Dynamics of antimicrobial resistance in intestinal Escherichia coli from children in community settings in South Asia and sub-Saharan Africa.

The dynamics of antimicrobial resistance (AMR) in developing countries are poorly understood, especially in community settings, due to a sparsity of data on AMR prevalence and genetics. We used a combination of phenotyping, genomics and antimicrobial usage data to investigate patterns of AMR amongst atypical enteropathogenic Escherichia coli (aEPEC) strains isolated from children younger than five years old in seven developing countries (four in sub-Saharan Africa and three in South Asia) over a three-year period. We detected high rates of AMR, with 65% of isolates displaying resistance to three or more drug classes. Whole-genome sequencing revealed a diversity of known genetic mechanisms for AMR that accounted for >95% of phenotypic resistance, with comparable rates amongst aEPEC strains associated with diarrhoea or asymptomatic carriage. Genetic determinants of AMR were associated with the geographic location of isolates, not E. coli lineage, and AMR genes were frequently co-located, potentially enabling the acquisition of multi-drug resistance in a single step. Comparison of AMR with antimicrobial usage data showed that the prevalence of resistance to fluoroquinolones and third-generation cephalosporins was correlated with usage, which was higher in South Asia than in Africa. This study provides much-needed insights into the frequency and mechanisms of AMR in intestinal E. coli in children living in community settings in developing countries

Atypical Enteropathogenic Escherichia coli Infection and Prolonged Diarrhea in Children

Infection of children with atypical EPEC is associated with prolonged diarrhea

Contribution of Plasmid-Encoded Fimbriae and Intimin to Capacity of Rabbit-Specific Enteropathogenic <i>Escherichia coli</i> To Attach to and Colonize Rabbit Intestine

ABSTRACT Attachment to the intestinal mucosa is an essential step in the pathogenesis of diarrhea caused by enteropathogenic Escherichia coli (EPEC). Fimbriae and intimin, the outer membrane protein product of the chromosomal eae gene, contribute to this process, but their relative roles and the nature of their interaction are not known. The aim of this study was to determine the relative contribution of plasmid-encoded fimbriae, termed Ral, and intimin to the capacity of rabbit-specific EPEC (REPEC) to attach to the intestinal mucosa of rabbits. To achieve this, we constructed a series of mutants in REPEC strain 83/39 (O15:H−), in which the ralE and eae genes were insertionally inactivated. These strains were then inoculated into ligated loops of rabbit ileum, which were resected 18 h later and examined by light and electron microscopy. The results showed that intimin, but not Ral, is essential for the elicitation of attaching-effacing lesions by REPEC. Nevertheless, a Δ eae Ral-bearing mutant adhered to the intestinal epithelium to the same extent as its eae -positive parent and far more extensively than an eae + Δral strain. To examine the contribution of Ral and intimin to colonization of rabbit intestine, we fed these strains to weanling rabbits, which were killed 4 days later, so that the number of bacteria in various regions of the intestine could be determined. The results indicated that strain 83/39 requires both Ral and intimin to colonize the intestine successfully and that a Δ eae ΔralE double mutant was incapable of colonizing the intestine. Taken together, these findings indicate that Ral and intimin act independently as adhesion factors of REPEC strain 83/39 and that this strain carries no other significant colonization factor. When both Ral and intimin are present, they appear to act cooperatively, with Ral-mediated adhesion preceding that mediated by intimin. </jats:p

The type II secretion system and its ubiquitous lipoprotein substrate, SsIE are required for biofilm formation and virulence of enteropathogenic escherichia coli

Enteropathogenic Escherichia coli (EPEC) is a major cause of diarrhea in infants in developing countries. We have identified a functional type II secretion system (T2SS) in EPEC that is homologous to the pathway responsible for the secretion of heat-labile enterotoxin by enterotoxigenic E. coli. The wild-type EPEC T2SS was able to secrete a heat-labile enterotoxin reporter, but an isogenic T2SS mutant could not. We showed that the major substrate of the T2SS in EPEC is SslE, an outer membrane lipoprotein (formerly known as YghJ), and that a functional T2SS is essential for biofilm formation by EPEC. T2SS and SslE mutants were arrested at the microcolony stage of biofilm formation, suggesting that the T2SS is involved in the development of mature biofilms and that SslE is a dominant effector of biofilm development. Moreover, the T2SS was required for virulence, as infection of rabbits with a rabbit-specific EPEC strain carrying a mutation in either the T2SS or SslE resulted in significantly reduced intestinal colonization and milder disease

Escherichia coli and Community-acquired Gastroenteritis, Melbourne, Australia

Atypical strains of enteropathogenic E. coli are a leading cause of gastroenteritis in Melbourne

Characterisation of Shiga toxin-producing Escherichia coli O157 strains isolated from humans in Argentina, Australia and New Zealand

Background: Shiga toxin-producing Escherichia coli (STEC) is an important cause of bloody diarrhoea (BD), non-bloody diarrhoea (NBD) and the haemolytic uraemic syndrome (HUS). In Argentina and New Zealand, the most prevalent STEC serotype is O157:H7, which is responsible for the majority of HUS cases. In Australia, on the other hand, STEC O157:H7 is associated with a minority of HUS cases. The main aims of this study were to compare the phenotypic and genotypic characteristics of STEC O157 strains isolated between 1993 and 1996 from humans in Argentina, Australia and New Zealand, and to establish their clonal relatedness. Results: Seventy-three O157 STEC strains, isolated from HUS (n = 36), BD (n = 20), NBD (n = 10), or unspecified conditions (n = 7) in Argentina, Australia and New Zealand, were analysed. The strains were confirmed to be E. coli O157 by biochemical tests and serotyping. A multiplex polymerase chain reaction (PCR) was used to amplify the stx1, stx2 and rfbO157 genes and a genotyping method based on PCR-RFLP was used to determine stx1 and stx2 variants. This analysis revealed that the most frequent stx genotypes were stx2/stx 2c (vh-a) (91%) in Argentina, stx2 (89%) in New Zealand, and stx1/stx2 (30%) in Australia. No stx 1-postive strains were identified in Argentina or New Zealand. All strains harboured the eae gene and 72 strains produced enterohaemolysin (EHEC-Hly). The clonal relatedness of strains was investigated by phage typing and pulsed-field gel electrophoresis (PFGE). The most frequent phage types (PT) identified in Argentinian, Australian, and New Zealand strains were PT49 (n = 12), PT14 (n = 9), and PT2 (n = 15), respectively. Forty-six different patterns were obtained by XbaI-PFGE; 37 strains were grouped in 10 clusters and 36 strains showed unique patterns. Most clusters could be further subdivided by BlnI-PFGE. Conclusion: STEC O157 strains isolated in Argentina, Australia, and New Zealand differed from each other in terms of stx-genotype and phage type. Additionally, no common PFGE patterns were found in strains isolated in the three countries. International collaborative studies of the type reported here are needed to detect and monitor potentially hypervirulent STEC clones.Fil: Leotta, Gerardo Anibal. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorio e Instituto de Salud “Dr. C. G. Malbrán”; ArgentinaFil: Miliwebsky, Elizabeth S.. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorio e Instituto de Salud “Dr. C. G. Malbrán”; ArgentinaFil: Chinen, Isabel. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorio e Instituto de Salud “Dr. C. G. Malbrán”; ArgentinaFil: Espinosa, Estela M.. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorio e Instituto de Salud “Dr. C. G. Malbrán”; ArgentinaFil: Azzopardi, Kristy. University of Melbourne; AustraliaFil: Tennant, Sharon M.. University of Melbourne; AustraliaFil: Robins Browne, Roy M.. University of Melbourne; AustraliaFil: Rivas, Marta. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorio e Instituto de Salud “Dr. C. G. Malbrán”; Argentin

Characterisation of atypical enteropathogenic E. coli strains of clinical origin

BACKGROUND: Enteropathogenic E. coli (EPEC) is a prominent cause of diarrhoea, and is characterised in part by its carriage of a pathogenicity island: the locus for enterocyte effacement (LEE). EPEC is divided into two subtypes according to the presence of bundle-forming pili (BFP), a fimbrial adhesin that is a virulence determinant of typical EPEC (tEPEC), but is absent from atypical EPEC (aEPEC). Because aEPEC lack BFP, their virulence has been questioned, as they may represent LEE-positive Shiga toxin-producing E. coli (STEC) that have lost the toxin-encoding prophage, or tEPEC that have lost the genes for BFP. To determine if aEPEC isolated from humans in Australia or New Zealand fall into either of these categories, we undertook phylogenetic analysis of 75 aEPEC strains, and compared them with reference strains of EPEC and STEC. We also used PCR and DNA hybridisation to determine if aEPEC carry virulence determinants that could compensate for their lack of BFP. RESULTS: The results showed that aEPEC are highly heterogeneous. Multilocus sequence typing revealed that 61 of 75 aEPEC strains did not belong to known tEPEC or STEC clades, and of those that did, none expressed an O:H serotype that is frequent in tEPEC or STEC strains associated with disease. PCR for each of 18 known virulence-associated determinants of E. coli was positive in less than 15% of strains, apart from NleB which was detected in 30%. Type I fimbriae were expressed by all aEPEC strains, and 12 strains hybridised with DNA probes prepared from either bfpA or bfpB despite being negative in the PCR for bfpA. CONCLUSION: Our findings indicate that clinical isolates of aEPEC obtained from patients in Australia or New Zealand are not derived from tEPEC or STEC, and suggest that functional equivalents of BFP and possibly type I fimbriae may contribute to the virulence of some aEPEC strains

Are Escherichia coli Pathotypes Still Relevant in the Era of Whole-Genome Sequencing?

The empirical and pragmatic nature of diagnostic microbiology has given rise to several different schemes to subtype E.coli, including biotyping, serotyping, and pathotyping. These schemes have proved invaluable in identifying and tracking outbreaks, and for prognostication in individual cases of infection, but they are imprecise and potentially misleading due to the malleability and continuous evolution of E. coli. Whole genome sequencing can be used to accurately determine E. coli subtypes that are based on allelic variation or differences in gene content, such as serotyping and pathotyping. Whole genome sequencing also provides information about single nucleotide polymorphisms in the core genome of E. coli, which form the basis of sequence typing, and is more reliable than other systems for tracking the evolution and spread of individual strains. A typing scheme for E. coli based on genome sequences that includes elements of both the core and accessory genomes, should reduce typing anomalies and promote understanding of how different varieties of E. coli spread and cause disease. Such a scheme could also define pathotypes more precisely than current methods

Phasevarion Mediated Epigenetic Gene Regulation in Helicobacter pylori

Many host-adapted bacterial pathogens contain DNA methyltransferases (mod genes) that are subject to phase-variable expression (high-frequency reversible ON/OFF switching of gene expression). In Haemophilus influenzae and pathogenic Neisseria, the random switching of the modA gene, associated with a phase-variable type III restriction modification (R-M) system, controls expression of a phase-variable regulon of genes (a “phasevarion”), via differential methylation of the genome in the modA ON and OFF states. Phase-variable type III R-M systems are also found in Helicobacter pylori, suggesting that phasevarions may also exist in this key human pathogen. Phylogenetic studies on the phase-variable type III modH gene revealed that there are 17 distinct alleles in H. pylori, which differ only in their DNA recognition domain. One of the most commonly found alleles was modH5 (16% of isolates). Microarray analysis comparing the wild-type P12modH5 ON strain to a P12ΔmodH5 mutant revealed that six genes were either up- or down-regulated, and some were virulence-associated. These included flaA, which encodes a flagella protein important in motility and hopG, an outer membrane protein essential for colonization and associated with gastric cancer. This study provides the first evidence of this epigenetic mechanism of gene expression in H. pylori. Characterisation of H. pylori modH phasevarions to define stable immunological targets will be essential for vaccine development and may also contribute to understanding H. pylori pathogenesis