Characterization of Antibiotic and Biocide Resistance Genes and Virulence Factors of Staphylococcus Species Associated with Bovine Mastitis in Rwanda

The present study was conducted from July to August 2018 on milk samples taken at dairy farms in the Northern Province and Kigali District of Rwanda in order to identify Staphylococcus spp. associated with bovine intramammary infection. A total of 161 staphylococcal isolates originating from quarter milk samples of 112 crossbred dairy cattle were included in the study. Antimicrobial susceptibility testing was performed and isolates were examined for the presence of various resistance genes. Staphylococcus aureus isolates were also analyzed for the presence of virulence factors, genotyped by spa typing and further phenotypically subtyped for capsule expression using Fourier Transform Infrared (FTIR) spectroscopy. Selected S. aureus were characterized using DNA microarray technology, multi-locus sequence typing (MLST) and whole-genome sequencing. All mecA-positive staphylococci were further genotyped using dru typing. In total, 14 different staphylococcal species were detected, with S. aureus being most prevalent (26.7%), followed by S. xylosus (22.4%) and S. haemolyticus (14.9%). A high number of isolates was resistant to penicillin and tetracycline. Various antimicrobial and biocide resistance genes were detected. Among S. aureus, the Panton–Valentine leukocidin (PVL) genes, as well as bovine leukocidin (LukM/LukF-P83) genes, were detected in two and three isolates, respectively, of which two also carried the toxic shock syndrome toxin gene tsst-1 bovine variant. t1236 was the predominant spa type. FTIR-based capsule serotyping revealed a high prevalence of non-encapsulated S. aureus isolates (89.5%). The majority of the selected S. aureus isolates belonged to clonal complex (CC) 97 which was determined using DNA microarray based assignment. Three new MLST sequence types were detected

Molecular characterization of Listeria monocytogenes isolates from a small-scale meat processor in Montenegro, 2011-2014

The presence of Listeria monocytogenes was evaluated in a small-scale meat processing facility in Montenegro during 2011-2014. L. monocytogenes isolates from traditional meat products and environmental swabs were subjected to a) molecular characterization b) serotyping by both multiplex PCR and next generation sequencing (NGS) c) potential antimicrobial resistance (AMR) was assessed by extraction of specific genes from NGS data and d) screening for the presence of some disinfectant resistance markers. Overall, traditional meat products were contaminated, most likely from incoming raw materials, with 4 major specific STs of L. monocytogenes (ST515, ST8, ST21, ST121) representing 4 clonal complexes (CC1, CC8, CC21, CC121) identified during the four-year period. These strains belonged to serogroup IIa which predominated, followed by IVb (ST515, CC1). The strains from environmental swabs belonged, exclusively, to ST21 and were isolated from cutting board and floor swabs in 2011. Furthermore, we found Tn6188, a novel transposon conferring tolerance to BC, to be specific to sequence type ST121. In addition, antimicrobial resistance genes mprF and fosX were present in clonal complexes CC21 and CC121, while complexes CC8 and CC1 exclusively harbored the mprF antimicrobial resistance gene.This is the peer-reviewed version of the article: Zuber, I.; Lakićević, B.; Pietzka, A.; Milanov, D.; Đorđević, V.; Karabasil, N.; Teodorović, V.; Ruppitsch, W.; Dimitrijević, M. Molecular Characterization of Listeria Monocytogenes Isolates from a Small-Scale Meat Processor in Montenegro, 2011-2014. Food Microbiology 2019, 79, 116–122. [https://doi.org/10.1016/j.fm.2018.12.005

Urban brown rats (Rattus norvegicus) as possible source of multidrug-resistant Enterobacteriaceae and meticillin-resistant Staphylococcus spp., Vienna, Austria, 2016 and 2017

Background: Brown rats (Rattus norvegicus) are an important wildlife species in cities, where they live in close proximity to humans. However, few studies have investigated their role as reservoir of antimicrobial-resistant bacteria. Aim: We intended to determine whether urban rats at two highly frequented sites in Vienna, Austria, carry extended-spectrum β-lactamase-producing Enterobacteriaceae, fluoroquinolone-resistant Enterobacteriaceae and meticillin-resistant (MR) Staphylococcus spp. (MRS). Methods: We surveyed the presence of antimicrobial resistance in 62 urban brown rats captured in 2016 and 2017 in Vienna, Austria. Intestinal and nasopharyngeal samples were cultured on selective media. We character-ised the isolates and their antimicrobial properties using microbiological and genetic methods including disk diffusion, microarray analysis, sequencing, and detection and characterisation of plasmids. Results: Eight multidrug-resistant Escherichia coli and two extensively drug-resistant New Delhi metallo-β-lactamases-1 (NDM-1)-producing Enterobacter xiangfangensis ST114 (En. cloacae complex) were isolated from nine of 62 rats. Nine Enterobacteriaceae isolates harboured the blaCTX-M gene and one carried a plasmid-encoded ampC gene (blaCMY-2). Forty-four MRS were isolated from 37 rats; they belonged to seven different staphylococcal species: S. fleuret-tii, S. sciuri, S. aureus, S. pseudintermedius, S. epidermidis, S. haemolyticus (all mecA-positive) and mecC-positive S. xylosus. Conclusion: Our findings suggest that brown rats in cities are a potential source of multidrug-resistant bacteria, including carbapenem-resistant En. xiangfangensis ST114. Considering the increasing worldwide urbanisation, rodent control remains an important priority for health in modern cities. © 2019, European Centre for Disease Prevention and Control (ECDC). All rights reserved

Dogs as carriers of virulent and resistant genotypes of Clostridioides difficile

Abstract While previous research on zoonotic transmission of community-acquired Clostridioides difficile infection (CA-CDI) focused on food-producing animals, the present study aimed to investigate whether dogs are carriers of resistant and/or virulent C. difficile strains. Rectal swabs were collected from 323 dogs and 38 C. difficile isolates (11.8%) were obtained. Isolates were characterized by antimicrobial susceptibility testing, whole-genome sequencing (WGS) and a DNA hybridization assay. Multilocus sequence typing (MLST), core genome MLST (cgMLST) and screening for virulence and antimicrobial resistance genes were performed based on WGS. Minimum inhibitory concentrations for erythromycin, clindamycin, tetracycline, vancomycin and metronidazole were determined by E-test. Out of 38 C. difficile isolates, 28 (73.7%) carried genes for toxins. The majority of isolates belonged to MLST sequence types (STs) of clade I and one to clade V. Several isolates belonged to STs previously associated with human CA-CDI. However, cgMLST showed low genetic relatedness between the isolates of this study and C. difficile strains isolated from humans in Austria for which genome sequences were publicly available. Four isolates (10.5%) displayed resistance to three of the tested antimicrobial agents. Isolates exhibited resistance to erythromycin, clindamycin, tetracycline and metronidazole. These phenotypic resistances were supported by the presence of the resistance genes erm(B), cfr(C) and tet(M). All isolates were susceptible to vancomycin. Our results indicate that dogs may carry virulent and antimicrobial-resistant C. difficile strains.1 Introduction 2 Methods 2.1 Sampling and ethics 2.2 Isolation and identification of Clostridioides difficile 2.3 Antimicrobial susceptibility testing 2.4 Whole-genome sequencing and comparative genomic analysis 2.5 Statistical analysis 3 Results 3.1 Prevalence of Clostridioides difficile and risk factors for shedding 3.2 Antimicrobial susceptibility testing and detection of antimicrobial resistance determinants 3.3 Genomic characterization of canine Clostridioides difficile 3.4 Genome annotation and comparison 4 Discussio

Characterization of antibiotic and biocide resistance genes and virulence factors of staphylococcus species associated with bovine mastitis in Rwanda

The present study was conducted from July to August 2018 on milk samples taken at dairy farms in the Northern Province and Kigali District of Rwanda in order to identify Staphylococcus spp. associated with bovine intramammary infection. A total of 161 staphylococcal isolates originating from quarter milk samples of 112 crossbred dairy cattle were included in the study. Antimicrobial susceptibility testing was performed and isolates were examined for the presence of various resistance genes. Staphylococcus aureus isolates were also analyzed for the presence of virulence factors, genotyped by spa typing and further phenotypically subtyped for capsule expression using Fourier Transform Infrared (FTIR) spectroscopy. Selected S. aureus were characterized using DNA microarray technology, multi-locus sequence typing (MLST) and whole-genome sequencing. All mecA-positive staphylococci were further genotyped using dru typing. In total, 14 different staphylococcal species were detected, with S. aureus being most prevalent (26.7%), followed by S. xylosus (22.4%) and S. haemolyticus (14.9%). A high number of isolates was resistant to penicillin and tetracycline. Various antimicrobial and biocide resistance genes were detected. Among S. aureus, the Panton–Valentine leukocidin (PVL) genes, as well as bovine leukocidin (LukM/LukF-P83) genes, were detected in two and three isolates, respectively, of which two also carried the toxic shock syndrome toxin gene tsst-1 bovine variant. t1236 was the predominant spa type. FTIR-based capsule serotyping revealed a high prevalence of non-encapsulated S. aureus isolates (89.5%). The majority of the selected S. aureus isolates belonged to clonal complex (CC) 97 which was determined using DNA microarray based assignment. Three new MLST sequence types were detected. © 2019 by the authors. Licensee MDPI, Basel, Switzerland

Comparison of Automated Ribotyping, spa Typing, and MLST in 108 Clinical Isolates of Staphylococcus aureus from Orthopedic Infections

108 isolates of Staphylococcus aureus, belonging to six large ribogroups according to the automated Ribo-Printer® system, were studied with two highly used molecular methods for epidemiological studies, namely multi-locus sequence typing (MLST) and spa typing, followed by BURP and eBURST v3 analysis for clustering spa types and sequence (ST) types. The aim was to evaluate whether automated ribotyping could be considered a useful screening tool for identifying S. aureus genetic lineages with respect to spa typing and MLST. Clarifying the relationship of riboprinting with these typing methods and establishing whether ribogroups fit single clonal complexes were two main objectives. Further information on the genetic profile of the isolates was obtained from agr typing and the search for the mecA, tst genes, and the IS256 insertion sequence. Automated ribotyping has been shown to predict spa clonal complexes and MLST clonal complexes. The high cost and lower discriminatory power of automated ribotyping compared to spa and MSLT typing could be an obstacle to fine genotyping analyzes, especially when high discriminatory power is required. On the other hand, numerous advantages such as automation, ease and speed of execution, stability, typeability and reproducibility make ribotyping a reliable method to be juxtaposed to gold standard methods

Characterizing Antimicrobial Resistance in Clinically Relevant Bacteria Isolated at the Human/Animal/Environment Interface Using Whole-Genome Sequencing in Austria

This article belongs to the Collection Feature Papers in Molecular Microbiology.Antimicrobial resistance (AMR) is a public health issue attributed to the misuse of antibiotics in human and veterinary medicine. Since AMR surveillance requires a One Health approach, we sampled nine interconnected compartments at a hydrological open-air lab (HOAL) in Austria to obtain six bacterial species included in the WHO priority list of antibiotic-resistant bacteria (ARB). Whole genome sequencing-based typing included core genome multilocus sequence typing (cgMLST). Genetic and phenotypic characterization of AMR was performed for all isolates. Eighty-nine clinically-relevant bacteria were obtained from eight compartments including 49 E. coli, 27 E. faecalis, 7 K. pneumoniae and 6 E. faecium. Clusters of isolates from the same species obtained in different sample collection dates were detected. Of the isolates, 29.2% were resistant to at least one antimicrobial. E. coli and E. faecalis isolates from different compartments had acquired antimicrobial resistance genes (ARGs) associated with veterinary drugs such as aminoglycosides and tetracyclines, some of which were carried in conjugative and mobilizable plasmids. Three multidrug resistant (MDR) E. coli isolates were found in samples from field drainage and wastewater. Early detection of ARGs and ARB in natural and farm-related environments can identify hotspots of AMR and help prevent its emergence and dissemination along the food/feed chain.This research was partially funded by the European Union’s Horizon 2020 Research and Innovation program, grant agreement No 773830: One Health European Joint Program.info:eu-repo/semantics/publishedVersio

An epidemic CC1-MRSA-IV clone yields false-negative test results in molecular MRSA identification assays: a note of caution, Austria, Germany, Ireland, 2020

We investigated why a clinical meticillin-resistant Staphylococcus aureus (MRSA) isolate yielded false-negative results with some commercial PCR tests for MRSA detection. We found that an epidemic European CC1-MRSA-IV clone generally exhibits this behaviour. The failure of the assays was attributable to a large insertion in the orfX/SCCmec integration site. To ensure the reliability of molecular MRSA tests, it is vital to monitor emergence of new SCCmec types and junction sites

The Pheno- and Genotypic Characterization of Porcine Escherichia coli Isolates

Escherichia (E.) coli is the main causative pathogen of neonatal and post-weaning diarrhea and edema disease in swine production. There is a significant health concern due to an increasing number of human infections associated with food and/or environmental-borne pathogenic and multidrug-resistant E. coli worldwide. Monitoring the presence of pathogenic and antimicrobial-resistant E. coli isolates is essential for sustainable disease management in livestock and human medicine. A total of 102 E. coli isolates of diseased pigs were characterized by antimicrobial and biocide susceptibility testing. Antimicrobial resistance genes, including mobile colistin resistance genes, were analyzed by PCR and DNA sequencing. The quinolone resistance-determining regions of gyrA and parC in ciprofloxacin-resistant isolates were analyzed. Clonal relatedness was investigated by two-locus sequence typing (CH clonotyping). Phylotyping was performed by the Clermont multiplex PCR method. Virulence determinants were analyzed by customized DNA-based microarray technology developed in this study for fast and economic molecular multiplex typing. Thirty-five isolates were selected for whole-genome sequence-based analysis. Most isolates were resistant to ampicillin and tetracycline. Twenty-one isolates displayed an ESBL phenotype and one isolate an AmpC β-lactamase-producing phenotype. Three isolates had elevated colistin minimal inhibitory concentrations and carried the mcr-1 gene. Thirty-seven isolates displayed a multi-drug resistance phenotype. The most predominant β-lactamase gene classes were blaTEM-1 (56%) and blaCTX-M-1 (13.71%). Mutations in QRDR were observed in 14 ciprofloxacin-resistant isolates. CH clonotyping divided all isolates into 51 CH clonotypes. The majority of isolates belonged to phylogroup A. Sixty-four isolates could be assigned to defined pathotypes wherefrom UPEC was predominant. WGS revealed that the most predominant sequence type was ST100, followed by ST10. ST131 was detected twice in our analysis. This study highlights the importance of monitoring antimicrobial resistance and virulence properties of porcine E. coli isolates. This can be achieved by applying reliable, fast, economic and easy to perform technologies such as DNA-based microarray typing. The presence of high-risk pathogenic multi-drug resistant zoonotic clones, as well as those that are resistant to critically important antibiotics for humans, can pose a risk to public health. Improved protocols may be developed in swine farms for preventing infections, as well as the maintenance and distribution of the causative isolates