Raw meat-based diet for pets: a neglected source of human exposure to Salmonella and pathogenic Escherichia coli clones carrying mcr, Portugal, September 2019 to January 2020.

BackgroundThe pet industry is expanding worldwide, particularly raw meat-based diets (RMBDs). There are concerns regarding the safety of RMBDs, especially their potential to spread clinically relevant antibiotic-resistant bacteria or zoonotic pathogens.AimWe aimed to investigate whether dog food, including RMBD, commercially available in Portugal can be a source of Salmonella and/or other Enterobacteriaceae strains resistant to last-line antibiotics such as colistin.MethodsFifty-five samples from 25 brands (21 international ones) of various dog food types from 12 suppliers were screened by standard cultural methods between September 2019 and January 2020. Isolates were characterised by phenotypic and genotypic methods, including whole genome sequencing and comparative genomics.ResultsOnly RMBD batches were contaminated, with 10 of 14 containing polyclonal multidrug-resistant (MDR) Escherichia coli and one MDR Salmonella. One turkey-based sample contained MDR Salmonella serotype 1,4,[5],12:i:- ST34/cgST142761 with similarity to human clinical isolates occurring worldwide. This Salmonella exhibited typical antibiotic resistance (bla TEM + strA-strB + sul2 + tet(B)) and metal tolerance profiles (pco + sil + ars) associated with the European epidemic clone. Two samples (turkey/veal) carried globally dispersed MDR E. coli (ST3997-complexST10/cgST95899 and ST297/cgST138377) with colistin resistance (minimum inhibitory concentration: 4 mg/L) and mcr-1 gene on IncX4 plasmids, which were identical to other IncX4 circulating worldwide.ConclusionSome RMBDs from European brands available in Portugal can be a vehicle for clinically relevant MDR Salmonella and pathogenic E. coli clones carrying genes encoding resistance to the last-line antibiotic colistin. Proactive actions within the One Health context, spanning regulatory, pet-food industry and consumer levels, are needed to mitigate these public health risks.

Decoding Klebsiella pneumoniae in poultry chain: unveiling genetic landscape, antibiotic resistance, and biocide tolerance in non-clinical reservoirs.

The rise of antibiotic resistance in the food chain is influenced by the use of antimicrobial agents, such as antibiotics, metals, and biocides, throughout the entire farm-to-fork continuum. Besides, non-clinical reservoirs potentially contribute to the transmission of critical pathogens such as multidrug-resistant (MDR) Klebsiella pneumoniae. However, limited knowledge exists about the population structure and genomic diversity of K. pneumoniae circulating in conventional poultry production. We conducted a comprehensive characterization of K. pneumoniae across the whole chicken production chain (7 farms; 14 flocks + environment + meat, 56 samples; 2019-2022), exploring factors beyond antibiotics, like copper and quaternary ammonium compounds (QACs). Clonal diversity and adaptive features of K. pneumoniae were characterized through cultural, molecular (FT-IR), and whole-genome-sequencing (WGS) approaches. All except one flock were positive for K. pneumoniae with a significant increase (p < 0.05) from early (n = 1/14) to pre-slaughter (n = 11/14) stages, most (n = 6/7) persisting in chicken meat batches. Colistin-resistant K. pneumoniae rates were low (4%-n = 1/24 positive samples), while most samples carried MDR strains (67%-n = 16/24) and copper-tolerant isolates (63%-n = 15/24, with sil and pco gene clusters; MICCuSO4 ≥ 16 mM), particularly at pre-slaughter. Benzalkonium chloride consistently exhibited activity against K. pneumoniae (MIC/MBC range = 4-64 mg/L) from representative strains independently of the presence or absence of genes linked to QACs tolerance. A polyclonal K. pneumoniae population, discriminated by FT-IR and WGS, included various lineages dispersed throughout the chicken's lifecycle at the farm (ST29-KL124, ST11-KL106, ST15-KL19, ST1228-KL38), until the meat (ST1-KL19, ST11-KL111, ST6405-KL109, and ST6406-CG147-KL111), or over years (ST631-49 KL109, ST6651-KL107, ST6406-CG147-KL111). Notably, some lineages were identical to those from human clinical isolates. WGS also revealed F-type multireplicon plasmids carrying sil + pco (copper) co-located with qacEΔ1 ± qacF (QACs) and antibiotic resistance genes like those disseminated in humans. In conclusion, chicken farms and their derived meat are significant reservoirs for diverse K. pneumoniae clones enriched in antibiotic resistance and metal tolerance genes, some exhibiting genetic similarities with human clinical strains. Further research is imperative to unravel the factors influencing K. pneumoniae persistence and dissemination within poultry production, contributing to improved food safety risk management. This study underscores the significance of understanding the interplay between antimicrobial control strategies and non-clinical sources to effectively address the spread of antimicrobial resistance.

VirulenceFinder for Enterococcus faecium and Enterococcus lactis: an enhanced database for detection of putative virulence markers by using whole-genome sequencing data.

Enterococcus faecium (Efm) is a leading cause of hospital-associated (HA) infections, often enriched in putative virulence markers (PVMs). Recently, the Efm clade B was assigned as Enterococcus lactis (Elts), which usually lack HA-Efm infection markers. Available databases for extracting PVM are incomplete and/or present an intermix of genes from Efm and Enterococcus faecalis, with distinct virulence profiles. In this study, we constructed a new database containing 27 PVMs [acm, scm, sgrA, ecbA, fnm, sagA, hylEfm, ptsD, orf1481, fms15, fms21-fms20 (pili gene cluster 1, PGC-1), fms14-fms17-fms13 (PGC-2), empA-empB-empC (PGC-3), fms11-fms19-fms16 (PGC-4), ccpA, bepA, gls20-glsB1, and gls33-glsB] from nine reference genomes (seven Efm + two Elts). The database was validated against these reference genomes and further evaluated using a collection of well-characterized Efm (n = 43) and Elts (n = 7) control strains, by assessing PVM presence/absence and its variants together with a genomic phylogeny constructed as single-nucleotide polymorphisms. We found a high concordance between the phylogeny and in silico findings of the PVM, with Elts clustering separately and mostly carrying Elts-specific PVM gene variants. Based on our validation results, we recommend using the database with raw reads instead of assemblies to avoid missing gene variants. This newly constructed database of 27 PVMs will enable a more comprehensive characterization of Efm and Elts based on WGS data. The developed database exhibits scalability and boasts a range of applications in public health, including diagnostics, outbreak investigations, and epidemiological studies. It can be further used in risk assessment for distinguishing between safe and unsafe enterococci.IMPORTANCEThe newly constructed database, consisting of 27 putative virulence markers, is highly scalable and serves as a valuable resource for the comprehensive characterization of these closely related species using WGS data. It holds significant potential for various public health applications, including hospital outbreak investigations, surveillance, and risk assessment for probiotics and feed additives.

The population-level impact of Enterococcus faecalis genetics on intestinal colonization and extraintestinal infection.

IMPORTANCE: Enterococcus faecalis causes life-threatening invasive hospital- and community-associated infections that are usually associated with multidrug resistance globally. Although E. faecalis infections cause opportunistic infections typically associated with antibiotic use, immunocompromised immune status, and other factors, they also possess an arsenal of virulence factors crucial for their pathogenicity. Despite this, the relative contribution of these virulence factors and other genetic changes to the pathogenicity of E. faecalis strains remain poorly understood. Here, we investigated whether specific genomic changes in the genome of E. faecalis isolates influence its pathogenicity-infection of hospitalized and nonhospitalized individuals and the propensity to cause extraintestinal infection and intestinal colonization. Our findings indicate that E. faecalis genetics partially influence the infection of hospitalized and nonhospitalized individuals and the propensity to cause extraintestinal infection, possibly due to gut-to-bloodstream translocation, highlighting the potential substantial role of host and environmental factors, including gut microbiota, on the opportunistic pathogenic lifestyle of this bacterium.

Unraveling Enterococcus susceptibility to quaternary ammonium compounds: genes, phenotypes, and the impact of environmental conditions.

Quaternary ammonium compounds (QACs) have been extensively used in the community, healthcare facilities, and food chain, in concentrations between 20 and 30,000 mg/L. Enterococcus faecalis and Enterococcus faecium are ubiquitous in these settings and are recognized as nosocomial pathogens worldwide, but QACs' activity against strains from diverse epidemiological and genomic backgrounds remained largely unexplored. We evaluated the role of Enterococcus isolates from different sources, years, and clonal lineages as hosts of QACs tolerance genes and their susceptibility to QACs in optimal, single-stress and cross-stress growth conditions. Only 1% of the Enterococcus isolates included in this study and 0.5% of publicly available Enterococcus genomes carried qacA/B, qacC, qacG, qacJ, qacZ, qrg, bcrABC or oqxAB genes, shared with >60 species of Bacillota, Pseudomonadota, Actinomycetota, or Spirochaetota. These genes were generally found within close proximity of antibiotics and/or metals resistance genes. The minimum inhibitory concentrations (MIC) and minimum bactericidal concentrations (MBC) of benzalkonium chloride (BC) and didecyldimethylammonium chloride ranged between 0.5 and 4 mg/L (microdilution: 37°C/20 h/pH = 7/aerobiosis) for 210 E. faecalis and E. faecium isolates (two isolates carrying qacZ). Modified growth conditions (e.g., 22°C/pH = 5) increased MICBC/MBCBC (maximum of eightfold and MBCBC = 16 mg/L) and changed bacterial growth kinetics under BC toward later stationary phases in both species, including in isolates without QACs tolerance genes. In conclusion, Enterococcus are susceptible to in-use QACs concentrations and rarely carry QACs tolerance genes. However, their potential gene exchange with different microbiota, the decreased susceptibility to QACs under specific environmental conditions, and the presence of subinhibitory QACs concentrations in various settings may contribute to the selection of particular strains and, thus, require a One Health strategy to maintain QACs effectiveness. IMPORTANCE Despite the increasing use of quaternary ammonium compounds (QACs), the susceptibility of pathogens to these antimicrobials remains largely unknown. Enterococcus faecium and Enterococcus faecalis are susceptible to in-use QACs concentrations and are not main hosts of QACs tolerance genes but participate in gene transfer pathways with diverse bacterial taxa exposed to these biocides. Moreover, QACs tolerance genes often share the same genetic contexts with antibiotics and/or metals resistance genes, raising concerns about potential co-selection events. E. faecium and E. faecalis showed increased tolerance to benzalkonium chloride under specific environmental conditions (22°C, pH = 5), suggesting that strains might be selected in settings where they occur along with subinhibitory QACs concentrations. Transcriptomic studies investigating the cellular mechanisms of Enterococcus adaptation to QACs tolerance, along with longitudinal metadata analysis of tolerant populations dynamics under the influence of diverse environmental factors, are essential and should be prioritized within a One Health strategy.

Exploring Peracetic Acid and Acidic pH Tolerance of Antibiotic-Resistant Non-Typhoidal Salmonella and Enterococcus faecium from Diverse Epidemiological and Genetic Backgrounds.

Acid stress poses a common challenge for bacteria in diverse environments by the presence of inorganic (e.g., mammals' stomach) or organic acids (e.g., feed additives; acid-based disinfectants). Limited knowledge exists regarding acid-tolerant strains of specific serotypes, clonal lineages, or sources in human/animal pathogens: namely, non-typhoidal Salmonella enterica (NTS) and Enterococcus faecium (Efm). This study evaluated the acidic pH (Mueller-Hinton acidified with HCl) and peracetic acid (PAA) susceptibility of Efm (n = 72) and NTS (n = 60) from diverse epidemiological/genetic backgrounds and with multiple antibiotic resistance profiles. Efm minimum growth/survival pH was 4.5-5.0/3.0-4.0, and for NTS it was 4.0-4.5/3.5-4.0. Efm distribution among acidic pH values showed that only isolates of clade-non-A1 (non-hospital associated) or the food chain were more tolerant to acidic pH compared to clade-A1 (hospital-associated clones) or clinical isolates (p < 0.05). In the case of NTS, multidrug-resistant (MDR) isolates survived better in acidic pH (p < 0.05). The PAA MIC/MBC for Efm was 70-120/80-150 mg/L, and for NTS, it was 50-70/60-100 mg/L. The distribution of Efm among PAA concentrations showed that clade-A1 or MDR strains exhibited higher tolerance than clade-non-A1 or non-MDR ones (p < 0.05). NTS distribution also showed higher tolerance to PAA among non-MDR and clinical isolates than food chain ones (p < 0.05) but there were no differences among different serogroups. This unique study identifies specific NTS or Efm populations more tolerant to acidic pH or PAA, emphasizing the need for further research to tailor controlled measures of public health and food safety within a One Health framework.

Unraveling the Role of Metals and Organic Acids in Bacterial Antimicrobial Resistance in the Food Chain.

Antimicrobial resistance (AMR) has a significant impact on human, animal, and environmental health, being spread in diverse settings. Antibiotic misuse and overuse in the food chain are widely recognized as primary drivers of antibiotic-resistant bacteria. However, other antimicrobials, such as metals and organic acids, commonly present in agri-food environments (e.g., in feed, biocides, or as long-term pollutants), may also contribute to this global public health problem, although this remains a debatable topic owing to limited data. This review aims to provide insights into the current role of metals (i.e., copper, arsenic, and mercury) and organic acids in the emergence and spread of AMR in the food chain. Based on a thorough literature review, this study adopts a unique integrative approach, analyzing in detail the known antimicrobial mechanisms of metals and organic acids, as well as the molecular adaptive tolerance strategies developed by diverse bacteria to overcome their action. Additionally, the interplay between the tolerance to metals or organic acids and AMR is explored, with particular focus on co-selection events. Through a comprehensive analysis, this review highlights potential silent drivers of AMR within the food chain and the need for further research at molecular and epidemiological levels across different food contexts worldwide.

Uncovering the effects of copper feed supplementation on the selection of copper-tolerant and antibiotic-resistant Enterococcus in poultry production for sustainable environmental practices.

The use of antibiotics in animal production is linked to the emergence and spread of antibiotic-resistant bacteria, a threat to animal, environmental and human health. Copper (Cu) is an essential element in poultry diets and an alternative to antibiotics, supplementing inorganic or organic trace mineral feeds (ITMF/OTMF). However, its contribution to select multidrug-resistant (MDR) and Cu tolerant Enterococcus, a bacteria with a human-animal-environment-food interface, remains uncertain. We evaluated whether feeding chickens with Cu-ITMF or Cu-OTMF contributes to the selection of Cu tolerant and MDR Enterococcus from rearing to slaughter. Animal faeces [2-3-days-old (n = 18); pre-slaughter (n = 16)] and their meat (n = 18), drinking-water (n = 14) and feed (n = 18) from seven intensive farms with ITMF and OTMF flocks (10.000-64.000 animals each; 2019-2020; Portugal) were sampled. Enterococcus were studied by cultural, molecular and whole-genome sequencing methods and Cu concentrations by ICP-MS. Enterococcus (n = 477; 60 % MDR) were identified in 80 % of the samples, with >50 % carrying isolates resistant to tetracycline, quinupristin-dalfopristin, erythromycin, streptomycin, ampicillin or ciprofloxacin. Enterococcus with Cu tolerance genes, especially tcrB ± cueO, were mainly found in faeces (85 %; E. faecium/E. lactis) of ITMF/OTMF flocks. Similar occurrence and load of tcrB ± cueO Enterococcus in the faeces was detected throughout the chickens' lifespan in the ITMF/OTMF flocks, decreasing in meat. Most of the polyclonal MDR Enterococcus population carrying tcrB ± cueO or only cueO (67 %) showed a wild-type phenotype (MICCuSO4 ≤ 12 mM) linked to absence of tcrYAZB or truncated variants, also detected in 85 % of Enterococcus public genomes from poultry. Finally, < 65 µg/g Cu was found in all faecal and meat samples. In conclusion, Cu present in ITMF/OTMF is not selecting Cu tolerant and MDR Enterococcus during chickens' lifespan. However, more studies are needed to assess the minimum concentration of Cu required for MDR bacterial selection and horizontal transfer of antibiotic resistance genes, which would support sustainable practices mitigating antibiotic resistance spread in animal production and the environment beyond.

From Farm to Fork: Persistence of Clinically Relevant Multidrug-Resistant and Copper-Tolerant Klebsiella pneumoniae Long after Colistin Withdrawal in Poultry Production.

Concerns about colistin-resistant bacteria in animal food-environmental-human ecosystems prompted the poultry sector to implement colistin restrictions and explore alternative trace metals/copper feed supplementation. The impact of these strategies on the selection and persistence of colistin-resistant Klebsiella pneumoniae in the whole poultry production chain needs clarification. We assessed colistin-resistant and copper-tolerant K. pneumoniae occurrence in chickens raised with inorganic and organic copper formulas from 1-day-old chicks to meat (7 farms from 2019 to 2020), after long-term colistin withdrawal (>2 years). Clonal diversity and K. pneumoniae adaptive features were characterized by cultural, molecular, and whole-genome-sequencing (WGS) approaches. Most chicken flocks (75%) carried K. pneumoniae at early and preslaughter stages, with a significant decrease (P < 0.05) in meat batches (17%) and sporadic water/feed contamination. High rates (>50%) of colistin-resistant/mcr-negative K. pneumoniae were observed among fecal samples, independently of feed. Most samples carried multidrug-resistant (90%) and copper-tolerant (81%; silA and pcoD positive and with a MICCuSO4 of ≥16 mM) isolates. WGS revealed accumulation of colistin resistance-associated mutations and F type multireplicon plasmids carrying antibiotic resistance and metal/copper tolerance genes. The K. pneumoniae population was polyclonal, with various lineages dispersed throughout poultry production. ST15-KL19, ST15-KL146, and ST392-KL27 and IncF plasmids were similar to those from global human clinical isolates, suggesting chicken production as a reservoir/source of clinically relevant K. pneumoniae lineages and genes with potential risk to humans through food and/or environmental exposure. Despite the limited mcr spread due to the long-term colistin ban, this action was ineffective in controlling colistin-resistant/mcr-negative K. pneumoniae, regardless of feed. This study provides crucial insights into the persistence of clinically relevant K. pneumoniae in the poultry production chain and highlights the need for continued surveillance and proactive food safety actions within a One Health perspective. IMPORTANCE The spread of bacteria resistant to last-resort antibiotics such as colistin throughout the food chain is a serious concern for public health. The poultry sector has responded by restricting colistin use and exploring alternative trace metals/copper feed supplements. However, it is unclear how and to which extent these changes impact the selection and persistence of clinically relevant Klebsiella pneumoniae throughout the poultry chain. We found a high occurrence of copper-tolerant and colistin-resistant/mcr-negative K. pneumoniae in chicken flocks, regardless of inorganic and organic copper formulas use and a long-term colistin ban. Despite the high K. pneumoniae isolate diversity, the occurrence of identical lineages and plasmids across samples and/or clinical isolates suggests poultry as a potential source of human K. pneumoniae exposure. This study highlights the need for continued surveillance and proactive farm-to-fork actions to mitigate the risks to public health, relevant for stakeholders involved in the food industry and policymakers tasked with regulating food safety.

Inverted classes as a successful tool to overcome teaching and learning challenges imposed by COVID-19 lockdown to food microbiology laboratory classes.

This study evaluated if the online inverted classes (IC) model maintained high students' learning engagement and performance on the laboratory component of Food Microbiology during an academic year dominated by COVID-19-lockdown, compared to pre-pandemic years. Porto University students from 2 courses (n = 36-Pharmaceutical Sciences; n = 59-Nutrition Sciences) were engaged. A high rate of students answered they prefer the IC rather than lecture-only style delivery mode and were involved in the IC learning process (prepared classes asynchronously; participated in case-studies resolution during synchronous classes). Very good laboratory final performances were maintained as in pre-pandemic years. Variable perceptions about work volume and adaptation to face-to-face laboratory classes were observed among both groups, potentially related to different course organization and defined learning outcomes/competences.

Enterococcus spp. from chicken meat collected 20 years apart overcome multiple stresses occurring in the poultry production chain: Antibiotics, copper and acids.

Poultry meat has been a vehicle of antibiotic resistant bacteria and genes. Yet, the diversity of selective pressures associated with their maintenance in the poultry-production chain remains poorly explored. We evaluated the susceptibility of Enterococcus spp. from chicken meat collected 20 years apart to antibiotics, metals, acidic pH and peracetic acid-PAA. Contemporary chicken-meat samples (n = 53 batches, each including a pool of neck skin from 10 single carcasses) were collected in a slaughterhouse facility using PAA as disinfectant (March-August 2018, North of Portugal). Broilers were raised in intensive farms (n = 29) using CuSO4 and organic acids as feed additives. Data were compared with that of 67 samples recovered in the same region during 1999-2001. All 2018 samples had multidrug resistant-MDR isolates, with >45 % carrying Enterococcus faecalis, Enterococcus faecium or Enterococcus gallinarum resistant to tetracycline, erythromycin, ampicillin, quinupristin-dalfopristin, ciprofloxacin, chloramphenicol or aminoglycosides. Resistance rates were similar (P > 0.05) to those of 1999-2001 samples for all but five antibiotics. The decrease of samples carrying vancomycin-resistant isolates from 46 % to 0 % between 1999-2001 and 2018 was the most striking difference. Isolates from both periods were similarly susceptible to acid pH [minimum-growth pH (4.5-5.0), minimum-survival pH (3.0-4.0)] and to PAA (MIC90 = 100-120 mg/L/MBC90 = 140-160 mg/L; below concentrations used in slaughterhouse). Copper tolerance genes (tcrB and/or cueO) were respectively detected in 21 % and 4 % of 2018 and 1999-2001 samples. The tcrB gene was only detected in E. faecalis (MICCuSO4 > 12 mM), and their genomes were compared with other international ones of chicken origin (PATRIC database), revealing a polyclonal population and a plasmid or chromosomal location for tcrB. The tcrB plasmids shared diverse genetic modules, including multiple antimicrobial resistance genes (e.g. to tetracyclines, chloramphenicol, macrolide-lincosamide-streptogramin B-MLSB, aminoglycosides, bacitracin, coccidiostats). When in chromosome, the tcrB gene was co-located closely to merA (mercury) genes. Chicken meat remains an important vehicle of MDR Enterococcus spp. able to survive under diverse stresses (e.g. copper, acid) potentially contributing to these bacteria maintenance and flux among animal-environment-humans.

Distinction between Enterococcus faecium and Enterococcus lactis by a gluP PCR-Based Assay for Accurate Identification and Diagnostics.

It was recently proposed that Enterococcus faecium colonizing the human gut (previous clade B) actually corresponds to Enterococcus lactis. Our goals were to develop a PCR assay to rapidly differentiate these species and to discuss the main phenotypic and genotypic differences from a clinical perspective. The pan-genome of 512 genomes of E. faecium and E. lactis strains was analyzed to assess diversity in genes between the two species. Sequences were aligned to find the best candidate gene for designing species-specific primers, and their accuracy was tested with a collection of 382 enterococci. E. lactis isolates from clinical origins were further characterized by whole-genome sequencing (Illumina). Pan-genome analysis resulted in 12 gene variants, with gene gluP (rhomboid protease) being selected as the candidate for species differentiation. The nucleotide sequence of gluP diverged by 90 to 92% between sets, which allowed species identification through PCR with 100% specificity and no cross-reactivity. E. lactis strains were greatly pan-susceptible and not host specific. Hospital E. lactis isolates were susceptible to clinically relevant antibiotics, lacked infection-associated virulence markers, and were associated with patients presenting risk factors for enhanced bacterial translocation. Here, we propose a PCR-based assay using gluP for easy routine differentiation between E. faecium and E. lactis that could be implemented in different public health contexts. We further suggest that E. lactis, a dominant human gut species, can cross the gut barrier in severely ill, immunodeficient, and surgical patients. Knowing that bacterial translocation may be a sepsis promoter, the relevance of infections caused by E. lactis strains, even if they are pan-susceptible, should be explored. IMPORTANCE Enterococcus faecium is a WHO priority pathogen that causes severe and hard-to-treat human infections. It was recently proposed that E. faecium colonizing the human gut (previous clade B) actually corresponds to Enterococcus lactis; therefore, some of the human infections occurring globally are being misidentified. In this work, we developed a PCR-based rapid identification method for the differentiation of E. faecium and E. lactis and discussed the main phenotypic and genotypic differences of these species from a clinical perspective. We identified the gluP gene as the best candidate, based on the phylogenomic analysis of 512 published pan-genomes, and validated the PCR assay with a comprehensive collection of 382 enterococci obtained from different sources. Further detailed analysis of clinical E. lactis strains showed that they are highly susceptible to antibiotics and lack the typical virulence markers of E. faecium but are able to cause severe human infections in immunosuppressed patients, possibly in part due to gut barrier translocation.

Evolution of Chlorhexidine Susceptibility and of the EfrEF Operon among Enterococcus faecalis from Diverse Environments, Clones, and Time Spans.

Chlorhexidine (CHX) is widely used to control the spread of pathogens (e.g., human/animal clinical settings, ambulatory care, food industry). Enterococcus faecalis, a major nosocomial pathogen, is broadly distributed in diverse hosts and environments facilitating its exposure to CHX over the years. Nevertheless, CHX activity against E. faecalis is understudied. Our goal was to assess CHX activity and the variability of ChlR-EfrEF proteins (associated with CHX tolerance) among 673 field isolates and 1,784 E. faecalis genomes from the PATRIC database from different sources, time spans, clonal lineages, and antibiotic-resistance profiles. The CHX MIC (MICCHX) and minimum bactericidal concentration (MBCCHX) against E. faecalis presented normal distributions (0.5 to 64 mg/L). However, more CHX-tolerant isolates were detected in the food chain and recent human infections, suggesting an adaptability of E. faecalis populations in settings where CHX is heavily used. Heterogeneity in ChlR-EfrEF sequences was identified, with isolates harboring incomplete ChlR-EfrEF proteins, particularly the EfrE identified in the ST40 clonal lineage, showing low MICCHX (≤1mg/L). Distinct ST40-E. faecalis subpopulations carrying truncated and nontruncated EfrE were detected, with the former being predominant in human isolates. This study provides a new insight about CHX susceptibility and ChlR-EfrEF variability within diverse E. faecalis populations. The MICCHX/MBCCHX of more tolerant E. faecalis (MICCHX = 8 mg/L; MBCCHX = 64 mg/L) remain lower than in-use concentrations of CHX (≥500 mg/L). However, increased CHX use, combined with concentration gradients occurring in diverse environments, potentially selecting multidrug-resistant strains with different CHX susceptibilities, signals the importance of monitoring the trends of E. faecalis CHX tolerance within a One Health approach. IMPORTANCE Chlorhexidine (CHX) is a disinfectant and antiseptic used since the 1950s and included in the World Health Organization's list of essential medicines. It has been widely applied in hospitals, the community, the food industry, animal husbandry and pets. CHX tolerance in Enterococcus faecalis, a ubiquitous bacterium and one of the leading causes of human hospital-acquired infections, remains underexplored. Our study provides novel and comprehensive insights about CHX susceptibility within the E. faecalis population structure context, revealing more CHX-tolerant subpopulations from the food chain and recent human infections. We further show a detailed analysis of the genetic diversity of the efrEF operon (previously associated with E. faecalis CHX tolerance) and its correlation with CHX phenotypes. The recent strains with a higher tolerance to CHX and the multiple sources where bacteria are exposed to this biocide alert us to the need for the continuous monitoring of E. faecalis adaptation toward CHX tolerance within a One Health approach.

Safety and Quality of Canned Sardines after Opening: A Shelf-Stability Study.

This study aimed to define the shelf life of canned sardines after opening to increase consumer awareness of their quality and safety and reduce food waste. For this purpose, canned sardines (Sardina pilchardus) packed with different sauces were opened and stored at 4 °C for 7 days. Microbiological, sensorial, physical and chemical stability was monitored daily by standard methodologies. Results show that the overall quality and safety are highly dependent on the sauce type. To preserve their full quality, sardines in brine and in vegetable oil should be consumed up to 1 day after opening, while sardines in tomato sauce were stable for up to 3 days, although none were considered nonedible up to the 7th day. Many parameters demonstrated statistical differences and correlations with storage, although they were not as decisive as sensory evaluation. This integrated approach should be adopted by the food industry and regulating authorities to provide information to consumers regarding the quality and safety of handled goods.

High-Resolution Genotyping Unveils Identical Ampicillin-Resistant Enterococcus faecium Strains in Different Sources and Countries: A One Health Approach.

Multidrug-resistant (MDR) Enterococcus faecium (Efm) infections continue to increase worldwide, although epidemiological studies remain scarce in lower middle-income countries. We aimed to explore which strains circulate in E. faecium causing human infections in Tunisian healthcare institutions in order to compare them with strains from non-human sources of the same country and finally to position them within the global E. faecium epidemiology by genomic analysis. Antibiotic susceptibility testing was performed and transfer of vancomycin-vanA and ampicillin-pbp5 resistance was performed by conjugation. WGS-Illumina was performed on Tunisian strains, and these genomes were compared with Efm genomes from other regions present in the GenBank/NCBI database (n = 10,701 Efm genomes available May 2021). A comparison of phenotypes with those predicted by the recent ResFinder 4.1-CGE webtool unveiled a concordance of 88%, with discordant cases being discussed. cgMLST revealed three clusters [ST18/CT222 (n = 13), ST17/CT948 strains (n = 6), and ST203/CT184 (n = 3)], including isolates from clinical, healthy-human, retail meat, and/or environmental sources in different countries over large time spans (10-12 years). Isolates within each cluster showed similar antibiotic resistance, bacteriocin, and virulence genetic patterns. pbp5-AmpR was transferred by VanA-AmpR-ST80 (clinical) and AmpR-ST17-Efm (bovine meat). Identical chromosomal pbp5-platforms carrying metabolic/virulence genes were identified between ST17/ST18 strains of clinical, farm animal, and retail meat sources. The overall results emphasize the role of high-resolution genotyping as provided by WGS in depicting the dispersal of MDR-Efm strains carrying relevant adaptive traits across different hosts/regions and the need of a One Health task force to curtail their spread.

Enterococcus spp. as a Producer and Target of Bacteriocins: A Double-Edged Sword in the Antimicrobial Resistance Crisis Context.

Enterococcus spp. are one of the most frequent producers of bacteriocins (enterocins), which provides them with an advantage to compete in their natural environment, which is the gut of humans and many animals. The enterocins' activity against microorganisms from different phylogenetic groups has raised interest in Enterococcus spp. in different contexts throughout the last decades, especially in the food industry. Nevertheless, some species can also cause opportunistic life-threatening infections and are frequently multidrug-resistant (MDR). Vancomycin-resistant Enterococcus (VRE), in particular, are an ongoing global challenge given the lack of therapeutic options. In this scenario, bacteriocins can offer a potential solution to this persistent threat, either alone or in combination with other antimicrobials. There are a handful of studies that demonstrate the advantages and applications of bacteriocins, especially against VRE. The purpose of this review is to present a current standpoint about the dual role of Enterococcus spp., from important producers to targets needed to be controlled, and the crucial role that enterocins may have in the expansion of enterococcal populations. Classification and distribution of enterocins, the current knowledge about the bacteriocinome of clinical enterococci, and the challenges of bacteriocin use in the fight against VRE infections are particularly detailed.

Fitness cost of vancomycin-resistant Enterococcus faecium plasmids associated with hospital infection outbreaks.

BACKGROUND: Vancomycin resistance is mostly associated with Enterococcus faecium due to Tn1546-vanA located on narrow- and broad-host plasmids of various families. This study's aim was to analyse the effects of acquiring Tn1546-carrying plasmids with proven epidemicity in different bacterial host backgrounds. METHODS: Widespread Tn1546-carrying plasmids of different families RepA_N (n = 5), Inc18 (n = 4) and/or pHTß (n = 1), and prototype plasmids RepA_N (pRUM) and Inc18 (pRE25, pIP501) were analysed. Plasmid transferability and fitness cost were assessed using E. faecium (GE1, 64/3) and Enterococcus faecalis (JH2-2/FA202/UV202) recipient strains. Growth curves (Bioscreen C) and Relative Growth Rates were obtained in the presence/absence of vancomycin. Plasmid stability was analysed (300 generations). WGS (Illumina-MiSeq) of non-evolved and evolved strains (GE1/64/3 transconjugants, n = 49) was performed. SNP calling (Breseq software) of non-evolved strains was used for comparison. RESULTS: All plasmids were successfully transferred to different E. faecium clonal backgrounds. Most Tn1546-carrying plasmids and Inc18 and RepA_N prototypes reduced host fitness (-2% to 18%) while the cost of Tn1546 expression varied according to the Tn1546-variant and the recipient strain (9%-49%). Stability of Tn1546-carrying plasmids was documented in all cases, often with loss of phenotypic resistance and/or partial plasmid deletions. SNPs and/or indels associated with essential bacterial functions were observed on the chromosome of evolved strains, some of them linked to increased fitness. CONCLUSIONS: The stability of E. faecium Tn1546-carrying plasmids in the absence of selective pressure and the high intra-species conjugation rates might explain the persistence of vancomycin resistance in E. faecium populations despite the significant burden they might impose on bacterial host strains.

Linezolid- and Multidrug-Resistant Enterococci in Raw Commercial Dog Food, Europe, 2019-2020.

We describe enterococci in raw-frozen dog food commercialized in Europe as a source of genes encoding resistance to the antibiotic drug linezolid and of strains and plasmids enriched in antibiotic-resistance and virulence genes in hospitalized patients. Whole-genome sequencing was fundamental to linking isolates from dog food to human cases across Europe.

Industrial dog food is a vehicle of multidrug-resistant enterococci carrying virulence genes often linked to human infections.

The increase in the number of pets in recent years has been followed by an exponential growth of the industrial pet food sector, which has been accompanied by new food safety risks, namely antibiotic resistance. The aim of this study was to investigate whether dog food commercially available in Portugal is a reservoir of clinically-relevant antibiotic resistant Enterococcus. Fifty-five samples (25 brands; 22 wet, 14 raw frozen, 8 dry, 7 treats and 4 semi-wet) were collected on 9 commercial surfaces in the Porto region (September 2019 to January 2020). Most samples were obtained from brands that are commercialized worldwide (n = 21/25). Sample (25 g) processing included pre-enrichment and enrichment steps in culture media without/with 3 antibiotics, and then plating into selective media without/with the same antibiotics. Susceptibility was studied for 13 antibiotics (disk diffusion; Etest; microdilution) according to EUCAST/CLSI. Clinically-relevant species (E. faecium and E. faecalis), antibiotic resistance (vanA, vanB, optrA, poxtA) and virulence (e.g. ptsD, esp, sgrA) genes were identified by PCR. Other species of Enterococcus were identified by MALDI-TOF MS. Clonality was established by MLST in selected isolates. Enterococcus (n = 184; 7 species; >85% E. faecium and E. faecalis) were detected in 30 samples (54%) of different types (14 raw, 16 heat treated-7 dry, 6 wet, 3 treats). E. faecium and E. faecalis were more frequent in dry and wet samples, respectively. More than 40% of enterococci recovered were resistant to erythromycin, tetracycline, quinupristin-dalfopristin, streptomycin, gentamicin, chloramphenicol, ampicillin or ciprofloxacin, and to a lesser extent to linezolid (23%; optrA, poxtA) or vancomycin and teicoplanin (2% each; vanA). Multidrug-resistant isolates (31%), including to vancomycin and linezolid, were obtained mostly from raw foods, although also detected in wet samples or treats, and mainly from culture media supplemented with antibiotics. Samples subjected to thermal treatment mostly carried non-MDR isolates. The variety of clones observed included strains previously identified in hospitalized patients (E. faecium ST17/ST80; E. faecalis ST40), farm animals, pets and environmental strains. This study shows that dog food from international brands is a vehicle of clinically-relevant enterococci carrying resistance to last resort antibiotics and relevant virulence genes, thus positioning pet food as an important source of antibiotic resistance spread within the One Health context. The high incidence of Enterococcus in a variety of dog food samples indicates the need to review selection of raw materials, manufacturing and hygiene practices in an emerging food sector growing worldwide.