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- ItemAn epidemic CC1-MRSA-IV clone yields false-negative test results in molecular MRSA identification assays: a note of caution, Austria, Germany, Ireland, 2020(Stockholm : European Centre for Disease Prevention and Control, 2020) Monecke, Stefan; König, Elisabeth; Earls, Megan R.; Leitner, Eva; Müller, Elke; Wagner, Gabriel E.; Poitz, David M.; Jatzwauk, Lutz; Vremerǎ, Teodora; Dorneanu, Olivia S.; Simbeck, Alexandra; Ambrosch, Andreas; Zollner-Schwetz, Ines; Krause, Robert; Ruppitsch, Werner; Schneider-Brachert, Wulf; Coleman, David C.; Steinmetz, Ivo; Ehricht, RalfWe 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.
- ItemMelioidosis DS rapid test: A standardized serological dipstick assay with increased sensitivity and reliability due to multiplex detection(Lawrence, Kan. : PLoS, 2020) Wagner, Gabriel E.; Föderl-Höbenreich, Esther; Assig, Karoline; Lipp, Michaela; Berner, Andreas; Kohler, Christian; Lichtenegger, Sabine; Stiehler, Julia; Karoonboonyanan, Wisansanee; Thanapattarapairoj, Nida; Promkong, Chidchanok; Koosakulnirand, Sirikamon; Chaichana, Panjaporn; Ehricht, Ralf; Gad, Marie; Söffing, Hans H.; Dunachie, Susanna J.; Chantratita, Narisara; Steinmetz, IvoBackground Melioidosis, caused by Burkholderia pseudomallei, is a severe infectious disease with high mortality rates, but is under-recognized worldwide. In endemic areas, there is a great need for simple, low-cost and rapid diagnostic tools. In a previous study we showed, that a protein multiplex array with 20 B. pseudomallei-specific antigens detects antibodies in melioidosis patients with high sensitivity and specificity. In a subsequent study the high potential of anti-B. pseudomallei antibody detection was confirmed using a rapid Hcp1 single protein-based assay. Our protein array also showed that the antibody profile varies between patients, possibly due to a combination of host factors but also antigen variations in the infecting B. pseudomallei strains. The aim of this study was to develop a rapid test, combining Hcp1 and the best performing antigens BPSL2096, BPSL2697 and BPSS0477 from our previous study, to take advantage of simultaneous antibody detection. Methods and principal findings The 4-plex dipstick was validated with sera from 75 patients on admission plus control groups, achieving 92% sensitivity and 97–100% specificity. We then re-evaluated melioidosis sera with the 4-plex assay that were previously misclassified by the monoplex Hcp1 rapid test. 12 out of 55 (21.8%) false-negative samples were positive in our new dipstick assay. Among those, 4 sera (7.3%) were Hcp1 positive, whereas 8 (14.5%) sera remained Hcp1 negative but gave a positive reaction with our additional antigens. Conclusions Our dipstick rapid test represents an inexpensive, standardized and simple diagnostic tool with an improved serodiagnostic performance due to multiplex detection. Each additional band on the test strip makes a false-positive result more unlikely, contributing to its reliability. Future prospective studies will seek to validate the gain in sensitivity and specificity of our multiplex rapid test approach in different melioidosis patient cohorts.
- ItemPhenotypic and Molecular Detection of Biofilm Formation in Staphylococcus aureus Isolated from Different Sources in Algeria(Basel : MDPI, 2020) Achek, Rachid; Hotzel, Helmut; Nabi, Ibrahim; Kechida, Souad; Mami, Djamila; Didouh, Nassima; Tomaso, Herbert; Neubauer, Heinrich; Ehricht, Ralf; Monecke, Stefan; El-Adawy, HosnyStaphylococcus aureus is an opportunistic bacterium causing a wide variety of diseases. Biofilm formation of Staphylococcus aureus is of primary public and animal health concern. The purposes of the present study were to investigate the ability of Staphylococcus aureus isolated from animals, humans, and food samples to form biofilms and to screen for the presence of biofilmassociated and regulatory genes. In total, 55 Staphylococcus aureus isolated from sheep mastitis cases (n = 28), humans (n = 19), and from food matrices (n = 8) were identified using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The ability of Staphylococcus aureus for slime production and biofilm formation was determined quantitatively. A DNA microarray examination was performed to detect adhesion genes (icaACD and biofilmassociated protein gene (bap)), genes encoding microbial surface components recognizing adhesive matrix molecules (MSCRAMMs), regulatory genes (accessory gene regulator (agr) and staphylococcal accessory regulator (sarA)), and the staphylococcal cassette chromosome mec elements (SCCmec). Out of 55 Staphylococcus aureus isolates, 39 (71.0%) and 23 (41.8%) were producing slime and biofilm, respectively. All Staphylococcus aureus strains isolated from food showed biofilm formation ability. 52.6% of the Staphylococcus aureus strains isolated from sheep with mastitis, and 17.9% of isolates from humans, were able to form a biofilm. Microarray analysis typed the Staphylococcus aureus into 15 clonal complexes. Among all Staphylococcus aureus isolates, four of the human isolates (21.1%) harbored the mecA gene (SCCmec type IV) typed into 2 clonal complexes (CC22-MRSA-IV and CC80-MRSA-IV) and were considered as methicillin-resistant, while two of them were slime-producing. None of the isolates from sheep with mastitis harbored the cna gene which is associated with biofilm production. The fnbB gene was found in 100%, 60% and 40% of biofilm-producing Staphylococcus aureus isolated from food, humans, and sheep with mastitis, respectively. Three agr groups were present and agr group III was predominant with 43.6%, followed by agr group I (38.2%), and agr group II (18.2%). This study revealed the capacity of Staphylococcus aureus isolates to form biofilms and highlighted the genetic background displayed by Staphylococcus aureus isolates from different sources in Algeria. © 2020 by the authors. Licensee MDPI, Basel, Switzerland.
- ItemCharacterization of antibiotic and biocide resistance genes and virulence factors of staphylococcus species associated with bovine mastitis in Rwanda(Basel : MDPI AG, 2020) Antók, Fruzsina Irén; Mayrhofer, Rosa; Marbach, Helene; Masengesho, Jean Claude; Keinprecht, Helga; Nyirimbuga, Vedaste; Fischer, Otto; Lepuschitz, Sarah; Ruppitsch, Werner; Ehling-Schulz, Monika; Feßler, Andrea T.; Schwarz, Stefan; Monecke, Stefan; Ehricht, Ralf; Grunert, Tom; Spergser, Joachim; Loncaric, IgorThe 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.
- ItemStaphylococcus aureus and methicillin resistant S. Aureus in nepalese primates: Resistance to antimicrobials, virulence, and genetic lineages(Basel : MDPI AG, 2020) Roberts, Marilyn C.; Joshi, Prabhu Raj; Monecke, Stefan; Ehricht, Ralf; Müller, Elke; Gawlik, Darius; Diezel, Celia; Braun, Sascha D.; Paudel, Saroj; Acharya, Mahesh; Khanal, Laxman; Koju, Narayan P.; Chalise, Mukesh; Kyes, Randall C.Staphylococcus aureus is a ubiquitous pathogen and colonizer in humans and animals. There are few studies on the molecular epidemiology of S. aureus in wild monkeys and apes. S. aureus carriage in rhesus macaques (Macaca mulatta) and Assam macaques (Macaca assamensis) is a species that has not previously been sampled and lives in remote environments with limited human contact. Forty Staphylococcus aureus isolates including 33 methicillin-susceptible S. aureus (MSSA) and seven methicillin-resistant S. aureus (MRSA) were characterized. Thirty-four isolates were from rhesus macaques and six isolates (five MSSA, one MRSA) were from Assam macaques. Isolates were characterized using StaphyType DNA microarrays. Five of the MRSA including one from Assam macaque were CC22 MRSA-IV (PVL+/tst+), which is a strain previously identified in Nepalese rhesus. One MRSA each were CC6 MRSA-IV and CC772 MRSA-V (PVL+). One MSSA each belonged to CC15, CC96, and CC2990. Six MRSA isolates carried the blaZ, while ten known CC isolates (seven MRSA, three MSSA) carried a variety of genes including aacA-aphD, aphA3, erm(C), mph(C), dfrA, msrA, and/or sat genes. The other 30 MSSA isolates belonged to 17 novel clonal complexes, carried no antibiotic resistance genes, lacked Panton–Valentine Leukocidin (PVL), and most examined exotoxin genes. Four clonal complexes carried egc enterotoxin genes, and four harbored edinB, which is an exfoliative toxin homologue. © 2020 by the authors. Licensee MDPI, Basel, Switzerland.
- ItemUse 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(London : BioMed Central, 2020) Loreck, Katharina; Mitrenga, Sylvia; Heinze, Regina; Ehricht, Ralf; Engemann, Claudia; Lueken, Caroline; Ploetz, Madeleine; Greiner, Matthias; Meemken, DianaBackground: 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. © 2020 The Author(s).
- ItemMolecular investigations on a chimeric strain of Staphylococcus aureus sequence type 80(San Francisco, California, US : PLOS, 2020) Gawlik, Darius; Ruppelt-Lorz, Antje; Müller, Elke; Reißig, Annett; Hotzel, Helmut; Braun, Sascha D.; Söderquist, Bo; Ziegler-Cordts, Albrecht; Stein, Claudia; Pletz, Mathias W.; Ehricht, Ralf; Monecke, StefanA PVL-positive, methicillin-susceptible Staphylococcus aureus was cultured from pus from cervical lymphadenitis of a patient of East-African origin. Microarray hybridisation assigned the isolate to clonal complex (CC) 80 but revealed unusual features, including the presence of the ORF-CM14 enterotoxin homologue and of an ACME-III element as well as the absence of etD and edinB. The isolate was subjected to both, Illumina and Nanopore sequencing allowing characterisation of deviating regions within the strain´s genome. Atypical features of this strain were attributable to the presence of two genomic regions that originated from other S. aureus lineages and that comprised, respectively, 3% and 1.4% of the genome. One deviating region extended from walJ to sirB. It comprised ORF-CM14 and the ACME-III element. A homologous but larger fragment was also found in an atypical S. aureus CC1/ST567 strain whose lineage might have served as donor of this genomic region. This region itself is a chimera comprising fragments from CC1 as well as fragments of unknown origin. The other deviating region comprised the region from htsB to ecfA2, i.e., another 3% of the genome. It was very similar to CC1 sequences. Either this suggests an incorporation of CC1 DNA into the study strain, or alternatively a recombination event affecting “canonical” CC80. Thus, the study strain bears witness of several recombination events affecting supposedly core genomic genes. Although the exact mechanism is not yet clear, such chimerism seems to be an additional pathway in the evolution of S. aureus. This could facilitate also a transmission of virulence and resistance factors and therefore offer an additional evolutionary advantage.