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- ItemShared MRSA Strains among Nepalese Rhesus macaques (Macaca mulatta), their Environment and Hospitalized Patients(Lausanne : Frontiers Media, 2019) Roberts, Marilyn C.; Joshi, Prabhu Raj; Monecke, Stefan; Ehricht, Ralf; Müller, Elke; Gawlik, Darius; Paudel, Saroj; Acharya, Mahesh; Bhattarai, Sankalpa; Pokharel, Sujana; Tuladhar, Reshma; Chalise, Mukesh K.; Kyes, Randall C.This study looked at 227 saliva samples from Rhesus macaques (Macaca mulatta) and 218 samples from the surrounding environments. From these samples, MRSA isolates were collected from Rhesus saliva samples (n = 13) and environmental samples (n = 19) near temple areas in Kathmandu, Nepal. For comparison, selected MRSA isolates (n = 5) were obtained from patients with wound infections from a Kathmandu hospital. All isolates were characterized using Abbott StaphyType® DNA microarrays. Eighteen isolates (62%) from monkeys (n = 4; 31%) and environmental samples (n = 14; 74%), were CC22-MRSA-IV. Most (n = 16) of them carried both, the PVL locus and toxic shock toxin gene (tst1), an unusual combination which is the same as in previously characterized strain from Nepalese macaques and pigs. The five human isolates also belonged to that strain type. Eight monkey MRSA isolates were CC361-MRSA-IV. One MRSA from a monkey and one from an environmental sample, were CC88-MRSA-V. Other environmental MRSA included one each, CC121-MRSA-VT, and CC772 -MRSA-V. Two were CC779-MRSA-VT, potentially a novel clone. All MRSA carried the blaZ gene. The aacA–aphD, dfrA, and erm (C) genes were very common in isolates from all sources. One macaque MRSA carried the resistance genes aphA3 and sat, neither previously identified in primate MRSA isolates. This current study suggests that humans could be a potential source of the MRSA in the macaques/environment and transmission may be linked to humans feeding the primates and/or living in close proximity to each other.This study looked at 227 saliva samples from Rhesus macaques (Macaca mulatta) and 218 samples from the surrounding environments. From these samples, MRSA isolates were collected from Rhesus saliva samples (n = 13) and environmental samples (n = 19) near temple areas in Kathmandu, Nepal. For comparison, selected MRSA isolates (n = 5) were obtained from patients with wound infections from a Kathmandu hospital. All isolates were characterized using Abbott StaphyType® DNA microarrays. Eighteen isolates (62%) from monkeys (n = 4; 31%) and environmental samples (n = 14; 74%), were CC22-MRSA-IV. Most (n = 16) of them carried both, the PVL locus and toxic shock toxin gene (tst1), an unusual combination which is the same as in previously characterized strain from Nepalese macaques and pigs. The five human isolates also belonged to that strain type. Eight monkey MRSA isolates were CC361-MRSA-IV. One MRSA from a monkey and one from an environmental sample, were CC88-MRSA-V. Other environmental MRSA included one each, CC121-MRSA-VT, and CC772 -MRSA-V. Two were CC779-MRSA-VT, potentially a novel clone. All MRSA carried the blaZ gene. The aacA–aphD, dfrA, and erm (C) genes were very common in isolates from all sources. One macaque MRSA carried the resistance genes aphA3 and sat, neither previously identified in primate MRSA isolates. This current study suggests that humans could be a potential source of the MRSA in the macaques/environment and transmission may be linked to humans feeding the primates and/or living in close proximity to each other.
- ItemThe Pheno- and Genotypic Characterization of Porcine Escherichia coli Isolates(Basel : MDPI, 2021) Bernreiter-Hofer, Tanja; Schwarz, Lukas; Müller, Elke; Cabal-Rosel, Adriana; Korus, Maciej; Misic, Dusan; Frankenfeld, Katrin; Abraham, Kerstin; Grünzweil, Olivia; Weiss, Astrid; Feßler, Andrea T.; Allerberger, Franz; Schwarz, Stefan; Szostak, Michael P.; Ruppitsch, Werner; Ladinig, Andrea; Spergser, Joachim; Braun, Sascha D.; Monecke, Stefan; Ehricht, Ralf; Loncaric, IgorEscherichia (E.) coli is the main causative pathogen of neonatal and post-weaning diarrhea and edema disease in swine production. There is a significant health concern due to an increasing number of human infections associated with food and/or environmental-borne pathogenic and multidrug-resistant E. coli worldwide. Monitoring the presence of pathogenic and antimicrobial-resistant E. coli isolates is essential for sustainable disease management in livestock and human medicine. A total of 102 E. coli isolates of diseased pigs were characterized by antimicrobial and biocide susceptibility testing. Antimicrobial resistance genes, including mobile colistin resistance genes, were analyzed by PCR and DNA sequencing. The quinolone resistance-determining regions of gyrA and parC in ciprofloxacin-resistant isolates were analyzed. Clonal relatedness was investigated by two-locus sequence typing (CH clonotyping). Phylotyping was performed by the Clermont multiplex PCR method. Virulence determinants were analyzed by customized DNA-based microarray technology developed in this study for fast and economic molecular multiplex typing. Thirty-five isolates were selected for whole-genome sequence-based analysis. Most isolates were resistant to ampicillin and tetracycline. Twenty-one isolates displayed an ESBL phenotype and one isolate an AmpC β-lactamase-producing phenotype. Three isolates had elevated colistin minimal inhibitory concentrations and carried the mcr-1 gene. Thirty-seven isolates displayed a multi-drug resistance phenotype. The most predominant β-lactamase gene classes were blaTEM-1 (56%) and blaCTX-M-1 (13.71%). Mutations in QRDR were observed in 14 ciprofloxacin-resistant isolates. CH clonotyping divided all isolates into 51 CH clonotypes. The majority of isolates belonged to phylogroup A. Sixty-four isolates could be assigned to defined pathotypes wherefrom UPEC was predominant. WGS revealed that the most predominant sequence type was ST100, followed by ST10. ST131 was detected twice in our analysis. This study highlights the importance of monitoring antimicrobial resistance and virulence properties of porcine E. coli isolates. This can be achieved by applying reliable, fast, economic and easy to perform technologies such as DNA-based microarray typing. The presence of high-risk pathogenic multi-drug resistant zoonotic clones, as well as those that are resistant to critically important antibiotics for humans, can pose a risk to public health. Improved protocols may be developed in swine farms for preventing infections, as well as the maintenance and distribution of the causative isolates.
- ItemSequence Analysis of Novel Staphylococcus aureus Lineages from Wild and Captive Macaques(Basel : Molecular Diversity Preservation International (MDPI), 2022) Monecke, Stefan; Roberts, Marilyn C.; Braun, Sascha D.; Diezel, Celia; Müller, Elke; Reinicke, Martin; Linde, Jörg; Joshi, Prabhu Raj; Paudel, Saroj; Acharya, Mahesh; Chalise, Mukesh K.; Feßler, Andrea T.; Hotzel, Helmut; Khanal, Laxman; Koju, Narayan P.; Schwarz, Stefan; Kyes, Randall C.; Ehricht, RalfStaphylococcus 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.
- ItemLong-Term Sinonasal Carriage of Staphylococcus aureus and Anti-Staphylococcal Humoral Immune Response in Patients with Chronic Rhinosinusitis(Basel : MDPI, 2021) Thunberg, Ulrica; Hugosson, Svante; Ehricht, Ralf; Monecke, Stefan; Müller, Elke; Cao, Yang; Stegger, Marc; Söderquist, BoWe investigated Staphylococcus aureus diversity, genetic factors, and humoral immune responses against antigens via genome analysis of S. aureus isolates from chronic rhinosinusitis (CRS) patients in a long-term follow-up. Of the 42 patients who provided S. aureus isolates and serum for a previous study, 34 could be included for follow-up after a decade. Clinical examinations were performed and bacterial samples were collected from the maxillary sinus and nares. S. aureus isolates were characterized by whole-genome sequencing, and specific anti-staphylococcal IgG in serum was determined using protein arrays. S. aureus was detected in the nares and/or maxillary sinus at both initial inclusion and follow-up in 15 of the 34 respondents (44%). Three of these (20%) had S. aureus isolates from the same genetic lineage as at inclusion. A low number of single-nucleotide polymorphisms (SNPs) were identified when comparing isolates from nares and maxillary sinus collected at the same time point. The overall change of antibody responses to staphylococcal antigens over time showed great variability, and no correlation was found between the presence of genes encoding antigens and the corresponding anti-staphylococcal IgG in serum; thus our findings did not support a role, in CRS, of the specific S. aureus antigens investigated.
- ItemPhylodynamic signatures in the emergence of community-associated MRSA(Washington, DC : National Acad. of Sciences, 2022) Steinig, Eike; Aglua, Izzard; Duchene, Sebastian; Meehan, Michael T.; Yoannes, Mition; Firth, Cadhla; Jaworski, Jan; Drekore, Jimmy; Urakoko, Bohu; Poka, Harry; Wurr, Clive; Ebos, Eri; Nangen, David; Müller, Elke; Mulvey, Peter; Jackson, Charlene; Blomfeldt, Anita; Aamot, Hege Vangstein; Laman, Moses; Manning, Laurens; Earls, Megan; Coleman, David C.; Greenhill, Andrew; Ford, Rebecca; Stegger, Marc; Syed, Muhammad Ali; Jamil, Bushra; Monecke, Stefan; Ehricht, Ralf; Smith, Simon; Pomat, William; Horwood, Paul; Tong, Steven Y. C.; McBryde, EmmaCommunity-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.
- ItemPresence of β-Lactamase-producing Enterobacterales and Salmonella Isolates in Marine Mammals(Basel : Molecular Diversity Preservation International (MDPI), 2021) Grünzweil, Olivia M.; Palmer, Lauren; Cabal, Adriana; Szostak, Michael P.; Ruppitsch, Werner; Kornschober, Christian; Korus, Maciej; Misic, Dusan; Bernreiter-Hofer, Tanja; Korath, Anna D. J.; Feßler, Andrea T.; Allerberger, Franz; Schwarz, Stefan; Spergser, Joachim; Müller, Elke; Braun, Sascha D.; Monecke, Stefan; Ehricht, Ralf; Walzer, Chris; Smodlaka, Hrvoje; Loncaric, IgorMarine 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 β-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 β-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-β-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.
- ItemProtein Microarray-Guided Development of a Highly Sensitive and Specific Dipstick Assay for Glanders Serodiagnostics(Washington, DC : American Society for Microbiology, 2022) Wagner, Gabriel E.; Berner, Andreas; Lipp, Michaela; Kohler, Christian; Assig, Karoline; Lichtenegger, Sabine; Saqib, Muhammad; Müller, Elke; Trinh, Trung T.; Gad, Anne-Marie; Söffing, Hans Hermann; Ehricht, Ralf; Laroucau, Karine; Steinmetz, IvoBurkholderia 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.