Core genome multilocus sequence typing (cgMLST) applicable to the monophyletic Klebsiella oxytoca species complex.

The environmental bacterium Klebsiella oxytoca displays an alarming increase of antibiotic-resistant strains that frequently cause outbreaks in intensive care units. Due to its prevalence in the environment and opportunistic presence in humans, molecular surveillance (including resistance marker screening) and high-resolution cluster analysis are of high relevance. Furthermore, K. oxytoca previously described in studies is rather a species complex (KoSC) than a single species comprising at least six closely related species that are not easily differentiated by standard typing methods. To reach a discriminatory power high enough to identify and resolve clusters within these species, whole genome sequencing is necessary. The resolution is achievable with core genome multilocus sequence typing (cgMLST) extending typing of a few housekeeping genes to thousands of core genome genes. CgMLST is highly standardized and provides a nomenclature enabling cross laboratory reproducibility and data exchange for routine diagnostics. Here, we established a cgMLST scheme not only capable of resolving the KoSC species but also producing reliable and consistent results for published outbreaks. Our cgMLST scheme consists of 2,536 core genome and 2,693 accessory genome targets, with a percentage of good cgMLST targets of 98.31% in 880 KoSC genomes downloaded from the National Center for Biotechnology Information (NCBI). We also validated resistance markers against known resistance gene patterns and successfully linked genetic results to phenotypically confirmed toxic strains carrying the til gene cluster. In conclusion, our novel cgMLST enables highly reproducible typing of four different clinically relevant species of the KoSC and thus facilitates molecular surveillance and cluster investigations.

A multicenter study on accuracy and reproducibility of nanopore sequencing-based genotyping of bacterial pathogens.

Nanopore sequencing has shown the potential to democratize genomic pathogen surveillance due to its ease of use and low entry cost. However, recent genotyping studies showed discrepant results compared to gold-standard short-read sequencing. Furthermore, although essential for widespread application, the reproducibility of nanopore-only genotyping remains largely unresolved. In our multicenter performance study involving five laboratories, four public health-relevant bacterial species were sequenced with the latest R10.4.1 flow cells and V14 chemistry. Core genome MLST analysis of over 500 data sets revealed highly strain-specific typing errors in all species in each laboratory. Investigation of the methylation-related errors revealed consistent DNA motifs at error-prone sites across participants at read level. Depending on the frequency of incorrect target reads, this either leads to correct or incorrect typing, whereby only minimal frequency deviations can randomly determine the final result. PCR preamplification, recent basecalling model updates and an optimized polishing strategy notably diminished the non-reproducible typing. Our study highlights the potential for new errors to appear with each newly sequenced strain and lays the foundation for computational approaches to reduce such typing errors. In conclusion, our multicenter study shows the necessity for a new validation concept for nanopore sequencing-based, standardized bacterial typing, where single nucleotide accuracy is critical.

Enterococcus montenegrensis sp. nov., isolated from artisanal Montenegrin dry sausage.

A novel, Gram-positive, facultative anaerobe, coccoid and non-motile bacterium, designated as CoE-012-22T was isolated from dried beef sausage (the original name in Montenegro is Govedji Kulen) manufactured in the municipality of Rozaje (Montenegro) in 2021. Cells of this strain were oxidase- and catalase-negative. Growth occurred at 4-50 °C, at pH 5.0-8.0 and with 0-6.5 % (w/v) NaCl in diverse growth media. MALDI-TOF analysis identified the strain as Enterococcus canintestini (log score 2). Phylogenetic analysis of the 16S rRNA gene and whole genome sequences assigned the strain to the genus Enterococcus. The closest relatives were E. canintestini DSM 21207T and E. dispar ATCC 51266T with 16S rRNA gene sequence pairwise similarities of 99.34 and 98.59 %, respectively. The average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values between isolate CoE-012-22T and other enterococci species were below the thresholds for species delineation thresholds (95.0 % ANI; 70.0 % dDDH) with maximum identities of 84.13 % (ANIb), 86.43 % (ANIm) and 28.4 % (dDDH) to E. saigonensis JCM 31193T and 70.97 % (ANIb), 88.99 % (ANIm) and 32.4 % (dDDH) to E. malodoratus ATCC 43197T. Two unknown Enterococcus isolates, Enterococcus sp. MJM12 and Enterococcus SMC-9, showed identities of 99.87 and 99.94 % (16S rRNA), 98.57 and 98.65 % (ANIb), 98.93 and 99.02 % (ANIm), and 89.8 and 90.0 % (dDDH) to strain CoE-012-22T and can therefore be regarded as the same species. Based on the characterization results, strain CoE-012-22T was considered to represent a novel species, for which the name Enterococcus montenegrensis sp. nov. is proposed. The type strain is CoE-012-22T (=DSM 115843T=NCIMB 15468T).

Diversity of Staphylococcus aureus isolated from nares of ruminants.

AIMS: To examine the diversity of Staphylococcus aureus isolated from nasal swabs of ruminants in Rwanda. METHODS AND RESULTS: A total of 454 nasal swabs from 203 cows, 170 goats, and 81 sheep were examined for the presence of S. aureus, and 30 S. aureus isolates were detected and characterized pheno- and genotypically. Resistance to penicillin and/or tetracycline was observed. The isolates were assigned to eight different spa types (t21057 (novel), t10103, t18853, t20842, t318, t355, t458, and t9432) belonging to six clonal complexes (CCs) (CC152, CC30, CC3591, CC3666, CC522, and CC97). Panton-Valentine leukocidin (PVL) genes (lukF-PV/lukS-PV), the bovine leukocidin genes (lukM/lukF-P83), and the human and bovine variants of the toxic shock syndrome toxin gene tst-1 variants were detected. CONCLUSION: These findings demonstrate that the nares of ruminants in Rwanda are colonized with mastitis-associated S. aureus, including lineages that are also carried by humans, underscoring the zoonotic risk, especially for livestock keepers. These results highlight the crucial importance of hygiene measures when handling livestock.

Antibiotic Resistance Hotspot: Comparative Genomics Reveals Multiple Strains of Multidrug-Resistant Citrobacter portucalensis in Edible Snails.

The demand for terrestrial snails as a food source is still on the increase globally, yet this has been overlooked in disease epidemiology and the spread of antimicrobial resistance. This study conducted genomic analyses of twenty Citrobacter portucalensis strains isolated from live edible snails traded in two hubs. The isolates were subjected to MALDI-TOF MS, antimicrobial resistance testing, whole genome sequencing, and analyses for in-depth characterization. The findings disclosed that seventeen strains across the two trading hubs were distinct from previously reported ones. Four isolates were found to share the same sequence type (ST881). Genome-based comparison suggests a clonal transmission of strains between snails traded in these hubs. All the isolates across the two hubs harbored similar variety of antimicrobial resistance genes, with notable ones being blaCMY and qnrB. Sixteen isolates (80%) expressed phenotypic resistance to second-generation cephalosporins, while eleven isolates (55%) exhibited resistance to third-generation cephalosporins. This report of multi-drug-resistant C. portucalensis strains in edible snails highlights significant concerns for food safety and clinical health because of the potential transmission to humans. Enhanced surveillance and stringent monitoring by health authorities are essential to evaluate the impact of these strains on the burden of antimicrobial resistance and to address the associated risk.

Real-Time Nanopore Q20+ Sequencing Enables Extremely Fast and Accurate Core Genome MLST Typing and Democratizes Access to High-Resolution Bacterial Pathogen Surveillance.

Next-generation whole-genome sequencing is essential for high-resolution surveillance of bacterial pathogens, for example, during outbreak investigations or for source tracking and escape variant analysis. However, current global sequencing and bioinformatic bottlenecks and a long time to result with standard technologies demand new approaches. In this study, we investigated whether novel nanopore Q20+ long-read chemistry enables standardized and easily accessible high-resolution typing combined with core genome multilocus sequence typing (cgMLST). We set high requirements for discriminatory power by using the slowly evolving bacterium Bordetella pertussis as a model pathogen. Our results show that the increased raw read accuracy enables the description of epidemiological scenarios and phylogenetic linkages at the level of gold-standard short reads. The same was true for our variant analysis of vaccine antigens, resistance genes, and virulence factors, demonstrating that nanopore sequencing is a legitimate competitor in the area of next-generation sequencing (NGS)-based high-resolution bacterial typing. Furthermore, we evaluated the parameters for the fastest possible analysis of the data. By combining the optimized processing pipeline with real-time basecalling, we established a workflow that allows for highly accurate and extremely fast high-resolution typing of bacterial pathogens while sequencing is still in progress. Along with advantages such as low costs and portability, the approach suggested here might democratize modern bacterial typing, enabling more efficient infection control globally.

Analysis of the HbpA Protein from Corynebacterium diphtheriae Clinical Isolates and Identification of a Putative Hemoglobin-Binding Site on HbpA.

The Corynebacterium diphtheriae hemoglobin-binding protein HbpA is critical for the acquisition of iron from the hemoglobin-haptoglobin complex (Hb-Hp). Previous studies using C. diphtheriae strain 1737 showed that large aggregates formed by HbpA are associated with iron transport activity and enhanced binding to Hb-Hp; however, specific regions within HbpA required for Hb-Hp binding or iron uptake have not been identified. In this study, we characterized two clinical isolates from Austria, designated 07-18 and 09-15, which express HbpA proteins that share only 53% and 44% sequence identity, respectively, to the strain 1737 HbpA protein. The HbpA proteins expressed by the Austrian strains had functional and structural properties similar to those of the HbpA protein in strain 1737 despite the limited sequence similarity. These shared characteristics between the HbpA proteins included similar cellular localization, aggregate formation, and Hb and Hb-Hp binding. Additionally, the Austrian strains were able to acquire iron from Hb and Hb-Hp, and deletion of the hbpA gene from these two clinical isolates reduced their ability to use Hb-Hp as an iron source. A sequence comparison between the HbpA proteins from 1737 and the Austrian strains assisted in the identification of a putative Hb-binding site that shared similar characteristics with the Hb-binding regions in Staphylococcus aureus NEAT domains. Amino acid substitutions within this conserved Hb-binding region significantly reduced Hb and Hb-Hp binding and diminished the hemin-iron uptake function of HbpA. These findings represent important advances in our understanding of the interaction of HbpA with human hemoproteins. IMPORTANCE Hemoglobin (Hb) is the primary source of iron in humans, and the acquisition of hemin-iron from Hb is critical for many bacterial pathogens to infect and survive in the human host. In this study, we have examined the C. diphtheriae Hb-binding protein HbpA in two clinical isolates and show that these proteins, despite limited sequence similarity, are functionally equivalent to the previously described HbpA protein in strain 1737. A sequence comparison between these three strains led to the identification of a conserved Hb-binding site, which will further our understanding of how this novel protein functions in hemin-iron transport and, more generally, will expand our knowledge on how Hb interacts with proteins.

Association of Phylogenomic Relatedness among Neisseria gonorrhoeae Strains with Antimicrobial Resistance, Austria, 2016-2020.

We investigated genomic determinants of antimicrobial resistance in 1,318 Neisseria gonorrhoeae strains isolated in Austria during 2016-2020. Sequence type (ST) 9363 and ST11422 isolates had high rates of azithromycin resistance, and ST7363 isolates correlated with cephalosporin resistance. These results underline the benefit of genomic surveillance for antimicrobial resistance monitoring.

Outbreak of Cronobacter turicensis in European brown hares (Lepus europaeus).

This is the first report of acute deaths in five European brown hares (Lepus europaeus) attributed to mucoid and necrotizing typhlocolitis caused by genetically different Cronobacter (C.) turicensis strains in northeastern Austria. As this opportunistic pathogen is mainly known for causing disease in immunocompromised humans and neonates, this previously unrecognized potential for a spill over from a wildlife reservoir to humans warrants further attention.

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.

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.

Isolate-Based Surveillance of Bordetella pertussis, Austria, 2018-2020.

Pertussis is a vaccine-preventable disease, and its recent resurgence might be attributable to the emergence of strains that differ genetically from the vaccine strain. We describe a novel pertussis isolate-based surveillance system and a core genome multilocus sequence typing scheme to assess Bordetella pertussis genetic variability and investigate the increased incidence of pertussis in Austria. During 2018-2020, we obtained 123 B. pertussis isolates and typed them with the new scheme (2,983 targets and preliminary cluster threshold of <6 alleles). B. pertussis isolates in Austria differed genetically from the vaccine strain, both in their core genomes and in their vaccine antigen genes; 31.7% of the isolates were pertactin-deficient. We detected 8 clusters, 1 of them with pertactin-deficient isolates and possibly part of a local outbreak. National expansion of the isolate-based surveillance system is needed to implement pertussis-control strategies.

Assessing the Genetic Diversity of Austrian Corynebacterium diphtheriae Clinical Isolates, 2011 to 2019.

Diphtheria is a vaccine-preventable disease with a high potential for reemergence. One of its causative agents is Corynebacterium diphtheriae, with some strains producing diphtheria toxin. From 2011 to 2019, 57 clinical C. diphtheriae strains were isolated in Austria, either from the respiratory tract or from skin infections. The aim of this study was to investigate the genetic diversity of these C. diphtheriae isolates using whole-genome sequencing. Isolates were characterized by genome-wide comparisons using single nucleotide polymorphism analysis or core genome multilocus sequence typing and by searching sequence data for antimicrobial resistance genes and genes involved in diphtheria toxin production. The genetic diversity among the isolates was high, with no clear distribution over time or place. Corynebacterium belfantii isolates were separated from other strains and were strongly associated with respiratory infections (odds ratio [OR] = 57). Two clusters, limited in time and space, were identified. Almost 40% of strains carried resistance genes against tetracycline or sulfonamides, mostly from skin infections. Microbiological tests showed that 55% of isolates were resistant to penicillin but did not carry genes conferring ß-lactam resistance. A diphtheria toxin gene with no nonsynonymous mutation was found in three isolates only. This study showed that sequencing can provide valuable information complementing routine microbiological and epidemiological investigations. It allowed us to identify unknown clusters, evaluate antimicrobial resistance more broadly, and support toxigenicity results obtained by PCR. For these reasons, C. diphtheriae surveillance could strongly benefit from the routine implementation of whole-genome sequencing.

Presence of ß-Lactamase-producing Enterobacterales and Salmonella Isolates in Marine Mammals.

Marine mammals have been described as sentinels of the health of marine ecosystems. Therefore, the aim of this study was to investigate (i) the presence of extended-spectrum ß-lactamase (ESBL)- and AmpC-producing Enterobacterales, which comprise several bacterial families important to the healthcare sector, as well as (ii) the presence of Salmonella in these coastal animals. The antimicrobial resistance pheno- and genotypes, as well as biocide susceptibility of Enterobacterales isolated from stranded marine mammals, were determined prior to their rehabilitation. All E. coli isolates (n = 27) were screened for virulence genes via DNA-based microarray, and twelve selected E. coli isolates were analyzed by whole-genome sequencing. Seventy-one percent of the Enterobacterales isolates exhibited a multidrug-resistant (MDR) pheno- and genotype. The gene blaCMY (n = 51) was the predominant ß-lactamase gene. In addition, blaTEM-1 (n = 38), blaSHV-33 (n = 8), blaCTX-M-15 (n = 7), blaOXA-1 (n = 7), blaSHV-11 (n = 3), and blaDHA-1 (n = 2) were detected. The most prevalent non-ß-lactamase genes were sul2 (n = 38), strA (n = 34), strB (n = 34), and tet(A) (n = 34). Escherichia coli isolates belonging to the pandemic sequence types (STs) ST38, ST167, and ST648 were identified. Among Salmonella isolates (n = 18), S. Havana was the most prevalent serotype. The present study revealed a high prevalence of MDR bacteria and the presence of pandemic high-risk clones, both of which are indicators of anthropogenic antimicrobial pollution, in marine mammals.

Virulence characterization and comparative genomics of Listeria monocytogenes sequence type 155 strains.

BACKGROUND: Listeria (L.) monocytogenes strains show a high diversity regarding stress tolerance and virulence potential. Genome studies have mainly focused on specific sequence types (STs) predominantly associated with either food or human listeriosis. This study focused on the prevalent ST155, showing equal distribution among clinical and food isolates. We evaluated the virulence potential of 20 ST155 strains and performed comparative genomic analysis of 130 ST155 strains isolated from food, food processing environments and human listeriosis cases in different countries and years. RESULTS: The in vitro virulence assays using human intestinal epithelial Caco2 and hepatocytic HEPG2 cells showed an impaired virulence phenotype for six of the 20 selected ST155 strains. Genome analysis revealed no distinct clustering of strains from the same source category (food, food processing environment, and clinical isolates). All strains harbored an intact inlA and inlB locus, except four strains, which had an internal deletion in the inlA gene. All strains harbored LIPI-1, but prfA was present in a longer variant in six strains, all showing impaired virulence. The longer PrfA variant resulted in lower expression of inlA, inlB, and prfA, and no expression of hly and actA. Regarding stress-related gene content, SSI-1 was present, whereas qacH was absent in all strains. 34.6% of the strains harbored a plasmid. All but one ST155 plasmids showed high conservation and harbored cadA2, bcrABC, and a triphenylmethane reductase. CONCLUSIONS: This study contributes to an enhanced understanding of L. monocytogenes ST155 strains, being equally distributed among isolates from humans, food, and food processing environments. The conservation of the present genetic traits and the absence of unique inherent genetic features makes these types of STs especially interesting since they are apparently equally adapted to the conditions in food processing environments, as well as in food as to the human host environment. However, a ST155-specific mutation resulting in a longer PrfA variant impaired the virulence potential of several ST155 strains.

Large Nationwide Outbreak of Invasive Listeriosis Associated with Blood Sausage, Germany, 2018-2019.

Invasive listeriosis is a severe foodborne infection in humans and is difficult to control. Listeriosis incidence is increasing worldwide, but some countries have implemented molecular surveillance programs to improve recognition and management of listeriosis outbreaks. In Germany, routine whole-genome sequencing, core genome multilocus sequence typing, and single nucleotide polymorphism calling are used for subtyping of Listeria monocytogenes isolates from listeriosis cases and suspected foods. During 2018-2019, an unusually large cluster of L. monocytogenes isolates was identified, including 134 highly clonal, benzalkonium-resistant sequence type 6 isolates collected from 112 notified listeriosis cases. The outbreak was one of the largest reported in Europe during the past 25 years. Epidemiologic investigations identified blood sausage contaminated with L. monocytogenes highly related to clinical isolates; withdrawal of the product from the market ended the outbreak. We describe how epidemiologic investigations and complementary molecular typing of food isolates helped identify the outbreak vehicle.

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.

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.

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.

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.