Antimicrobial activity and pathogen mutation prevention of originator and generics of cefepime, linezolid and piperacillin/tazobactam against clinical isolates of Staphylococcus aureus.

OBJECTIVES: Although generic medicinal products are required to have the same qualitative and quantitative composition of the active substance as their reference originator product, patients and health care professionals express concerns about their interchangeability and safety. Therefore, the present study investigated the antimicrobial activity and pathogen mutation prevention of original and generic cefepime, linezolid and piperacillin/tazobactam against Staphylococcus aureus. METHODS: Two generic formulations of cefepime, linezolid and piperacillin/tazobactam were tested against their respective originator products. Susceptibility testing was performed with twenty-one clinical isolates of S. aureus and ATCC-29213 using broth microdilution. Time kill curves (TKC) were performed with ATCC-29213 at drug concentrations above and below the respective minimum inhibitory concentrations (MIC). Mutation prevention concentration was determined for each drug formulation against ATCC-29213. All experiments were performed in triplicate. Mutant colonies from mutation prevention concentration (MPC) experiments were genotypically tested by sequence analysis. RESULTS: MIC ratios between contiguous originator and generic drugs were similar for each isolate. No visual differences were observed in TKCs between originator and generic substances. The MPC did not differ between different formulations of the same substance. Although sequence analysis of mutant colonies revealed genomic differences compared with the original ATCC-29213, no differences in mutation frequencies were observed between clinical isolates and ATCC-29213 treated with originator or generic substances. CONCLUSIONS: Similar antimicrobial activity and pathogen mutation prevention was observed between contiguous substances. These results support the interchangeability of generic and originator drug formulations with the same active ingredient.

Genomic Characterization of Cronobacter spp. and Salmonella spp. Strains Isolated From Powdered Infant Formula in Chile.

This study characterized five Cronobacter spp. and six Salmonella spp. strains that had been isolated from 155 samples of powdered infant formula (PIF) sold in Chile and manufactured in Chile and Mexico in 2018-2020. Two strains of Cronobacter sakazakii sequence type (ST) ST1 and ST31 (serotypes O:1 and O:2) and one strain of Cronobacter malonaticus ST60 (O:1) were identified. All Salmonella strains were identified as Salmonella Typhimurium ST19 (serotype O:4) by average nucleotide identity, ribosomal multilocus sequence typing (rMLST), and core genome MLST (cgMLST). The C. sakazakii and C. malonaticus isolates were resistant to cephalothin, whereas the Salmonella isolates were resistant to oxacillin and ampicillin. Nineteen antibiotic resistance genes were detected in the C. sakazakii and C. malonaticus isolates; the most prevalent were mcr-9.1, blaCSA , and blaCMA . In Salmonella, 30 genes encoding for aminoglycoside and cephalosporin resistance were identified, including aac(6')-Iaa, ß-lactamases ampH, ampC1, and marA. In the Cronobacter isolates, 32 virulence-associated genes were detected by WGS and clustered as flagellar proteins, outer membrane proteins, chemotaxis, hemolysins, invasion, plasminogen activator, colonization, transcriptional regulator, survival in macrophages, use of sialic acid, and toxin-antitoxin genes. In the Salmonella strains, 120 virulence associated genes were detected, adherence, magnesium uptake, resistance to antimicrobial peptides, secretion system, stress protein, toxin, resistance to complement killing, and eight pathogenicity islands. The C. sakazakii and C. malonaticus strains harbored I-E and I-F CRISPR-Cas systems and carried Col(pHHAD28) and IncFIB(pCTU1) plasmids, respectively. The Salmonella strains harbored type I-E CRISPR-Cas systems and carried IncFII(S) plasmids. The presence of C. sakazakii and Salmonella in PIF is a health risk for infants aged less than 6 months. For this reason, sanitary practices should be reinforced for its production and retail surveillance.

Culture-Free Detection of Antibiotic Resistance Markers from Native Patient Samples by Hybridization Capture Sequencing.

The increasing incidence of antimicrobial resistance (AMR) is a major global challenge. Routine techniques for molecular AMR marker detection are largely based on low-plex PCR and detect dozens to hundreds of AMR markers. To allow for comprehensive and sensitive profiling of AMR markers, we developed a capture-based next generation sequencing (NGS) workflow featuring a novel AMR marker panel based on the curated AMR database ARESdb. Our primary objective was to compare the sensitivity of target enrichment-based AMR marker detection to metagenomics sequencing. Therefore, we determined the limit of detection (LOD) in synovial fluid and urine samples across four key pathogens. We further demonstrated proof-of-concept for AMR marker profiling from septic samples using a selection of urine samples with confirmed monoinfection. The results showed that the capture-based workflow is more sensitive and requires lower sequencing depth compared with metagenomics sequencing, allowing for comprehensive AMR marker detection with an LOD of 1000 CFU/mL. Combining the ARESdb AMR panel with 16S rRNA gene sequencing allowed for the culture-free detection of bacterial taxa and AMR markers directly from septic patient samples at an average sensitivity of 99%. Summarizing, the newly developed ARESdb AMR panel may serve as a valuable tool for comprehensive and sensitive AMR marker detection.

Profiling the Virulence and Antibiotic Resistance Genes of Cronobacter sakazakii Strains Isolated From Powdered and Dairy Formulas by Whole-Genome Sequencing.

Cronobacter sakazakii is an enteropathogen that causes neonatal meningitis, septicemia, and necrotizing enterocolitis in preterm infants and newborns with a mortality rate of 15 to 80%. Powdered and dairy formulas (P-DF) have been implicated as major transmission vehicles and subsequently the presence of this pathogen in P-DF led to product recalls in Chile in 2017. The objective of this study was to use whole genome sequencing (WGS) and laboratory studies to characterize Cronobacter strains from the contaminated products. Seven strains were identified as C. sakazakii, and the remaining strain was Franconibacter helveticus. All C. sakazakii strains adhered to a neuroblastoma cell line, and 31 virulence genes were predicted by WGS. The antibiograms varied between strains. and included mcr-9.1 and bla CSA genes, conferring resistance to colistin and cephalothin, respectively. The C. sakazakii strains encoded I-E and I-F CRISPR-Cas systems, and carried IncFII(pECLA), Col440I, and Col(pHHAD28) plasmids. In summary, WGS enabled the identification of C. sakazakii strains and revealed multiple antibiotic resistance and virulence genes. These findings support the decision to recall the contaminated powdered and dairy formulas from the Chilean market in 2017.

Draft Genome Sequences of Seven Cronobacter sakazakii Strains Carrying the mcr 9.1 Gene Isolated in Chile.

Cronobacter sakazakii is a pathogen that causes severe diseases such as meningitis and necrotizing enterocolitis in infants under 12 months, associated with the consumption of contaminated rehydrated powdered infant formula (PIF). We present seven C. sakazakii genome sequences isolated from PIF and dairy products in Chile in 2017.

Genomic and Phenotypic Analysis of Linezolid-Resistant Staphylococcus epidermidis in a Tertiary Hospital in Innsbruck, Austria.

Whole genome sequencing is a useful tool to monitor the spread of resistance mechanisms in bacteria. In this retrospective study, we investigated genetic resistance mechanisms, sequence types (ST) and respective phenotypes of linezolid-resistant Staphylococcus epidermidis (LRSE, n = 129) recovered from a cohort of patients receiving or not receiving linezolid within a tertiary hospital in Innsbruck, Austria. Hereby, the point mutation G2603U in the 23S rRNA (n = 91) was the major resistance mechanism followed by the presence of plasmid-derived cfr (n = 30). The majority of LRSE isolates were ST2 strains, followed by ST5. LRSE isolates expressed a high resistance level to linezolid with a minimal inhibitory concentration of ≥256 mg/L (n = 83) in most isolates, particularly in strains carrying the cfr gene (p < 0.001). Linezolid usage was the most prominent (but not the only) trigger for the development of linezolid resistance. However, administration of linezolid was not associated with a specific resistance mechanism. Restriction of linezolid usage and the monitoring of plasmid-derived cfr in LRSE are potential key steps to reduce linezolid resistance and its transmission to more pathogenic Gram-positive bacteria.

Virulence and Antibiotic Resistance Genes in Listeria monocytogenes Strains Isolated From Ready-to-Eat Foods in Chile.

Listeria monocytogenes is causing listeriosis, a rare but severe foodborne infection. Listeriosis affects pregnant women, newborns, older adults, and immunocompromised individuals. Ready-to-eat (RTE) foods are the most common sources of transmission of the pathogen This study explored the virulence factors and antibiotic resistance in L. monocytogenes strains isolated from ready-to-eat (RTE) foods through in vitro and in silico testing by whole-genome sequencing (WGS). The overall positivity of L. monocytogenes in RTE food samples was 3.1% and 14 strains were isolated. L. monocytogenes ST8, ST2763, ST1, ST3, ST5, ST7, ST9, ST14, ST193, and ST451 sequence types were identified by average nucleotide identity, ribosomal multilocus sequence typing (rMLST), and core genome MLST. Seven isolates had serotype 1/2a, five 1/2b, one 4b, and one 1/2c. Three strains exhibited in vitro resistance to ampicillin and 100% of the strains carried the fosX, lin, norB, mprF, tetA, and tetC resistance genes. In addition, the arsBC, bcrBC, and clpL genes were detected, which conferred resistance to stress and disinfectants. All strains harbored hlyA, prfA, and inlA genes almost thirty-two the showed the bsh, clpCEP, hly, hpt, iap/cwhA, inlA, inlB, ipeA, lspA, mpl, plcA, pclB, oat, pdgA, and prfA genes. One isolate exhibited a type 11 premature stop codon (PMSC) in the inlA gene and another isolate a new mutation (deletion of A in position 819). The Inc18(rep25), Inc18(rep26), and N1011A plasmids and MGEs were found in nine isolates. Ten isolates showed CAS-Type II-B systems; in addition, Anti-CRISPR AcrIIA1 and AcrIIA3 phage-associated systems were detected in three genomes. These virulence and antibiotic resistance traits in the strains isolated in the RTE foods indicate a potential public health risk for consumers.

Molecular Characterization of Cronobacter sakazakii Strains Isolated from Powdered Milk.

Cronobacter spp. are opportunistic pathogens of the Enterobacteriaceae family. The organism causes infections in all age groups, but the most serious cases occur in outbreaks related to neonates with meningitis and necrotizing enterocolitis. The objective was to determine the in silico and in vitro putative virulence factors of six Cronobacter sakazakii strains isolated from powdered milk (PM) in the Czech Republic. Strains were identified by MALDI-TOF MS and whole-genome sequencing (WGS). Virulence and resistance genes were detected with the Ridom SeqSphere+ software task template and the Comprehensive Antibiotic Resistance Database (CARD) platform. Adherence and invasion ability were performed using the mouse neuroblastoma (N1E-115 ATCCCRL-2263) cell line. The CRISPR-Cas system was searched with CRISPRCasFinder. Core genome MLST identified four different sequence types (ST1, ST145, ST245, and ST297) in six isolates. Strains 13755-1B and 1847 were able to adhere in 2.2 and 3.2 × 106 CFU/mL, while 0.00073% invasion frequency was detected only in strain 1847. Both strains 13755-1B and 1847 were positive for three (50.0%) and four virulence genes, respectively. The cpa gene was not detected. Twenty-eight genes were detected by WGS and grouped as flagellar or outer membrane proteins, chemotaxis, hemolysins, and invasion, plasminogen activator, colonization, transcriptional regulator, and survival in macrophages. The colistin-resistance-encoding mcr-9.1 and cephalothin-resis-encoding blaCSA genes and IncFII(pECLA) and IncFIB(pCTU3) plasmids were detected. All strains exhibited CRISPR matrices and four of them two type I-E and I-F matrices. Combined molecular methodologies improve Cronobacter spp. decision-making for health authorities to protect the population.

Fecal Klebsiella pneumoniae Carriage Is Intermittent and of High Clonal Diversity.

The Klebsiella pneumoniae complex comprises several closely related entities, which are ubiquitous in the natural environment, including in plants, animals, and humans. K. pneumoniae is the major species within this complex. K. pneumoniae strains are opportunistic pathogens and a common cause of healthcare-associated infections. K. pneumoniae can colonize the human gastrointestinal tract, which may become a reservoir for infection. The aim of this study was to investigate the fecal K. pneumoniae carriage in six healthy individuals during a 1 year period. Stool samples were obtained once a week. Using direct and pre-enriched cultures streaked on ampicillin-supplemented agar plates, up to eight individual colonies per positive sample were selected for further characterization. Whole genome sequencing (WGS) was performed for strain characterization. Sequence type (ST), core genome complex type (CT), K and O serotypes, virulence traits, antibiotic resistance profiles, and plasmids were extracted from WGS data. In total, 80 K. pneumoniae isolates were obtained from 48 positive cultures of 278 stool samples from five of the six test subjects. The samples of the five colonized volunteers yielded at most two, three, four (two persons), and five different strains, respectively. These 80 K. pneumoniae isolates belonged to 60 STs, including nine new STs; they were of 70 CTs, yielded 48 K serotypes, 11 O serotypes, and 39 wzc and 51 wzi alleles. Four of the five subjects harbored serotypes K20 and K47, as well as STs ST37, ST101, ST1265, and ST20, which had previously been linked to high-risk K. pneumoniae clones. In total, 25 genes conferring antibiotic resistance and 42 virulence genes were detected among all 80 isolates. Plasmids of 15 different types were found among 65 of the isolates. Fecal carriage of individual strains was of short duration: 70 strains were found on a single sampling day only, and 5 strains were isolated in samples collected over two consecutive weeks. Two of the five colonized individuals-working colleagues having meals together-shared identical K. pneumoniae types four times during the study period. Our findings point toward the potential role of food as a reservoir for K. pneumoniae in humans.

Analytical Performance Validation of Next-Generation Sequencing Based Clinical Microbiology Assays Using a K-mer Analysis Workflow.

Next-generation sequencing (NGS) enables clinical microbiology assays such as molecular typing of bacterial isolates which is now routinely applied for infection control and epidemiology. Additionally, feasibility for NGS-based identification of antimicrobial resistance (AMR) markers as well as genetic prediction of antibiotic susceptibility testing results has been demonstrated. Various bioinformatics approaches enabling NGS-based clinical microbiology assays exist, but standardized, computationally efficient and scalable sample-to-results workflows including validated quality control parameters are still lacking. Bioinformatics analysis workflows based on k-mers have been shown to allow for fast and efficient analysis of large genomics data sets as obtained from microbial sequencing applications. We here demonstrate applicability of k-mer based clinical microbiology assays for whole-genome sequencing (WGS) including variant calling, taxonomic identification, bacterial typing as well as AMR marker detection. The wet-lab and dry-lab workflows were developed and validated in line with Clinical Laboratory Improvement Act (CLIA) guidelines for laboratory-developed tests (LDTs) on multi-drug resistant ESKAPE pathogens. The developed k-mer based workflow demonstrated ≥99.39% repeatability, ≥99.09% reproducibility and ≥99.76% accuracy for variant calling and applied assays as determined by intra-day and inter-day triplicate measurements. The limit of detection (LOD) across assays was found to be at 20× sequencing depth and 15× for AMR marker detection. Thorough benchmarking of the k-mer based workflow revealed analytical performance criteria are comparable to state-of-the-art alignment based workflows across clinical microbiology assays. Diagnostic sensitivity and specificity for multilocus sequence typing (MLST) and phylogenetic analysis were 100% for both approaches. For AMR marker detection, sensitivity and specificity were 95.29 and 99.78% for the k-mer based workflow as compared to 95.17 and 99.77% for the alignment-based approach. Summarizing, results illustrate that k-mer based analysis workflows enable a broad range of clinical microbiology assays, potentially not only for WGS-based typing and AMR gene detection but also genetic prediction of antibiotic susceptibility testing results.

Mycoplasma nasistruthionis sp. nov. and Mycoplasma struthionis sp. nov. isolated from ostriches with respiratory disease.

Twelve Mycoplasma (M.) strains isolated from the nose, the trachea, and the lung of ostriches (Struthio camelus) displaying respiratory disease were investigated. Analysis of 16S rRNA gene sequences placed five of these strains within the M. synoviae cluster, and seven strains within the M. hominis cluster of genus Mycoplasma, which was further confirmed by analyses of the 16S-23S rRNA intergenic spacer region, and partial rpoB gene and amino acid sequences. Genomic information as well as phenotypic features obtained by matrix-assisted laser desorption ionization time of flight (MALDI-ToF) mass spectrometry analysis and serological reactions indicated that the strains examined are representatives of two hitherto unclassified species of genus Mycoplasma, for which the names Mycoplasma nasistruthionis sp. nov., with type strain 2F1AT (= ATCC BAA-1893T = DSM 22456T), and Mycoplasma struthionis sp. nov., with type strain 237IAT (= ATCC BAA-1890T = DSM 22453T), are proposed.

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.

Phenotypic and Genotypic Antimicrobial Resistance Traits of Vibrio cholerae Non-O1/Non-O139 Isolated From a Large Austrian Lake Frequently Associated With Cases of Human Infection.

Vibrio cholerae belonging to serogroups other than O1 and O139 are opportunistic pathogens which cause infections with a variety of clinical symptoms. Due to the increasing number of V. cholerae non-O1/non-O139 infections in association with recreational waters in the past two decades, they have received increasing attention in recent literature and by public health authorities. Since the treatment of choice is the administration of antibiotics, we investigated the distribution of antimicrobial resistance properties in a V. cholerae non-O1/non-O139 population in a large Austrian lake intensively used for recreation and in epidemiologically linked clinical isolates. In total, 82 environmental isolates - selected on the basis of comprehensive phylogenetic information - and nine clinical isolates were analyzed for their phenotypic antimicrobial susceptibility. The genomes of 46 environmental and eight clinical strains were screened for known genetic antimicrobial resistance traits in CARD and ResFinder databases. In general, antimicrobial susceptibility of the investigated V. cholerae population was high. The environmental strains were susceptible against most of the 16 tested antibiotics, except sulfonamides (97.5% resistant strains), streptomycin (39% resistant) and ampicillin (20.7% resistant). Clinical isolates partly showed additional resistance to amoxicillin-clavulanic acid. Genome analysis showed that crp, a regulator of multidrug efflux genes, and the bicyclomycin/multidrug efflux system of V. cholerae were present in all isolates. Nine isolates additionally carried variants of bla CARB-7 and bla CARB-9, determinants of beta-lactam resistance and six isolates carried catB9, a determinant of phenicol resistance. Three isolates had both bla CARB-7 and catB9. In 27 isolates, five out of six subfamilies of the MATE-family were present. For all isolates no genes conferring resistance to aminoglycosides, macrolides and sulfonamides were detected. The apparent lack of either known antimicrobial resistance traits or mobile genetic elements indicates that in cholera non-epidemic regions of the world, V. cholerae non-O1/non-O139 play a minor role as a reservoir of resistance in the environment. The discrepancies between the phenotypic and genome-based antimicrobial resistance assessment show that for V. cholerae non-O1/non-O139, resistance databases are currently inappropriate for an assessment of antimicrobial resistance. Continuous collection of both data over time may solve such discrepancies between genotype and phenotype in the future.

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

BackgroundBrown 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.AimWe intended to determine whether urban rats at two highly frequented sites in Vienna, Austria, carry extended-spectrum ß-lactamase-producing Enterobacteriaceae, fluoroquinolone-resistant Enterobacteriaceae and meticillin-resistant (MR) Staphylococcus spp. (MRS).MethodsWe 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 characterised the isolates and their antimicrobial properties using microbiological and genetic methods including disk diffusion, microarray analysis, sequencing, and detection and characterisation of plasmids.ResultsEight multidrug-resistant Escherichia coli and two extensively drug-resistant New Delhi metallo-ß-lactamases-1 (NDM-1)-producing Enterobacter xiangfangensis ST114 (En. cloacae complex) were isolated from nine of 62 rats. Nine Enterobacteriaceae isolates harboured the bla CTX-M gene and one carried a plasmid-encoded ampC gene (bla CMY-2). Forty-four MRS were isolated from 37 rats; they belonged to seven different staphylococcal species: S. fleurettii, S. sciuri, S. aureus, S. pseudintermedius, S. epidermidis, S. haemolyticus (all mecA-positive) and mecC-positive S. xylosus.ConclusionOur 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.

Increased genetic diversity of methicillin-resistant Staphylococcus aureus (MRSA) isolated from companion animals.

The aim of the present study was to investigate the diversity of methicillin-resistant Staphylococcus aureus (MRSA) that originated from Austrian companion animals during the last five-year period. A total of 90 non-repetitive MRSA isolates were obtained during diagnostic activities from autumn 2013 to autumn 2018. They originated from horses (n = 62), cats (n = 13), dogs (n = 10), rabbits (n = 2), a domestic canary, a zoo-kept hammer-headed bat (Hypsignathus monstrosus) and a semi-captive northern bald ibis (Geronticus eremita). Antimicrobial susceptibility testing was performed. All isolates were mecA-positive and mecC-negative. The isolates were genotyped by SCCmec, spa and dru typing, Multiple-Locus Variable number of tandem repeat Analyses (MLVA), S. aureus DNA microarray, and whole-genome sequencing (WGS). Eight sequence types (STs - ST398, ST5275 (new ST), ST225, ST8, ST22, ST152, ST1, and ST45), three SCCmec types (II, IV, and V), sixteen spa types (t003, t008, t011, t015, t032, t034, t1381, t1928, t1985, t223, t334, t355, t430, t6447, t6867, and t7105), fourteen dru types (dt10a, dt10az, dt10q, dt10r, dt11a, dt5e, dt6j, dt9a, dt9ak, dt9g, and four new types dt8as, dt7ak, dt4j, dt14n), and thirty-five MLVA types were detected. WGS-based core genome MLST (cgMLST) displayed five main clusters. Compared to the time period 2004-2013, the results of the present study show not only a higher diversity among the MRSA isolates within the population of Austrian companion animals, but also the introduction of new clones. Although ST398 isolates remained predominant, mainly due to high presence of this lineage among horses, increasing isolation rates of human-associated MRSA clones were observed in cats and dogs.

Whole Genome Sequencing Based Surveillance of L. monocytogenes for Early Detection and Investigations of Listeriosis Outbreaks.

In Austria, all laboratories are legally obligated to forward human and food/environmental L. monocytogenes isolates to the National Reference Laboratory/Center (NRL) for Listeria. Two invasive human isolates of L. monocytogenes serotype 1/2a of the same pulsed-field gel electrophoresis (PFGE) pattern, previously unknown in Austria, were cultured for the first time in January 2016. Five further human isolates, obtained from patients with invasive listeriosis between April 2016 and September 2017, showed this PFGE pattern. In Austria the NRL started to use whole-genome sequencing (WGS) based typing in 2016, using a core genome MLST (cgMLST) scheme developed by Ruppitsch et al. 2015, which contains 1701 target genes. Sequence data are submitted to a publicly available nomenclature server (Ridom GmbH, Münster, Germany) for allocation of the core genome complex type (CT). The seven invasive human isolates differed from each other with zero to two alleles and were allocated to CT1234 (declared as outbreak strain). Among the Austrian strain collection of about 6,000 cgMLST-characterized non-human isolates (i.e., food/environmental isolates) 90 isolates shared CT1234. Out of these, 83 isolates were traced back to one meat processing-company. They differed from the outbreak strain by up to seven alleles; one isolate originated from the company's industrial slicer. The remaining seven CT1234-isolates were obtained from food products of four other companies (five fish-products, one ready-to-eat dumpling and one deer-meat) and differed from the outbreak strain by six to eleven alleles. The outbreak described shows the considerable potential of WGS to identify the source of a listeriosis outbreak. Compared to PFGE analysis, WGS-based typing has higher discriminatory power, yields better data accuracy, and allows higher laboratory through-put at lower cost. Utilization of WGS-based typing results of human and food/ environmental L. monocytogenes isolates by appropriate public health analysts and epidemiologists is indispensable to support a successful outbreak investigation.

Characterization of mecC gene-carrying coagulase-negative Staphylococcus spp. isolated from various animals.

The presence of the methicillin resistance gene mecC in coagulase-negative Staphylococcus spp. (CoNS) is scarce. The aim of this study was to characterize mecC-positive CoNS isolated from various wild and domestic animals. The presence of the mecC gene was screened in 4299 samples from wild animals and domestic animals. Fifteen coagulase-negative staphylococci, that displayed a cefoxitin-resistant phenotype, were tested mecC-positive by PCR. Antimicrobial susceptibility testing was performed for all isolates. The 15 isolates were genotyped by sequencing of the entire class E mec gene complex (blaZ-mecC-mecR1-mecI), the ccrA and ccrB recombinase genes and other determinants within the type XI SCCmec element. DNA microarray analysis was performed and five selected isolates were additionally whole genome sequenced and analyzed. S. stepanovicii (n = 3), S. caprae (n = 1), S. warneri (n = 1), S. xylosus (n = 1) and S. sciuri (n = 9) were detected. All but the S. sciuri isolates were found to be susceptible to all non-beta lactams. The entire class E mec gene complex was detected in all isolates but ccrA and ccrB genes were not identified in S. stepanovicii and S. xylosus. The genes erm(B) and fexA (n = 4, each) were the most predominant non-beta lactam resistance genes detected in the S. sciuri isolates. Even though the presence of the mecC gene among CoNS is a rare observation, this study further expands our knowledge by showing that the mecC gene, including its allotypes, are present in more staphylococcal species from different animal species than has been previously described.

Multicenter Study of Cronobacter sakazakii Infections in Humans, Europe, 2017.

Cronobacter sakazakii has been documented as a cause of life-threating infections, predominantly in neonates. We conducted a multicenter study to assess the occurrence of C. sakazakii across Europe and the extent of clonality for outbreak detection. National coordinators representing 24 countries in Europe were requested to submit all human C. sakazakii isolates collected during 2017 to a study center in Austria. Testing at the center included species identification by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, subtyping by whole-genome sequencing (WGS), and determination of antimicrobial resistance. Eleven countries sent 77 isolates, including 36 isolates from 2017 and 41 historical isolates. Fifty-nine isolates were confirmed as C. sakazakii by WGS, highlighting the challenge of correctly identifying Cronobacter spp. WGS-based typing revealed high strain diversity, indicating absence of multinational outbreaks in 2017, but identified 4 previously unpublished historical outbreaks. WGS is the recommended method for accurate identification, typing, and detection of this pathogen.

Whole genome sequencing reveals resemblance between ESBL-producing and carbapenem resistant Klebsiella pneumoniae isolates from Austrian rivers and clinical isolates from hospitals.

In 2016, the Austrian Agency for Health and Food Safety started a pilot project to investigate antimicrobial resistance in surface water. Here we report on the characterisation of carbapenem resistant and ESBL-producing K. pneumoniae isolates from Austrian river water samples compared to 95 clinical isolates recently obtained in Austrian hospitals. Ten water samples were taken from four main rivers, collected upstream and downstream of major cities in 2016. For subtyping and comparison, public core genome multi locus sequence typing (cgMLST) schemes were used. The presence of AMR genes, virulence genes and plasmids was extracted from whole genome sequence (WGS) data. In total three ESBL-producing strains and two carbapenem resistant strains were isolated. WGS based comparison of these five water isolates to 95 clinical isolates identified three clusters. Cluster 1 (ST11) and cluster 2 (ST985) consisted of doublets of carbapenem resistant strains (one water and one clinical isolate each). Cluster 3 (ST405) consisted of three ESBL-producing strains isolated from one water sample and two clinical specimens. The cities, in which patient isolates of cluster 2 and 3 were collected, were in concordance with the water sampling locations downstream from these cities. The genetic concordance between isolates from river water samples and patient isolates raises concerns regarding the release of wastewater treatment plant effluents into surface water. From a public health perspective these findings demand attention and strategies are required to minimize the spread of multiresistant strains to the environment.

Whole-Genome Analysis of a Human Enterobacter mori Isolate Carrying a blaIMI-2 Carbapenemase in Austria.

Enterobacterales belonging to the genus Enterobacter are well-known human pathogens and blaIMI carrying strains have been described in several countries. From Austria, we report on the first human Enterobacter mori isolate, typically a plant pathogen, which showed an undescribed multilocus sequence type and carried a blaIMI-2 carbapenemase.