Broad-spectrum cephalosporin-resistant and/or fluoroquinolone-resistant enterobacterales associated with canine and feline urogenital infections

The aim of the present study was to characterize Enterobacterales resistant to 3rd and 4th generation cephalosporins, carbapenems and/or fluoroquinolones, isolated from dogs and cats with urogenital infections. In total, 36 strains (Escherichia coli (n = 28), Klebsiella pneumoniae (n = 3), Serratia marcescens, Raoultella ornithinolytica, Proteus mirabilis, Citrobacter portucalensis and Enterobacter cloacae (each n = 1)) were included in the present study, 28 from Austria and 8 from Serbia. Isolates were characterized by a polyphasic approach including susceptibility pheno-and genotyping and microarray-based assays. Escherichia (E.) coli isolates were additionally characterized by two-locus (fumC and fimH) sequence phylotyping and multi-locus sequence typing (MLST) of selected isolates. MLST of carbapenem-resistant Enterobacter cloacae isolates was also performed. Among E. coli, the most dominant phylogenetic group was B1 (27.8%), followed by C, (16.6%), A and Clade II (5.5% each), B2 and F (2.77% each). The most predominant β-lactam resistance genes were blaTEM (70%) and blaCTX-M (38.8%), blaCMY (25%). blaNDM was detected in one carbapenem-resistant Enterobacter cloacae ST114. The most common ST among selected E. coli was 744 (10.7% isolates). The pandemic clones ST131 and ST648 carrying CTX-M-15 were also detected. Remaining STs belonged to 469, 1287, 1463 and 1642. E. coli clonotyping revealed 20 CH types. Based on the presence of certain virulence genes, three isolates were categorized as ExPEC/UPEC. The most prevalent virulence factors were fimH detected in 61%, iucD and iss both in 55%, iroN in 27.8%, papC in 13.8% and sat in 8.3% isolates

Diversity of Staphylococcus aureus associated with mastitis from dairy cows in Rwanda

Objectives The objective of the present study was to examine the diversity of Staphylococcus aureus from mastitis milk samples of cows in Rwanda. Methods A total of 1080 quarter milk samples from 279 dairy cows were collected in 80 different farms from all five provinces of Rwanda. In total, 135 S. aureus isolates were obtained and subjected to genotyping (spa typing, DNA microarray, whole-genome sequencing (WGS)), antimicrobial susceptibility testing (AST) and phenotypic profiling by Fourier Transform Infrared (FTIR) spectroscopy (including capsular serotyping). Results Resistance to penicillin and/or tetracycline was most frequently observed. Ten sequence types (STs) (ST1, ST151, ST152, ST5477, ST700, ST7110, ST7983, ST7984, ST8320, ST97) belonging to seven clonal complexes (CCs) (CC1, CC130, CC152, CC3591, CC3666, CC705, CC97) were detected. The Panton–Valentine leukocidin (PVL) genes (lukF-PV/lukS-PV), the bovine leukocidin genes (lukM/lukF-P83) and the human and bovine toxic shock syndrome toxin gene tst-1 variants were detected. FTIR-based capsular serotyping showed CC-specific differences. Most CC97 (cap5 allele) isolates were primarily nonencapsulated (82%), whereas isolates of CC3591 and CC3666 (cap8 allele) were mostly encapsulated (86.4% and 57.8%, respectively). Our results underline the widespread global distribution of cattle-adapted CC97. Conclusion The presence of CC3591 and CC3666 in bovine mastitis suggests an important role in cattle health and dairy production in Rwanda. The results of the present study support the need for a rigorous One-Health Surveillance program of the bovine–human interface

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

Selection for Replicases in Protocells

PMCID: PMC3649988This 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

The First Report of mcr-1-Carrying Escherichia coli Originating from Animals in Serbia

The aim of this study was continuous monitoring of the presence of mcr-1 to mcr-5 genes in Enterobacterales isolated from cattle, pigs, and domestic poultry at intensive breeding facilities in Northern Vojvodina, Serbia, from 1 January 1 to 1 October 2020. Out of 2167 examined samples, mcr-1 was observed in five E. coli isolates originating from healthy turkeys. Four isolates belonged to the phylogenetic group B1, and one isolate to the phylogenetic group A. Detected E. coli serogenotypes (somatic O and flagellar H antigens) were O8:H25 and O29:H25. Core-genome multi-locus sequence typing (cgMLST) revealed three ST58 isolates clustering together in Clonal Complex (CC) 155 and two singletons of ST641-CC86 and ST410-CC23, respectively. Clonotyping revealed CH4-32 (n = 3), CH6-53 (n = 1) and CH4-24 (n = 1). In all isolates, the mcr-1 gene was located on a large IncX4 replicon type plasmid. Eight virulence-associated genes (VAGs) typical of avian pathogenic E. coli (APEC) (fyuA, fimH, hlyF, iss, ompT, sitA, traT, iroN) were detected in four isolates. These isolates were investigated for susceptibility to four biocides and revealed MIC values of 0.125% for glutardialdehyde, of 0.00003–0.00006% for chlorohexidine, of 4–6% for isopropanol and of 0.001–0.002% for benzalkonium chloride. All obtained MIC values of the tested biocides were comparable to the reference strain, with no indication of possible resistance. This is the first report of mcr-1.1-carrying E. coli from Serbia. Although only samples from turkeys were mcr-positive in this study, continuous monitoring of livestock samples is advised to prevent a spill-over from animals to humans

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

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

The Arabidopsis BLAP75/Rmi1 Homologue Plays Crucial Roles in Meiotic Double-Strand Break Repair

In human cells and in Saccharomyces cerevisiae, BLAP75/Rmi1 acts together with BLM/Sgs1 and TopoIIIα/Top3 to maintain genome stability by limiting crossover (CO) formation in favour of NCO events, probably through the dissolution of double Holliday junction intermediates (dHJ). So far, very limited data is available on the involvement of these complexes in meiotic DNA repair. In this paper, we present the first meiotic study of a member of the BLAP75 family through characterisation of the Arabidopsis thaliana homologue. In A. thaliana blap75 mutants, meiotic recombination is initiated, and recombination progresses until the formation of bivalent-like structures, even in the absence of ZMM proteins. However, chromosome fragmentation can be detected as soon as metaphase I and is drastic at anaphase I, while no second meiotic division is observed. Using genetic and imunolocalisation studies, we showed that these defects reflect a role of A. thaliana BLAP75 in meiotic double-strand break (DSB) repair—that it acts after the invasion step mediated by RAD51 and associated proteins and that it is necessary to repair meiotic DSBs onto sister chromatids as well as onto the homologous chromosome. In conclusion, our results show for the first time that BLAP75/Rmi1 is a key protein of the meiotic homologous recombination machinery. In A. thaliana, we found that this protein is dispensable for homologous chromosome recognition and synapsis but necessary for the repair of meiotic DSBs. Furthermore, in the absence of BLAP75, bivalent formation can happen even in the absence of ZMM proteins, showing that in blap75 mutants, recombination intermediates exist that are stable enough to form bivalent structures, even when ZMM are absent

Meiotic Recombination Intermediates Are Resolved with Minimal Crossover Formation during Return-to-Growth, an Analogue of the Mitotic Cell Cycle

Accurate segregation of homologous chromosomes of different parental origin (homologs) during the first division of meiosis (meiosis I) requires inter-homolog crossovers (COs). These are produced at the end of meiosis I prophase, when recombination intermediates that contain Holliday junctions (joint molecules, JMs) are resolved, predominantly as COs. JM resolution during the mitotic cell cycle is less well understood, mainly due to low levels of inter-homolog JMs. To compare JM resolution during meiosis and the mitotic cell cycle, we used a unique feature of Saccharomyces cerevisiae, return to growth (RTG), where cells undergoing meiosis can be returned to the mitotic cell cycle by a nutritional shift. By performing RTG with ndt80 mutants, which arrest in meiosis I prophase with high levels of interhomolog JMs, we could readily monitor JM resolution during the first cell division of RTG genetically and, for the first time, at the molecular level. In contrast to meiosis, where most JMs resolve as COs, most JMs were resolved during the first 1.5–2 hr after RTG without producing COs. Subsequent resolution of the remaining JMs produced COs, and this CO production required the Mus81/Mms4 structure-selective endonuclease. RTG in sgs1-ΔC795 mutants, which lack the helicase and Holliday junction-binding domains of this BLM homolog, led to a substantial delay in JM resolution; and subsequent JM resolution produced both COs and NCOs. Based on these findings, we suggest that most JMs are resolved during the mitotic cell cycle by dissolution, an Sgs1 helicase-dependent process that produces only NCOs. JMs that escape dissolution are mostly resolved by Mus81/Mms4-dependent cleavage that produces both COs and NCOs in a relatively unbiased manner. Thus, in contrast to meiosis, where JM resolution is heavily biased towards COs, JM resolution during RTG minimizes CO formation, thus maintaining genome integrity and minimizing loss of heterozygosity