Food-producing animals are indicated as a reservoir of antibiotic resistance genes and a potential vector for transmission of plasmid-encoded antibiotic resistance genes by conjugation to the human ...intestinal microbiota. In this study, transfer of an antibiotic resistance plasmid from a commensal E. coli originating from a broiler chicken towards the human intestinal microbiota was assessed by using a Mucosal Simulator of the Human Intestinal Microbial Ecosystem (M-SHIME). This in vitro model mimics the human intestinal ecosystem and received a single dose of 109E. coli MB6212, which harbors a plasmid known to confer resistance towards several antibiotics including tetracycline, sulfamethoxazole and cefotaxime. Since the degree of stress imposed by stomach pH and bile acids vary with the consumed meal size, the effect of meal size on E. coli donor survival and on plasmid transfer towards lumen and mucosal coliforms and anaerobes was determined.
The administered commensal E. coli strain survived stomach acid and bile salt stress and was able to grow in the colon environment during the timeframe of the experiment (72 h). Transfer of antibiotic resistance was observed rapidly since cultivable transconjugant coliforms and anaerobes were already detected in the lumen and mucosa after 2 h in the simulated proximal colon. The presence of the resistance plasmid in the transconjugants was confirmed by PCR. Differences in meal size and adapted digestion had neither a detectable impact on antibiotic resistance transfer, nor on the survival of the E. coli donor strain, nor on short chain fatty acid profiles. The median number of resistant indigenous coliforms in the lumen of the inoculated colon vessels was 5.00 × 105 cfu/ml min - max: 3.47 × 104–3.70 × 108 cfu/ml, and on the mucosa 1.44 × 107 cfu/g min-max: 4.00 × 103–4.00 × 108 cfu/g. Exact quantification of the anaerobic transconjugants was difficult, as (intrinsic) resistant anaerobic background microbiota were present. QPCR data supported the observation of plasmid transfer in the simulated colon. Moreover, inoculation of E. coli MB6212 had no significant impact on the microbial diversity in the lumen as determined by 16 S ribosomal gene based next generation sequencing on lumen samples.
This study demonstrates that a commensal, antibiotic resistant E. coli strain present in food can transfer its antibiotic resistance plasmid relatively quickly to intestinal microbiota in the M-SHIME. The spread and persistence of antibiotic resistance genes and resistant bacteria in our intestinal system is an alarming scenario which might present clinical challenges, since it implies a potential reservoir for dissemination to pathogenic bacteria.
•Resistance transfer observed after 2h in simulated colon•Transfer of resistance genes to luminal and mucosal coliforms and anaerobes•Meal size and subsequent digestion had no impact on antibiotic resistance transfer•Inoculation of resistant E.coli donor had no effect on Short Chain Fatty Acid profiles and community diversity
Hepatitis E virus is a prominent cause of viral hepatitis worldwide. In Western countries, most infections are asymptomatic. However, acute self-limiting hepatitis and chronic cases in ...immunocompromised individuals can occur. Studying HEV is challenging due to its difficulty to grow in cell culture. Consequently, the detection of the virus mainly relies on RT-qPCR, which cannot differentiate between infectious and non-infectious particles. To overcome this problem, methods assessing viral integrity offer a possible solution to differentiate between intact and damaged viruses. This study aims at optimizing existing HEV cell culture models and RT-qPCR-based assays for selectively detecting intact virions to establish a reliable model for assessing HEV infectivity. In conclusion, these newly developed methods hold promise for enhancing food safety by identifying approaches for inactivating HEV in food processing, thereby increasing food safety measures.
Along with (in) direct contact with animals and a contaminated environment, humans are exposed to antibiotic-resistant bacteria by consumption of food. The implications of ingesting ...antibiotic-resistant commensal bacteria are unknown, as dose-response data on resistance transfer and spreading in our gut is lacking. In this study, transfer of a resistance plasmid (IncF), harbouring several antibiotic resistance genes, from a commensal
strain towards human intestinal microbiota was assessed using a Mucosal Simulator of the Human Intestinal Ecosystem (M-SHIME). More specifically, the effect of the initial
plasmid donor concentration (10
and 10
CFU/meal), antibiotic treatment (cefotaxime) and human individual (n = 6) on plasmid transfer towards lumen coliforms and anaerobes was determined. Transfer of the resistance plasmid to luminal coliforms and anaerobes was observed shortly after the donor strain arrived in the colon and was independent of the ingested dose. Transfer occurred in all six simulated colons and despite their unique microbial community composition, no differences could be detected in antibiotic resistance transfer rates between the simulated human colons. After 72 h, resistant coliform transconjugants levels ranged from 7.6 × 10
to 7.9 × 10
CFU
/Ml colon lumen. Presence of the resistance plasmid was confirmed and quantified by PCR and qPCR. Cefotaxime treatment led to a significant reduction (85%) in resistant coliforms, however no significant effect on the total number of cultivable coliforms and anaerobes was observed.
The aim of this study was metagenomics analyses of acquired antibiotic-resistance genes (ARGs) in the intestinal microbiome of two important food-animal species in Hungary from a One Health ...perspective. Intestinal content samples were collected from 12 domestic pigs (Sus scrofa) and from a common carp (Cyprinus carpio). Shotgun metagenomic sequencing of DNA purified from the intestinal samples was performed on the Illumina platform. The ResFinder database was applied for detecting acquired ARGs in the assembled metagenomic contigs. Altogether, 59 acquired ARG types were identified, 51 genes from domestic pig and 12 genes from the carp intestinal microbiome. ARG types belonged to the antibiotic classes aminoglycosides (27.1%), tetracyclines (25.4%), β-lactams (16.9%), and others. Of the identified ARGs, tet(E), a blaOXA-48-like β-lactamase gene, as well as cphA4, ampS, aadA2, qnrS2, and sul1, were identified only in carp but not in swine samples. Several of the detected acquired ARGs have not yet been described from food animals in Hungary. The tet(Q), tet(W), tet(O), and mef(A) genes detected in the intestinal microbiome of domestic pigs had also been identified from free-living wild boars in Hungary, suggesting a possible relationship between the occurrence of acquired ARGs in domestic and wild animal populations.
Zoonotic hepatitis E virus (HEV) genotype 3 infections are the predominant cause of acute viral hepatitis in Europe, mostly associated with the consumption of HEV contaminated pork meat. In this ...study we looked at the HEV RNA positivity rate of pork meat products readily available from Belgian supermarkets and evaluated the overall HEV consumer exposure in a Belgian context.
Two basic assessments were performed in a ‘worst-case’ scenario setting: one solely focusing on the contamination level of the product itself (ingredients and processing parameters) and another estimating the overall consumer exposure, taking into account consumption habits in Belgium. Non-thermal-processed ready-to-eat (i.e. ready for consumption without additional cooking step by consumer) pork meat products (e.g. raw dried sausages), had a high estimated HEV contamination level, while thermal-processed ready-to-eat pork meat products (e.g. pork liver pâté) had the highest overall consumer exposure estimates.
Following these assessments, pork liver pâtés, raw dried hams and raw dried sausages (n = 54) were purchased from Belgian supermarkets (n = 3) and analyzed for HEV RNA by RT-PCR. In total, 31 % (n = 17) products tested positive. HEV RNA was found in 65 % of the pork liver pâtés, 15 % of raw dried hams and 0 % of raw dried sausages. Phylogenetic analysis of four isolates (all gt3c) from pork liver pâté samples showed similarities with human clinical cases from Germany and Belgium.
•HEV product contamination and overall exposure was estimated.•Ready-to-eat pork products have the highest HEV contamination estimate.•Ready-to-eat pork meat products were tested for HEV RNA.•HEV RNA (ORF2) was detected in 17/54 (31 %) of samples.•All positive samples belonged to genotype 3, subtype 3c.
The production of cysts, an integral part of the life cycle of many free-living protozoa, allows these organisms to survive adverse environmental conditions. Given the prevalence of free-living ...protozoa in food-related environments, it is hypothesized that these organisms play an important yet currently underinvestigated role in the epidemiology of foodborne pathogenic bacteria. Intracystic bacterial survival is highly relevant, as this would allow bacteria to survive the stringent cleaning and disinfection measures applied in food-related environments. The present study shows that strains of widespread and important foodborne bacteria (Salmonella enterica, Escherichia coli, Yersinia enterocolitica, and Listeria monocytogenes) survive inside cysts of the ubiquitous amoeba Acanthamoeba castellanii, even when exposed to either antibiotic treatment (100 μg/ml gentamicin) or highly acidic conditions (pH 0.2) and resume active growth in broth media following excystment. Strain- and species-specific differences in survival periods were observed, with Salmonella enterica surviving up to 3 weeks inside amoebal cysts. Up to 53% of the cysts were infected with pathogenic bacteria, which were located in the cyst cytosol. Our study suggests that the role of free-living protozoa and especially their cysts in the persistence and epidemiology of foodborne bacterial pathogens in food-related environments may be much more important than hitherto assumed.
The formation of robust resting cysts enables
to resist harsh environmental conditions. This study investigated to what extent these cysts are resistant to physical and chemical stresses as applied ...in food industry cleaning and disinfection procedures. Moreover, it was assessed whether certain intracystic meat-borne bacterial pathogens are more stress resistant than free-living bacterial monocultures and if intracystic passage and subsequent association with trophozoites induces cross-tolerance toward other stressors. Several physical and chemical stressors (NaCl, H
O
, benzalkonium chloride, 55°C, heating until boiling, ethanol, dishwashing detergent, and sodium hypochlorite) frequently used in domestic and industrial food-related environments were tested against (i)
cysts, (ii) single strains of bacterial monocultures, (iii) intracystic bacteria, and (iv) bacteria after intracystic passage (cyst-primed bacteria). Only heating until boiling and hypochlorite treatment were cysticidal. After boiling, no viable trophozoites could be recovered from the cysts, and hypochlorite treatment caused a 1.34- to 4.72-log
cells/ml reduction in cyst viability. All treatments were effective in reducing or even eliminating the tested bacterial monocultures, whereas bacteria residing inside cysts were more tolerant toward these stressors. All cyst-primed bacteria exhibited an increased tolerance toward subsequent H
O
(>92% decrease in median log
CFU/ml reduction) and 70% ethanol (>99% decrease) treatments. Moreover, intracystic passage significantly increased the survival of
(74% decrease in median log
reduction),
(58%), and
(48%) after NaCl treatment and of
(96%),
(99%), and
(99%) after sodium hypochlorite treatment compared with that of nonprimed bacteria.
The results from this study demonstrated that both viable and nonviable amoebal cysts can protect internalized bacteria against stressful conditions. Moreover, cyst passage can induce cross-tolerance in bacteria, increasing their survival when exposed to selected stressors. These findings underscore the potential importance of free-living amoebae in food-related environments and their impact on the persistence of meat-borne bacterial pathogens.
Food-producing animals represent one of the sources of antibiotic resistant commensal bacteria. There is an increasing awareness that these bacteria might have the potential to transfer their ...resistance genes to other (pathogenic) bacteria. In this study, 50 commensal Escherichia coli strains originating from food-producing animals and resistant to the "highest priority, critically important antibiotics" cefotaxime and/or ciprofloxacin, were selected for further characterization. For each strain (i) an antibiogram, (ii) the phylogenetic group, (iii) plasmid replicon type, (iv) presence and identification of integrons, and (v) antibiotic resistance transfer ratios were determined. Forty-five of these strains were resistant to 5 or more antibiotics, and 6 strains were resistant to 10 or more antibiotics. Resistance was most common to ampicillin (100%), sulfamethoxazole, ciprofloxacin (82%), trimethoprim, tetracycline (74%), cefotaxime, (70%) and ceftazidime (62%). Phylogenetic groups A (62%) and B1 (26%) were most common, followed by C (8%) and E (4%). In 43 strains, more than 1 replicon type was detected, with FII (88%), FIB (70%), and I1 (48%) being the most encountered types. Forty strains, positive for integrons, all harbored a class I integron and seven of them contained an additional class II integron. No class III integrons were detected. The antibiotic resistance transfer was assessed by liquid mating experiments. The transfer ratio, expressed as the number of transconjugants per recipient, was between 10
and 10
for cefotaxime resistance and between 10
and 10
for ciprofloxacin resistance. The results of the current study prove that commensal E. coli in food-production animals can be a source of multiple resistance genes and that these bacteria can easily spread their ciprofloxacin and cefotaxime resistance.
Cysts of free-living protozoa have an impact on the ecology and epidemiology of bacteria because they may act as a transmission vector or shelter the bacteria against hash environmental conditions. ...Detection and localization of intracystic bacteria and examination of the en- and excystment dynamics is a major challenge because no detailed protocols for ultrastructural analysis of cysts are currently available. Transmission electron microscopy (TEM) is ideally suited for those analyses; however, conventional TEM protocols are not satisfactory for cysts of free-living protozoa. Here we report on the design and testing of four protocols for TEM sample preparation of cysts. Two protocols, one based on chemical fixation in coated well plates and one on high-pressure freezing, were selected as the most effective for TEM-based ultrastructural studies of cysts. Our protocols will enable improved analysis of cyst structure and a better understanding of bacterial survival mechanisms in cysts.
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