reservoir of active pathogenic E. coli and thus represent a risk for human health. To do this, we analyzed (i) the abundance and distribution of TC and FC; (ii) the abundance and distribution of E. coli strains, along with their genetic relatedness; and (iii) the presence of extraintestinal pathogenic E. coli carrying virulence gene factors. To determine bacterial abundance, culture-dependent (the membrane filtration [MF] technique) and culture-independent (the FISH technique) approaches were used. Finally, to identify the factors potentially responsible for the accumulation and survival of E. coli in the benthic environment, we investigated the environmental variables possibly related to the distribution of FIB.     

Prevalence of Virulence Genes Associated with Pathogenic Escherichia coli Strains Isolated from Domestically Harvested Rainwater during Low- and High-Rainfall Periods

The possible health risks associated with the consumption of harvested rainwater remains one of the major obstacles hampering its large-scale implementation in water limited countries such as South Africa. Rainwater tank samples collected on eight occasions during the low- and high-rainfall periods (March to August 2012) in Kleinmond, South Africa, were monitored for the presence of virulence genes associated with Escherichia coli. The identity of presumptive E. coli isolates in rainwater samples collected from 10 domestic rainwater harvesting (DRWH) tanks throughout the sampling period was confirmed through universal 16S rRNA PCR with subsequent sequencing and phylogenetic analysis. Species-specific primers were also used to routinely screen for the virulent genes, aggR, stx, eae, and ipaH found in enteroaggregative E. coli (EAEC), enterohemorrhagic E. coli (EHEC), enteropathogenic E. coli (EPEC), and enteroinvasive E. coli, respectively, in the rainwater samples. Of the 92 E. coli strains isolated from the rainwater using culture based techniques, 6% were presumptively positively identified as E. coli O157:H7 using 16S rRNA. Furthermore, virulent pathogenic E. coli genes were detected in 3% (EPEC and EHEC) and 16% (EAEC) of the 80 rainwater samples collected during the sampling period from the 10 DRWH tanks. This study thus contributes valuable information to the limited data available regarding the ongoing prevalence of virulent pathotypes of E. coli in harvested rainwater during a longitudinal study in a high-population-density, periurban setting.     

Virulence Profiling of Shiga Toxin-Producing Escherichia coli O111:NM Isolates from Cattle

Shiga toxin-producing Escherichia coli (STEC) O111:NM is an important serotype that has been incriminated in disease outbreaks in the United States. This study characterized cattle STEC O111:NM for virulence factors and markers by PCR. Major conclusions are that STEC O111:NM characterized in this study lacks stx₂ and the full spectrum of nle gene markers, and it has an incomplete OI-122.     

Cryptic Genes for Cellobiose Utilization in Natural Isolates of Escherichia coli

The ECOR collection of natural Escherichia coli isolates was screened to determine the proportion of strains that carried functional, cryptic and nonfunctional genes for utilization of the three beta-glucoside sugars, arbutin, salicin and cellobiose. None of the 71 natural isolates utilized any of the beta-glucosides. Each strain was subjected to selection for utilization of each of the sugars. Only five of the isolates were incapable of yielding spontaneous beta-glucoside-utilizing mutants. Forty-five strains yielded cellobiose+ mutants, 62 yielded arbutin+ mutants, and 58 strains yielded salicin+ mutants. A subset of the mutants was screen by mRNA hybridization to determine whether they were expressing either the cel or the bgl beta-glucoside utilization operons of E. coli K12. Two cellobiose+ and two arbutin+-salicin+ strains failed to express either of these known operons. It is concluded that there are at least four gene clusters specifying beta-glucoside utilization functions in E. coli populations, and that all of these are normally cryptic. It is estimated that in any random isolate the probability of any particular cluster having been irreversibly inactivated by the accumulation of random mutations is about 0.5.     

Prevalence of Clonal Complexes and Virulence Genes among Commensal and Invasive Staphylococcus aureus Isolates in Sweden

Staphylococcus aureus encodes a remarkable number of virulence factors which may contribute to its pathogenicity and ability to cause invasive disease. The main objective of this study was to evaluate the association between S. aureus invasiveness and bacterial genotype, in terms of the presence of virulence genes and affiliation to clonal complexes. Also, the significance of different virulence genes, mainly adhesins, for the development of infective endocarditis was investigated.DNA microarray technology was used to analyze 134 S. aureus isolates, all methicillin-susceptible, derived from three groups of clinically well-characterized patients: nasal carriers (n=46), bacteremia (n=55), and bacteremia with infective endocarditis (n=33).Invasive isolates were dominant in four of the major clonal complexes: 5, 8, 15, and 25. Of the 170 virulence genes examined, those encoding accessory gene regulator group II (agr II), capsule polysaccharide serotype 5 (cap5), and adhesins such as S. aureus surface protein G (sasG) and fibronectin-binding protein B (fnbB) were found to be associated with invasive disease. The same was shown for the leukocidin genes lukD/lukE, as well as the genes encoding serine protease A and B (splA/splB), staphylococcal complement inhibitor (scn) and the staphylococcal exotoxin-like protein (setC or selX). In addition, there was a trend of higher prevalence of certain genes or gene clusters (sasG, agr II, cap5) among isolates causing infective endocarditis compared to other invasive isolates. In most cases, the presence of virulence genes was linked to clonal complex affiliation.In conclusion, certain S. aureus clonal lineages harboring specific sets of virulence genes seem to be more successful in causing invasive disease.