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- 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.
- 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.
- ItemCharacterisation of a novel composite SCCmec-SCCfus element in an emerging Staphylococcus aureus strain from the Arabian Gulf region(San Francisco : Public Library of Science, 2019) Senok, Abiola; Slickers, Peter; Hotzel, Helmut; Boswihi, Samar; Braun, Sascha D.; Gawlik, Darius; Müller, Elke; Nabi, Anju; Nassar, Rania; Nitschke, Hedda; Reißig, Annett; Ruppelt-Lorz, Antje; Mafofo, Joseph; Somili, Ali M.; Udo, Edet; Ehricht, Ralf; Monecke, StefanFusidic acid is a steroid antibiotic known since the 1960s. It is frequently used in topical preparations, i.e., ointments, for the treatment of skin and soft tissue infections caused by Staphylococcus aureus. There is an increasing number of methicillin-resistant S. aureus (MRSA) strains that harbour plasmid-borne fusB/far1 or fusC that is localised on SCC elements. In this study we examined a series of related CC30-MRSA isolates from the Arabian Gulf countries that presented with SCCmec elements and fusC, including a variant that—to the best of our knowledge—has not yet formally been described. It consisted of a class B mec complex and ccrA/B-4 genes. The fusidic acid resistance gene fusC was present, but contrary to the previously sequenced element of HDE288, it was not accompanied by tirS. This element was identified in CC30 MRSA from Kuwait, Saudi Arabia and the United Arab Emirates that usually also harbour the Panton-Valentin leukocidin (PVL) genes. It was also identified in CC8 and ST834 isolates. In addition, further CC30 MRSA strains with other SCCmec VI elements harbouring fusC were found to circulate in the Arabian Gulf region. It can be assumed that MRSA strains with SCCmec elements that include fusC have a selective advantage in both hospital and community settings warranting a review of the use of topical antibiotics and indicating the necessity of reducing over-the-counter sale of antibiotics, including fusidic acid, without prescription.Fusidic acid is a steroid antibiotic known since the 1960s. It is frequently used in topical preparations, i.e., ointments, for the treatment of skin and soft tissue infections caused by Staphylococcus aureus. There is an increasing number of methicillin-resistant S. aureus (MRSA) strains that harbour plasmid-borne fusB/far1 or fusC that is localised on SCC elements. In this study we examined a series of related CC30-MRSA isolates from the Arabian Gulf countries that presented with SCCmec elements and fusC, including a variant that—to the best of our knowledge—has not yet formally been described. It consisted of a class B mec complex and ccrA/B-4 genes. The fusidic acid resistance gene fusC was present, but contrary to the previously sequenced element of HDE288, it was not accompanied by tirS. This element was identified in CC30 MRSA from Kuwait, Saudi Arabia and the United Arab Emirates that usually also harbour the Panton-Valentin leukocidin (PVL) genes. It was also identified in CC8 and ST834 isolates. In addition, further CC30 MRSA strains with other SCCmec VI elements harbouring fusC were found to circulate in the Arabian Gulf region. It can be assumed that MRSA strains with SCCmec elements that include fusC have a selective advantage in both hospital and community settings warranting a review of the use of topical antibiotics and indicating the necessity of reducing over-the-counter sale of antibiotics, including fusidic acid, without prescription.
- ItemFast, economic and simultaneous identification of clinically relevant Gram-negative species with multiplex real-time PCR(London : Future Medicine Ltd, 2019) Weiss, Daniel; Gawlik, Darius; Hotzel, Helmut; Engelmann, Ines; Mueller, Elke; Slickers, Peter; Braun, Sascha D.; Monecke, Stefan; Ehricht, RalfAim: A newly designed multiplex real-time PCR (rt-PCR) was validated to detect four clinically relevant Gram-negative bacteria (Escherichia coli, Acinetobacter baumannii, Klebsiella pneumoniae and Pseudomonas aeruginosa). Materials & methods: Serial dilutions of genomic DNA were used to determine the limit of detection. Colony PCR was performed with isolates of the four selected species and other species as negative controls. Isolates were characterized genotypically and phenotypically to evaluate the assay. Results: Specific signals of all target genes were detected with diluted templates comprising ten genomic equivalents. Using colony rt-PCR, all isolates of the target species were identified correctly. All negative control isolates were negative. Conclusion: The genes gad, basC, khe and ecfX can reliably identify these four species via multiplex colony rt-PCR. © 2018 Daniel Weiss.
- ItemAnti-Staphylococcal Humoral Immune Response in patients with chronic rhinosinusitis(Amsterdam : European Rhinologic Society, 2019) Thunberg, Ulrica; Hugosson, Svante; Fredlund, Hans; Cao, Yang; Ehricht, Ralf; Monecke, Stefan; Mueller, Elke; Engelmann, Susanne; Söderquist, BoBackground: Staphylococcus aureus (S. aureus) can behave both as a harmless commensal and as a pathogen. Its significance in the pathogenesis of chronic rhinosinusitis (CRS) is not yet fully understood. This study aimed to determine serum antibody responses to specific staphylococcal antigens in patients with CRS and healthy controls, and to investigate the correlation between specific antibody response and severity of symptoms. Methodology: Serum samples from 39 patients with CRS and 56 healthy controls were analysed using a protein microarray to investigate the antibody response to S. aureus specific antigens, with a focus on immunoglobulin G (IgG) directed toward staphylococcal components accessible to the immune system. Holm-Bonferroni corrections were applied in all analyses. Information about growth of S. aureus in nares and maxillary sinus was taken from a previous study based on the same individuals. Clinical symptoms were assessed using a scoring system. Results: IgG antibody levels toward staphylococcal TSST-1 and LukF-PV were significantly higher in the CRS patient group compared to healthy controls, and levels of anti-TSST-1 antibodies were significantly higher in the CRS patient group with S. aureus in maxillary sinus than in controls. There were no correlations between the severity of symptoms and levels of serum anti-staphylococcal IgG antibody levels for LukF-PV and TSST-1. Conclusions: TSST-1 and LukF-PV could be interesting markers for future studies of the pathogenesis of CRS.
- ItemExploring the evolution and epidemiology of European CC1-MRSA-IV: tracking a multidrug-resistant community-associated meticillin-resistant Staphylococcus aureus clone(London : Soc., 2021) Earls, Megan R.; Steinig, Eike J.; Monecke, Stefan; Samaniego Castruita, José A.; Simbeck, Alexandra; Schneider-Brachert, Wulf; Vremerǎ, Teodora; Dorneanu, Olivia S.; Loncaric, Igor; Bes, Michèle; Lacoma, Alicia; Prat Aymerich, Cristina; Wernery, Ulrich; Armengol-Porta, Marc; Blomfeldt, Anita; Duchene, Sebastian; Bartels, Mette D.; Ehricht, Ralf; Coleman, David C.This study investigated the evolution and epidemiology of the community-associated and multidrug-resistant Staphylococcus aureus clone European CC1-MRSA-IV. Whole-genome sequences were obtained for 194 European CC1-MRSA-IV isolates (189 of human and 5 of animal origin) from 12 countries, and 10 meticillin-susceptible precursors (from North-Eastern Romania; all of human origin) of the clone. Phylogenetic analysis was performed using a maximum-likelihood approach, a time-measured phylogeny was reconstructed using Bayesian analysis, and in silico microarray genotyping was performed to identify resistance, virulence-associated and SCCmec (staphylococcal cassette chromosome mec) genes. Isolates were typically sequence type 1 (190/204) and spa type t127 (183/204). Bayesian analysis indicated that European CC1-MRSA-IV emerged in approximately 1995 before undergoing rapid expansion in the late 1990s and 2000s, while spreading throughout Europe and into the Middle East. Phylogenetic analysis revealed an unstructured meticillin-resistant S. aureus (MRSA) population, lacking significant geographical or temporal clusters. The MRSA were genotypically multidrug-resistant, consistently encoded seh, and intermittently (34/194) encoded an undisrupted hlb gene with concomitant absence of the lysogenic phage-encoded genes sak and scn. All MRSA also harboured a characteristic ~5350 nt insertion in SCCmec adjacent to orfX. Detailed demographic data from Denmark showed that there, the clone is typically (25/35) found in the community, and often (10/35) among individuals with links to South-Eastern Europe. This study elucidated the evolution and epidemiology of European CC1-MRSA-IV, which emerged from a meticillin-susceptible lineage prevalent in North-Eastern Romania before disseminating rapidly throughout Europe.
- ItemCharacterisation of Methicillin-Resistant Staphylococcus aureus from Alexandria, Egypt(Basel : MDPI, 2023) Monecke, Stefan; Bedewy, Amira K.; Müller, Elke; Braun, Sascha D.; Diezel, Celia; Elsheredy, Amel; Kader, Ola; Reinicke, Martin; Ghazal, Abeer; Rezk, Shahinda; Ehricht, RalfThe present study aims to characterise clinical MRSA isolates from a tertiary care centre in Egypt’s second-largest city, Alexandria. Thirty isolates collected in 2020 were genotypically characterised by microarray to detect their resistance and virulence genes and assign them to clonal complexes (CC) and strains. Isolates belonged to 11 different CCs and 14 different strains. CC15-MRSA-[V+fus] (n = 6), CC1-MRSA-[V+fus+tir+ccrA/B-1] (PVL+) (n = 5) as well as CC1-MRSA-[V+fus+tir+ccrA/B-1] and CC1153-MRSA-[V+fus] (PVL+) (both with n = 3) were the most common strains. Most isolates (83%) harboured variant or composite SCCmec V or VI elements that included the fusidic acid resistance gene fusC. The SCCmec [V+fus+tir+ccrA/B-1] element of one of the CC1 isolates was sequenced, revealing a presence not only of fusC but also of blaZ, aacA-aphD and other resistance genes. PVL genes were also common (40%). The hospital-acquired MRSA CC239-III strain was only found twice. A comparison to data from a study on strains collected in 2015 (Montelongo et al., 2022) showed an increase in fusC and PVL carriage and a decreasing prevalence of the CC239 strain. These observations indicate a diffusion of community-acquired strains into hospital settings. The beta-lactam use in hospitals and the widespread fusidic acid consumption in the community might pose a selective pressure that favours MRSA strains with composite SCCmec elements comprising mecA and fusC. This is an unsettling trend, but more MRSA typing data from Egypt are required.
- ItemEmergence of novel methicillin resistant Staphylococcus aureus strains in a tertiary care facility in Tiyadh, Saudi Arabia(Macclesfield, UK : Dove Medical Press, 2019) Senok, Abiola; Somili, Ali M.; Nassar, Rania; Garaween, Ghada; Kim Sing, Garwin; Müller, Elke; Reißig, Annett; Gawlik, Darius; Ehricht, Ralf; Monecke, StefanPurpose: There is a need for continuous surveillance of methicillin-resistant Staphylococcus aureus (MRSA) to identify emergence of new strains. We hypothesize that MRSA strains are evolving with ongoing acquisition of SCCmec elements. This study was carried out to evaluate the evolution of MRSA at a tertiary care facility in Saudi Arabia. Methods: MRSA isolates associated with invasive clinical infection, which were identified in 2017 at the microbiology laboratory, King Khalid University Hospital (KKUH) in Riyadh, Saudi Arabia, were studied. The molecular characterization of isolates was carried out using StaphyType DNA microarray (Alere Technologies GmbH/Abbott, Jena, Germany). Results: The 125 MRSA isolates studied belonged to 18 clonal complexes (CC) which were distributed into 32 strain assignments. The predominant CC were CC5 (n=30), CC6 (n=17), CC80 (n=13), CC22 (n=12), CC361 (n=12). The findings demonstrated the first identification of CC152, CC361 and CC1153 MRSA as well as ST5-MRSA-[I+fus], “Geraldine Clone”, CC6-MRSA-IV (PVL+) and CC88-MRSA-V (PVL+), WA MRSA-117 in Saudi Arabia. Four novel variants were identified: CC5-MRSA-[VI+fus+tirS], CC22-MRSA-[V/VT+fus](PVL+), CC152-MRSA-[V+fus](PVL+) and CC361-MRSA-[VT+fus]. Fifty-four isolates (n/N=54/125; 43.2%) including the novel strains carried the Q6GD50 SCCfusC gene while the Panton-Valentine leukocidin genes were present in 30.4% (n/N=38/125). Conclusion: The findings demonstrate an expanding MRSA repertoire in our setting including emergence of previously unreported clonal complexes and novel strains. The high carriage of fusC gene suggests a role for fusidic acid misuse in driving the evolution of the MRSA genome and underscores the need for increased monitoring of antibiotic use.Purpose: There is a need for continuous surveillance of methicillin-resistant Staphylococcus aureus (MRSA) to identify emergence of new strains. We hypothesize that MRSA strains are evolving with ongoing acquisition of SCCmec elements. This study was carried out to evaluate the evolution of MRSA at a tertiary care facility in Saudi Arabia. Methods: MRSA isolates associated with invasive clinical infection, which were identified in 2017 at the microbiology laboratory, King Khalid University Hospital (KKUH) in Riyadh, Saudi Arabia, were studied. The molecular characterization of isolates was carried out using StaphyType DNA microarray (Alere Technologies GmbH/Abbott, Jena, Germany). Results: The 125 MRSA isolates studied belonged to 18 clonal complexes (CC) which were distributed into 32 strain assignments. The predominant CC were CC5 (n=30), CC6 (n=17), CC80 (n=13), CC22 (n=12), CC361 (n=12). The findings demonstrated the first identification of CC152, CC361 and CC1153 MRSA as well as ST5-MRSA-[I+fus], “Geraldine Clone”, CC6-MRSA-IV (PVL+) and CC88-MRSA-V (PVL+), WA MRSA-117 in Saudi Arabia. Four novel variants were identified: CC5-MRSA-[VI+fus+tirS], CC22-MRSA-[V/VT+fus](PVL+), CC152-MRSA-[V+fus](PVL+) and CC361-MRSA-[VT+fus]. Fifty-four isolates (n/N=54/125; 43.2%) including the novel strains carried the Q6GD50 SCCfusC gene while the Panton-Valentine leukocidin genes were present in 30.4% (n/N=38/125). Conclusion: The findings demonstrate an expanding MRSA repertoire in our setting including emergence of previously unreported clonal complexes and novel strains. The high carriage of fusC gene suggests a role for fusidic acid misuse in driving the evolution of the MRSA genome and underscores the need for increased monitoring of antibiotic use.
- 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.
- ItemClonal Complexes Distribution of Staphylococcus aureus Isolates from Clinical Samples from the Caribbean Islands(Basel : MDPI, 2023) Monecke, Stefan; Akpaka, Patrick Eberechi; Smith, Margaret R.; Unakal, Chandrashekhar G.; Thoms Rodriguez, Camille-Ann; Ashraph, Khalil; Müller, Elke; Braun, Sascha D.; Diezel, Celia; Reinicke, Martin; Ehricht, RalfThe aim of this study was to comprehensively characterise S. aureus from the Caribbean Islands of Trinidad and Tobago, and Jamaica. A total of 101 S. aureus/argenteus isolates were collected in 2020, mainly from patients with skin and soft tissue infections. They were characterised by DNA microarray allowing the detection of ca. 170 target genes and assignment to clonal complexes (CC)s and strains. In addition, the in vitro production of Panton–Valentine leukocidin (PVL) was examined by an experimental lateral flow assay. Two isolates were identified as S. argenteus, CC2596. The remaining S. aureus isolates were assigned to 21 CCs. The PVL rate among methicillin-susceptible S. aureus (MSSA) isolates was high (38/101), and 37 of the 38 genotypically positive isolates also yielded positive lateral flow results. The isolate that did not produce PVL was genome-sequenced, and it was shown to have a frameshift mutation in agrC. The high rate of PVL genes can be attributed to the presence of a known local CC8–MSSA clone in Trinidad and Tobago (n = 12) and to CC152–MSSA (n = 15). In contrast to earlier surveys, the USA300 clone was not found, although one MSSA isolate carried the ACME element, probably being a mecA-deficient derivative of this strain. Ten isolates, all from Trinidad and Tobago, were identified as MRSA. The pandemic ST239–MRSA–III strain was still common (n = 7), but five isolates showed a composite SCCmec element not observed elsewhere. Three isolates were sequenced. That showed a group of genes (among others, speG, crzC, and ccrA/B-4) to be linked to its SCC element, as previously found in some CC5– and CC8–MRSA, as well as in S. epidermidis. The other three MRSA belonged to CC22, CC72, and CC88, indicating epidemiological connections to Africa and the Middle East.