Phylodynamic signatures in the emergence of community-associated MRSA.

Community-associated, methicillin-resistant Staphylococcus aureus (MRSA) lineages have emerged in many geographically distinct regions around the world during the past 30 y. Here, we apply consistent phylodynamic methods across multiple community-associated MRSA lineages to describe and contrast their patterns of emergence and dissemination. We generated whole-genome sequencing data for the Australian sequence type (ST) ST93-MRSA-IV from remote communities in Far North Queensland and Papua New Guinea, and the Bengal Bay ST772-MRSA-V clone from metropolitan communities in Pakistan. Increases in the effective reproduction number (Re) and sustained transmission (Re > 1) coincided with spread of progenitor methicillin-susceptible S. aureus (MSSA) in remote northern Australian populations, dissemination of the ST93-MRSA-IV genotype into population centers on the Australian East Coast, and subsequent importation into the highlands of Papua New Guinea and Far North Queensland. Applying the same phylodynamic methods to existing lineage datasets, we identified common signatures of epidemic growth in the emergence and epidemiological trajectory of community-associated S. aureus lineages from America, Asia, Australasia, and Europe. Surges in Re were observed at the divergence of antibiotic-resistant strains, coinciding with their establishment in regional population centers. Epidemic growth was also observed among drug-resistant MSSA clades in Africa and northern Australia. Our data suggest that the emergence of community-associated MRSA in the late 20th century was driven by a combination of antibiotic-resistant genotypes and host epidemiology, leading to abrupt changes in lineage-wide transmission dynamics and sustained transmission in regional population centers.

Sequencing a CC239-MRSA-III with a novel composite SCC mec element from Kuwait.

Staphylococcus aureus CC239-MRSA-III is an ancient pandemic strain of hospital-associated, methicillin-resistant S. aureus that spread globally for decades and that still can be found in some parts of the world. In Kuwait, microarray-based surveillance identified from 2019 to 2022 a series of isolates of a hitherto unknown variant of this strain that carried a second set of recombinase genes, ccrA/B-2. To elucidate the structure of its SCCmec element, two isolates were subjected to nanopore sequencing. This revealed, in addition to ccrA/B-2, several SCC-associated genes including speG (spermidine N acetyltransferase) and a gene encoding a large "E-domain containing protein" (dubbed as edcP-SCC). This gene contained three regions consisting of multiple repeating units. In terms of sequence and structure it was similar but not identical to the biofilm-related aap gene from S. epidermidis. A review of published sequences identified edcP-SCC in eighteen genome sequences of S. aureus, S. epidermidis and S. capitis, and frequently it appears in a similar cluster of genes as in the strains sequenced herein. Isolates also carried a prophage with the adhesion factor sasX/sesI and aminoglycoside resistance genes. This is consistent with an affiliation to the "South-East Asian" Clade of CC239. The emergence of edcP-SCC and sasX-positive CC239 strain shows that, against a global trend towards community-associated MRSA, the ancient pandemic CC239 hospital strain still continues to evolve and to cause outbreaks.

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.

Reduced Glycolysis and Cytotoxicity in Staphylococcus aureus Isolates from Chronic Rhinosinusitis as Strategies for Host Adaptation.

Chronic rhinosinusitis (CRS) is a multifactorial infection of the nasal cavity and sinuses. In this study, nasal swabs from control donors (N = 128) and patients with CRS (N = 246) were analysed. Culture methods and metagenomics revealed no obvious differences in the composition of the bacterial communities between the two groups. However, at the functional level, several metabolic pathways were significantly enriched in the CRS group compared to the control group. Pathways such as carbohydrate transport metabolism, ATP synthesis, cofactors and vitamins, photosynthesis and transcription were highly enriched in CRS. In contrast, pathways related to lipid metabolism were more representative in the control microbiome. As S. aureus is one of the main species found in the nasal cavity, staphylococcal isolates from control and CRS samples were analysed by microarray and functional assays. Although no significant genetic differences were detected by microarray, S. aureus from CRS induced less cytotoxicity to lung cells and lower rates of glycolysis in host cells than control isolates. These results suggest the differential modulation of staphylococcal virulence by the environment created by other microorganisms and their interactions with host cells in control and CRS samples. These changes were reflected in the differential expression of cytokines and in the expression of Agr, the most important quorum-sensing regulator of virulence in S. aureus. In addition, the CRS isolates remained stable in their cytotoxicity, whereas the cytotoxic activity of S. aureus isolated from control subjects decreased over time during in vitro passage. These results suggest that host factors influence the virulence of S. aureus and promote its adaptation to the nasal environment during CRS.

Protein Microarray-Guided Development of a Highly Sensitive and Specific Dipstick Assay for Glanders Serodiagnostics.

Burkholderia mallei, the causative agent of glanders, is a clonal descendant of Burkholderia pseudomallei, the causative agent of melioidosis, which has lost its environmental reservoir and has a restricted host range. Despite limitations in terms of sensitivity and specificity, complement fixation is still the official diagnostic test for glanders. Therefore, new tools are needed for diagnostics and to study the B. mallei epidemiology. We recently developed a highly sensitive serodiagnostic microarray test for human melioidosis based on the multiplex detection of B. pseudomallei proteins. In this study, we modified our array tests by using anti-horse IgG conjugate and tested sera from B. mallei-infected horses (n = 30), negative controls (n = 39), and horses infected with other pathogens (n = 14). Our array results show a sensitivity of 96.7% (confidence interval [CI] 85.5 to 99.6%) and a specificity of 100.0% (CI, 95.4 to 100.0%). The reactivity pattern of the positive sera on our array test allowed us to identify a set of 12 highly reactive proteins of interest for glanders diagnosis. The B. mallei variants of the three best protein candidates were selected for the development of a novel dipstick assay. Our point-of-care test detected glanders cases in less than 15 min with a sensitivity of 90.0% (CI, 75.7 to 97.1%) and a specificity of 100.0% (CI, 95.4 to 100.0%). The microarray and dipstick can easily be adopted for the diagnosis of both B. mallei and B. pseudomallei infections in different animals. Future studies will show whether multiplex serological testing has the potential to differentiate between these pathogens.

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.

Sequence Analysis of Novel Staphylococcus aureus Lineages from Wild and Captive Macaques.

Staphylococcus aureus is a widespread and common opportunistic bacterium that can colonise or infect humans as well as a wide range of animals. There are a few studies of both methicillin-susceptible S. aureus (MSSA) and methicillin-resistant S. aureus (MRSA) isolated from monkeys, apes, and lemurs, indicating a presence of a number of poorly or unknown lineages of the pathogen. In order to obtain insight into staphylococcal diversity, we sequenced strains from wild and captive individuals of three macaque species (Macaca mulatta, M. assamensis, and M. sylvanus) using Nanopore and Illumina technologies. These strains were previously identified by microarray as poorly or unknown strains. Isolates of novel lineages ST4168, ST7687, ST7688, ST7689, ST7690, ST7691, ST7692, ST7693, ST7694, ST7695, ST7745, ST7746, ST7747, ST7748, ST7749, ST7750, ST7751, ST7752, ST7753, and ST7754 were sequenced and characterised for the first time. In addition, isolates belonging to ST2990, a lineage also observed in humans, and ST3268, a MRSA strain already known from macaques, were also included into the study. Mobile genetic elements, genomic islands, and carriage of prophages were analysed. There was no evidence for novel host-specific virulence factors. However, a conspicuously high rate of carriage of a pathogenicity island harbouring edinB and etD2/etE as well as a higher number of repeat units within the gene sasG (encoding an adhesion factor) than in human isolates were observed. None of the strains harboured the genes encoding Panton-Valentine leukocidin. In conclusion, wildlife including macaques may harbour an unappreciated diversity of S. aureus lineages that may be of clinical relevance for humans, livestock, or for wildlife conservation, given the declining state of many wildlife populations.

Molecular characterisation of extended-spectrum ß-lactamase producing Escherichia coli in wild birds and cattle, Ibadan, Nigeria.

BACKGROUND: Antimicrobial resistance (AMR) is an increasing global health concern reducing options for therapy of infections and also for perioperative prophylaxis. Many Enterobacteriaceae cannot be treated anymore with third generation cephalosporins (3GC) due to the production of certain 3GC hydrolysing enzymes (extended spectrum beta-lactamases, ESBLs). The role of animals as carriers and vectors of multi-resistant bacteria in different geographical regions is poorly understood. Therefore, we investigated the occurrence and molecular characteristics of ESBL-producing Escherichia coli (E. coli) in wild birds and slaughtered cattle in Ibadan, Nigeria. Cattle faecal samples (n = 250) and wild bird pooled faecal samples (cattle egrets, Bubulcus ibis, n = 28; white-faced whistling duck, Dendrocygna viduata, n = 24) were collected and cultured on cefotaxime-eosin methylene blue agar. Antimicrobial susceptibility was determined by agar diffusion assays and all 3GC resistant isolates were genotypically characterised for AMR genes, virulence associated genes (VAGs) and serotypes using DNA microarray-based assays. RESULTS: All 3GC resistant isolates were E. coli: cattle (n = 53), egrets (n = 87) and whistling duck (n = 4); cultured from 32/250 (12.8%), 26/28 (92.9%), 2/24(8.3%), cattle, egrets and whistling duck faecal samples, respectively. blaCTX-M gene family was prevalent; blaCTX-M15 (83.3%) predominated over blaCTX-M9 (11.8%). All were susceptible to carbapenems. The majority of isolates were resistant to at least one of the other tested antimicrobials; multidrug resistance was highest in the isolates recovered from egrets. The isolates harboured diverse repositories of other AMR genes (including strB and sul2), integrons (predominantly class 1) and VAGs. The isolates recovered from egrets harboured more AMR genes; eight were unique to these isolates including tetG, gepA, and floR. The prevalent VAGs included hemL and iss; while 14 (including sepA) were unique to certain animal isolates. E. coli serotypes O9:H9, O9:H30 and O9:H4 predominated. An identical phenotypic microarray profile was detected in three isolates from egrets and cattle, indicative of a clonal relationship amongst these isolates. CONCLUSION: Wild birds and cattle harbour diverse ESBL-producing E. coli populations with potential of inter-species dissemination and virulence. Recommended guidelines to balance public health and habitat conservation should be implemented with continuous surveillance.

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.

Rapid culture-based identification of Shiga toxin-producing Escherichia coli and Shigella spp./Enteroinvasive E. coli using the eazyplex® EHEC complete assay.

Shiga toxin-producing Escherichia coli (STEC) and Shigella spp./enteroinvasive E. coli (EIEC) are common diarrheagenic bacteria that cause sporadic diseases and outbreaks. Clinical manifestations vary from mild symptoms to severe complications. For microbiological diagnosis, culture confirmation of a positive stool screening PCR test is challenging because of time-consuming methods for isolation of strains, wide variety of STEC pathotypes, and increased emergence of non-classical strains with unusual serotypes. Therefore, molecular assays for the rapid identification of suspect colonies growing on selective media are very useful. In this study, the performance of the newly introduced eazyplex® EHEC assay based on loop-mediated isothermal amplification (LAMP) was evaluated using 18 representative STEC and Shigella strains and 31 isolates or positive-enrichment broths that were collected from clinical stool samples following screening by BD MAX™ EBP PCR. Results were compared to real-time PCR as a reference standard. Overall, sensitivities and specificities of the eazyplex® EHEC were as follows: 94.7% and 100% for Shiga toxin 1 (stx1), 100% and 100% for stx2, 93.3% and 97.1% for intimin (eae), 100% and 100% for enterohemolysin A (ehlyA), and 100% and 100% for invasion-associated plasmid antigen H (ipaH) as Shigella spp./EIEC target, respectively. Sample preparation for LAMP took only some minutes, and the time to result of the assay ranged from 8.5 to 13 min. This study shows that eazyplex® EHEC is a very fast and easy to perform molecular assay that provides reliable results as a culture confirmation assay for the diagnosis of STEC and Shigella spp./EIEC infections.

Genotyping of methicillin-resistant Staphylococcus aureus from sepsis patients in Pakistan and detection of antibodies against staphylococcal virulence factors.

In order to obtain more information on the MRSA population structure in the border region of Afghanistan and Pakistan, we collected and genotyped MRSA causing bloodstream infections from a tertiary care hospital in Peshawar, Pakistan, that serves the local population as well as Afghan immigrants and refugees. Thirty-one MRSA isolates from 30 patients were included and characterized by microarray hybridisation. For 25 patients, serum samples were tested using protein microarrays in order to detect antibodies against staphylococcal virulence factors. The most conspicuous result was the high rate of PVL-positive MRSA. Twenty-two isolates (71%) harboured lukF/S-PV genes. The most common lineage was CC772-MRSA-V/VT (PVL+) to which eleven isolates were assigned. The second most common strain was, surprisingly, CC8-MRSA-[IV+ACME] (PVL+), "USA300" (9 isolates). Two isolates were tst1 positive CC22-MRSA-IV, matching the Middle Eastern "Gaza Epidemic Strain". Another two were PVL-positive CC30-MRSA-IV. The remaining isolates belonged to, possibly locally emerging, CC1, CC5, and CC8 strains with SCC mec IV elements. Twenty-three patient sera were positive for anti-PVL-IgG antibodies. Several questions arise from the present study. It can be assumed that MRSA and high rates of PVL-positive S. aureus/MRSA are a public health issue in the Afghanistan/Pakistan border region. A possible emergence of the "USA300" clone as well as of the CC772 lineage warrants further investigation.

Use of meat juice and blood serum with a miniaturised protein microarray assay to develop a multi-parameter IgG screening test with high sample throughput potential for slaughtering pigs.

BACKGROUND: Serological screening of pig herds at the abattoir is considered a potential tool to improve meat inspection procedures and herd health management. Therefore, we previously reported the feasibility of a miniaturised protein microarray as a new serological IgG screening test for zoonotic agents and production diseases in pigs. The present study investigates whether the protein microarray-based assay is applicable for high sample throughput using either blood serum or meat juice. MATERIAL AND METHODS: Microarrays with 12 different antigens were produced by Abbott (formerly Alere Technologies GmbH) Jena, Germany in a previously offered 'ArrayTube' platform and in an 'ArrayStrip' platform for large-scale use. A test protocol for the use of meat juice on both microarray platforms was developed. Agreement between serum and meat juice was analysed with 88 paired samples from three German abattoirs. Serum was diluted 1:50 and meat juice 1:2. ELISA results for all tested antigens from a preceding study were used as reference test to perform Receiver Operating Characteristic analysis for both test specimens on both microarray platforms. RESULTS: High area under curve values (AUC > 0.7) were calculated for the analysis of T. gondii (0.87), Y. enterocolitica (0.97), Mycoplasma hyopneumoniae (0.84) and Actinobacillus pleuropneumoniae (0.71) with serum as the test specimen and for T. gondii (0.99), Y. enterocolitica (0.94), PRRSV (0.88), A. pleuropneumoniae (0.78) and Salmonella spp. (0.72) with meat juice as the test specimen on the ArrayStrip platform. Cohens kappa values of 0.92 for T. gondii and 0.82 for Y. enterocolitica were obtained for the comparison between serum and meat juice. When applying the new method in two further laboratories, kappa values between 0.63 and 0.94 were achieved between the laboratories for these two pathogens. CONCLUSION: Further development of a miniaturised pig-specific IgG protein microarray assay showed that meat juice can be used on microarray platforms. Two out of twelve tested antigens (T. gondii, Y. enterocolitica) showed high test accuracy on the ArrayTube and the ArrayStrip platform with both sample materials.

Short communication: Diversity of staphylococci isolated from sheep mastitis in northern Algeria.

Mastitis in ruminants is an important disease with major effects on both the economy and animal welfare. It is caused by major pathogens such as Staphylococcus aureus and minor pathogens such as coagulase-negative staphylococci. The objective of this study was to identify and characterize staphylococci as a cause of sheep mastitis in Algeria. In this study, 123 milk samples were collected directly from the udder of sheep suffering from clinical mastitis in 2 provinces in Algeria. Recovered isolates were identified using MALDI-TOF mass spectrometry. Virulence-associated and antimicrobial resistance genes as well as clonal complexes (CC) of S. aureus were determined using microarray-based analysis. A total of 45 staphylococci isolates were cultivated from sheep milk samples, and 28 S. aureus were identified as methicillin susceptible (62.2%). Seventeen other Staphylococcus isolates of different species were identified using MALDI-TOF mass spectrometry. Subsequent microarray analysis typed the methicillin-susceptible S. aureus to 6 CC: CC8-MSSA, CC97-MSSA, CC130/521-MSSA, CC479-MSSA, CC522-MSSA, and CC705-MSSA. The accessory gene regulator agrIII and the ruminant leukocidin genes lukF-P83 and lukM were found in all isolates of CC130/521, CC479, CC522, and CC705. The toxic shock syndrome toxin gene tst1 was detected exclusively in CC130/521. Additionally, virulence-associated genes (sea, sed, sak, hld, hlgA, edinB, and others) were detected. The presence of antibiotic resistance genes [blaZ, erm(B), and tet(K)] was detected in small numbers of staphylococci. Staphylococci possessing these genes are considered potential hazards for farm animals, farmers, and consumers. Data concerning the prevalence and diversity of staphylococci causing mastitis in sheep from Algeria are lacking. Presented results on different aspects about staphylococci in Algerian sheep populations should at least partially close that gap. However, further extensive studies covering more geographical regions are needed to assess the epidemiological risk.

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.

Microarray-based detection of resistance genes in coagulase-negative staphylococci isolated from cattle and buffalo with mastitis in Egypt.

The present study aimed to provide a detailed characterization of coagulase-negative staphylococci (CoNS) isolated from cows and buffaloes with mastitis. The study included seventy-five CoNS isolates (60 came from cattle and 15 from buffaloes) originating from 68 individual quarters of 67 dairy cows (53 cattle and 14 buffaloes). The animals belonged to five different small holding dairy herds (n = 140 cows) that show clinical or subclinical mastitis. CoNS isolates were phenotypically characterized using MALDI-TOF-MS and were further genotypically characterized by microarray-based assays. Furthermore, the antimicrobial susceptibility of CoNS strains which carried the mecA gene was examined by broth microdilution. The occurrence of CoNS in the respective five herds was 10.5%, 14.7%, 14.8%, 12.8%, and 9.9%, with an average of 12.4%. Six different CoNS species were identified: S. sciuri (n = 37; 30 from cattle and 7 from buffaloes), S. chromogenes (n = 14; 8 from cattle and 6 from buffaloes), S. haemolyticus (n = 10; nine from cattle and one buffalo), S. xylosus (n = 10; nine from cattle and one buffalo), S. hyicus (n = 2), S. warneri (n = 1), and unidentified CoNS (n = 1). Twenty percent (20%) of CoNS isolates (17.3% of cattle origin) carried at least one antimicrobial resistance gene, while 4% of the isolate including two isolates of S. haemolyticus and one S. warneri of cattle origin carried the mecA gene and were phenotypically identified as methicillin-resistant strains. The genes detected were blaZ (16%), followed by tet(K) (8%), aacA-aphD (4%), aphA3 (2.6%), msr(A) (2.6%), [far1 (2.6%), and fusC (2.6%)], sat (2.6%), and cat (1.3%) conferring resistance to penicillin, tetracycline, gentamicin, neomycin/kanamycin, erythromycin, fusidic acid, streptothricin, and chloramphenicol, respectively. The majority of investigated CoNS strains displayed considerably low prevalence of resistance genes, while resistance to more than three antibiotics was found in S. haemolyticus and S. warneri. Implementing effective preventive measures is, therefore, important for limiting the transmission of CoNS, rather than using antibiotics to control mastitis in bovines.

Controlling intestinal colonization of high-risk haematology patients with ESBL-producing Enterobacteriaceae: a randomized, placebo-controlled, multicentre, Phase II trial (CLEAR).

OBJECTIVES: We assessed the efficacy and safety of an oral antimicrobial regimen for short- and long-term intestinal eradication of ESBL-producing Escherichia coli and Klebsiella pneumoniae (ESBL-EC/KP) in immunocompromised patients. METHODS: We performed a randomized (2:1), double-blind multicentre Phase II study in four haematology-oncology departments. Patients colonized with ESBL-EC/KP received a 7 day antimicrobial regimen of oral colistin (2 × 106 IU 4×/day), gentamicin (80 mg 4×/day) and fosfomycin (three administrations of 3 g every 72 h), or placebo. Faecal, throat and urine specimens were collected on day 0, 6 ± 2, 11 ± 2, 28 ± 4 and 42 ± 4 after treatment initiation, and the quantitative burden of ESBL-EC/KP, resistance genes and changes in intestinal microbiota were analysed. Clinicaltrials.gov: NCT01931592. RESULTS: As the manufacture of colistin powder was suspended worldwide, the study was terminated prematurely. Overall, 29 (18 verum/11 placebo) out of 47 patients were enrolled. The short-term intestinal eradication was marginal at day 6 (verum group 15/18, 83.3% versus placebo 2/11, 18.2%; relative risk 4.58, 95% CI 1.29-16.33; Fisher's exact test P = 0.001) and not evident at later timepoints. Quantitative analysis showed a significant decrease of intestinal ESBL-EC/KP burden on day 6. Sustained intestinal eradication (day 28 + 42) was not achieved (verum, 38.9% versus placebo, 27.3%; P = 0.299). In the verum group, mcr-1 genes were detected in two faecal samples collected after treatment. Microbiome analysis showed a significant decrease in alpha diversity and a shift in beta diversity. CONCLUSIONS: In this prematurely terminated study of a 7 day oral antimicrobial eradication regimen, short-term ESBL-EC/KP suppression was marginal, while an altered intestinal microbiota composition was clearly apparent.

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.

Related carbapenemase-producing Klebsiella isolates detected in both a hospital and associated aquatic environment in Sweden.

Carbapenem antibiotics are one of the last-resort agents against multidrug-resistant (MDR) bacteria. The occurrence of carbapenemase-producing Enterobacteriaceae (CPE) in wastewater and aquatic environments is an indication of MDR bacteria in the community. This study evaluated CPE in aquatic environments and compared them to the local hospital isolates in Sweden. Phenotypic and genotypic analyses of antibiotic resistance of environmental and clinical CPE were performed. The relatedness of the isolates and possible clonal dissemination was evaluated using phylogenetic and phyloproteomic analysis. Klebsiella oxytoca carrying carbapenemase genes (blaVIM-1, blaIMP-29) were isolated from wastewater and the recipient river, while K. oxytoca (blaVIM-1) and Klebsiella pneumoniae (blaVIM-1, blaOXA-48, blaNDM-1, blaKPC-3) were isolated from patients at the local clinics or hospital. The K. oxytoca classified as sequence type 172 (ST172) isolated from the river was genotypically related to two clinical isolates recovered from patients. The similarity between environmental and clinical isolates suggests the dispersion of blaVIM-1 producing K. oxytoca ST172 from hospital to aquatic environment and the likelihood of its presence in the community. This is the first report of CPE in aquatic environments in Sweden; therefore, surveillance of aquatic and hospital environments for CPE in other urban areas is important to determine the major transfer routes in order to formulate strategies to prevent the spread of MDR bacteria.

Hospital-acquired methicillin-resistant Staphylococcus aureus (MRSA) from Pakistan: molecular characterisation by microarray technology.

The prevalence of methicillin-resistant Staphylococcus aureus (MRSA) in Pakistan is known to be high, but very few studies have described the molecular epidemiology of the different MRSA clones circulating in the country. Forty-four MRSA isolates were collected from two tertiary care hospitals of the Rawalpindi district of Pakistan. All strains were identified by a conventional phenotypic method and then subjected to genotyping by microarray hybridisation. Six clonal complexes (CCs) and 19 strains were identified. The most commonly identified strains were: (i) Panton-Valentine leucocidin (PVL)-positive CC772-MRSA-V, "Bengal Bay Clone" (ten isolates; 22.3%), (ii) ST239-MRSA [III + ccrC] (five isolates) and (iii) a CC8-MRSA-IV strain, as well as CC6-MRSA-IV (both with four isolates; 9.1% each). Several of the strains detected indicated epidemiological links to the Middle Eastern/Arabian Gulf region. Further studies are needed to type MRSA from countries with less known epidemiology and to monitor the distribution and spread of strains, as well as possible links to global travel, migration and commerce.

Serogenotyping and antimicrobial susceptibility testing of Salmonella spp. isolated from retail meat samples in Lagos, Nigeria.

Microarray-based serogenotyping, antimicrobial susceptibility tests and the detection of relevant resistance genes were performed on isolates of Salmonella spp. from retail meat samples obtained in Lagos, Nigeria. Out of 151 meat samples, 33 Salmonella isolates were obtained. Nine different Salmonella serovars (S. Amoutive, S. Bargny, S. Drac, S. Ealing, S. Urbana, S. Hadar, S. Nyborg, S. Anatum and S. Havana) were identified by microarray-based serogenotyping and confirmed afterwards using classical serotyping. Antibiotic susceptibility tests with 17 antibiotics showed that almost all isolates were fully susceptible to this panel. The results of this study indicated a high prevalence of Salmonella in retail meat, the presence of some previously rather rarely described Serovars in retail meat samples from Lagos, and a need to monitor for Salmonella and their antibiotic resistance determinants. The microarray-based system used herein proved to be perfectly suited as epidemiological tool to replace classical serotyping.