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

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

Background: Brown 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. Aim: We intended to determine whether urban rats at two highly frequented sites in Vienna, Austria, carry extended-spectrum β-lactamase-producing Enterobacteriaceae, fluoroquinolone-resistant Enterobacteriaceae and meticillin-resistant (MR) Staphylococcus spp. (MRS). Methods: We 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 character-ised the isolates and their antimicrobial properties using microbiological and genetic methods including disk diffusion, microarray analysis, sequencing, and detection and characterisation of plasmids. Results: Eight multidrug-resistant Escherichia coli and two extensively drug-resistant New Delhi metallo-β-lactamases-1 (NDM-1)-producing Enterobacter xiangfangensis ST114 (En. cloacae complex) were isolated from nine of 62 rats. Nine Enterobacteriaceae isolates harboured the blaCTX-M gene and one carried a plasmid-encoded ampC gene (blaCMY-2). Forty-four MRS were isolated from 37 rats; they belonged to seven different staphylococcal species: S. fleuret-tii, S. sciuri, S. aureus, S. pseudintermedius, S. epidermidis, S. haemolyticus (all mecA-positive) and mecC-positive S. xylosus. Conclusion: Our 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. © 2019, European Centre for Disease Prevention and Control (ECDC). All rights reserved

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. © 2019 by the authors. Licensee MDPI, Basel, Switzerland

Antibiotikaresistenz und One Health: Vorkommen multiresistenter humanpathogener Bakterien in Lebensmitteln und in der Umwelt.

Abweichender Titel nach Übersetzung der Verfasserin/des VerfassersKumulative Dissertation aus vierzehn ArtikelnIm Laufe der Evolution hat sich das Spektrum der Krankheitserreger, die eine Bedrohung für die Gesundheit und das Leben des Menschen darstellen, konstant verändert. Um deren Verbreitung einzudämmen, ist eine ständige Überwachung infektiöser Erreger als Maßnahme im Bereich der öffentlichen Gesundheit unerlässlich. Das Auftreten beziehungsweise Wiederauftreten von Infektionskrankheiten kann durch unterschiedliche Faktoren verursacht werden. Der übermäßige Gebrauch und Missbrauch antimikrobieller Substanzen sind für eine zunehmende Antibiotika Resistenz der Krankheitserreger verantwortlich und erschweren notwendige medizinische Behandlungen damit erheblich. Sowohl Gram positive als auch Gram negative Bakterien entwickeln zunehmende Resistenzen gegen die jeweiligen Antibiotikatherapien. Reservoire von Krankheitserregern sind nicht nur auf Krankenhäuser, in denen der selektive Druck durch Antibiotika besonders hoch ist, beschränkt, sondern befinden sich auch in der Umwelt (z. B. Wasser, Lebensmittelproduktion, Landwirtschaft). Insbesondere lebensmittelbedingte Krankheitserreger sind eine weitere treibende Kraft für die Ausbreitung von Infektionskrankheiten. Durch behördliche Kontrollen können Kontaminationsquellen zwar reduziert werden, jedoch können lebensmittelbedingte Infektionen in allen Altersgruppen auftreten und sind nach wie vor weit verbreitet. Daher sind regelmäßige Kontrollen und zugleich eine genaue Typisierung von Bakterien notwendig, um die Risiken einer Infektion und deren Ausbreitung zu minimieren. Ziel dieses Projekts war es, natürliche Reservoirs und Quellen pathogener Mikroorganismen zu definieren. Dabei lag ein besonderer Schwerpunkt auf multiresistenten Mikroorganismen in Gewässern. Dazu wurden österreichische Flüsse auf die Präsenz humanpathogener, multiresistenter Erreger untersucht. Weiters wurde das Vorkommen eines Lebensmittelassoziierten Erregers namens Cronobacter sakazakii, der besonders schwere Krankheitsausbrüche bei Säuglingen verursachen kann, im Zuge einer europäischen Multicenter-Studie im Jahr 2017 untersucht. Mittels Ganzgenomsequenzierung wurde eine detaillierte Charakterisierung und Subtypisierung pathogener klinischer und lebensmittelbedingter Erreger durchgeführt und der jeweilige Antibiotikaresistenzund Virulenz-Status bestimmt. Die Ergebnisse des ersten Projekts zeigen, dass multiresistente Erreger, die in Krankenhäusern vorkommen auch im österreichischen Oberflächengewässer nachgewiesen werden können. Es gelang der Nachweis eines Methicillin-resistenten Staphylococcus aureus USA300 Isolats und der Nachweis von multiresistenten Klebsiella pneumoniae Isolaten, die mit den aus Krankenhäusern erhaltenen Isolaten eine genombasierte Übereinstimmung zeigten. Der Nachweis humaner Krankheitserreger, wie dem Staphylococcus aureus USA300 Isolat und den Klebsiella pneumoniae Isolaten in österreichischen Flüssen hat gezeigt, dass Antibiotikaresistenzen nicht nur auf Krankenhäuser beschränkt sind, sondern von dort auch in die Umwelt gelangen können. Die anthropogene Verschmutzung durch Kläranlagen der jeweiligen Flüsse, wurde durch den Nachweis von multiresistenten Klebsiella pneumoniae Isolaten bestätigt, da sie von in Krankenhäusern gesammelten klinischen Isolaten nicht unterschieden werden konnten. Da Antibiotikagaben zur primären Behandlungsmethode gegen bakterielle Infektionen gehören, ist die zunehmende Ineffektivität alarmierend und erfordert Strategien, um eine Ausbreitung multiresistenter Stämme in der Umwelt zu minimieren. Die europäische Cronobacter sakazakii Multicenter-Studie ermöglichte einen Überblick über die Situation der Cronobacter Infektionen in ganz Europa. Die Studie hat gezeigt, dass in Ländern, die 2017 Cronobacter sakazakii Isolate nachweisen konnten, keine europaweiten Ausbrüche vorlagen. Die korrekte Identifizierung von Cronobacter Spezies stellte sich als eine große Herausforderung für die teilnehmenden Laboratorien heraus und trug daher zu einer unterschätzten Verbreitung in ganz Europa bei. Die Etablierung eines Typisierungsschemas für Ausbruchsuntersuchungen ermöglichte die Entdeckung von vier bisher unveröffentlichten historischen Ausbrüchen (vor 2016). Die Typisierung von Bakterien mittels Ganzgenomsequenzierung erlaubt eine Unterscheidung der Isolate in höchstmöglicher Auflösung und ist daher die Methode der Wahl, um Übertragungswege und Ausbrüche durch pathogene Erreger aufzudecken. Darüber hinaus ermöglichen genombasierten Sequenzdaten die Analyse und Charakterisierung von Genen, welche Antibiotikaresistenzen und Virulenzeigenschaften verursachen. Die Ergebnisse dieser Arbeit bestätigen, dass Ganzgenomsequenzierung besonders zur Überwachung, Erkennung von Ausbrüchen und zur Beobachtung der Entwicklung von Krankheitserregern geeignet ist.Throughout evolution, the spectrum of pathogens affecting human health and the thereby associated infectious diseases changed. To decrease dissemination, disease and death, control via constant public health surveillance is essential. Emergence respectively re-emergence of infectious diseases can be promoted by diverse factors. Amongst others, the use, overuse and misuse of antimicrobials lead to the growing emergence of infections, caused by increased antibiotic resistance of pathogens to medical treatment. Gram positive as well as Gram negative bacteria progressively resist antibiotic therapy. Reservoirs of such bacteria are not restricted to hospitals where the selective pressure is particularly high, but can also be found in the environment (e.g. water, food-production, husbandry). In addition, foodborne pathogens are a driving force for infectious diseases. Although regulatory controls can reduce sources of contamination, infections transmitted through food still remain common. Outbreaks caused by foodborne pathogens can appear in all age groups, therefore reliable regulatory control and accurate bacterial typing is important to reduce the risk of infection and dissemination. The aim of this project was to define the natural reservoirs and sources of pathogenic bacterial organisms. One special focus was on multiresistant strains in the water environment. Main Austrian rivers were screened for the presence of multiresistant pathogens. Second, the occurrence of the foodborne pathogen Cronobacter sakazakii, which is associated with outbreaks of severe infections in infants, was investigated by a European multi-centre study in 2017. Clinical and foodborne pathogens were analysed in detail determining their antimicrobial resistanceand virulence status as well as characterization and subtyping of pathogenic isolates by whole genome sequencing (WGS). Results of the first project revealed that human pathogens, which were simultaneously occurring in hospitals, have been detectable in Austrian surface water. This included the detection of a community acquired methicillin resistant Staphylococcus aureus USA300 isolate and the detection of multiresistant Klebsiella pneumoniae isolates, which resembled isolates from specimens obtained in hospitals. Overall, the detection of human pathogens, such as the Staphylococcus aureus USA300 isolate in Austrian river water, shows that antimicrobial resistance is not restricted to hospitals, but that antibiotic resistant isolates spill over into the environment. Further, the occurrence of anthropogenic pollution by wastewater treatment plants has been affirmed by the detection of multiresistant Klebsiella pneumoniae isolates, which were indistinguishable with human isolates collected in hospitals. Since antibiotics belong to the treatment of choice against bacterial infections, the increasing ineffectiveness is alarming and demands attention and strategies to minimize the spread of multiresistant strains to the environment. The European multi-centre study of Cronobacter sakazakii infections in humans allowed a comparison of the situation for Cronobacter infections across Europe. This study revealed the absence of European wide outbreaks in countries submitting Cronobacter sakazakii isolates in 2017. Species identification turned out to be a major challenge for participating laboratories, contributing to an underestimated prevalence across Europe. The establishment of a typing scheme for outbreak investigations enabled the detection of four previously unpublished historical outbreaks (before 2016). Strain typing using WGS data discriminates isolates with the highest possible resolution and is therefore the method of choice for uncovering chains of transmission and outbreak investigations. In addition, WGS data allow the analysis and characterization of genes conferring antibiotic resistance and virulence. The findings corroborate, WGS as the recommended tool for surveillance, outbreak detection and for monitoring the evolution of pathogens.11

Subtyping of livestock-associated methicillin-resistant Staphylococcus aureus CC398 isolates by next generation sequencing

Mit dem Beginn des neuen Jahrtausends kam es zum Auftreten eines neuen Methicillin resistenten Staphylococcus aureus (MRSA) Stamm, welcher zum klonalen Komplex 398 gehört (CC398). Dieser führt in Menschen, die Kontakt zu Tieren haben, zu Infektionen (Nutztier-assoziierte (LA) - MRSA). In Österreich wurden im Jahr 2004 die ersten Infektionen durch CC398 beobachtet, als das Nationale Referenzlabor für Staphylokokken (NRLS) in der Österreichischen Agentur für Gesundheit und Ernährungssicherheit (AGES) mit der Typisierung von MRSA Isolaten begann. Von 2004 bis heute beobachtete das NRLS eine stetige Zunahme an Infektionen durch CC398 Isolate von 0.2% im Jahr 2004 auf 7.9% im Jahr 2015. Alle LA-MRSA Isolate des NRLS gehörten zu CC398 und hatten dazugehörige spa- Typen (t011, t034, t108), wobei t011 der vorherrschende spa-Typ (87.6%) war. Aufgrund der limitierten Differenzierungsmöglichkeit von konventionellen Subtypisierungsmethoden, wie multilocus sequence typing (MLST) und der Sequenzierung der variablen X-Region des Protein A Gens von Staphylocuccus aureus (spa-Typisierung), wurde Next Generation Sequencing (NGS) zur hochauflösenden Typisierung des österreichischen LA-MRSA Bestandes angewendet. Insgesamt wurden 153 LA-MRSA Isolate und 47 zufällig gewählte HA- (Krankenhaus-assoziierte) MRSA Isolate mittels NGS untersucht. Dabei wurde eine Gen-für-Gen Vorgehensweise zum Vergleich der Genome auf der Basis eines vordefinierten Core-Genoms (cgMLST) mit 1861 Genen verwendet. Die Typisierung mit NGS ermöglichte eine Unterscheidung zwischen allen LA-MRSA und HA-MRSA Isolaten. HA-MRSA Isolate unterschieden sich von LA-MRSA Isolaten in mindestens 1717 Allelen. Das Maximum an Allel Unterschieden betrug 1658 Allele zwischen HA- MRSA und 131 zwischen LA-MRSA Isolaten. Allgemein war der genetische Unterschied zwischen unterschiedlichen spa-Typen größer, als zwischen Isolaten des Selben spa-Typs. Damit wurde bestätigt, dass cgMLST sich hervorragend für die hochauflösende Typisierung von LA-MRSA Isolaten desselben spa-Typs eignet.With the beginning of the new millennium a new methicillin-resistant Staphylococcus aureus (MRSA) clonal complex 398 (CC398) has emerged as a cause of infections in humans with contact to livestock (livestock-associated MRSA, LA-MRSA). In Austria, first infections caused by CC398 isolates were observed in 2004 when the National Reference Laboratory for Staphylococci (NRLS) at the Austrian Agency for Health and Food Safety (AGES) started typing MRSA isolates. From 2004 till today the NRLS observed a steady increase of infections caused by CC398 isolates from 0.2% in 2004 to 7.9% in 2015. All LA-MRSA isolates from the NRLS harboured CC398 corresponding spa-types (t011, t034, t108), whereby t011 was the most prevalent spa-type (87.6%). Due to the limited discriminatory power of the subtyping methods multilocus sequence typing (MLST) and sequencing of the variable X-region of staphylococcal protein A gene (spa- typing), next generation sequencing (NGS) was applied for high resolution typing of 153 LA-MRSA isolates and an arbitrary collection of 47 hospital acquired (HA)-MRSA isolates belonging to 12 classical MLSTs for comparison. A gene-by-gene approach was used for comparison of genomes based on a predefined core genome (cgMLST) comprising 1861 genes. NGS based typing allowed a differentiation between all investigated LA-MRSA and HA-MRSA isolates. HA-MRSA isolates differed in at least 1717 alleles from LA-MRSA isolates. The maximal allelic difference was 1658 among HA-MRSA isolates and 131 among LA-MRSA isolates. In general the genetic difference between spa-types was higher than within isolates of a certain spa-type. In conclusion cgMLST is highly suitable for high resolution typing of LA-MRSA isolates with the same spa-type

Austria-wide survey on resistant, potentially pathogenic bacteria at Austrian bathing sites, 2017

There is growing concern about human-induced antibiotic resistance and on the occurrence of antibiotic-resistant, potentially pathogenic bacteria in the environment. The aim of this study was to investigate the incidence of resistant, clinically relevant bacteria at bathing sites. In total, 27 of 263 bathing sites authorized under the EU Bathing Water Directive (3 per Austrian state) were sampled during the summer of 2017. Samples were tested for antibiotic-resistant bacteria by enrichment in thioglycollate broth and cultivation on chromogenic media. The screening for potentially pathogenic antibiotic-resistant bacteria was negative in 23 of the 27 samples. Antibiotic-resistant bacteria were detected from 4 of the 27 bathing sites: one Pseudomonas aeruginosa and three resistant Enterobacteriaceae (piperacillin/tazobactam-resistant Enterobacter cloacae with high-level expression of AmpC beta-lactamase, carbapenem-resistant Enterobacter mori, extended-spectrum beta-lactamase-producing Escherichia coli). Despite the occurrence of resistant bacteria, we consider the public health risk at Austrian bathing sites as low

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

ABSTRACT: 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

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

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

Background Brown 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. Aim We intended to determine whether urban rats at two highly frequented sites in Vienna, Austria, carry extended-spectrum β-lactamase-producing Enterobacteriaceae, fluoroquinolone-resistant Enterobacteriaceae and meticillin-resistant (MR) Staphylococcus spp. (MRS). Methods We 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. Results Eight multidrug-resistant Escherichia coli and two extensively drug-resistant New Delhi metallo-β-lactamases-1 (NDM-1)-producing Enterobacter xiangfangensis ST114 (En. cloacae complex) were isolated from nine of 62 rats. Nine Enterobacteriaceae isolates harboured the blaCTX-M gene and one carried a plasmid-encoded ampC gene (blaCMY-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. Conclusion Our 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