Alan McNally

Comparison of the biotypes of Yersinia enterocolitica isolated from pigs, cattle and sheep at slaughter and from humans with yersiniosis in Great Britain during 1999-2000

Aims:  To investigate the relationship between livestock carriage of Yersinia enterocolitica and human disease. The biotypes/serotypes of strains recovered from the faeces of pigs, cattle and sheep at slaughter during a national survey in Great Britain in 1999–2000, were compared with those of strains isolated from human cases of yersiniosis during the same period.

Differences in Levels of Secreted Locus of Enterocyte Effacement Proteins between Human Disease-Associated and Bovine Escherichia coli O157

ABSTRACT Ongoing extensive epidemiological studies of verotoxin-carryingEscherichia coli O157 (stx+eae+) have shown this bacterial pathogen to be common in cattle herds in the United States and the United Kingdom. However, the incidence of disease in humans due to this pathogen is still very low. This study set out to investigate if there is a difference between strains isolated from human disease cases and those isolated from asymptomatic cattle which would account for the low disease incidence of such a ubiquitous organism. The work presented here has compared human disease strains from both sporadic and outbreak cases with a cross-section, as defined by pulsed-field gel electrophoresis, of E. coli O157 strains from cattle. Human (n = 22) and bovine (n = 31) strains were genotyped for carriage of the genes for Shiga-like toxin types 1, 2, and 2c; E. coli secreted protein genesespA, espB, and espP; the enterohemolysin gene; eae (intimin); ast(enteroaggregative E. coli stable toxin [EAST]); and genes for common E. coli adhesins. Strains were also phenotyped for hemolysin, EspP, Tir, and EspD expression as well as production of actin and cytoskeletal rearrangement associated with attaching and effacing (A/E) lesions on HeLa cells. The genotyping confirmed that there was little difference between the two groups, including carriage of stx2 andstx2c, which was similar in both sets.ast alleles were confirmed to all contain mutations that would prevent EAST expression. espP mutations were found only in cattle strains (5 of 30). Clear differences were observed in the expression of locus of enterocyte effacement (LEE)-encoded factors between strains and in different media. EspD, as an indicator of LEE4 (espA, -B, and -D) expression, and Tir levels in supernatants were measured. Virtually all strains from both sources could produce EspD in Luria-Bertani broth, although at very different levels. Standard trichloroacetic acid precipitation of secreted proteins from tissue culture medium produced detectable levels of EspD from the majority of strains of human origin (15 of 20) compared with only a few (4 of 20) bovine strains (P < 0.001), which is indicative of much higher levels of protein secretion from the human strains. Addition of bovine serum albumin carrier protein before precipitation and enhanced detection techniques confirmed that EspD could be detected after growth in tissue culture medium for all strains, but levels from strains of human origin were on average 90-fold higher than those from strains of bovine origin. In general, levels of secretion also correlated with ability to form A/E lesions on HeLa cells, with only the high-level protein secretors in tissue culture medium exhibiting a localized adherence phenotype. This research shows significant differences between human- and bovine-derived E. coliO157 (stx+eae+) strains and their production of certain LEE-encoded virulence factors. These data support the recent finding of Kim et al. (J. Kim, J. Nietfeldt, and A. K. Benson, Proc. Natl. Acad. Sci. USA 96:13288–13293, 1999) proposing different E. coli O157 lineages in cattle and humans and extend the differential to the regulation of virulence factors. Potentially only a subset of E. coli O157 isolates (stx+eae+) in cattle may be capable of causing severe disease in humans.

Parallel independent evolution of pathogenicity within the genus Yersinia.

Significance Our past understanding of pathogen evolution has been fragmented because of tendencies to study human clinical isolates. To understand the evolutionary trends of pathogenic bacteria though, we need the context of their nonpathogenic relatives. Our unique and detailed dataset allows description of the parallel evolution of two key human pathogens: the causative agents of plague and Yersinia diarrhea. The analysis reveals an emerging pattern where few virulence-related functions are found in all pathogenic lineages, representing key “foothold” moments that mark the emergence of these pathogens. Functional gene loss and metabolic streamlining are equally complementing the evolution of Yersinia across the pathogenic spectrum. The genus Yersinia has been used as a model system to study pathogen evolution. Using whole-genome sequencing of all Yersinia species, we delineate the gene complement of the whole genus and define patterns of virulence evolution. Multiple distinct ecological specializations appear to have split pathogenic strains from environmental, nonpathogenic lineages. This split demonstrates that contrary to hypotheses that all pathogenic Yersinia species share a recent common pathogenic ancestor, they have evolved independently but followed parallel evolutionary paths in acquiring the same virulence determinants as well as becoming progressively more limited metabolically. Shared virulence determinants are limited to the virulence plasmid pYV and the attachment invasion locus ail. These acquisitions, together with genomic variations in metabolic pathways, have resulted in the parallel emergence of related pathogens displaying an increasingly specialized lifestyle with a spectrum of virulence potential, an emerging theme in the evolution of other important human pathogens.

Molecular epidemiology of extraintestinal pathogenic Escherichia coli isolates from a regional cohort of elderly patients highlights the prevalence of ST131 strains with increased antimicrobial resistance in both community and hospital care settings

OBJECTIVES To assess the molecular epidemiology and prevalence of antibiotic resistance in Escherichia coli causing urinary tract infections of elderly patients from community and hospital settings. Also, to determine whether the possession of antibiotic resistance and virulence-associated genes can be linked to patient location or the clonal group of the organisms in question. METHODS E. coli were isolated from the urine samples of elderly patients from the Nottingham area, and subjected to antibiotic susceptibility testing, virulence gene detection by PCR and multilocus sequence typing. RESULTS No correlation was observed between community- or hospital-derived strains with regard to antibiotic resistance levels or virulence gene profiles. E. coli ST131 (where ST stands for sequence type) was the predominant ST found in both hospital and community samples, and demonstrated high levels of antibiotic resistance to the test panel, but did not possess a significantly larger array of virulence genes or a specific gene profile compared with other STs. CONCLUSIONS The level of antibiotic resistance or virulence gene possession in uropathogenic E. coli is not directly associated with the healthcare setting of the patient, but there is a variation in antibiotic resistance and virulence gene possession depending on clonal group. ST131 is highly virulent and demonstrates high levels of antibiotic resistance, but its virulence does not appear to be attributable to the possession of a specific virulence-associated gene set or the possession of any virulence-associated gene in significantly higher levels than in any other ST.

Recombination is a key driver of genomic and phenotypic diversity in a Pseudomonas aeruginosa population during cystic fibrosis infection

The Cystic Fibrosis (CF) lung harbors a complex, polymicrobial ecosystem, in which Pseudomonas aeruginosa is capable of sustaining chronic infections, which are highly resistant to multiple antibiotics. Here, we investigate the phenotypic and genotypic diversity of 44 morphologically identical P. aeruginosa isolates taken from a single CF patient sputum sample. Comprehensive phenotypic analysis of isolates revealed large variances and trade-offs in growth, virulence factors and quorum sensing (QS) signals. Whole genome analysis of 22 isolates revealed high levels of intra-isolate diversity ranging from 5 to 64 SNPs and that recombination and not spontaneous mutation was the dominant driver of diversity in this population. Furthermore, phenotypic differences between isolates were not linked to mutations in known genes but were statistically associated with distinct recombination events. We also assessed antibiotic susceptibility of all isolates. Resistance to antibiotics significantly increased when multiple isolates were mixed together. Our results highlight the significant role of recombination in generating phenotypic and genetic diversification during in vivo chronic CF infection. We also discuss (i) how these findings could influence how patient-to-patient transmission studies are performed using whole genome sequencing, and (ii) the need to refine antibiotic susceptibility testing in sputum samples taken from patients with CF.

Why prokaryotes have pangenomes

The existence of large amounts of within-species genome content variability is puzzling. Population genetics tells us that fitness effects of new variants—either deleterious, neutral or advantageous—combined with the long-term effective population size of the species determines the likelihood of a new variant being removed, spreading to fixation or remaining polymorphic. Consequently, we expect that selection and drift will reduce genetic variation, which makes large amounts of gene content variation in some species so puzzling. Here, we amalgamate population genetic theory with models of horizontal gene transfer and assert that pangenomes most easily arise in organisms with large long-term effective population sizes, as a consequence of acquiring advantageous genes, and that the focal species has the ability to migrate to new niches. Therefore, we suggest that pangenomes are the result of adaptive, not neutral, evolution. Amalgamation of population genetic theory and models of horizontal gene transfer suggest that pangenomes in prokaryotes result from adaptive, not neutral, evolution.

Increased human pathogenic potential of Escherichia coli from polymicrobial urinary tract infections in comparison to isolates from monomicrobial culture samples

The current diagnostic standard procedure outlined by the Health Protection Agency for urinary tract infections (UTIs) in clinical laboratories does not report bacteria isolated from samples containing three or more different bacterial species. As a result many UTIs go unreported and untreated, particularly in elderly patients, where polymicrobial UTI samples are especially prevalent. This study reports the presence of the major uropathogenic species in mixed culture urine samples from elderly patients, and of resistance to front-line antibiotics, with potentially increased levels of resistance to ciprofloxacin and trimethoprim. Most importantly, the study highlights that Escherichia coli present in polymicrobial UTI samples are statistically more invasive (P<0.001) in in vitro epithelial cell infection assays than those isolated from monomicrobial culture samples. In summary, the results of this study suggest that the current diagnostic standard procedure for polymicrobial UTI samples needs to be reassessed, and that E. coli present in polymicrobial UTI samples may pose an increased risk to human health.

Infection dynamics of highly pathogenic avian influenza and virulent avian paramyxovirus type 1 viruses in chickens, turkeys and ducks

A range of virus doses were used to infect 3-week-old chickens, turkeys and ducks intranasally/intraocularly, and infection was confirmed by the detection of virus shedding from the buccal or cloacal route by analysis of swabs collected using real-time reverse transcriptase-polymerase chain reaction assays. The median infectious dose (ID50) and the median lethal dose (LD50) values for two highly pathogenic avian influenza (HPAI) viruses of H5N1 and H7N1 subtypes and one virulent Newcastle disease virus (NDV) were determined for each virus and host combination. For both HPAI viruses, turkeys were >100-fold more susceptible to infection than chickens, while both these hosts were >10-fold more susceptible to H5N1 virus than the H7N1 virus. All infected chickens and turkeys died. Ducks were also much more readily infected with the H5N1 virus (ID50≤101 median embryo infective dose [EID50]) than the H7N1 virus (ID50=104.2 EID50). However, the most notable difference between the two viruses was their virulence for ducks, with a LD50 of 103 EID50 for the H5N1 virus, but no deaths in ducks being attributed to infection with H7N1 virus even at the highest dose (106 EID50). For both HPAI virus infections of ducks, the ID50 was lower than the LD50, indicating that infected birds were able to survive and thus excrete virus over a longer period than chickens and turkeys. The NDV strain used did not appear to establish infection in ducks even at the highest dose used (106 EID50). Some turkeys challenged with 106 EID50, but not other doses, of NDV excreted virus for a number of days (ID50=104.6 EID50), but none died. In marked contrast, chickens were shown to be extremely susceptible to infection and all infected chickens died (ID50/LD50=101.9 EID50).

'Add, stir and reduce': Yersinia spp. as model bacteria for pathogen evolution

Pathogenic species in the Yersinia genus have historically been targets for research aimed at understanding how bacteria evolve into mammalian pathogens. The advent of large-scale population genomic studies has greatly accelerated the progress in this field, and Yersinia pestis, Yersinia pseudotuberculosis and Yersinia enterocolitica have once again acted as model organisms to help shape our understanding of the evolutionary processes involved in pathogenesis. In this Review, we highlight the gene gain, gene loss and genome rearrangement events that have been identified by genomic studies in pathogenic Yersinia species, and we discuss how these findings are changing our understanding of pathogen evolution. Finally, as these traits are also found in the genomes of other species in the Enterobacteriaceae, we suggest that they provide a blueprint for the evolution of enteropathogenic bacteria.

The Evolutionary Path to Extraintestinal Pathogenic, Drug-Resistant Escherichia coli Is Marked by Drastic Reduction in Detectable Recombination within the Core Genome

Escherichia coli is a highly diverse group of pathogens ranging from commensal of the intestinal tract, through to intestinal pathogen, and extraintestinal pathogen. Here, we present data on the population diversity of E. coli, using Bayesian analysis to identify 13 distinct clusters within the population from multilocus sequence typing data, which map onto a whole-genome-derived phylogeny based on 62 genome sequences. Bayesian analysis of recombination within the core genome identified reduction in detectable core genome recombination as one moves from the commensals, through the intestinal pathogens down to the multidrug-resistant extraintestinal pathogenic clone E. coli ST131. Our data show that the emergence of a multidrug-resistant, extraintestinal pathogenic lineage of E. coli is marked by substantial reduction in detectable core genome recombination, resulting in a lineage which is phylogenetically distinct and sexually isolated in terms of core genome recombination.