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.

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.

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.

Diversity of metal and antibiotic resistance genes in Enterococcus spp. from the last century reflects multiple pollution and genetic exchange among phyla from overlapping ecosystems.

Arsenic (As), mercury (Hg), and copper (Cu) are among the major historical and contemporary metal pollutants linked to global anthropogenic activities. Enterococcus have been considered indicators of fecal pollution and antibiotic resistance for years, but its largely underexplored metallome precludes understanding their role as metal pollution bioindicators as well. Our goal was to determine the occurrence, diversity, and phenotypes associated with known acquired genes/operons conferring tolerance to As, Hg or Cu among Enterococcus and to identify their genetic context (381 field isolates from diverse epidemiological and genetic backgrounds; 3547 enterococcal genomes available in databases representing a time span during 1900-2019). Genes conferring tolerance to As (arsA), Hg (merA) or Cu (tcrB) were used as biomarkers of widespread metal tolerance operons. Different variants of metal tolerance (MeT) genes (13 arsA, 6 merA, 1 tcrB) were more commonly recovered from the food-chain (arsA, tcrB) or humans (merA), and were shared with 49 other bacterial taxa. Comparative genomics analysis revealed that MeT genes occurred in heterogeneous operons, at least since the 1900s, with an increasing accretion of antibiotic resistance genes since the 1960's, reflecting diverse antimicrobial pollution. Multiple MeT genes were co-located on the chromosome or conjugative plasmids flanked by elements with high potential for recombination, often along with antibiotic resistance genes. Phenotypic analysis of some isolates carrying MeT genes revealed up to 128× fold increase in the minimum inhibitory concentrations to metals. The main distribution of functional MeT genes among Enterococcus faecium and Enterococcus faecalis from different sources, time spans, and clonal lineages, and their ability to acquire diverse genes from multiple taxa bacterial communities places these species as good candidates to be used as model organisms in future projects aiming at the identification and quantification of bioindicators of metal polluted environments by anthropogenic activities.

MicroMundo@UPorto: an experimental microbiology project fostering student's antimicrobial resistance awareness and personal and social development.

Antimicrobial resistance (AMR) is a global societal challenge requiring the contribution of professionals along with general community citizens for their containment. Portugal is one of the European countries where a lack of knowledge on the correct use of antimicrobials and AMR problematic is preeminent. Moreover, youth demotivation to pursue science careers is emerging. To address these problems an innovative experimental service-learning pedagogical strategy, MicroMundo@UPorto, was implemented in Portugal during 2018 through University of Porto as a partner of the global Citizen Science project 'Tiny Earth' responding to the AMR crisis. In this first edition of MicroMundo@UPorto, university students (n = 41; Pharmaceutical Sciences and Nutrition Sciences) organized in eight teams tutored by university professors/researchers (n = 13) on Microbiology and AMR theoretical and practical aspects as well on communication skills to enable their guidance of younger school students (n = 140/3 schools) in experiments to discover antimicrobial-producing microorganisms while exploring the soil microbial diversity. Post-survey-based evaluation revealed that this project allowed university students to acquire diverse personal, social and scientific skills while increasing AMR awareness, in the One-Health perspective, and interest for science in school students. This University to Society approach can be successfully extended across Portugal and for education in Microbiology in general, with benefits for the future generations contributing to socially responsible and scientifically-literate citizens.

Atypical Non-H2S-Producing Monophasic Salmonella Typhimurium ST3478 Strains from Chicken Meat at Processing Stage Are Adapted to Diverse Stresses.

Poultry products are still an important cause of Salmonella infections worldwide, with an increasingly reported expansion of less-frequent serotypes or atypical strains that are frequently multidrug-resistant. Nevertheless, the ability of Salmonella to survive antimicrobials promoted in the context of antibiotic reducing/replacing and farming rethinking (e.g., organic acids and copper in feed/biocides) has been scarcely explored. We investigated Salmonella occurrence (conventional and molecular assays) among chicken meat at the processing stage (n = 53 batches/29 farms) and characterized their tolerance to diverse stress factors (antibiotics, copper, acid pH, and peracetic acid). Whole-genome sequencing was used to assess adaptive features and to perform comparative analysis. We found a low Salmonella occurrence (4%) and identified S. Enteritidis/ST11 plus atypical non-H2S-producing S. 1,4,[5],12:i:-/ST3478. The ST3478 presented the ability to grow under diverse stresses (antibiotics, copper, and acid-pH). Comparative genomics among ST3478 isolates showed similar antibiotic/metal resistance gene repertoires and identical nonsense phsA thiosulfate reductase mutations (related to H2S-negative phenotype), besides their close phylogenetic relationship by cgMLST and SNPs. This study alerts for the ongoing national and international spread of an emerging monophasic Salmonella Typhimurium clonal lineage with an enlarged ability to survive to antimicrobials/biocides commonly used in poultry production, being unnoticed by conventional Salmonella detection approaches due to an atypical non-H2S-producing phenotype.

Tolerance to arsenic contaminant among multidrug-resistant and copper-tolerant Salmonella successful clones is associated with diverse ars operons and genetic contexts.

Emergence and expansion of frequent multidrug-resistant (MDR) major Salmonella clones/serotypes has been a significant threat in the last years. Metal compounds, such as copper, commonly used in animal-production settings, have been pointed out as possible contributors for the selection of such strains/clones. However, the scarcity of studies limits our understanding of the impact of other metal environmental contaminants as arsenic (used in insecticides/herbicides/coccidiostats). We analysed arsenic tolerance (AsT) dispersion by phenotypic and genotypic (PCR/sequencing/I-CeuI/S1/XbaI-PFGE/hybridization) assays among Salmonella with diverse epidemiological and genetic backgrounds. Then, to better understand ars operon genetic contexts, the whole genome of five representative strains was sequenced. We found a high dispersion of ars operons conferring AsT, especially among copper-tolerant and relevant serotypes/clones related to pig-production setting. The acr3-type was found dispersed in the chromosome of diverse serotypes, including the emergent S. Rissen. Conversely, arsBII was almost confined to the MDR ST34 European clone of S. Typhimurium/S. 4,[5],12:i:-, always along with copper/silver tolerance sil + pco clusters in an integrative conjugative element. These data suggest that AsT is an essential adaptive feature for the ecological success of these epidemic clones/serotypes and alerts for global strategies to reduce arsenic-based compounds' impact thus preventing environmental/food contamination with frequent MDR foodborne pathogens.