A novel metagenomic approach uncovers phage genes as markers for increased disinfectant tolerance in mixed Listeria monocytogenes communities.

Listeria monocytogenes is an important human pathogen with a high mortality rate. Consumption of contaminated ready-to-eat food is the main mode of transmission to humans. Disinfectant-tolerant L. monocytogenes have emerged, which are believed to have increased persistence potential. Elucidating the mechanisms of L. monocytogenes disinfectant tolerance has been the focus of previous studies using pure cultures. A limitation of such approach is the difficulty to identify strains with reduced susceptibility due to inter-strain variation and the need to screen large numbers of strains and genes. In this study, we applied a novel metagenomic approach to detect genes associated with disinfectant tolerance in mixed L. monocytogenes planktonic communities. Two communities, consisting of 71 and 80 isolates each, were treated with the food industry disinfectants benzalkonium chloride (BC, 1.75 mg/L) or peracetic acid (PAA, 38 mg/L). The communities were subjected to metagenomic sequencing and differences in individual gene abundances between biocide-free control communities and biocide-treated communities were determined. A significant increase in the abundance of Listeria phage-associated genes was observed in both communities after treatment, suggesting that prophage carriage could lead to an increased disinfectant tolerance in mixed L. monocytogenes planktonic communities. In contrast, a significant decrease in the abundance of a high-copy emrC-harbouring plasmid pLmN12-0935 was observed in both communities after treatment. In PAA-treated community, a putative ABC transporter previously found to be necessary for L. monocytogenes resistance to antimicrobial agents and virulence, was among the genes with the highest weight for differentiating treated from control samples. The undertaken metagenomic approach in this study can be applied to identify genes associated with increased tolerance to other antimicrobials in mixed bacterial communities.

Evaluation and validation of laboratory procedures for the surveillance of ESBL-, AmpC-, and carbapenemase-producing Escherichia coli from fresh meat and caecal samples.

Introduction: Extended-spectrum ß-lactamase- (ESBL) and AmpC- ß-lactamase-producing Enterobacterales are widely distributed and emerging in both human and animal reservoirs worldwide. A growing concern has emerged in Europe following the appearance of carbapenemase-producing Escherichia coli (E. coli) in the primary production of food animals. In 2013, the European Commission (EC) issued the Implementing Decision on the monitoring and reporting of antimicrobial resistance in zoonotic and commensal bacteria. The European Union Reference Laboratory for Antimicrobial Resistance (EURL-AR) was tasked with providing two laboratory protocols for samples derived from meat and caecal content, respectively, for the isolation of ESBL- and AmpC-producing E. coli (part 1) and carbapenemase-producing (CP) E. coli (part 2). In this study, we describe the current protocols, including the preparatory work for the development. Methods: Up to nine laboratory procedures were tested using minced meat as the matrix from beef, pork, and chicken as well as six procedures for the caecal content of cattle, pigs, and chicken. Variables included sample volume, pre-enrichment volume, pre-enrichment broth with and without antimicrobial supplementation, and incubation time/temperature. The procedures were evaluated against up to nine E. coli strains harboring different AMR genes and belonging to the three ß-lactamase groups. Results and discussion: The laboratory procedures tested revealed that the most sensitive and specific methodologies were based on a Buffered Peptone Water pre-enrichment of 225 ml to 25 g or 9 ml to 1 g for minced meat and caecal content, respectively, incubated at 37°C overnight, followed by inoculation onto MacConkey agar supplemented with 1 mg/L cefotaxime for detecting ESBL- and AmpC-producing E. coli and Chrom ID SMART (Chrom ID CARBA and OXA) for CP E. coli, incubated overnight at 37 and 44°C, respectively. We provided two isolation protocols for the EU-specific monitoring of ESBL- and AmpC- producing E. coli (part 1) and CP E. coli (part 2) from fresh meat (protocol 1) and caecal (protocol 2) samples, which have been successfully implemented by all EU Member States for the monitoring period 2014-2027 (EU 2020/1729).

One Health surveillance-A cross-sectoral detection, characterization, and notification of foodborne pathogens.

Introduction: Several Proficiency Test (PT) or External Quality Assessment (EQA) schemes are currently available for assessing the ability of laboratories to detect and characterize enteropathogenic bacteria, but they are usually targeting one sector, covering either public health, food safety or animal health. In addition to sector-specific PTs/EQAs for detection, cross-sectoral panels would be useful for assessment of the capacity to detect and characterize foodborne pathogens in a One Health (OH) perspective and further improving food safety and interpretation of cross-sectoral surveillance data. The aims of the study were to assess the cross-sectoral capability of European public health, animal health and food safety laboratories to detect, characterize and notify findings of the foodborne pathogens Campylobacter spp., Salmonella spp. and Yersinia enterocolitica, and to develop recommendations for future cross-sectoral PTs and EQAs within OH. The PT/EQA scheme developed within this study consisted of a test panel of five samples, designed to represent a theoretical outbreak scenario. Methods: A total of 15 laboratories from animal health, public health and food safety sectors were enrolled in eight countries: Denmark, France, Italy, the Netherlands, Poland, Spain, Sweden, and the United Kingdom. The laboratories analyzed the samples according to the methods used in the laboratory and reported the target organisms at species level, and if applicable, serovar for Salmonella and bioserotype for Yersinia. Results: All 15 laboratories analyzed the samples for Salmonella, 13 for Campylobacter and 11 for Yersinia. Analytical errors were predominately false negative results. One sample (S. Stockholm and Y. enterocolitica O:3/BT4) with lower concentrations of target organisms was especially challenging, resulting in six out of seven false negative results. These findings were associated with laboratories using smaller sample sizes and not using enrichment methods. Detection of Salmonella was most commonly mandatory to notify within the three sectors in the eight countries participating in the pilot whereas findings of Campylobacter and Y. enterocolitica were notifiable from human samples, but less commonly from animal and food samples. Discussion: The results of the pilot PT/EQA conducted in this study confirmed the possibility to apply a cross-sectoral approach for assessment of the joint OH capacity to detect and characterize foodborne pathogens.

Genomic analysis of sewage from 101 countries reveals global landscape of antimicrobial resistance.

Antimicrobial resistance (AMR) is a major threat to global health. Understanding the emergence, evolution, and transmission of individual antibiotic resistance genes (ARGs) is essential to develop sustainable strategies combatting this threat. Here, we use metagenomic sequencing to analyse ARGs in 757 sewage samples from 243 cities in 101 countries, collected from 2016 to 2019. We find regional patterns in resistomes, and these differ between subsets corresponding to drug classes and are partly driven by taxonomic variation. The genetic environments of 49 common ARGs are highly diverse, with most common ARGs carried by multiple distinct genomic contexts globally and sometimes on plasmids. Analysis of flanking sequence revealed ARG-specific patterns of dispersal limitation and global transmission. Our data furthermore suggest certain geographies are more prone to transmission events and should receive additional attention.

Multicentre evaluation of a selective isolation protocol for detection of mcr-positive E. coli and Salmonella spp. in food-producing animals and meat.

This study was conducted to evaluate the performance of a screening protocol to detect and isolate mcr-positive Escherichia coli and Salmonella spp. from animal caecal content and meat samples. We used a multicentre approach involving 12 laboratories from nine European countries. All participants applied the same methodology combining a multiplex PCR performed on DNA extracted from a pre-enrichment step, followed by a selective culture step on three commercially available chromogenic agar plates. The test panel was composed of two negative samples and four samples artificially contaminated with E. coli and Salmonella spp. respectively harbouring mcr-1 or mcr-3 and mcr-4 or mcr-5 genes. PCR screening resulted in a specificity of 100% and a sensitivity of 83%. Sensitivity of each agar medium to detect mcr-positive colistin-resistant E. coli or Salmonella spp. strains was 86% for CHROMID® Colistin R, 75% for CHROMagarTM COL-APSE and 70% for COLISTIGRAM. This combined method was effective to detect and isolate most of the E. coli or Salmonella spp. strains harbouring different mcr genes from food-producing animals and food products and might thus be used as a harmonized protocol for the screening of mcr genes in food-producing animals and food products in Europe.

A European multicenter evaluation study to investigate the performance on commercially available selective agar plates for the detection of carbapenemase producing Enterobacteriaceae.

The European Food Safety Authority (EFSA) advised to prioritize monitoring carbapenemase producing Enterobacteriaceae (CPE) in food producing animals. Therefore, this study evaluated the performance of different commercially available selective agars for the detection of CPE using spiked pig caecal and turkey meat samples and the proposed EFSA cultivation protocol. Eleven laboratories from nine countries received eight samples (four caecal and four meat samples). For each matrix, three samples contained approximately 100 CFU/g CPE, and one sample lacked CPE. After overnight enrichment in buffered peptone water, broths were spread upon Brilliance™ CRE Agar (1), CHROMID® CARBA (2), CHROMagar™ mSuperCARBA™ (3), Chromatic™ CRE (4), CHROMID® OXA-48 (5) and Chromatic™ OXA-48 (6). From plates with suspected growth, one to three colonies were selected for species identification, confirmation of carbapenem resistance and detection of carbapenemase encoding genes, by methods available at participating laboratories. Of the eleven participating laboratories, seven reported species identification, susceptibility tests and genotyping on isolates from all selective agar plates. Agars 2, 4 and 5 performed best, with 100% sensitivity. For agar 3, a sensitivity of 96% was recorded, while agar 1 and 6 performed with 75% and 43% sensitivity, respectively. More background flora was noticed for turkey meat samples than pig caecal samples. Based on this limited set of samples, most commercially available agars performed adequately. The results indicate, however, that OXA-48-like and non-OXA-48-like producers perform very differently, and one should consider which CPE strains are of interest to culture when choosing agar type.

Exit tunnel modulation as resistance mechanism of S. aureus erythromycin resistant mutant.

The clinical use of the antibiotic erythromycin (ery) is hampered owing to the spread of resistance genes that are mostly mutating rRNA around the ery binding site at the entrance to the protein exit tunnel. Additional effective resistance mechanisms include deletion or insertion mutations in ribosomal protein uL22, which lead to alterations of the exit tunnel shape, located 16 Å away from the drug's binding site. We determined the cryo-EM structures of the Staphylococcus aureus 70S ribosome, and its ery bound complex with a two amino acid deletion mutation in its ß hairpin loop, which grants the bacteria resistance to ery. The structures reveal that, although the binding of ery is stable, the movement of the flexible shorter uL22 loop towards the tunnel wall creates a wider path for nascent proteins, thus enabling bypass of the barrier formed by the drug. Moreover, upon drug binding, the tunnel widens further.

Microbiota encompassing putative spoilage bacteria in retail packaged broiler meat and commercial broiler abattoir.

It is well established, that certain bacteria within the Brochothrix, Carnobacterium, Lactobacillus, Lactococcus, and Leuconostoc genera have an important role in the spoilage of chill stored poultry meat packaged in modified atmosphere. However, little is known about the role of microorganisms that are difficult to culture and the microbiota during poultry spoilage. We combined traditional cultivation and culture-independent 16S rRNA amplicon sequencing to investigate the microbiota encompassing putative bacteria of whole broiler meat, packaged in modified atmosphere, during and exceeding shelf-life. Samples were taken from 6 flocks during independent slaughter days. Additional samples were analysed from the production line. There was a significant difference in the microbial community structure of 80%O2/20%CO2 retail packaged broiler meat during different times of shelf-life, mainly due to an increase of species within the Brochothrix, Carnobacterium, Vagococcus, and Janthinobacterium genera. These genera were already detected four to eight days after slaughter. However, no significant difference between flocks with respect to the microbiota encompassing putative spoilage bacteria was observed when examined in retail packaged broilers, slaughtered at the same abattoir on different days. Our study also showed that lactic acid bacteria within the Vagococcus genus can constitute a dominating part of the later shelf-life microbiota in fresh whole broiler meat packaged in 80%O2/20%CO2 modified atmosphere. A single operational taxonomic unit (OTU) assigned as Janthinobacterium lividum, an occasional spoiler of meat products, was identified as a major part of the microbiota in late shelf life broiler meat and swab samples in the cooling facility at the slaughter house production line. The combination of traditional cultivation and culture-independent methods provided a great insight into the microbiota of broiler meat during shelf-life and identified a potential point of contamination in the production line for cold tolerant Janthinobacterium.

Global monitoring of antimicrobial resistance based on metagenomics analyses of urban sewage.

Antimicrobial resistance (AMR) is a serious threat to global public health, but obtaining representative data on AMR for healthy human populations is difficult. Here, we use metagenomic analysis of untreated sewage to characterize the bacterial resistome from 79 sites in 60 countries. We find systematic differences in abundance and diversity of AMR genes between Europe/North-America/Oceania and Africa/Asia/South-America. Antimicrobial use data and bacterial taxonomy only explains a minor part of the AMR variation that we observe. We find no evidence for cross-selection between antimicrobial classes, or for effect of air travel between sites. However, AMR gene abundance strongly correlates with socio-economic, health and environmental factors, which we use to predict AMR gene abundances in all countries in the world. Our findings suggest that global AMR gene diversity and abundance vary by region, and that improving sanitation and health could potentially limit the global burden of AMR. We propose metagenomic analysis of sewage as an ethically acceptable and economically feasible approach for continuous global surveillance and prediction of AMR.

Multiplex PCR for detection of plasmid-mediated colistin resistance determinants, mcr-1, mcr-2, mcr-3, mcr-4 and mcr-5 for surveillance purposes.

Background and aimPlasmid-mediated colistin resistance mechanisms have been identified worldwide in the past years. A multiplex polymerase chain reaction (PCR) protocol for detection of all currently known transferable colistin resistance genes (mcr-1 to mcr-5, and variants) in Enterobacteriaceae was developed for surveillance or research purposes. Methods: We designed four new primer pairs to amplify mcr-1, mcr-2, mcr-3 and mcr-4 gene products and used the originally described primers for mcr-5 to obtain a stepwise separation of ca 200 bp between amplicons. The primer pairs and amplification conditions allow for single or multiple detection of all currently described mcr genes and their variants present in Enterobacteriaceae. The protocol was validated testing 49 European Escherichia coli and Salmonella isolates of animal origin. Results: Multiplex PCR results in bovine and porcine isolates from Spain, Germany, France and Italy showed full concordance with whole genome sequence data. The method was able to detect mcr-1, mcr-3 and mcr-4 as singletons or in different combinations as they were present in the test isolates. One new mcr-4 variant, mcr-4.3, was also identified. Conclusions: This method allows rapid identification of mcr-positive bacteria and overcomes the challenges of phenotypic detection of colistin resistance. The multiplex PCR should be particularly interesting in settings or laboratories with limited resources for performing genetic analysis as it provides information on the mechanism of colistin resistance without requiring genome sequencing.

Cross-border outbreak of listeriosis caused by cold-smoked salmon, revealed by integrated surveillance and whole genome sequencing (WGS), Denmark and France, 2015 to 2017.

In August 2017, an outbreak of six listeriosis cases in Denmark was traced to cold-smoked salmon, using epidemiological investigations and whole-genome sequencing (WGS) analyses. Exchange of genome sequences allowed identification in France of a food isolate from a salmon-derived product and a human isolate from 2016 within the same cgMLST cluster as the Danish isolates (L2-SL8-ST8-CT771). The salmon product came from a third European Union country. WGS can rapidly link human cases and food isolates across Europe.

The Ribosomal Protein uL22 Modulates the Shape of the Protein Exit Tunnel.

Erythromycin is a clinically useful antibiotic that binds to an rRNA pocket in the ribosomal exit tunnel. Commonly, resistance to erythromycin is acquired by alterations of rRNA nucleotides that interact with the drug. Mutations in the ß hairpin of ribosomal protein uL22, which is rather distal to the erythromycin binding site, also generate resistance to the antibiotic. We have determined the crystal structure of the large ribosomal subunit from Deinococcus radiodurans with a three amino acid insertion within the ß hairpin of uL22 that renders resistance to erythromycin. The structure reveals a shift of the ß hairpin of the mutated uL22 toward the interior of the exit tunnel, triggering a cascade of structural alterations of rRNA nucleotides that propagate to the erythromycin binding pocket. Our findings support recent studies showing that the interactions between uL22 and specific sequences within nascent chains trigger conformational rearrangements in the exit tunnel.

Methicillin-resistant and -susceptible Staphylococcus aureus from retail meat in Denmark.

Livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) is increasingly related to human infections. Farmers and veterinarians have the highest risk, but infections have also occurred in individuals without prior contact to livestock. Clonal complex (CC) 398 is the predominant LA-MRSA lineage causing human infections, and although pigs are the major source of CC398 worldwide, poultry and other animals are also reservoirs. This raises concern for transmission of MRSA via meat. In this study, the occurrence and characteristics of S. aureus isolated from Danish retail meat were examined with main focus on chicken meat. A total of 145 meat samples from Danish supermarkets were examined, including chicken (Danish, n=102), turkey (non-Danish origin; n=23), and pork (Danish, n=20). S. aureus was detected in 69% of the meat samples. MRSA was detected in 19 meat samples (13%), resulting in MRSA prevalence of 4% of chicken, 52% of turkey, and 15% of pork. Three MRSA positive samples were obtained by direct plating (Brilliance MRSA2), whereas 16 MRSA positive samples were detected only after enrichment (TSB+6.5% NaCl and Brilliance MRSA2). Based on spa typing, 68% of MRSA isolates belonged to CC398 (spa t034, t011, t2582, t108), and hereof one isolate derived from chicken (1%). Further findings were spa type t1430 (CC9) in turkey samples (16%) and the human-associated t008 (CC8) in chicken samples (16%). In conclusion, S. aureus was readily detected in Danish retail meat, but presence of MRSA in chicken meat is rare and it is unlikely to be an important transmission factor of MRSA to humans.

Multilocus sequence typing and biocide tolerance of Arcobacter butzleri from Danish broiler carcasses.

BACKGROUND: Arcobacter spp. have in recent years received increasing interest as potential emerging enteropathogens and zoonotic agents. They are associated with various animals including poultry and can be isolated from meat products. The possibilities of persistence and cross-contamination in slaughterhouses during meat processing are not well established. We have evaluated the occurrence and persistence of Arcobacter spp. in a Danish slaughterhouse and determined the sensitivity of isolates to sodium hypochlorite, a commonly used biocide. RESULTS: Arcobacter contamination was examined in a broiler slaughterhouse by selective enrichment of 235 swabs from the processing line during two production days and after sanitizing in between. In total 13.6% of samples were positive for A. butzleri with the majority (29 of 32 isolates) originating from the evisceration machine. No Arcobacter spp. was isolated after cleaning. A. butzleri isolates confirmed by PCR were typed by multilocus sequence typing (MLST) resulting in 10 new sequence types (STs). Two sequence types were isolated on both processing days. Minimum inhibitory concentration (MIC) to sodium hypochlorite was determined to 0.5% hypochlorite biocide (500 ppm chlorine) for most isolates, which allows growth of A. butzleri within the working concentration of the biocide (0.2 - 0.5%). CONCLUSIONS: A. butzleri was readily isolated from a Danish broiler slaughterhouse, primarily in the evisceration machine. Typing by MLST showed high strain variability but the recurrence of two STs indicate that some persistence or cross-contamination takes place. Importantly, the isolates tolerated sodium hypochlorite, a biocide commonly employed in slaughterhouse sanitizing, at levels close to the disinfection concentration, and thus, A. butzleri may survive the disinfection process although this was not observed in our study.

Residual antibiotics disrupt meat fermentation and increase risk of infection.

UNLABELLED: Fermented sausages, although presumed safe for consumption, sometimes cause serious bacterial infections in humans that may be deadly. Not much is known about why and when this is the case. We tested the hypothesis that residual veterinary antibiotics in meat can disrupt the fermentation process, giving pathogenic bacteria a chance to survive and multiply. We found that six commercially available starter cultures were susceptible to commonly used antibiotics, namely, oxytetracycline, penicillin, and erythromycin. In meat, statutorily tolerable levels of oxytetracycline and erythromycin inhibited fermentation performance of three and five of the six starter cultures, respectively. In model sausages, the disruption of meat fermentation enhanced survival of the pathogens Escherichia coli O157:H7 and Salmonella enterica serovar Typhimurium compared to successful fermentations. Our work reveals an overlooked risk associated with the presence of veterinary drugs in meat. IMPORTANCE: Antibiotics have for a long time been used as growth promoters in farm animals, and while they are banned as such in Europe, their clinical use in farm animals still accounts for the majority of consumption. Here, we examined how acceptable levels of antibiotics in meat influence fermentation. Our results show that commonly used bacterial starter cultures are sensitive to residual antibiotics at or near statutorily tolerable levels, and as a result, processed sausages may indeed contain high levels of pathogens. Our findings provide a possible explanation for outbreaks and disease cases associated with consumption of fermented sausages and offer yet another argument for limiting the use of antimicrobials in farm animals.

Method enabling gene expression studies of pathogens in a complex food matrix.

We describe a simple method for stabilizing and extracting high-quality prokaryotic RNA from meat. Heat and salt stress of Escherichia coli and Salmonella spp. in minced meat reproducibly induced dnaK and otsB expression, respectively, as observed by quantitative reverse transcription-PCR (>5-fold relative changes). Thus, the method is applicable in studies of bacterial gene expression in a meat matrix.

Growth and survival at chiller temperatures of Arcobacter butzleri.

Arcobacter butzleri is prevalent on chicken products. Arcobacter spp. are generally isolated in only low numbers from the chicken gut, so chicken carcasses may be contaminated by A. butzleri that proliferate in the slaughterhouse environment. To address this issue, we examined the behaviour of A. butzleri ATCC 49616 and newly isolated A. butzleri strains under conditions likely to prevail in the slaughterhouse environment using a chicken meat juice medium (CMJ). CMJ supported growth of A. butzleri at 15 degrees C, the recognised minimal growth temperature of this organism, and at 10 degrees C. At 5 degrees C, CMJ enhanced survival of A. butzleri as compared with survival in Brain Heart Infusion with less than a one log reduction after 77 days incubation. Lastly, we examined the ability of A. butzleri to form biofilms and found that the organism produces biofilm at temperatures ranging from 5 to 37 degrees C. Given the ability to survive, multiply and form biofilm under chilled conditions A. butzleri appears well suited for establishment in food processing and slaughterhouse environments.

Microbial changes and growth of Listeria monocytogenes during chilled storage of brined shrimp (Pandalus borealis).

Thirteen storage trials and ten challenge tests were carried out to examine microbial changes, spoilage and the potential growth of Listeria monocytogenes in brined shrimp (Pandalus borealis). Shrimp in brine as well as brined and drained shrimp in modified atmosphere packaging (MAP) were produced and studied. Different recipes were used to study the effect of preserving parameters (organic acids, pH and NaCl) on growth of microorganisms and shelf life at 7-8 degrees C or 12 degrees C. Particularly, brines with different concentrations of (i) benzoic, citric and sorbic acids or (ii) acetic, citric and lactic acids were studied. Furthermore, the effect of adding diacetate to brined shrimp was evaluated. A single batch of cooked and peeled shrimp was used to study both industrially and manually processed brined shrimp with respect to the effect of process hygiene on microbial changes and the shelf life of products. Concentrations of microorganisms on newly produced brined shrimp from an industrial scale processing line were 1.0-2.3 log (CFU g(-1)) higher than comparable concentrations in manually processed samples. This resulted in a substantially shorter shelf life and a more diverse spoilage microflora of the industrially processed brined shrimp. In addition, shelf life of brined shrimp was affected by the types and concentrations of organic acids and by the storage temperature as expected. The effect of MAP was less pronounced. Eighty-two isolates from the spoilage microflora of brined shrimp were identified and they included 53 lactic acid bacteria, 6 coagulase negative Staphylococcus spp., 18 Pseudomonas fluorescens and 5 yeast isolates. After storage at 7 degrees C, P. fluorescens, Enterococcus-like isolates, E. malodoratus, Carnobacterium maltaromaticum, coagulase negative Staphylococcus spp. and Lactobacillus sakei constituted the dominating microflora of shrimp in brines that contained benzoic, citric and sorbic acids as preservatives. L. sakei dominated the spoilage microflora of brined and drained MAP shrimp, and of brined shrimp preserved using acetic, citric and lactic acids, irrespective of packaging conditions. Shrimp in brine with benzoic, citric and sorbic acids prevented growth of L. monocytogenes during more than 40 days at 7 degrees C when the preserving parameters resembled those of commercial products. However, small changes in the preserving parameters and, particularly, reduced concentrations of benzoic acid led to growth of L. monocytogenes in brined shrimp. The present study provides significant new information on microbial changes, shelf life and growth of L. monocytogenes in brined shrimp. This information can facilitate development of new and safe brined shrimp products.