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.

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.

Unveiling a Listeria monocytogenes Outbreak in a Rabbit Farm: Clinical Manifestation, Antimicrobial Resistance, Genomic Insights and Environmental Investigation.

Listeria monocytogenes poses a threat to both human and animal health. This work describes an L. monocytogenes outbreak in a Portuguese rabbit farm, detailing the isolates' clinical manifestations, necropsy findings, and phenotypic and genomic profiles. Clinical signs, exclusively observed in does, included lethargy and reproductive signs. Post-mortem examination of does revealed splenomegaly, hepatomegaly with a reticular pattern, pulmonary congestion, and haemorrhagic lesions in the uterus, with thickening of the uterine wall and purulent greyish exudates. Positive L. monocytogenes samples were identified in fattening and maternity units across different samples, encompassing does and environmental samples. Core-genome Multi Locus Sequence Typing (cgMLST) analysis confirmed the outbreak, with the 16 sequenced isolates (lineage II, CC31, and ST325) clustering within a ≤2 allelic difference (AD) threshold. Antimicrobial susceptibility testing for five antibiotics revealed that 15 out of 19 outbreak isolates were resistant to sulfamethoxazole-trimethoprim (SXT). Concordantly, all SXT-resistant sequenced isolates were found to exclusively harbour a plasmid containing a trimethoprim-resistance gene (dfrD), along with loci linked to resistance to lincosamides (lnuG), macrolides (mphB), and polyether ionophores (NarAB operon). All sequenced outbreak isolates carried the antibiotic resistance-related genes tetM, fosX, lin, norB, lmrB, sul, and mprF. The outbreak cluster comprises isolates from does and the environment, which underscores the ubiquitous presence of L. monocytogenes and emphasizes the importance of biosecurity measures. Despite limited data on listeriosis in rabbit farming, this outbreak reveals its significant impact on animal welfare and production.

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.

Occurrence of Multidrug-Resistant Bacteria Resulting from the Selective Pressure of Antibiotics: A Comprehensive Analysis of ESBL K. pneumoniae and MRSP Isolated in a Dog with Rhinorrhea.

Because of public health concerns, much greater scrutiny is now placed on antibiotic use in pets, especially for antimicrobial agents that have human analogs. Therefore, this study aimed to characterize the phenotypic and genotypic profiles of multidrug-resistant bacteria isolated from nasal swabs samples taken from a one-year-old male Serra da Estrela dog with rhinorrhea that was treated with amikacin. An extended-spectrum ß-lactamases (ESBL) Klebsiella pneumoniae was isolated in the first sample taken from the left nasal cavity of the dog. Seven days later, methicillin-resistant (MRSP) Staphylococcus pseudintermedius was also isolated. Nevertheless, no alterations to the therapeutic protocol were performed. Once the inhibitory action of the antibiotic disappeared, the competitive advantage of the amikacin-resistant MRSP was lost, and only commensal flora was observed on both nasal cavities. The genotypic profile of extended-spectrum ß-lactamase (ESBL)-producing Klebsiella pneumoniae revealed the same characteristics and close relation to other strains, mainly from Estonia, Slovakia and Romania. Regarding MRSP isolates, although resistance to aminoglycosides was present in the first MRSP, the second isolate carried aac(6')-aph(2″), which enhanced its resistance to amikacin. However, the veterinary action was focused on the treatment of the primary agent (ESBL K. pneumoniae), and the antibiotic applied was according to its phenotypic profile, which may have led to the resolution of the infectious process. Therefore, this study highlights the importance of targeted therapy, proper clinical practice and laboratory-hospital communication to safeguard animal, human and environmental health.

Gulls in Porto Coastline as Reservoirs for Salmonella spp.: Findings from 2008 and 2023.

Gulls act as intermediaries in the exchange of microorganisms between the environment and human settlements, including Salmonella spp. This study assessed the antimicrobial resistance and molecular profiles of Salmonella spp. isolates obtained from fecal samples of gulls in the city of Porto, Portugal, in 2008 and 2023 and from water samples in 2023. Antimicrobial susceptibility profiling revealed an improvement in the prevalence (71% to 17%) and antimicrobial resistance between the two collection dates. Two isolate collections from both 2008 and 2023 underwent serotyping and whole-genome sequencing, revealing genotypic changes, including an increased frequency in the monophasic variant of S. Typhimurium. qacE was identified in 2008 and 2023 in both water and fecal samples, with most isolates exhibiting an MDR profile. The most frequently observed plasmid types were IncF in 2008 (23%), while IncQ1 predominated in 2023 (43%). Findings suggest that Salmonella spp. circulate between humans, animals, and the environment. However, the genetic heterogeneity among the isolates from the gulls' feces and the surface water may indicate a complex ecological and evolutionary dynamic shaped by changing conditions. The observed improvements are likely due to measures to reduce biological contamination and antimicrobial resistance. Nevertheless, additional strategies must be implemented to reduce the public health risk modeled by the dissemination of pathogens by gulls.

High diversity of pathogenic Escherichia coli clones carrying mcr-1 among gulls underlines the need for strategies at the environment-livestock-human interface.

The expansion of mcr-carrying bacteria is a well-recognized public health problem. Measures to contain mcr spread have mainly been focused on the food-animal production sector. Nevertheless, the spread of MCR producers at the environmental interface particularly driven by the increasing population of gulls in coastal cities has been less explored. Occurrence of mcr-carrying Escherichia coli in gull's colonies faeces on a Portuguese beach was screened over 7 months. Cultural, molecular and genomic approaches were used to characterize their diversity, mcr plasmids and adaptive features. Multidrug-resistant mcr-1-carrying E. coli were detected for 3 consecutive months. Over time, multiple strains were recovered, including zoonotic-related pathogenic E. coli clones (e.g. B2-ST131-H22, A-ST10 and B1-ST162). Diverse mcr-1 genetic environments were mainly associated with ST2/ST4-HI2 (ST10, ST131, ST162, ST354 and ST4204) but also IncI2 (ST12990) plasmids or in the chromosome (ST656). Whole-genome sequencing revealed enrichment of these strains on antibiotic resistance, virulence and metal tolerance genes. Our results underscore gulls as important spreaders of high-priority bacteria and genes that may affect the environment, food-animals and/or humans, potentially undermining One-Health strategies to reduce colistin resistance.