The risk to import ESBL-producing Enterobacteriaceae and Staphylococcus aureus through chicken meat trade in Gabon

A main export market for chicken meat from industrialized countries is sub-Saharan Africa. We hypothesized that antibiotic resistant bacteria could be exported to developing countries through chicken meat trade. The objective was to investigate the occurrence and molecular types of ESBL-producing Enterobacteriaceae and Staphylococcus aureus in chicken meat in Gabon and to assess their dissemination among humans. Frozen chicken meat samples imported from industrialized countries to Gabon (n = 151) were screened for ESBL-producing Enterobacteriaceae and S. aureus. Genotypes and resistance genes (SHV, TEM, CTX-M, CMY-2) of isolates from meat were compared with isolates derived from humans. The contamination rate per chicken part (i. e. leg, wing) with ESBL-producing Escherichia coli (ESBL E. coli, no other ESBL-producing Enterobacteriaceae were found) and S. aureus was 23% and 3%, respectively. The beta-lactamase CTX-M 1 was predominant in ESBL E. coli from meat samples but was not found in isolates from cases of human colonization or infection. S. aureus belonging to spa type t002 (multilocus sequence type ST5) were found both in chicken meat and humans. There is a risk to import ESBL E. coli to Gabon but molecular differences between isolates from humans and chicken meat argue against a further dissemination. No MRSA isolate was detected in imported chicken mea

Lability of the pAA Virulence Plasmid in <i>Escherichia coli</i> O104:H4: Implications for Virulence in Humans

<div><p>Background</p><p><i>Escherichia coli</i> O104:H4 that caused the large German outbreak in 2011 is a highly virulent hybrid of enterohemorrhagic (EHEC) and enteroaggregative (EAEC) <i>E. coli</i>. The strain displays “stacked-brick” aggregative adherence to human intestinal epithelial cells mediated by aggregative adherence fimbriae I (AAF/I) encoded on the pAA plasmid. The AAF/I-mediated augmented intestinal adherence might facilitate systemic absorption of Shiga toxin, the major virulence factor of EHEC, presumably enhancing virulence of the outbreak strain. However, the stability of pAA in the outbreak strain is unknown. We therefore tested outbreak isolates for pAA, monitored pAA loss during infection, and determined the impact of pAA loss on adherence and clinical outcome of infection.</p><p>Methodology/Principal Findings</p><p><i>E. coli</i> O104:H4 outbreak isolates from 170 patients (128 with hemolytic uremic syndrome [HUS] and 42 with diarrhea without HUS) were tested for pAA using polymerase chain reaction and plasmid profiling. pAA-harboring bacteria in stool samples were quantified using colony blot hybridization, and adherence to HCT-8 cells was determined. Isolates from 12 (7.1%) patients lacked pAA. Analyses of sequential stool samples demonstrated that the percentages of pAA-positive populations in the initial stools were significantly higher than those in the follow-up stools collected two to eight days later in disease (<i>P</i>≤0.01). This indicates a rapid loss of pAA during infections of humans. The pAA loss was associated with loss of the aggregative adherence phenotype and significantly reduced correlation with HUS (<i>P</i>  = 0.001).</p><p>Conclusions/Significance</p><p>The pAA plasmid can be lost by <i>E. coli</i> O104:H4 outbreak strain in the human gut in the course of disease. pAA loss might attenuate virulence and diminish the ability to cause HUS. The pAA instability has clinical, diagnostic, epidemiologic, and evolutionary implications.</p></div

The risk to import ESBL-producing Enterobacteriaceae and Staphylococcus aureus through chicken meat trade in Gabon

Background: A main export market for chicken meat from industrialized countries is sub-Saharan Africa. We hypothesized that antibiotic resistant bacteria could be exported to developing countries through chicken meat trade. The objective was to investigate the occurrence and molecular types of ESBL-producing Enterobacteriaceae and Staphylococcus aureus in chicken meat in Gabon and to assess their dissemination among humans. Results: Frozen chicken meat samples imported from industrialized countries to Gabon (n = 151) were screened for ESBL-producing Enterobacteriaceae and S. aureus. Genotypes and resistance genes (SHV, TEM, CTX-M, CMY-2) of isolates from meat were compared with isolates derived from humans. The contamination rate per chicken part (i. e. leg, wing) with ESBL-producing Escherichia coli (ESBL E. coli, no other ESBL-producing Enterobacteriaceae were found) and S. aureus was 23% and 3%, respectively. The beta-lactamase CTX-M 1 was predominant in ESBL E. coli from meat samples but was not found in isolates from cases of human colonization or infection. S. aureus belonging to spa type t002 (multilocus sequence type ST5) were found both in chicken meat and humans. Conclusion: There is a risk to import ESBL E. coli to Gabon but molecular differences between isolates from humans and chicken meat argue against a further dissemination. No MRSA isolate was detected in imported chicken meat.<br

Virulence from vesicles : Novel mechanisms of host cell injury by Escherichia coli O104:H4 outbreak strain

The highly virulent Escherichia coli O104:H4 that caused the large 2011 outbreak of diarrhoea and haemolytic uraemic syndrome secretes blended virulence factors of enterohaemorrhagic and enteroaggregative E. coli, but their secretion pathways are unknown. We demonstrate that the outbreak strain releases a cocktail of virulence factors via outer membrane vesicles (OMVs) shed during growth. The OMVs contain Shiga toxin (Stx) 2a, the major virulence factor of the strain, Shigella enterotoxin 1, H4 flagellin, and O104 lipopolysaccharide. The OMVs bind to and are internalised by human intestinal epithelial cells via dynamin-dependent and Stx2a-independent endocytosis, deliver the OMV-associated virulence factors intracellularly and induce caspase-9-mediated apoptosis and interleukin-8 secretion. Stx2a is the key OMV component responsible for the cytotoxicity, whereas flagellin and lipopolysaccharide are the major interleukin-8 inducers. The OMVs represent novel ways for the E. coli O104: H4 outbreak strain to deliver pathogenic cargoes and injure host cells

pAA is lost by the outbreak strain in the course of infection.

<p>Enrichment cultures of the initial and follow-up stool samples from patients A to G diluted to give rise to 150–200 well-separated colonies were plated on ESBL agar and pAA-positive colonies were identified using colony hybridization with the pCVD432 probe. The percentages of pCVD432-positive colonies among all colonies grown on the plates were calculated. The numbers above the columns indicate the time interval (days) between collection of the initial (black bars) and follow-up (white bars) stool samples. In all cases the percentage of pAA-positive colonies in the follow-up stool is significantly lower than that in the initial stool (<i>P</i>≤0.01; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066717#pone-0066717-t002" target="_blank">Table 2</a>).</p

Characteristics of pAA-positive and pAA-negative EHEC O104:H4 isolates^a and clinical outcomes of infection in the respective patients.

a<p>The identity of all isolates as EHEC O104:H4 outbreak strain was confirmed using the multiplex real-time PCR targeting <i>rfb</i>_O104, <i>fliC</i>_H4, and <i>stx</i>_2a<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066717#pone.0066717-Zhang1" target="_blank">[24]</a> and multilocus sequence typing, which demonstrated that all belong to ST678 typical for the outbreak strain <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066717#pone.0066717-Bielaszewska1" target="_blank">[8]</a>.</p>b<p>HUS, hemolytic uremic syndrome; BD, bloody diarrhea; D, diarrhea without visible blood.</p>c<p>1, initial isolate; 2, follow-up isolate; the number in parenthesis indicates the time interval between recovery of the initial and the follow-up isolate.</p>d<p>+/+, PCR amplicon of corresponding size and hybridization signal on the 75-kb plasmid present; −/−, no PCR amplicon, no hybridization signal present.</p>e<p>Sizes of plasmids in kilobase pairs (kb).</p>f<p>Stx2a titers were defined as the highest dilutions of sterile culture filtrates that caused cytotoxicity in 50% Vero cells after 72 h.</p>g<p>AA, aggregative adherence pattern (HCT-8 cells).</p

Qualitative and quantitative analyses of pAA-positive bacteria in the paired initial and follow-up stool samples.

a<p>HUS, hemolytic uremic syndrome; BD, bloody diarrhea; D, diarrhea without visible blood.</p>b<p>1, initial stool sample; 2, follow-up stool sample; the number in parenthesis indicates the time interval between collection of the initial and the follow-up stool sample.</p>c<p>+, the isolate contained all pAA-encoded virulence genes (<i>aatA</i>, <i>aggR</i>, <i>aggC</i>, <i>aap</i>, <i>sepA</i>) in PCR and harbored a 75-kb plasmid hybridizing with pCVD432, <i>aggR</i>, <i>aggC</i>, <i>aap</i>, and <i>sepA</i> probes. -, the isolate lacked all pAA-encoded virulence genes in PCR and lacked the 75-kb plasmid in plasmid profiling.</p>d<p>+, an amplicon of corresponding size was obtained from the whole stool culture harvested from ESBL agar in PCR with primers pCVD432/start and pCVD432/stop <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066717#pone.0066717-Schmidt1" target="_blank">[30]</a>;</p><p>-, no PCR amplicon from the whole stool culture was obtained with these primers.</p>e<p>Determined by colony blot hybridization of stool cultures plated on ESBL agar with the pCVD432 probe and calculated from the total numbers of colonies grown on the plates.</p>f<p>Paired Student's <i>t</i> test (<i>P</i><0.05 considered significant); n.a., not applicable.</p

Loss of pAA by EHEC O104:H4 outbreak strain as demonstrated by plasmid profiling.

<p>Isolated plasmids were separated using 0.6% agarose gel, visualized by staining with Midori green and photographed. Lane M, molecular size marker (plasmids from <i>E. coli</i> 39r861). In lanes 1 to 10, plasmid profiles of EHEC O104:H4 outbreak isolates from the following patients are shown (patients’ designations refer to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066717#pone-0066717-t001" target="_blank">Table 1</a>): Lane 1, initial isolate from patient A (pAA-positive); lane 2, follow-up isolate from patient A (pAA-negative); lane 3, initial isolate from patient C (pAA-positive); lane 4, follow-up isolate from patient C (pAA-negative); lane 5, initial isolate from patient G (pAA-positive); lane 6, follow-up isolate from patient G (pAA-negative); lane 7, reference EHEC O104:H4 outbreak isolate LB226692 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066717#pone.0066717-Bielaszewska1" target="_blank">[8]</a>; lane 8, initial isolate from patient H (pAA-negative); lane 9, follow-up isolate from patient H (pAA-negative). Sizes of the pAA (75 kb) and pESBL (88 kb) plasmids are indicated on the right side.</p

Model of intracellular trafficking and action of OMV-associated EHEC-Hly.

<p>1. After its secretion by EHEC bacteria and association with OMVs, the OMV-associated EHEC-Hly is endocytosed by dynamin-dependent endocytosis and enters the endosomal compartments of target cells. 2. During endosome acidification via the H⁺-ATPase the neutral pH 7.4 of endosomes drops to pH 5.0, which induces separation of EHEC-Hly from OMVs. 3. The separated toxin plausibly interacts with the endosomal/lysosomal membrane and, as a pore-forming toxin, it damages lysosomal membrane by its pore-forming activity in order to release from lysosomes. As a consequence of the membrane damage, the proton gradient of lysosomes is disrupted leading to lysosomal pH increase. 4. EHEC-Hly released from lysosomes translocates by an unknown mechanism to mitochondria. 5. This results in cytochrome c release to the cytosol, which leads to activation of caspase-9 and caspase-3 and apoptotic cell death. 6. Presence of the proton ATPase inhibitor BafA1 inhibits endosomal acidification and thus prevents the toxin to be separated from OMVs. As a consequence, EHEC-Hly is trapped in endosomes/lysosomes and cannot translocate into mitochondria. The figure was produced using Servier Medical Art.</p