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Previous studies in Ghana indicated low prevalence of methicillin-resistant Staphylococcus aureus (MRSA) and predominance of ST152 methicillin-susceptible S. aureus (MSSA) among clinical isolates. ST152 MRSA clones are associated with severe infections and epidemics. Using whole genome sequencing (WGS), 159 S . aureus isolated from clinical sources (wound, blood, urine, ear, abscess, umbilical cord, eye, vaginal samples, and others) from 10 hospitals across Ghana were investigated. mecA (gene for methicillin resistance) was detected in 38% of the isolates. Panton-Valentine leucocidin toxin (PVL) gene occurred in 65% isolates, with 84% of the MRSA's harboring the PVL gene. ST152 was the major clone, with 74% harboring the mecA gene. Other MRSA clones detected were ST5, ST5204, ST852, and ST1. MSSA clones included ST3249, ST152, ST5, ST1, and ST8. Twenty-three genes encoding resistance to 12 antimicrobial classes were observed with blaZ (97%) being the most prevalent. Other predominant resistance genes included tetK (46%), cat (42%), and dfrG (36%) encoding resistance for tetracyclines, phenicols, and diaminopyrimidine, respectively. Virulence genes for enterotoxins, biofilms, toxic-shock-syndrome toxins, hemolysins , and leukotoxins were also detected. Phylogenetic analysis revealed a shift in the dominant clone from MSSA ST152 to MRSA ST152 over the past decade. The study provides valuable insights into the genomic content of S. aureus from clinical sources in Ghana. The finding of ST152 MRSA in high numbers suggests a shifting epidemiological landscape of these pathogens and continuous surveillance using robust tools like WGS is needed to monitor the rise and spread of these epidemic clones in the country.IMPORTANCESince its emergence in 1959, MRSA has been a significant public health concern, causing infections in both clinical and community settings. Patients with MRSA-related infections experience higher mortality rates due to its ability to evade antimicrobials and immune defenses. In Ghana, understanding the molecular epidemiology of MRSA has been hindered by the lack of appropriate laboratory infrastructure and the limited capacity for molecular data analysis. This study, the largest genomic study of S. aureus in Ghana, addresses this gap by utilizing whole genome sequencing to examine the diversity of circulating S. aureus strains from 10 hospitals. Our findings highlight the predominance of pandemic clones, particularly ST152, and the notable transition of ST152 MSSA to ST152 MRSA over the past decade. The findings from this study supports AMR surveillance efforts in Ghana and emphasize the importance of implementing genomic surveillance using WGS to comprehensively monitor the rise and spread of multi-drug-resitant organisms such as MRSA in the country.

Objectives: To characterize the genetic basis of azithromycin resistance in Escherichia coli and Salmonella collected within the EU harmonized antimicrobial resistance (AMR) surveillance programme in 2014-18 and the Danish AMR surveillance programme in 2016-19., Methods: WGS data of 1007 E. coli [165 azithromycin resistant (MIC > 16 mg/L)] and 269 Salmonella [29 azithromycin resistant (MIC > 16 mg/L)] were screened for acquired macrolide resistance genes and mutations in rplDV, 23S rRNA and acrB genes using ResFinder v4.0, AMRFinder Plus and custom scripts. Genotype-phenotype concordance was determined for all isolates. Transferability of mef(C)-mph(G)-carrying plasmids was assessed by conjugation experiments., Results: mph(A), mph(B), mef(B), erm(B) and mef(C)-mph(G) were detected in E. coli and Salmonella, whereas erm(C), erm(42), ere(A) and mph(E)-msr(E) were detected in E. coli only. The presence of macrolide resistance genes, alone or in combination, was concordant with the azithromycin-resistant phenotype in 69% of isolates. Distinct mph(A) operon structures were observed in azithromycin-susceptible (n = 50) and -resistant (n = 136) isolates. mef(C)-mph(G) were detected in porcine and bovine E. coli and in porcine Salmonella enterica serovar Derby and Salmonella enterica 1,4, [5],12:i:-, flanked downstream by ISCR2 or TnAs1 and associated with IncIγ and IncFII plasmids., Conclusions: Diverse azithromycin resistance genes were detected in E. coli and Salmonella from food-producing animals and meat in Europe. Azithromycin resistance genes mef(C)-mph(G) and erm(42) appear to be emerging primarily in porcine E. coli isolates. The identification of distinct mph(A) operon structures in susceptible and resistant isolates increases the predictive power of WGS-based methods for in silico detection of azithromycin resistance in Enterobacterales., (© The Author(s) 2024. Published by Oxford University Press on behalf of British Society for Antimicrobial Chemotherapy.)

In 2023, an increase of OXA-48-producing Klebsiella pneumoniae was noticed by the Lithuanian National Public Health Surveillance Laboratory. Whole genome sequencing (WGS) of 106 OXA-48-producing K. pneumoniae isolates revealed three distinct clusters of carbapenemase-producing K. pneumoniae high-risk clones, including sequence type (ST) 45 (n = 35 isolates), ST392 (n = 32) and ST395 (n = 28), involving six, six and nine hospitals in different regions, respectively. These results enabled targeted investigation and control, and underscore the value of national WGS-based surveillance for antimicrobial resistance.

We created a database of all currently known mobile colistin resistance genes and variants ( n = 115). It contains accession numbers of the gene and protein sequences, mutations between the protein variants and the main proteins, and additional metadata. It is accompanied by all genetic and protein sequences as two aggregated FASTA files., Competing Interests: The authors declare no conflict of interest.

Introduction: Salmonella is considered one of the most significant pathogens in public health since it is a bacterium that is frequently linked to food-borne illnesses in humans. Some Salmonella serovars are responsible for outbreaks that are connected to the consumption of animal products. Cattle are connected to humans through a shared environment and the food chain as a significant source of animal protein. In Nigeria, antimicrobial medications are easily accessible for use in food-producing animals. Abattoir environments are reservoirs of foodborne bacteria like non-typhoidal Salmonella enterica (NTS), that have become resistant to antibiotics used for prophylaxis or treatment in animals. This study investigated the prevalence and resistance patterns of Salmonella enterica serovars in abattoir employees, beef cattle and abattoir environments in Abuja and Lagos, Nigeria., Methods: A total of 448 samples were collected from healthy personnel, slaughtered cattle, and abattoir environments between May and December 2020. Using Kirby-Bauer disk diffusion method, the resistance profile of NTS isolates were determined. Multidrug resistance (MDR) was considered when NTS was resistant to ≥3 antimicrobial drug classes. We performed phenotypic and genotypic characterizations of all Salmonella isolates including serotyping. Descriptive statistics were used to analyze the data., Results: Twenty-seven (6%) NTS isolates were obtained. Prevalence of NTS was highest in abattoir environments (15.5%; 9/58), followed by cattle (4.8%;13/272) and abattoir employees (4.2%; 5/118). A high prevalence of resistance was observed for gentamicin (85.2%; 23/27) and tetracycline (77.8%; 21/27). Whole-genome sequencing of 22 NTS showed dissemination of aac(6')-laa (22/22), qnrB19 (1/22), fosA7 (1/22), and tetA (1/22) genes. Serovar diversity of NTS varied with source. S. Anatum, a rare serovar predominated with a prevalence of 18.2% (4/22). Chromosomal point mutations showed ParC T57S substitution in 22 NTS analyzed. Among 22 NTS, 131 mobile genetic elements (MGEs) were detected including insertion sequences (56.5%) and miniature inverted repeats (43.5%). Two integrating MGEs IS6 and IS21 were observed to carry the tetA gene + Incl-1 on the same contig in NTS originating from cattle. Rare serovars namely S. Abony and S. Stormont with MDR phenotypes recovered from cattle and abattoir environments were closely related with a pairwise distance of ≤5 SNPs., Conclusions: First report of rare serovars in Nigeria with MDR phenotypes in humans, cattle, and abattoir environments. This study demonstrates the spread of resistance in the abattoir environment possibly by MGEs and emphasizes the importance of genomic surveillance. Beef cattle may be a risk to public health because they spread a variety of rare Salmonella serovars. Therefore, encouraging hand hygiene among abattoir employees while processing beef cattle will further reduce NTS colonization in this population. This requires a One Health collaborative effort among various stakeholders in human health, animal health, and environmental health., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Aworh et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Background: Strengthening external quality assessment (EQA) services across the One Health sector supports implementation of effective antimicrobial resistance (AMR) control strategies. Here we describe and compare 2 different approaches for conducting virtual laboratory follow-up assessments within an EQA program to evaluate quality management system (QMS) and procedures for pathogen identification and antimicrobial susceptibility testing (AST)., Methods: During the coronavirus disease 2019 (COVID-19) pandemic in 2021 and 2022, 2 laboratory assessment approaches were introduced: virtual-based and survey-based methodologies. The evaluation of 2 underperforming Animal Health laboratories through a virtual-based approach occurred between May and August 2021. This evaluation encompassed the utilization of 3 online meetings and document reviews, performed subsequent to the execution of EQA procedures. Within a distinct group of laboratories, the survey-based assessment was implemented from December 2021 to February 2022, also following EQA procedures. This phase encompassed the dissemination of an online survey to 31 participating laboratories, alongside a sole online consultation meeting involving 4 specific underperforming laboratories., Results: The virtual-based assessment post-EQA aimed to identify gaps and areas for improvement in the laboratory's practices for pathogen identification and AST. This approach was, however, time-intensive, and, hence, only 2 laboratories were assessed. In addition, limited interactions in virtual platforms compromised the assessment quality. The survey-based post-EQA assessment enabled evaluation of 31 laboratories. Despite limitations for in-depth analysis of each procedure, gaps in QMS across multiple laboratories were identified and tailored laboratory-specific recommendations were provided., Conclusions: Reliable internet and plans for efficient time management, post-EQA virtual laboratory follow-up assessments are an effective alternative when conducting onsite evaluation is infeasible as observed during the COVID-19 pandemic, although the successful implementation of remediation plans will likely require in person assessments. We advocate application of hybrid approaches (both onsite and virtual) for targeted capacity building of AMR procedures with the ability to implement and oversee the process., Competing Interests: Potential conflicts of interest. The authors: No reported conflicts of interest. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest., (© The Author(s) 2023. Published by Oxford University Press on behalf of Infectious Diseases Society of America.)

Introduction: Enterococcus spp. have gradually evolved from commensals to causing life-threatening hospital-acquired infections globally due to their inherent antimicrobial resistance ability and virulence potential. Enterococcus spp. recovered from livestock and raw meat samples were characterized using antimicrobial susceptibility testing and whole-genome sequencing., Materials and Methods: Isolates were confirmed using the MALDI-ToF mass spectrometer, and antimicrobial susceptibility was determined using the Kirby-Bauer disk diffusion method. Whole genome sequencing was performed on isolates resistant to two or more antibiotics. Bioinformatics analysis was performed to determine sequence types, resistance and virulence gene content and evolutionary relationships between isolates from meat and livestock samples, and other enterococci genomes curated by PATRIC. eBURST analysis was used to assign genomes to clonal complexes., Results: Enterococcus spp. were predominantly E. faecalis (96/236; 41%) and E. faecium (89/236; 38%). Overall, isolates showed resistance to erythromycin (78/236; 33%), tetracycline (71/236; 30%), ciprofloxacin (20/236; 8%), chloramphenicol (12/236; 5%), linezolid (7/236; 3%), ampicillin (4/236; 2%) and vancomycin (1/236, 0.4%). Resistance to two or more antimicrobial agents was detected among 17% ( n  = 40) Enterococcus spp. Resistance genes for streptogramins [ lsa(A), lsa(E), msr(C) ], aminoglycosides [ aac(6')-Ii, aph(3')-III, ant(6)-Ia, aac(6')-aph(2″), str], amphenicol [ cat ], macrolides [ erm(B), erm(T), msr(C) ], tetracyclines [ tet(M), tet(L), tet(S) ] and lincosamides [ lsa(A), lsa(E), lnu(B) ] were detected among the isolates. Genes for biofilm formation, adhesins, sex pheromones, cytolysins, hyaluronidase, oxidative stress resistance, quorum-sensing and anti-phagocytic activity were also identified. Potential plasmids with replicon sequences ( rep1, rep2, repUS43, repUS47, rep9a, rep9b ) and other mobile genetic elements ( Tn917, cn_5536_ISEnfa1, Tn6009, ISEnfa1, ISEfa10 ) were detected. Clinically relevant E. faecium ST32 and ST416 clones were identified in meat samples., Conclusion: The occurrence of antimicrobial-resistant Enterococcus spp. in livestock and raw meat samples, carrying multiple resistance and virulence genes, including known clones associated with hospital-acquired infections, underscores the critical need for employing robust tools like whole genome sequencing. Such tools provide detailed data essential for ongoing surveillance efforts aimed at addressing the challenge of antimicrobial resistance with a focus on one health., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Amuasi, Dsani, Owusu-Nyantakyi, Owusu, Mohktar, Nilsson, Adu, Hendriksen and Egyir.)

Historically, epidemiological investigation and surveillance for bacterial antimicrobial resistance (AMR) has relied on low-resolution isolate-based phenotypic analyses undertaken at local and national reference laboratories. Genomic sequencing has the potential to provide a far more high-resolution picture of AMR evolution and transmission, and is already beginning to revolutionise how public health surveillance networks monitor and tackle bacterial AMR. However, the routine integration of genomics in surveillance pipelines still has considerable barriers to overcome. In 2022, a workshop series and online consultation brought together international experts in AMR and pathogen genomics to assess the status of genomic applications for AMR surveillance in a range of settings. Here we focus on discussions around the use of genomics for public health and international AMR surveillance, noting the potential advantages of, and barriers to, implementation, and proposing recommendations from the working group to help to drive the adoption of genomics in public health AMR surveillance. These recommendations include the need to build capacity for genome sequencing and analysis, harmonising and standardising surveillance systems, developing equitable data sharing and governance frameworks, and strengthening interactions and relationships among stakeholders at multiple levels., Competing Interests: Declaration of interests KSB reports funding from the Biotechnology and Biological Sciences Research Council and Medical Research Council (MRC) and partial salary cover from Wellcome Trust and the UK Health Security Agency (UKHSA) over the course of this work. EJ had partial salary cover from Wellcome Trust over the course of this work. SWL has received the Robert Austrian Research Award sponsored by Pfizer in 2022. DGA reports funding from the NIHR. INO reports funding from the Bill & Melinda Gates Foundation, Joint Programming Intiative on Antimicrobial Resistance, Wellcome Trust, Grand Challenges Africa Award, and UK MRC; royalties for Genetics: Genes, Genomes and Evolution (Oxford University Press), and Divining Without Seeds and for Antimicrobial Resistance in Developing Countries (Springer); consulting fees from Wellcome Trust; honoraria for Harvard University seminars; and the Peter Wildy Lecture Award 2023. DGA, BE, RSH, and INO report receiving funding from the UK Department of Health and Social Care in the form of a grant managed by the Fleming Fund and work performed under the auspices of the SEQAFRICA project. LS-B reports funding by Conselleria de Sanitat Universal i Salut Pública, Generalitat Valenciana, Valencia (Spain), under Plan GenT (reference number CDEI-06/20-B). NAF reports funding from the Gates Foundation, UK Research and Innovation, and NIHR. SJP is a member of the Scientific Advisory Board of Next Gen Diagnostics and was supported by Illumina to attend the European Congress of Clinical Microbiology and Infectious Disease conference. All other authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Ltd. This is an Open Access article under the CC BY 4.0 license. Published by Elsevier Ltd.. All rights reserved.)

Nearly a century after the beginning of the antibiotic era, which has been associated with unparalleled improvements in human health and reductions in mortality associated with infection, the dwindling pipeline for new antibiotic classes coupled with the inevitable spread of antimicrobial resistance (AMR) poses a major global challenge. Historically, surveillance of bacteria with AMR typically relied on phenotypic analysis of isolates taken from infected individuals, which provides only a low-resolution view of the epidemiology behind an individual infection or wider outbreak. Recent years have seen increasing adoption of powerful new genomic technologies with the potential to revolutionise AMR surveillance by providing a high-resolution picture of the AMR profile of the bacteria causing infections and providing real-time actionable information for treating and preventing infection. However, many barriers remain to be overcome before genomic technologies can be adopted as a standard part of routine AMR surveillance around the world. Accordingly, the Surveillance and Epidemiology of Drug-resistant Infections Consortium convened an expert working group to assess the benefits and challenges of using genomics for AMR surveillance. In this Series, we detail these discussions and provide recommendations from the working group that can help to realise the massive potential benefits for genomics in surveillance of AMR., Competing Interests: Declaration of interests KSB reports funding from the Biotechnology and Biological Sciences Research Council and Medical Research Council and partial salary cover from Wellcome Trust and the UK Health Security Agency (UKHSA) over the course of this work. EJ had partial salary cover from Wellcome Trust over the course of this work. RA reports funding unrelated to this study from Novo Nordisk, Roche, Novartis, and UICC, and honoraria (unrelated to this study) from Merck & Co, Novartis, and F Hoffmann-La Roche. BE and INO report receiving funding from the UK Department of Health and Social Care: with a grant managed by the Fleming Fund and work performed under the auspices of the SEQAFRICA project. INO reports funding from the Bill & Melinda Gates Foundation, Joint Programming Initiative in Antimicrobial Resistance, Wellcome Trust, Grand Challenges Africa Award, and UK Medical Research Council, royalties for Genetics: Genes, Genomes and Evolution (Oxford University Press) and Divining Without Seeds and for Antimicrobial Resistance in Developing Countries (Springer), consulting fees from Wellcome Trust, and honoraria for Harvard University seminars and Peter Wildy Lecture Award 2023. LYH reports funding from Pfizer and honoraria from BioMerieux for a lecture in 2022. DMM reports funding from the British Society for Antimicrobial Chemotherapy. NEW reports funding from Nuclear Threat Initiative, Medical Research Council, Open Philantropy, and Shionogi as well as consulting fees from Nuclear Threat Initiative. DMA reports funding from the National Institute for Health and Care Research. NAF reports funding from the Bill & Melinda Gates Foundation, UK Research and Innovation, and National Institute for Health and Care Research. SJP is a member of the scientific advisory board of Next Gen Diagnostics and was supported by Illumina to attend the European Congress of Clinical Microbiology and Infectious Disease conference. All other authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Ltd. This is an Open Access article under the CC BY 4.0 license. Published by Elsevier Ltd.. All rights reserved.)

The emergence and rapid spread of the plasmid-mediated colistin-resistant mcr-1 gene introduced a serious threat to public health. In 2021, a multi-drug resistant, mcr-1 positive Escherichia coli EC1945 strain, was isolated from pig caecal content in Croatia. Antimicrobial susceptibility testing and whole genome sequencing were performed. Bioinformatics tools were used to determine the presence of resistance genes, plasmid Inc groups, serotype, sequence type, virulence factors, and plasmid reconstruction. The isolated strain showed phenotypic and genotypic resistance to nine antimicrobial classes. It was resistant to colistin, gentamicin, ampicillin, cefepime, cefotaxime, ceftazidime, sulfamethoxazole, chloramphenicol, nalidixic acid, and ciprofloxacin. Antimicrobial resistance genes included mcr-1, bla TEM-1B , bla CTX-M-1 , aac(3)-IId , aph(3')-Ia , aadA5 , sul2 , catA1 , gyrA (S83L, D87N), and parC (A56T, S80I). The mcr-1 gene was located within the conjugative IncX4 plasmid. IncI1, IncFIB, and IncFII plasmids were also detected. The isolate also harbored 14 virulence genes and was classified as ST744 and O101:H10. ST744 is a member of the ST10 group which includes commensal, extraintestinal pathogenic E. coli isolates that play a crucial role as a reservoir of genes. Further efforts are needed to identify mcr-1 -carrying E. coli isolates in Croatia, especially in food-producing animals to identify such gene reservoirs.

The Two Weeks in the World research project has resulted in a dataset of 3087 clinically relevant bacterial genomes with pertaining metadata, collected from 59 diagnostic units in 35 countries around the world during 2020. A relational database is available with metadata and summary data from selected bioinformatic analysis, such as species prediction and identification of acquired resistance genes., (© 2023. Springer Nature Limited.)

Background: The Global Typhoid Genomics Consortium was established to bring together the typhoid research community to aggregate and analyse Salmonella enterica serovar Typhi (Typhi) genomic data to inform public health action. This analysis, which marks 22 years since the publication of the first Typhi genome, represents the largest Typhi genome sequence collection to date (n=13,000)., Methods: This is a meta-analysis of global genotype and antimicrobial resistance (AMR) determinants extracted from previously sequenced genome data and analysed using consistent methods implemented in open analysis platforms GenoTyphi and Pathogenwatch., Results: Compared with previous global snapshots, the data highlight that genotype 4.3.1 (H58) has not spread beyond Asia and Eastern/Southern Africa; in other regions, distinct genotypes dominate and have independently evolved AMR. Data gaps remain in many parts of the world, and we show the potential of travel-associated sequences to provide informal 'sentinel' surveillance for such locations. The data indicate that ciprofloxacin non-susceptibility (>1 resistance determinant) is widespread across geographies and genotypes, with high-level ciprofloxacin resistance (≥3 determinants) reaching 20% prevalence in South Asia. Extensively drug-resistant (XDR) typhoid has become dominant in Pakistan (70% in 2020) but has not yet become established elsewhere. Ceftriaxone resistance has emerged in eight non-XDR genotypes, including a ciprofloxacin-resistant lineage (4.3.1.2.1) in India. Azithromycin resistance mutations were detected at low prevalence in South Asia, including in two common ciprofloxacin-resistant genotypes., Conclusions: The consortium's aim is to encourage continued data sharing and collaboration to monitor the emergence and global spread of AMR Typhi, and to inform decision-making around the introduction of typhoid conjugate vaccines (TCVs) and other prevention and control strategies., Funding: No specific funding was awarded for this meta-analysis. Coordinators were supported by fellowships from the European Union (ZAD received funding from the European Union's Horizon 2020 research and innovation programme under the Marie Sklodowska-Curie grant agreement No 845681), the Wellcome Trust (SB, Wellcome Trust Senior Fellowship), and the National Health and Medical Research Council (DJI is supported by an NHMRC Investigator Grant [GNT1195210])., Competing Interests: MC, ZD, DI, AA, MA, MC, KC, JC, BH, KK, MM, CP, SV, HW, AA, AA, SA, JA, PA, BB, AB, JC, Kd, AD, Jd, PD, CD, SD, ME, LF, DG, GG, MG, AG, CG, MG, RH, RH, YH, JH, OI, JI, JJ, CJ, DJ, JJ, GK, AK, AK, AK, SK, RK, RK, AL, ML, RL, SL, GM, TM, CM, AM, GN, SN, SN, TN, SN, EN, IO, SP, AP, FQ, FQ, SR, SR, DR, PR, RR, TR, JR, SS, SS, KS, MS, JS, JS, VS, JS, RS, SS, MS, AS, AS, KT, DT, AT, MT, MT, RT, NT, ST, KV, MV, BV, FW, JW, GD, SA, JK, DA, SB, KH No competing interests declared, NF NAF chairs the Wellcome Surveillance and Epidemiology of Drug Resistant Infections (SEDRIC) group, which has a focus on antimicrobial resistance. This could be perceived as relevant although not a direct conflict, IB IB has consulted to BlueDot and the NHL Players' Association, AP AJP is chair of the UK Department of Health and Social Care's (DHSC) Joint Committee on Vaccination and Immunisation (JCVI) but does not take part in the JCVI COVID-19 committee. He was a member of WHO SAGE until 2022. AJPs employer, Oxford University has entered into a partnership with AstraZeneca for development of a COVID-19 vaccine. AJP has provided advice to Shionogi & Co., Ltd on development of a COVID19 vaccine, (© 2023, Carey et al.)

Introduction: Extended-spectrum β-lactamase- (ESBL) and AmpC- β-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 β-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)., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Hendriksen, Cavaco, Guerra, Bortolaia, Agersø, Svendsen, Nielsen, Kjeldgaard, Pedersen, Fertner and Hasman.)

The global surveillance and outbreak investigation of antimicrobial resistance (AMR) is amidst a paradigm shift from traditional biology to bioinformatics. This is due to developments in whole-genome-sequencing (WGS) technologies, bioinformatics tools, and reduced costs. The increased use of WGS is accompanied by challenges such as standardization, quality control (QC), and data sharing. Thus, there is global need for inter-laboratory WGS proficiency test (PT) schemes to evaluate laboratories' capacity to produce reliable genomic data. Here, we present the results of the first iteration of the Genomic PT (GPT) organized by the Global Capacity Building Group at the Technical University of Denmark in 2020. Participating laboratories sequenced two isolates and corresponding DNA of Salmonella enterica , Escherichia coli and Campylobacter coli , using WGS methodologies routinely employed at their laboratories. The participants' ability to obtain consistently good-quality WGS data was assessed based on several QC WGS metrics. A total of 21 laboratories from 21 European countries submitted WGS and meta-data. Most delivered high-quality sequence data with only two laboratories identified as overall underperforming. The QC metrics, N50 and number of contigs, were identified as good indicators for high-sequencing quality. We propose QC thresholds for N50 greater than 20 000 and 25 000 for Campylobacter coli and Escherichia coli, respectively, and number of contigs >200 bp greater than 225, 265 and 100 for Salmonella enterica , Escherichia coli and Campylobacter coli , respectively. The GPT2020 results confirm the importance of systematic QC procedures, ensuring the submission of reliable WGS data for surveillance and outbreak investigation to meet the requirements of the paradigm shift in methodology.

The Inter European Union Reference Laboratories (EURLs) Working Group on Next Generation Sequencing (NGS) involves eight EURLs for microbiological food and feed hazards and has been working since 2017 to promote the adoption of NGS by the National Reference Laboratories (NRLs) in the European Union. This work illustrates the results of the first 5 years of activity. By working together, the EURLs involved have released guidance documents for assisting NRLs in all the steps of NGS, helping the transition from classical molecular methods towards whole genome sequencing while ensuring harmonization, with the final aim of improving preparedness in the use of NGS to characterize microbial hazards and trace the sources of infection.

Antimicrobial resistant Salmonella enterica serovar Concord (S. Concord) is known to cause severe gastrointestinal and bloodstream infections in patients from Ethiopia and Ethiopian adoptees, and occasional records exist of S. Concord linked to other countries. The evolution and geographical distribution of S. Concord remained unclear. Here, we provide a genomic overview of the population structure and antimicrobial resistance (AMR) of S. Concord by analysing genomes from 284 historical and contemporary isolates obtained between 1944 and 2022 across the globe. We demonstrate that S. Concord is a polyphyletic serovar distributed among three Salmonella super-lineages. Super-lineage A is composed of eight S. Concord lineages, of which four are associated with multiple countries and low levels of AMR. Other lineages are restricted to Ethiopia and horizontally acquired resistance to most antimicrobials used for treating invasive Salmonella infections in low- and middle-income countries. By reconstructing complete genomes for 10 representative strains, we demonstrate the presence of AMR markers integrated in structurally diverse IncHI2 and IncA/C2 plasmids, and/or the chromosome. Molecular surveillance of pathogens such as S. Concord supports the understanding of AMR and the multi-sector response to the global AMR threat. This study provides a comprehensive baseline data set essential for future molecular surveillance., (© 2023. The Author(s).)

Objectives: Analysing samples of municipal wastewater influent (before treatment) can help to map the status of antibiotic-resistant bacteria (ARB) at the population level in sewershed communities and may also help in predicting the public health risks of ARB in surface water because of the outfall of wastewater. In this study, we investigated the bacterial isolates carrying beta-lactamase genes in wastewater and compared their genotypic and phenotypic characteristics., Methods: A total of 399 bacterial isolates grown on CHROMagarESBL (n = 207) and CHROMagarKPC (n = 192) from composite wastewater influent samples (n = 7) from the Viikinmäki wastewater treatment plant (Helsinki) were subcultured, nucleic acid was extracted, and the prevalence of different beta-lactamase genes was screened with multiplex polymerase chain reaction (PCR). All PCR-positive isolates were identified with MALDI-TOF., Results: A total of 32.6% of isolates (130 of 399) were PCR positive for at least one resistance gene, and 13% of these positive isolates out of 130 had at least three resistance genes. Among the 22 detected genes, bla GES group was the most prevalent, at 25.8% (n = 198; many isolates carried multiple genes), followed by bla MOX (13.1%) and bla TEM (10.1%) as most frequently detected. Furthermore, out of 18 different bacterial species/genera detected as carrying beta-lactamase genes, A. hydrophila/caviae (28.5%), Enterobacter spp. (16.9%), and E. coli (14.6%) were the most prevalent. Enterobacter spp., Aeromonas spp., and K. cryocescens potentially carried AmpC genes, and E. coli carried ESBL genes., Conclusion: We recorded a huge variety of beta-lactamases (bla Amp C , bla ESBL , and bla CARBA ) genes in many potential pathogens that probably originated from both enteric and environmental sources., (Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Background: Commensal Escherichia coli residing in the guts of humans and animals are reservoirs of multidrug resistance (MDR) genes, including quinolone resistance genes, in humans and poultry. This study aimed to characterize quinolones resistance in E. coli recovered from poultry workers, chickens, and poultry farm/market environments in Abuja, Nigeria., Methods: This was a cross-sectional study conducted between December 2018 and April 2019 comprising poultry workers, chickens and their poultry farm/market environments. This study characterized E. coli isolates from stool, faecal and environmental samples using antimicrobial susceptibility testing and whole-genome sequencing methods. Core-genome multilocus sequences-based phylogeny was used to determine the relatedness between quinolone-resistant E. coli isolates. Data were analyzed using descriptive statistics., Results: Of 110 E. coli isolates, quinolone-resistant phenotypes were observed in 68.2% (n = 75) isolates. Whole-genome sequencing detected plasmid-mediated quinolone resistance (PMQR) genes in 63.6% (n = 70) isolates. The most prevalent PMQR gene detected in 56 of these 70 E. coli isolates was qnrS1, followed by qnrB19 in 14 isolates and aac(6')-lb-cr in two isolates. Fifteen ciprofloxacin and 19 nalidixic acid-resistant isolates respectively showed double mutations in the quinolone-resistance determining regions (QRDRs) of gyrA, with single or double mutations in parC, and a single mutation in parE. The most prevalent amino-acid substitutions observed were S83L + D87N in gyrA (46.5%, n = 20), S80I in parC (51.2%, n = 22) and S458A in parE (14%, n = 6). About 2.9% (2/70) of PMQR isolates were extended-spectrum beta-lactamase (ESBL) producers while 2.9% (2/70) had plasmid-mediated colistin resistance (PMCR) genes., Conclusions: PMQR genes were prevalent in E. coli isolates recovered from healthy humans, chickens and poultry farm/market environments. PMCR genes (mcr-1.1) occurred in PMQR-positive isolates recovered from manure and drinking water originating from poultry farm/market environments. It was found that the gene encoding ESBL coexisted with qnrS-positive isolates of human and avian origin. Horizontal transfer of PMQR genes among E. coli isolates in the human-poultry-environment interface has public health implications for the spread of antimicrobial resistance. Relevant government agencies should enforce regulations to restrict the use of critically important antimicrobials in poultry production., (© 2023. The Author(s).)

Whole-genome sequence data for clinically relevant Gram-negative bacteria from the African continent are scarce. In this report, we present the draft genome sequence data and antibiograms of four species, namely, Kerstersia gyiorum, Providencia vermicola, Providencia stuartii, and Alcaligenes faecalis, that were recovered from human soft tissue biopsy samples.

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., (© 2022. The Author(s).)

Bacteria of the Bacillus cereus group are Gram-positive rods and are widespread in nature, but little information is currently available about their presence in reptiles. Here, we report draft genome sequences of six Bacillus isolates belonging to three species, namely, Bacillus cereus, Bacillus paranthracis, and Bacillus toyonensis, isolated from pet reptiles in Poland.

A high burden of Salmonella enterica subspecies enterica serovar Typhi (S. Typhi) bacteremia has been reported from urban informal settlements in sub-Saharan Africa, yet little is known about the introduction of these strains to the region. Understanding regional differences in the predominant strains of S. Typhi can provide insight into the genomic epidemiology. We genetically characterized 310 S. Typhi isolates from typhoid fever surveillance conducted over a 12-year period (2007-2019) in Kibera, an urban informal settlement in Nairobi, Kenya, to assess the circulating strains, their antimicrobial resistance attributes, and how they relate to global S. Typhi isolates. Whole genome multi-locus sequence typing (wgMLST) identified 4 clades, with up to 303 pairwise allelic differences. The identified genotypes correlated with wgMLST clades. The predominant clade contained 290 (93.5%) isolates with a median of 14 allele differences (range 0-52) and consisted entirely of genotypes 4.3.1.1 and 4.3.1.2. Resistance determinants were identified exclusively in the predominant clade. Determinants associated with resistance to aminoglycosides were observed in 245 isolates (79.0%), sulphonamide in 243 isolates (78.4%), trimethoprim in 247 isolates (79.7%), tetracycline in 224 isolates (72.3%), chloramphenicol in 247 isolates (79.6%), β-lactams in 239 isolates (77.1%) and quinolones in 62 isolates (20.0%). Multidrug resistance (MDR) determinants (defined as determinants conferring resistance to ampicillin, chloramphenicol and cotrimoxazole) were found in 235 (75.8%) isolates. The prevalence of MDR associated genes was similar throughout the study period (2007-2012: 203, 76.3% vs 2013-2019: 32, 72.7%; Fisher's Exact Test: P = 0.5478, while the proportion of isolates harboring quinolone resistance determinants increased (2007-2012: 42, 15.8% and 2013-2019: 20, 45.5%; Fisher's Exact Test: P<0.0001) following a decline in S. Typhi in Kibera. Some isolates (49, 15.8%) harbored both MDR and quinolone resistance determinants. There were no determinants associated with resistance to cephalosporins or azithromycin detected among the isolates sequenced in this study. Plasmid markers were only identified in the main clade including IncHI1A and IncHI1B(R27) in 226 (72.9%) isolates, and IncQ1 in 238 (76.8%) isolates. Molecular clock analysis of global typhoid isolates and isolates from Kibera suggests that genotype 4.3.1 has been introduced multiple times in Kibera. Several genomes from Kibera formed a clade with genomes from Kenya, Malawi, South Africa, and Tanzania. The most recent common ancestor (MRCA) for these isolates was from around 1997. Another isolate from Kibera grouped with several isolates from Uganda, sharing a common ancestor from around 2009. In summary, S. Typhi in Kibera belong to four wgMLST clades one of which is frequently associated with MDR genes and this poses a challenge in treatment and control., Competing Interests: The authors have declared that no competing interests exist.

Antimicrobial resistance profiling of pathogens helps to identify the emergence of rare or new resistance threats and prioritize possible actions to be taken against them. The analysis of wastewater (WW) can reveal the circulation of antimicrobial-resistant bacteria (ARB) and antimicrobial resistance genes (ARG) among the catchment communities. Here, we analyzed WW influent samples to determine the prevalence of carbapenemase genes-carrying Gram-negative bacteria (Carba-GNB) in Helsinki, Finland. This study set important historical reference points from the very early stage of the carbapenemase era, during the period 2011-2012. A total of 405 bacterial isolates grown on CHROMagarKPC ( n  = 195) and CHROMagarESBL ( n  = 210) from WW influent samples were collected between October 2011 and August 2012 and were analyzed. The bacterial DNA from the isolates was extracted, and the prevalence of carbapenemases genes bla KPC , bla NDM , bla GES , bla OXA-48 , bla IMP , bla IMI , and bla VIM were screened with multiplexed PCR. All carbapenemase-positive isolates were identified taxonomically to species or genus level with matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). The nucleic acid extraction was successful for 399 isolates, of which 59 (14.8%) were found to carry carbapenemase genes. A total of 89.8% of the carbapenemase positive isolates (53 out of 59) were obtained from CHROMagarKPC plates and only 10.2% (six out of 59) were obtained from CHROMagar ESBL plates. Among the Carba-GNB isolates, 86.4% were bla GES (51 out of 59), 10.2% were bla KPC (six out of 59), and 3.4% were bla VIM (two out of 59). The most common carba-gene, bla GES , was carried by 10 different bacterial species, including Aeromonas spp., Enterobacter spp., and Kluyvera spp.; the bla KPC gene was carried by Escherichia coli , Klebsiella pneumoniae , and Kluyvera cryocescens ; and the bla VIM gene was carried by Aeromonas hydrophila / caviae and Citrobacter amalonaticus . This study emphasizes that wastewater surveillance (WWS) can be an additional tool for monitoring antimicrobial resistance (AMR) at the population level., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Tiwari, Paakkanen, Österblad, Kirveskari, Hendriksen and Heikinheimo.)

Introduction: Beef cattle, one of the food-producing animals, are linked to humans through a shared environment and the food chain as a major source of animal protein. Antimicrobial drugs are readily accessible for use in food animal production in Nigeria. Beef cattle and abattoir environments harbor pathogenic bacteria such as Escherichia coli ( E. coli ) which have developed resistance to antimicrobial agents used for prophylaxis or treatment. This study investigated the zoonotic transmission of extended-spectrum beta-lactamase-producing E. coli (ESBL-EC) among humans, beef cattle, and abattoir environments in Abuja and Lagos, Nigeria., Materials and Methods: We conducted a cross-sectional study among abattoir workers, beef cattle, and abattoir environments in Abuja and Lagos. Stool, cecal, and environmental samples were collected from apparently healthy workers, slaughtered cattle, and abattoir environments from May to December 2020. Data were collected electronically using open data kit app installed on a mobile phone. Antimicrobial susceptibility patterns were determined using the Kirby-Bauer disk diffusion method against a panel of 16 antimicrobial agents. Phenotypic and genotypic characterizations of the isolates were conducted. Data were analyzed with descriptive statistics., Results: From 21.7% ( n = 97) of 448 samples, ESBL-EC were isolated and further characterized. Prevalence of ESBL-EC was highest in cattle (45.4%; n = 44), abattoir workers (41.2%; n = 40), and abattoir environment (13.4%; n = 13). Whole-genome sequencing of ESBL-EC showed dissemination of bla CTX-M-15 (90.7%; n = 88); bla CTX-M-14 (5.2%; n = 5); and bla CTX-M-55 (2.1%; n = 2) genes. The bla CTX-M-15 coexisted with bla CTX-M-14 and bla TEM-1 genes in 2.1% ( n = 2) and 39.2% ( n = 38) of the isolates, respectively. The presence of bla CTX-M-14 and bla CTX-M-15 genes was significantly associated with isolates originating from abattoir workers when compared with beef cattle isolates ( p = 0.05; p < 0.01). The most prevalent sequence types (ST) were ST10 ( n = 11), ST215 ( n = 7), ST4684 ( n = 7), and ST2178 ( n = 6). ESBL-EC strain (ST 205 / B1 ) harbored mcr -1.1 and bla CTX-M15 and was isolated from a worker at Lagos abattoir. In 91 ESBL-EC isolates, 219 mobile genetic elements (MGEs) harbored resistance genes out of which β-lactam genes were carried on 64 different MGEs. Isolates showed equal distribution of insertion sequences and miniature inverted repeats although only a few composite transposons were detected (humans n = 12; cattle n = 9; environment n = 4). Two isolates of human and cattle origin (ST46/A) harboring ESBL genes and carried by MGEs were clonally related., Conclusions: This is the first report of bla CTX-M-55 gene in humans and cattle in Nigeria. This study demonstrates the horizontal transfer of ESBL genes possibly by MGEs and buttresses the importance of genomic surveillance. Healthcare workers should be sensitized that people working closely with cattle or in abattoir environments are a high-risk group for fecal carriage of ESBL-EC when compared with the general population., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Aworh, Ekeng, Nilsson, Egyir, Owusu-Nyantakyi and Hendriksen.)

Introduction: Non-typhoidal Salmonella are major foodborne pathogens causing serious challenges to public health and food safety worldwide. This study aimed to determine the resistance, virulence genes, sequence type, using multi-locus sequence typing, plasmids and Single Nucleotide Polymorphisms (SNPs) of Salmonella enterica subsp. enterica serovar Nigeria (S. Nigeria) from livestock in Ilorin, North central Nigeria., Methodology: A total of 1,500 samples from pig (feces; n = 600) and poultry (feces, postmortem samples; n = 900) were collected and analyzed between 2014 to 2017. Presumptive Salmonella isolates were characterized by Whole Genome Sequencing (WGS)., Results: We recovered nine S. Nigeria serovars. All the isolates harbored a single point mutation parC(T57S) in addition to qnrB19 and the tetA gene. Furthermore, two plasmids, Col(pHAD28) and IncQ1 predicted to encode qnrB19 and tetA genes, respectively, were detected in all the strains. All the isolates belonged to a single sequence type (ST) 4911, the SNP-based phylogeny showed all the isolates to be highly related, in addition two clinical isolates from the United Kingdom (UK) and Canada, collected outside of this study, also fell into this cluster. Twenty virulence genes were identified from Salmonella Pathogenicity Islands (SPI), chromosomal and fimbriae loci., Conclusions: This study highlights the roles of pig and poultry in the emergence and spread of S. Nigeria serovar in Nigeria, sub-Sahara Africa. It also highlighted the importance of WGS in clinical and epidemiological surveillance. There is the need for collaborative research studies to investigate the public health importance of Salmonella enterica serovar Nigeria., Competing Interests: No Conflict of Interest is declared, (Copyright (c) 2021 Ibrahim A Raufu, Olayiwola A Ahmed, Abdulfatai Aremu, James A Ameh, Ruth E Timme, Rene S Hendriksen, AbdulGaniyu Ambali.)

Introduction: Establishing effective external quality assessment (EQA) programmes is an important element in ensuring the quality of, and building capacity for, antimicrobial resistance (AMR) laboratory surveillance., Objectives: To understand the current coverage of, and challenges to participation in, EQAs in National Reference Laboratories (NRLs) across One Health (OH) sectors in Asia., Methods: Current EQA coverage was evaluated through desktop review, online surveys and interviews of both EQA participants and providers. EQA coverage was mapped and summarized by laboratory type and 'readiness' level and identified challenges evaluated qualitatively., Results: Of the 31 identified NRLs [16 Human Health (HH) and 15 Animal/Food Safety laboratories (A/FS)], 14 HH and 7 A/FS laboratories currently participated in international EQA schemes and several participated in two or more different schemes. Seven laboratories were currently not participating in any EQA scheme and two of these (one HH and one A/FS) do not currently perform microbiology; six HH NRLs provided national EQAs. Of the eight surveyed international EQA providers, three were based in Asia and all offered varying programmes in terms of pathogens, frequency and support mechanisms for reporting and follow-up. Only one provider currently served laboratories across all OH sectors., Conclusions: The current coverage of EQA programmes for AMR in Asia was heterogeneous across countries but especially across OH sectors. This updated overview of the coverage and challenges associated with participation in, and provision of, EQAs for AMR suggest the benefit and relevance of introducing one comprehensive and high-quality EQA programme across OH sectors in Asia., (© The Author(s) 2021. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy.)

Objectives: The COVID-19 pandemic has had a substantial impact on health systems. The WHO Antimicrobial Resistance (AMR) Surveillance and Quality Assessment Collaborating Centres Network conducted a survey to assess the effects of COVID-19 on AMR surveillance, prevention and control., Methods: From October to December 2020, WHO Global Antimicrobial Resistance and Use Surveillance System (GLASS) national focal points completed a questionnaire, including Likert scales and open-ended questions. Data were descriptively analysed, income/regional differences were assessed and free-text questions were thematically analysed., Results: Seventy-three countries across income levels participated. During the COVID-19 pandemic, 67% reported limited ability to work with AMR partnerships; decreases in funding were frequently reported by low- and middle-income countries (LMICs; P < 0.01). Reduced availability of nursing, medical and public health staff for AMR was reported by 71%, 69% and 64%, respectively, whereas 67% reported stable cleaning staff availability. The majority (58%) reported reduced reagents/consumables, particularly LMICs (P < 0.01). Decreased numbers of cultures, elective procedures, chronically ill admissions and outpatients and increased ICU admissions reported could bias AMR data. Reported overall infection prevention and control (IPC) improvement could decrease AMR rates, whereas increases in selected inappropriate IPC practices and antimicrobial prescribing could increase rates. Most did not yet have complete data on changing AMR rates due to COVID-19., Conclusions: This was the first survey to explore the global impact of COVID-19 on AMR among GLASS countries. Responses highlight important actions to help ensure that AMR remains a global health priority, including engaging with GLASS to facilitate reliable AMR surveillance data, seizing the opportunity to develop more sustainable IPC programmes, promoting integrated antibiotic stewardship guidance, leveraging increased laboratory capabilities and other system-strengthening efforts., (© The Author(s) 2021. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy.)

Background: A plasmid-mediated mechanism of bacterial resistance to polymyxin is a serious threat to public health worldwide. The present study aimed to determine the occurrence of plasmid-mediated colistin resistance genes and to conduct the molecular characterization of mcr-positive Escherichia coli strains isolated from Polish poultry., Methods: In this study, 318 E. coli strains were characterized by the prevalence of mcr1-mcr5 genes, antimicrobial susceptibility testing by minimal inhibitory concentration method, the presence of antimicrobial resistance genes was screened by PCR, and the biofilm formation ability was tested using the crystal violet staining method. Genetic relatedness of mcr-1-positive E. coli strains was evaluated by multilocus sequence typing method., Results: Among the 318 E. coli isolates, 17 (5.35%) harbored the mcr-1 gene. High antimicrobial resistance rates were observed for ampicillin (100%), tetracycline (88.24%), and chloramphenicol (82.35%). All mcr-1-positive E. coli strains were multidrug-resistant, and as many as 88.24% of the isolates contained the bla TEM gene, tetracycline (tetA and tetB), and sulfonamide (sul1, sul2, and sul3) resistance genes. Additionally, 41.18% of multidrug-resistant, mcr-1-positive E. coli isolates were moderate biofilm producers, while the rest of the strains showed weak biofilm production. Nine different sequence types were identified, and the dominant ST was ST93 (29.41%), followed by ST117 (17.65%), ST156 (11.76%), ST 8979 (11.76%), ST744 (5.88%), and ST10 (5.88%). Moreover, the new ST was identified in this study., Conclusions: Our results showed a low occurrence of mcr-1-positive E. coli strains isolated from Polish poultry; however, all the isolated strains were resistant to multiple antimicrobial agents and were able to form biofilms at low or medium level., (© 2021. The Author(s).)

The emergence and dissemination of resistance to third- and fourth-generation cephalosporins among Enterobacteriaceae from different sources impose a global public health threat. Here, we characterized by whole-genome sequencing four Escherichia coli strains harboring the bla CTX-M-65 gene identified among 49 isolates from beef and pork collected at retail. The genomic content was determined using the Center for Genomic Epidemiology web tools. Additionally, the prediction and reconstruction of plasmids were conducted, the genetic platform of the bla CTX-M-65 genes was investigated, and phylogenetic analysis was carried out using 17 other genomes with the same sequence type and harboring the bla CTX-M-65 gene. All strains harbored bla CTX-M-65 , bla OXA-1 , and bla TEM-1B , and one also carried the bla SHV-12 gene. Other resistance genes, namely, qnrS2 , aac(6')-Ib-c , dfrA14 , sul2 , tetA , and mphA , were present in all the genomes; the mcr -1.1 gene was identified in the colistin-resistant strains. They belong to sequence type 2179, phylogenetic group B1, and serotype O9:H9 and carried plasmids IncI, IncFIC(FII), and IncFIB. All strains share an identical genetic environment with IS 903 and IS Ecp1 flanking the bla CTX-M-65 gene. It seems likely that the bla CTX-M-65 gene is located in the chromosome in all isolates based on deep in silico analysis. Our findings showed that the strains are clonally related and belong to two sub-lineages. This study reports the emergence of CTX-M-65-producing E. coli in Portugal in food products of animal origin. The chromosomal location of the bla CTX-M-65 gene may ensure a stable spread of resistance in the absence of selective pressure., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Leão, Clemente, Moura, Seyfarth, Hansen, Hendriksen and Amaro.)

Salmonella enterica is a foodborne pathogen of global public health importance with developing countries mostly affected. Foodborne outbreaks are often attributed to pork consumption and Salmonella contamination of retail pork is directly linked to the Salmonella prevalence on farm. The widespread use of antimicrobials at different steps of swine production can favor resistant strains of Salmonella. The objectives of this study are to characterize the distribution, multilocus sequence typing (MLST), plasmid, virulence profiles and antimicrobial resistance of Salmonella serovars circulating in selected pig farms. Six hundred fecal samples were randomly collected from nine selected farms in Ilorin, Nigeria. Isolates were analyzed by cultural isolation using selective media, conventional biochemical characterization, serotyping, MLST and whole genome sequencing (WGS). Sixteen samples were positive for Salmonella sub-species, comprising of nine serovars. The antimicrobial susceptibility results revealed low-level resistance against 13 antimicrobial agents. Five strains exhibited resistance to nalidixic acid and intermediate resistance to ciprofloxacin with chromosomal (double) mutation at gyrA and parC while four strains possessed single mutation in parC. Salmonella Kentucky showed double mutation each at gyrA and parC. WGS analysis, revealed eight diverse sequence types (STs), the most common STs were ST-321 and ST-19 (n = 4) exhibited by S. Muenster and S. Typhimurium, respectively. Single Nucleotide Polymorphism (SNP)-based phylogeny analysis showed the 16 isolates to be highly related and fell into 8 existing clusters at NCBI Pathogen Detection. Curtailing the spread of resistant strains will require the establishment of continuous surveillance program at the state and national levels in Nigeria. This study provides useful information for further studies on antimicrobial resistance mechanisms in foodborne Salmonella species., (Copyright © 2021 Elsevier B.V. All rights reserved.)

The increasing occurrence of antimicrobial-resistant Escherichia coli in human and animal population has become a global public health problem that requires immediate intervention. We aimed to investigate prevalence and risk factors for faecal carriage of drug-resistant E. coli among slaughterhouse workers. We conducted this cross-sectional study among 118 apparently healthy workers in the largest slaughterhouses in Abuja and Lagos from July to December 2020. E. coli was isolated from stool samples of slaughterhouse workers and antimicrobial susceptibility testing performed using the Kirby-Bauer disk diffusion method. Multi-drug resistance (MDR) was defined as resistance to three or more classes of antibiotics. Majority were males: 88.1% (n = 104), aged > 41 years: 28.8% (n = 34), married: 70.3% (n = 83), and were butchers: 53.4% (n = 63). Prevalence of MDR E. coli was 50% (n = 59), highest among butchers compared to slaughterhouse cleaners. Of 75 E. coli isolates identified, 25.3% (n = 19) were ESBL producers; 78.7% (n = 59) were MDR. Keeping animals (p = 0.01); eating at the slaughterhouse (p = 0.03) and collecting waste (p = 0.02) remained independent risk factors for acquiring MDR E. coli. Prevalence of resistant E. coli was highest among butchers and associated with keeping animals at home, eating at work, and waste-collection. Hand-hygiene and responsible use of antibiotics among slaughterhouse workers should be encouraged.

Background: Typhoid fever, caused by S. enterica ser. Typhi, continues to be a substantial health burden in developing countries. Little is known of the genotypic diversity of S. enterica ser. Typhi in Zimbabwe, but this is key for understanding the emergence and spread of this pathogen and devising interventions for its control., Objectives: To report the molecular epidemiology of S. enterica ser. Typhi outbreak strains circulating from 2012 to 2019 in Zimbabwe, using comparative genomics., Methods: A review of typhoid cases records from 2012 to 2019 in Zimbabwe was performed. The phylogenetic relationship of outbreak isolates from 2012 to 2019 and emergence of antibiotic resistance was investigated by whole-genome sequence analysis., Results: A total 22 479 suspected typhoid cases, 760 confirmed cases were reported from 2012 to 2019 and 29 isolates were sequenced. The majority of the sequenced isolates were predicted to confer resistance to aminoglycosides, β-lactams, phenicols, sulphonamides, tetracycline and fluoroquinolones (including qnrS detection). The qnrS1 gene was associated with an IncN (subtype PST3) plasmid in 79% of the isolates. Whole-genome SNP analysis, SNP-based haplotyping and resistance determinant analysis showed that 93% of the isolates belonged to a single clade represented by multidrug-resistant H58 lineage I (4.3.1.1), with a maximum pair-wise distance of 22 SNPs., Conclusions: This study has provided detailed genotypic characterization of the outbreak strain, identified as S. Typhi 4.3.1.1 (H58). The strain has reduced susceptibility to ciprofloxacin due to qnrS carried by an IncN (subtype PST3) plasmid resulting from ongoing evolution to full resistance., (© The Author(s) 2020. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Background: Inappropriate use of antimicrobial agents in animal production has led to the development of antimicrobial resistance (AMR) in foodborne pathogens. Transmission of AMR foodborne pathogens from reservoirs, particularly chickens to the human population does occur. Recently, we reported that occupational exposure was a risk factor for multidrug-resistant (MDR) Escherichia coli (E. coli) among poultry-workers. Here we determined the prevalence and genetic relatedness among MDR E. coli isolated from poultry-workers, chickens, and poultry environments in Abuja, Nigeria. This study was conducted to address the gaps identified by the Nigerian AMR situation analysis., Methods: We conducted a cross-sectional study among poultry-workers, chickens, and poultry farm/live bird market (LBM) environments. The isolates were tested phenotypically for their antimicrobial susceptibility profiles, genotypically characterized using whole-genome sequencing (WGS) and in silico multilocus sequence types (MLST). We conducted a phylogenetic single nucleotide polymorphism (SNPs) analysis to determine relatedness and clonality among the isolates., Results: A total of 115 (26.8%) out of 429 samples were positive for E. coli. Of these, 110 isolates were viable for phenotypic and genotypic characterization. The selection comprised 47 (42.7%) isolates from poultry-workers, 36 (32.7%) from chickens, and 27 (24.5%) from poultry-farm or LBM environments. Overall, 101 (91.8%) of the isolates were MDR conferring resistance to at least three drug classes. High frequency of resistance was observed for tetracycline (n = 102; 92.7%), trimethoprim/sulfamethoxazole (n = 93; 84.5%), streptomycin (n = 87; 79.1%) and ampicillin (n = 88; 80%). Two plasmid-mediated colistin genes-mcr-1.1 harboured on IncX4 plasmids were detected in environmental isolates. The most prevalent sequence types (ST) were ST-155 (n = 8), ST-48 (n = 8) and ST-10 (n = 6). Two isolates of human and environmental sources with a SNPs difference of 6161 originating from the same farm shared a novel ST. The isolates had similar AMR genes and plasmid replicons., Conclusion: MDR E.coli isolates were prevalent amongst poultry-workers, poultry, and the poultry farm/LBM environment. The emergence of MDR E. coli with novel ST in two isolates may be plasmid-mediated. Competent authorities should enforce AMR regulations to ensure prudent use of antimicrobials to limit the risk of transmission along the food chain.

Background: A novel Brucella strain closely related to Brucella (B.) melitensis biovar (bv) 3 was found in Croatian cattle during testing within a brucellosis eradication programme., Case Presentation: Standardised serological, brucellin skin test, bacteriological and molecular diagnostic screening for Brucella infection led to positive detection in one dairy cattle herd. Three isolates from that herd were identified to species level using the Bruce ladder method. Initially, two strains were typed as B. melitensis and one as B. abortus, but multiplex PCR based on IS711 and the Suis ladder showed that all of them to belong to B. melitensis, and the combination of whole-genome and multi-locus sequencing as well as Multi-Locus Variable numbers of tandem repeats Analysis (MLVA) highlighted a strong proximity within the phylogenetic branch of B. melitensis strains previously isolated from Croatia, Albania, Kosovo and Bosnia and Herzegovina. Two isolates were determined to be B. melitensis bv. 3, while the third showed a unique phylogenetic profile, growth profile on dyes and bacteriophage typing results. This isolate contained the 609-bp omp31 sequence, but not the 723-bp omp31 sequence present in the two isolates of B. melitensis bv. 3., Conclusions: Identification of a novel Brucella variant in this geographic region is predictable given the historic endemicity of brucellosis. The emergence of a new variant may reflect a combination of high prevalence among domestic ruminants and humans as well as weak eradication strategies. The zoonotic potential, reservoirs and transmission pathways of this and other Brucella variants should be explored.

Antimicrobial resistance (AMR) in bacteria is a complex subject, why one need to look at this phenomenon from a wider and holistic perspective. The extensive use of the same antimicrobial classes in human and veterinary medicine as well as horticulture is one of the main drivers for the AMR selection. Here, we applied shotgun metagenomics to investigate the AMR epidemiology in several animal species including farm animals, which are often exposed to antimicrobial treatment opposed to an unique set of wild animals that seems not to be subjected to antimicrobial pressure. The comparison of the domestic and wild animals allowed to investigate the possible anthropogenic impact on AMR spread. Inclusion of animals with different feeding behaviors (carnivores, omnivores) enabled to further assess which AMR genes that thrives within the food chain. We tested fecal samples not only of intensively produced chickens, turkeys, and pigs, but also of wild animals such as wild boars, red foxes, and rodents. A multi-directional approach mapping obtained sequences to several databases provided insight into the occurrence of the different AMR genes. The method applied enabled also analysis of other factors that may influence AMR of intestinal microbiome such as diet. Our findings confirmed higher levels of AMR in farm animals than in wildlife. The results also revealed the potential of wildlife in the AMR dissemination. Particularly in red foxes, we found evidence of several AMR genes conferring resistance to critically important antimicrobials like quinolones and cephalosporins. In contrast, the lowest abundance of AMR was observed in rodents originating from natural environment with presumed limited exposure to antimicrobials. Shotgun metagenomics enabled us to demonstrate that discrepancies between AMR profiles found in the intestinal microbiome of various animals probably resulted from the different antimicrobial exposure, habitats, and behavior of the tested animal species., Competing Interests: The authors have declared that no competing interests exist.

An inter-laboratory proficiency test was organized to assess the ability of participants to perform shotgun metagenomic sequencing of cold smoked salmon, experimentally spiked with a mock community composed of six bacteria, one parasite, one yeast, one DNA, and two RNA viruses. Each participant applied its in-house wet-lab workflow(s) to obtain the metagenomic dataset(s), which were then collected and analyzed using MG-RAST. A total of 27 datasets were analyzed. Sample pre-processing, DNA extraction protocol, library preparation kit, and sequencing platform, influenced the abundance of specific microorganisms of the mock community. Our results highlight that despite differences in wet-lab protocols, the reads corresponding to the mock community members spiked in the cold smoked salmon, were both detected and quantified in terms of relative abundance, in the metagenomic datasets, proving the suitability of shotgun metagenomic sequencing as a genomic tool to detect microorganisms belonging to different domains in the same food matrix. The implementation of standardized wet-lab protocols would highly facilitate the comparability of shotgun metagenomic sequencing dataset across laboratories and sectors. Moreover, there is a need for clearly defining a sequencing reads threshold, to consider pathogens as detected or undetected in a food sample.

The application of high-throughput DNA sequencing technologies (WGS) data remain an increasingly discussed but vastly unexplored resource in the public health domain of quantitative microbial risk assessment (QMRA). This is due to challenges including high dimensionality of WGS data and heterogeneity of microbial growth phenotype data. This study provides an innovative approach for modeling the impact of population heterogeneity in microbial phenotypic stress response and integrates this into predictive models inputting a high-dimensional WGS data for increased precision exposure assessment using an example of Listeria monocytogenes . Finite mixture models were used to distinguish the number of sub-populations for each of the stress phenotypes, acid, cold, salt and desiccation. Machine learning predictive models were selected from six algorithms by inputting WGS data to predict the sub-population membership of new strains with unknown stress response data. An example QMRA was conducted for cultured milk products using the strains of unknown stress phenotype to illustrate the significance of the findings of this study. Increased resistance to stress conditions leads to increased growth, the likelihood of higher exposure and probability of illness. Neglecting within-species genetic and phenotypic heterogeneity in microbial stress response may over or underestimate microbial exposure and eventual risk during QMRA.

Metagenomics-based high-throughput sequencing (HTS) enables comprehensive detection of all species comprised in a sample with a single assay and is becoming a standard method for outbreak investigation. However, unlike real-time PCR or serological assays, HTS datasets generated for pathogen detection do not easily provide yes/no answers. Rather, results of the taxonomic read assignment need to be assessed by trained personnel to gain information thereof. Proficiency tests are important instruments of validation, harmonization, and standardization. Within the European Union funded project COMPARE [COllaborative Management Platform for detection and Analyses of (Re-) emerging and foodborne outbreaks in Europe], we conducted a proficiency test to scrutinize the ability to assess diagnostic metagenomics data. An artificial dataset resembling shotgun sequencing of RNA from a sample of contaminated trout was provided to 12 participants with the request to provide a table with per-read taxonomic assignments at species level and a report with a summary and assessment of their findings, considering different categories like pathogen, background, or contaminations. Analysis of the read assignment tables showed that the software used reliably classified the reads taxonomically overall. However, usage of incomplete reference databases or inappropriate data pre-processing caused difficulties. From the combination of the participants' reports with their read assignments, we conclude that, although most species were detected, a number of important taxa were not or not correctly categorized. This implies that knowledge of and awareness for potentially dangerous species and contaminations need to be improved, hence, capacity building for the interpretation of diagnostic metagenomics datasets is necessary., (Copyright © 2020 Höper, Grützke, Brinkmann, Mossong, Matamoros, Ellis, Deneke, Tausch, Cuesta, Monzón, Juliá, Petersen, Hendriksen, Pamp, Leijon, Hakhverdyan, Walsh, Cotter, Chandrasekaran, Tay, Schlundt, Sala, De Cesare, Nitsche, Beer and Wylezich.)

It is important that antibiotics prescriptions are based on antimicrobial susceptibility data to ensure effective treatment outcomes. The increasing availability of next-generation sequencing, bacterial whole genome sequencing (WGS) can facilitate a more reliable and faster alternative to traditional phenotyping for the detection and surveillance of AMR. This work proposes a machine learning approach that can predict the minimum inhibitory concentration (MIC) for a given antibiotic, here ciprofloxacin, on the basis of both genome-wide mutation profiles and profiles of acquired antimicrobial resistance genes. We analysed 704 Escherichia coli genomes combined with their respective MIC measurements for ciprofloxacin originating from different countries. The four most important predictors found by the model, mutations in gyrA residues Ser83 and Asp87, a mutation in parC residue Ser80 and presence of the qnrS1 gene, have been experimentally validated before. Using only these four predictors in a linear regression model, 65% and 93% of the test samples' MIC were correctly predicted within a two- and a four-fold dilution range, respectively. The presented work does not treat machine learning as a black box model concept, but also identifies the genomic features that determine susceptibility. The recent progress in WGS technology in combination with machine learning analysis approaches indicates that in the near future WGS of bacteria might become cheaper and faster than a MIC measurement.