Animal and environmental risk factors for sporadic Shiga toxin-producing Escherichia coli (STEC) infection in England: a case control study for O157, O26 and other STEC serotypes.

Most Shiga toxin-producing E. coli (STEC) infections are sporadic. Routine enhanced surveillance questionnaires of confirmed STEC cases in England contained promising data to conduct a case-control study to identify non-food exposures linked to the risk of becoming infected with different STEC serotypes, including O157, O26 and all others; this study pulled eligible cases from the recorded enhanced surveillance data. Controls were recruited from the general population and answered a comparable postal questionnaire. Logistic regression was performed to identify risk factors associated with STEC infection for O157, O26 and other serotype cases. In adjusted models, travel outside of the U.K. and childcare occupations raised the risk of infection for all serotypes. Day trips within the UK, exposure to dogs and contact with soil were linked to lower infection risk. Resident region within England was often linked to decreased risk. Summer season was linked to O157 and O26, but not other STEC. Swimming in the sea was linked to increased risk of infection by O157, but not other types of STEC. Correlations between exposures and infection were similar when the analysis was repeated excluding participants with a history of foreign travel. As the first case-control study in England to include sporadic non-O157 STEC, the varying risk factors between O157 and non-O157 cases suggest there are potentially unique reservoirs for different serotypes.

Outbreaks of Shiga Toxin-Producing Escherichia coli Linked to Sprouted Seeds, Salad, and Leafy Greens: A Systematic Review.

Shiga toxin-producing Escherichia coli (STEC) outbreaks involving ready-to-eat salad products have been described in the scientific literature since 1995. These products typically do not undergo a definitive control step such as cooking to eliminate pathogens. To reduce the number of STEC infections from salad products, efforts will need to focus on preventing and reducing contamination throughout the food chain. We performed a systematic review of STEC outbreaks involving sprouted seeds, salad, or leafy green products to determine whether there were recurrent features, such as availability of microbiological evidence or identification of the contamination event, which may inform future investigations and prevention and control strategies. Thirty-five STEC outbreaks linked to contaminated leafy greens were identified for inclusion. The outbreaks occurred from 1995 to 2018 and ranged from 8 to more than 8,500 cases. Detection of STEC in the food product was rare (4 of 35 outbreaks). For the remaining outbreaks, the determination of leafy greens as the source of the outbreak mainly relied on analytical epidemiology (20 of 35) or descriptive evidence (11 of 35). The traceback investigation in 21 of 32 outbreaks was not able to identify possible routes leading to where the STEC bacteria came from or how the leaves were contaminated. Investigations in eight outbreaks found poor practice during processing that may have contributed to the outbreak, such as insufficient postharvest disinfection of the product. Six outbreak investigations were able to identify the outbreak strain in animal feces near the growing fields; two of these were also able to find it in irrigation water on the farms, providing a likely route of contamination. These results highlight the limitations of relying on microbiological confirmation as a basis to initiate investigations of upstream production to understand the source of contamination. This review also demonstrates the importance of, and difficulties associated with, food-chain traceback studies to inform control measures and future prevention.

Risk factors for Cryptosporidium infection in low and middle income countries: A systematic review and meta-analysis.

BACKGROUND: Cryptosporidium infection causes gastrointestinal disease and has a worldwide distribution. The highest burden is in developing countries. OBJECTIVES: We sought to conduct a systematic review and meta-analysis to identify Cryptosporidium risk factors in Low and Middle Income countries (LMICs). METHODS: Medline Ovid and Scopus databases were searched with no restriction on year or language of publication. All references were screened independently in duplicate and were included if they presented data on at least 3 risk factors. Meta-analyses using random effects models were used to calculate overall estimates for each exposure. RESULTS: The most frequently reported risk factors in the 15 included studies were overcrowding, household diarrhoea, poor quality drinking water, animal contact, open defecation/ lack of toilet and breastfeeding. The combined odds ratio for animal contact was 1.98 (95%CI: 1.11-3.54) based on 11 studies and for diarrhoea in the household 1.98 (95%CI: 1.13-3.49) based on 4 studies. Open defecation was associated with a pooled odds ratio of 1.82 (95%CI: 1.19-2.8) based on 5 studies. Poor drinking water quality was not associated with a significant Cryptosporidium risk, odds ratio 1.06 (95%CI: 0.77-1.47). Breastfeeding was protective with pooled odds ratio 0.4 (95%CI: 0.13-1.22), which was not statistically significant. CONCLUSIONS: Based on the included studies, crowded living conditions, animal contact and open defecation are responsible for the majority of Cryptosporidium cases in LMICs. Future studies investigating Cryptosporidium risk factors should have a good study design and duration, include appropriate number of cases, select suitable controls, investigate multiple relevant risk factors, fully report data and perform multivariate analysis.

Salmonella enterica Serovar Typhi Lipopolysaccharide O-Antigen Modification Impact on Serum Resistance and Antibody Recognition.

Salmonella enterica serovar Typhi is a human-restricted Gram-negative bacterial pathogen responsible for causing an estimated 27 million cases of typhoid fever annually, leading to 217,000 deaths, and current vaccines do not offer full protection. The O-antigen side chain of the lipopolysaccharide is an immunodominant antigen, can define host-pathogen interactions, and is under consideration as a vaccine target for some Gram-negative species. The composition of the O-antigen can be modified by the activity of glycosyltransferase (gtr) operons acquired by horizontal gene transfer. Here we investigate the role of two gtr operons that we identified in the S Typhi genome. Strains were engineered to express specific gtr operons. Full chemical analysis of the O-antigens of these strains identified gtr-dependent glucosylation and acetylation. The glucosylated form of the O-antigen mediated enhanced survival in human serum and decreased complement binding. A single nucleotide deviation from an epigenetic phase variation signature sequence rendered the expression of this glucosylating gtr operon uniform in the population. In contrast, the expression of the acetylating gtrC gene is controlled by epigenetic phase variation. Acetylation did not affect serum survival, but phase variation can be an immune evasion mechanism, and thus, this modification may contribute to persistence in a host. In murine immunization studies, both O-antigen modifications were generally immunodominant. Our results emphasize that natural O-antigen modifications should be taken into consideration when assessing responses to vaccines, especially O-antigen-based vaccines, and that the Salmonellagtr repertoire may confound the protective efficacy of broad-ranging Salmonella lipopolysaccharide conjugate vaccines.

Transmission pathways for sporadic Shiga-toxin producing E. coli infections: A systematic review and meta-analysis.

BACKGROUND: Shiga-toxin E. coli infections remain a public health concern because of the severity of the gastrointestinal illness and associated complications. Transmission pathways are typically elucidated from outbreaks, with foodborne transmission the primary source. However, most STEC cases are sporadic. This systematic review aimed to identify the most common pathways for sporadic STEC transmission and quantify their importance. METHODS: We systematically reviewed epidemiological studies of sporadic (non-outbreak) STEC cases that investigated potential risk factors. Searches were run in Medline, EMBASE, and Scopus. Included studies needed to confirm STEC infection and investigate ≥20 cases. RESULTS: 31 studies were included, of which 25 were case-control or case-case studies. 62.5% found consumption of undercooked/raw meat associated with STEC infection while 70.4% found contact with animals or their environment a risk factor. Random-effects meta-analysis provided pooled odds ratios and population attributable fraction (PAF). The PAF was 19% for undercooked/raw meat, followed by person to person transmission at 15%. Contact with animals and visiting farm environments had PAFs of 14% and 12% respectively. CONCLUSIONS: Out of potential sources for STEC exposure, undercooked meat and contact with animals and their environment were the most frequently found transmission routes. Decreasing the chances of acquiring the bacteria by these methods would additionally cut down on the other major transmission route, person-to-person spread.

A BTP1 prophage gene present in invasive non-typhoidal Salmonella determines composition and length of the O-antigen of the lipopolysaccharide.

Salmonella Typhimurium isolate D23580 represents a recently identified ST313 lineage of invasive non-typhoidal Salmonellae (iNTS). One of the differences between this lineage and other non-iNTS S. Typhimurium isolates is the presence of prophage BTP1. This prophage encodes a gtrC gene, implicated in O-antigen modification. GtrC(BTP) (1) is essential for maintaining O-antigen length in isolate D23580, since a gtr(BTP) (1) mutant yields a short O-antigen. This phenotype can be complemented by gtrC(BTP) (1) or very closely related gtrC genes. The short O-antigen of the gtr(BTP) (1) mutant was also compensated by deletion of the BTP1 phage tailspike gene in the D23580 chromosome. This tailspike protein has a putative endorhamnosidase domain and thus may mediate O-antigen cleavage. Expression of the gtrC(BTP) (1) gene is, in contrast to expression of many other gtr operons, not subject to phase variation and transcriptional analysis suggests that gtrC is produced under a variety of conditions. Additionally, GtrC(BTP) (1) expression is necessary and sufficient to provide protection against BTP1 phage infection of an otherwise susceptible strain. These data are consistent with a model in which GtrC(BTP) (1) mediates modification of the BTP1 phage O-antigen receptor in lysogenic D23580, and thereby prevents superinfection by itself and other phage that uses the same O-antigen co-receptor.

Site-directed mutagenesis reveals key residue for O antigen chain length regulation and protein stability in Pseudomonas aeruginosa Wzz2.

The production of preferred lipopolysaccharide O antigen chain lengths is important for the survival of pathogenic Gram-negative bacteria in different environments, yet how Wzz proteins regulate these lengths is not well understood. The Wzz2 proteins from two different serotype O11 Pseudomonas aeruginosa strains are responsible for the expression of different very long chain lengths despite high sequence homology. Site-directed mutagenesis was performed to determine whether a specific amino acid was responsible for this difference in chain length; the residue present in position 321 within the second predicted coiled-coil region was able to determine which chain length was produced. A panel of site-directed mutants introducing different amino acids at this position implicated that the charge of the amino acid affected chain length, with positively charged residues associated with shorter chain lengths. Expression data also suggested this site was important for overall stability of the protein because mutants predicted to disrupt proper folding of the α helix led to lower protein levels. Cross-linking studies found that Wzz2 proteins producing shorter chain lengths had more stable higher-order oligomers. Mapping residue 321 onto the solved Escherichia coli Wzz FepE crystal structure predicted it to be located within α helix 8, which participates in intermonomeric interactions. These data further support the observation that Wzz oligomerization is necessary for chain length regulating activity but also provide evidence that differences in complex stability or changes in the conformation of the oligomer can lead to shifts in the length of the O antigen side chain.

Structural basis for the interaction of lipopolysaccharide with outer membrane protein H (OprH) from Pseudomonas aeruginosa.

Pseudomonas aeruginosa is a major nosocomial pathogen that infects cystic fibrosis and immunocompromised patients. The impermeability of the P. aeruginosa outer membrane contributes substantially to the notorious antibiotic resistance of this human pathogen. This impermeability is partially imparted by the outer membrane protein H (OprH). Here we have solved the structure of OprH in a lipid environment by solution NMR. The structure reveals an eight-stranded ß-barrel protein with four extracellular loops of unequal size. Fast time-scale dynamics measurements show that the extracellular loops are disordered and unstructured. It was previously suggested that the function of OprH is to provide increased stability to the outer membranes of P. aeruginosa by directly interacting with lipopolysaccharide (LPS) molecules. Using in vivo and in vitro biochemical assays, we show that OprH indeed interacts with LPS in P. aeruginosa outer membranes. Based upon NMR chemical shift perturbations observed upon the addition of LPS to OprH in lipid micelles, we conclude that the interaction is predominantly electrostatic and localized to charged regions near both rims of the barrel, but also through two conspicuous tyrosines in the middle of the bilayer. These results provide the first molecular structure of OprH and offer evidence for multiple interactions between OprH and LPS that likely contribute to the antibiotic resistance of P. aeruginosa.

Relating the physical properties of Pseudomonas aeruginosa lipopolysaccharides to virulence by atomic force microscopy.

Lipopolysaccharides (LPS) are an important class of macromolecules that are components of the outer membrane of Gram-negative bacteria such as Pseudomonas aeruginosa. P. aeruginosa contains two different sugar chains, the homopolymer common antigen (A band) and the heteropolymer O antigen (B band), which impart serospecificity. The characteristics of LPS are generally assessed after isolation rather than in the context of whole bacteria. Here we used atomic force microscopy (AFM) to probe the physical properties of the LPS of P. aeruginosa strain PA103 (serogroup O11) in situ. This strain contains a mixture of long and very long polymers of O antigen, regulated by two different genes. For this analysis, we studied the wild-type strain and four mutants, ΔWzz1 (producing only very long LPS), ΔWzz2 (producing only long LPS), DΔM (with both the wzz1 and wzz2 genes deleted), and Wzy::GM (producing an LPS core oligosaccharide plus one unit of O antigen). Forces of adhesion between the LPS on these strains and the silicon nitride AFM tip were measured, and the Alexander and de Gennes model of steric repulsion between a flat surface and a polymer brush was used to calculate the LPS layer thickness (which we refer to as length), compressibility, and spacing between the individual molecules. LPS chains were longest for the wild-type strain and ΔWzz1, at 170.6 and 212.4 nm, respectively, and these values were not statistically significantly different from one another. Wzy::GM and DΔM have reduced LPS lengths, at 34.6 and 37.7 nm, respectively. Adhesion forces were not correlated with LPS length, but a relationship between adhesion force and bacterial pathogenicity was found in a mouse acute pneumonia model of infection. The adhesion forces with the AFM probe were lower for strains with LPS mutations, suggesting that the wild-type strain is optimized for maximal adhesion. Our research contributes to further understanding of the role of LPS in the adhesion and virulence of P. aeruginosa.

Regulation of lipopolysaccharide O antigen expression in Pseudomonas aeruginosa.

Pseudomonas aeruginosa is a Gram-negative bacterium that is ubiquitously found in the environment. It is an important opportunistic pathogen in immunocompromised patients and causes life-threatening lung infections in individuals with cystic fibrosis. A prominent virulence factor for many Gram-negative bacteria, including P. aeruginosa, is lipopolysaccharide (LPS), which is an immunodominant antigen located in the outer portion of the outer membrane. P. aeruginosa produces two O antigens that are attached to lipid A + core: a B-band O antigen and an A-band O polysaccharide. The B-band O antigen-repeating unit of LPS is responsible for serotype specificity; strains lacking O antigen have been shown to be less virulent in animal models of infection. What is less well understood is how the O antigen chain length is regulated and why P. aeruginosa and some other bacteria show two preferred O antigen lengths. P. aeruginosa encodes two genes encoding O antigen chain length regulators. These genes, wzz1 and wzz2, influence the expression of the long and very long chain lengths, respectively. The long chain length appears more important for resistance to the action of sera and virulence in a mouse model of infection, while the very long chain length appears to be more sensitive to environmental stress conditions. Studies in other bacteria point to regulation at the level of transcription and complex formation as being involved in determining the O antigen chain length and may provide clues to the regulation in P. aeruginosa.

Lipopolysaccharide O-antigen chain length regulation in Pseudomonas aeruginosa serogroup O11 strain PA103.

The Wzz proteins are important for determining the length of the O-antigen side chain attached to lipopolysaccharide (LPS). Several bacteria, including Pseudomonas aeruginosa strain PAO1 (serogroup O5), produce two such proteins responsible for the preference of two different chain lengths on the surface. Our group has previously identified one wzz gene (wzz1) within the O-antigen locus of P. aeruginosa strain PA103 (serogroup O11). In this study we have identified the second wzz gene (wzz2), located in the same region of the genome and with 92% similarity to PAO1's wzz2 gene. Mutations were generated in both wzz genes by interruption with antibiotic resistance cassettes, and the effects of these mutations were characterized. Wild-type PA103 prefers two O-antigen chain lengths, referred to as long and very long. The expression of the long O-antigen chain length was reduced in the wzz1 mutant, indicating the Wzz1 protein is important for this chain length preference. The wzz2 mutant, on the other hand, was missing O-antigens of the very long chain length, indicating the Wzz2 protein is responsible for the production of very long O-antigen. The effects of the wzz mutations on virulence were also investigated. In both serum sensitivity assays and a mouse pneumonia model of infection, the wzz1 mutants exhibited greater defects in virulence compared to either wild-type PA103 or the wzz2 mutant, indicating the long chain length plays a greater role during these infectious processes.