A total of 215 isolates from infections of dogs and cats, including 49 Enterococcus faecalis, 37 Enterococcus faecium, 59 Escherichia coli, 56 Pseudomonas aeruginosa, and 14 Acinetobacter baumannii, were investigated for their susceptibility to 27 (Gram-positive bacteria) or 20 (Gram-negative bacteria) antimicrobial agents/combinations of antimicrobial agents by broth microdilution according to the recommendations of the Clinical and Laboratory Standards Institute. Moreover, all isolates were analysed for their susceptibility to the biocides benzalkonium chloride, chlorhexidine, polyhexanide, and octenidine by a recently published broth microdilution biocide susceptibility testing method. While the E. faecalis isolates did not show expanded resistances, considerable numbers of the E. faecium isolates were resistant to penicillins, macrolides, tetracyclines, and fluoroquinolones. Even a single vancomycin-resistant isolate that carried the vanA gene cluster was detected. Expanded multiresistance phenotypes were also detected among the E. coli isolates, including a single carbapenem-resistant, blaOXA-48-positive isolate. In addition, multiresistant A. baumannii isolates were detected. The minimal inhibitory concentrations of the biocides showed unimodal distributions but differed with respect to the biocide and the bacterial species investigated. Although there were no indications of a development of biocide resistance, some P. aeruginosa isolates exhibited benzalkonium MICs higher than the highest test concentration.

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Antimicrobial and Biocide Resistance among Canine and Feline Enterococcus faecalis, Enterococcus faecium, Escherichia coli, Pseudomonas aeruginosa, and Acinetobacter baumannii Isolates from Diagnostic Submissions

Every laboratory test needs validation by quality controls. For biocide susceptibility testing (BST), neither quality control (QC) strains nor QC ranges applicable to these strains are currently available. As QC strains, four well-defined laboratory reference strains (Staphylococcus aureus ATCC® 6538, Enterococcus hirae ATCC® 10541, Escherichia coli ATCC® 10536 and Pseudomonas aeruginosa ATCC® 15442), which have been used previously for biocide efficacy testing, were selected. In an interlaboratory trial with eleven participating laboratories, BST QC ranges should be developed for the aforementioned four strains and the four biocides benzalkonium chloride, chlorhexidine, octenidine and polyhexanide. The performance of three different lots of tryptic soy broth was explored using the broth microdilution method and the data were subsequently evaluated using the RangeFinder software. As a result, QC ranges were defined for all reference strain–biocide combinations, except for P. aeruginosa ATCC® 15442 with the two biocides chlorhexidine and polyhexanide. The development of the latter two QC ranges was not possible, due to the limited solubility of the biocides in the test range required for P. aeruginosa ATCC® 15442. The newly developed QC ranges comprise three to five dilution steps. The establishment of QC ranges will contribute to the validation of BST in the future.

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Development of Quality Control Ranges for Biocide Susceptibility Testing

There is a link between antibiotic resistance in humans, livestock and the environment. This study was carried out to characterize antibiotic resistant bovine and environmental Enterobacteriaceae isolates from Edo state, Nigeria. A total of 109 consecutive isolates of Enterobacteriaceae were isolated from March–May 2015 from 150 fecal samples of healthy bovine animals from three farms at slaughter in Edo state Nigeria. Similarly, 43 Enterobacteriaceae isolates were also obtained from a total of 100 environmental samples from different sources. Isolates were recovered and identified from samples using standard microbiological techniques. Recovered isolates were pre-identified by the Microbact Gram-Negative identification system and confirmed with Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry and ribosomal multilocus sequence typing (rMLST). Antibiotic susceptibility testing was carried out by Kirby-Bauer method for 14 antibiotics. Whole genome sequencing (WGS) was carried out for isolate characterization and identification of resistance determinants. Out of 109 animal and 43 environmental Enterobacteriaceae isolates, 18 (17%) and 8 (19%) isolates based on selection criteria showed antibiotic resistance and were further investigated by whole genome sequencing (WGS). Resistance genes were detected in all (100%) of the resistant bovine and environmental Enterobacteriaceae isolates. The resistance determinants included β-lactamase genes, aminoglycoside modifying enzymes, qnr genes, sulfonamide, tetracycline and trimethoprim resistance genes, respectively. Out of the 18 and 8 resistant animal and environmental isolates 3 (17%) and 2 (25%) were multidrug resistant (MDR) and had resistance determinants which included efflux genes, regulatory systems modulating antibiotic efflux and antibiotic target alteration genes. Our study shows the dissemination of antibiotic resistance especially MDR strains among Nigerian bovine and environmental Enterobacteriaceae isolates. The presence of these resistant strains in animals and the environment constitute a serious health concern indicated by the difficult treatment options of the infections caused by these organisms. To the best of our knowledge we report the first detailed genomic characterization of antibiotic resistance in bovine and environmental Enterobacteriaceae isolates for Nigeria.

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Genetic Characterization of Antibiotic Resistant Enterobacteriaceae Isolates From Bovine Animals and the Environment in Nigeria

This study aimed to investigate the prevalence and resistance mechanisms of colistin-resistant Acinetobacter baumannii (ColRAB) isolates in Serbia, assess their genetic relatedness to other circulating A. baumannii isolates in the neighbouring European countries, and analyse the global genomic epidemiology of ColRAB isolates. A total of 784 isolates of A. baumannii were recovered from hospitalised patients in Serbia between 2018 and 2021. The antimicrobial susceptibility testing was performed using disk diffusion and broth microdilution. All ColRAB isolates were subjected to DNA isolation and whole-genome sequencing (WGS). Overall, 3.94 % (n = 30) isolates were confirmed as ColRAB. Results of mutational and transcriptional analysis of genes associated with colistin resistance indicate the central role of the two-component regulating system, PmrAB, and increased expression of the pmrC gene in ColRAB. Most of the isolates (n = 29, 96.6 %) belonged to international clone II, with the most common sequence type being STPas2 (n = 23, 76.6 %). Based on the WGS analysis, ColRAB isolates belonging to the same ST isolated in various countries were grouped into the same clusters, indicating the global dissemination of several high-risk clonal lineages. Phylogenomic analysis of ColRAB isolates, together with all previously published A. baumannii genomes from South-Eastern European countries, showed that colistin resistance arose independently in several clonal lineages. Comparative genomic analysis revealed multiple genes with various roles (transcriptional regulation, transmembrane transport, outer membrane assembly, etc.), which might be associated with colistin resistance in A. baumannii. The obtained findings serve as the basis for further studies, contributing to a better understanding of colistin resistance mechanisms in A. baumannii. 

Comparative genomics and molecular epidemiology of colistin-resistant Acinetobacter baumannii

Non-fermenting Gram-negative bacilli (NFGNB), such as Pseudomonas aeruginosa and Acinetobacter baumannii, are among the major opportunistic pathogens involved in the global antibiotic resistance epidemic. They are designated as urgent/serious threats by the Centers for Disease Control and Prevention and are part of the World Health Organization’s list of critical priority pathogens. Also, Stenotrophomonas maltophilia is increasingly recognized as an emerging cause for healthcare-associated infections in intensive care units, life-threatening diseases in immunocompromised patients, and severe pulmonary infections in cystic fibrosis and COVID-19 individuals. The last annual report of the ECDC showed drastic differences in the proportions of NFGNB with resistance towards key antibiotics in different European Union/European Economic Area countries. The data for the Balkans are of particular concern, indicating more than 80% and 30% of invasive Acinetobacter spp. and P. aeruginosa isolates, respectively, to be carbapenem-resistant. Moreover, multidrug-resistant and extensively drug-resistant S. maltophilia from the region have been recently reported. The current situation in the Balkans includes a migrant crisis and reshaping of the Schengen Area border. This results in collision of diverse human populations subjected to different protocols for antimicrobial stewardship and infection control. The present review article summarizes the findings of whole-genome sequencing-based resistome analyses of nosocomial multidrug-resistant NFGNBs in the Balkan countries.

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Whole-Genome Sequencing-Based Resistome Analysis of Nosocomial Multidrug-Resistant Non-Fermenting Gram-Negative Pathogens from the Balkans

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.

Viktoria Milovanovic

Papers

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