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- 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.
- 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.
- ItemCharacterization of PVL-Positive MRSA Isolates in Northern Bavaria, Germany over an Eight-Year Period(Basel : MDPI, 2022) Szumlanski, Tobias; Neumann, Bernd; Bertram, Ralph; Simbeck, Alexandra; Ziegler, Renate; Monecke, Stefan; Ehricht, Ralf; Schneider-Brachert, Wulf; Steinmann, JoergPurpose: Community-acquired methicillin-resistant Staphylococcus aureus strains (CA-MRSA) are spread worldwide and often cause recurring and persistent infections in humans. CA-MRSA strains frequently carry Panton–Valentine leukocidin (PVL) as a distinctive virulence factor. This study investigates the molecular epidemiology, antibiotic resistance and clinical characteristics of PVL-positive MRSA strains in Northern Bavaria, Germany, isolated over an eight-year period. Methods: Strains were identified by MALDI-TOF MS and antibiotic susceptibility was tested by automated microdilution (VITEK 2) or disk diffusion. PVL-encoding genes and mecA were detected by PCR. MRSA clonal complexes (CC) and lineages were assigned by genotyping via DNA microarray and spa-typing. Results: In total, 131 PVL-positive MRSA were collected from five hospital sites between 2009 and 2016. Predominant lineages were CC8-MRSA-[IV+ACME], USA300 (27/131; 20.6%); CC30-MRSA-IV, Southwest Pacific Clone (26/131; 19.8%) and CC80-MRSA-IV (25/131; 19.1%). Other CCs were detected less frequently. Resistance against erythromycin and clindamycin was prevalent, whereas all strains were sensitive towards vancomycin and linezolid. In total, 100 cases (76.3%) were causally linked to an infection. The majority (102/131; 77.9%) of isolates were detected in skin swabs or swabs from surgical sites. Conclusions: During the sample period we found an increase in the PVL-positive MRSA lineages CC30 and CC1. Compared to less-abundant lineages CC1 or CC22, the predominant lineages CC8, CC30 and CC80 harbored a broader resistance spectrum. Furthermore, these lineages are probably associated with a travel and migration background. In the spatio-temporal setting we investigated, these were arguably drivers of diversification and change in the landscape of PVL-positive MRSA.
- ItemMolecular epidemiology of methicillin-susceptible and methicillin-resistant staphylococcus aureus in wild, captive and laboratory rats: Effect of habitat on the Nasal S. aureus Population(Basel : MDPI, 2020) Raafat, Dina; Mrochen, Daniel M.; Al’Sholui, Fawaz; Heuser, Elisa; Ryll, René; Pritchett-Corning, Kathleen R.; Jacob, Jens; Walther, Bernd; Matuschka, Franz-Rainer; Richter, Dania; Westerhüs, Uta; Pikula, Jiri; van den Brandt, Jens; Nicklas, Werner; Monecke, Stefan; Strommenger, Birgit; van Alen, Sarah; Becker, Karsten; Ulrich, Rainer G.; Holtfreter, SilvaRats are a reservoir of human- and livestock-associated methicillin-resistant Staphylococcus aureus (MRSA). However, the composition of the natural S. aureus population in wild and laboratory rats is largely unknown. Here, 144 nasal S. aureus isolates from free-living wild rats, captive wild rats and laboratory rats were genotyped and profiled for antibiotic resistances and human-specific virulence genes. The nasal S. aureus carriage rate was higher among wild rats (23.4%) than laboratory rats (12.3%). Freeliving wild rats were primarily colonized with isolates of clonal complex (CC) 49 and CC130 and maintained these strains even in husbandry. Moreover, upon livestock contact, CC398 isolates were acquired. In contrast, laboratory rats were colonized with many different S. aureus lineages-many of which are commonly found in humans. Five captive wild rats were colonized with CC398-MRSA. Moreover, a single CC30-MRSA and two CC130-MRSA were detected in free-living or captive wild rats. Rat-derived S. aureus isolates rarely harbored the phage-carried immune evasion gene cluster or superantigen genes, suggesting long-term adaptation to their host. Taken together, our study revealed a natural S. aureus population in wild rats, as well as a colonization pressure on wild and laboratory rats by exposure to livestock- and human-associated S. aureus, respectively. © 2020 by the authors.
- ItemClinical S. aureus Isolates Vary in Their Virulence to Promote Adaptation to the Host(Basel : MDPI, 2019) Tuchscherr, Lorena; Pöllath, Christine; Siegmund, Anke; Deinhardt-Emmer, Stefanie; Hoerr, Verena; Svensson, Carl-Magnus; Figge, Marc Thilo; Monecke, Stefan; Löffler, BettinaStaphylococcus aureus colonizes epithelial surfaces, but it can also cause severe infections. The aim of this work was to investigate whether bacterial virulence correlates with defined types of tissue infections. For this, we collected 10–12 clinical S. aureus strains each from nasal colonization, and from patients with endoprosthesis infection, hematogenous osteomyelitis, and sepsis. All strains were characterized by genotypic analysis, and by the expression of virulence factors. The host–pathogen interaction was studied through several functional assays in osteoblast cultures. Additionally, selected strains were tested in a murine sepsis/osteomyelitis model. We did not find characteristic bacterial features for the defined infection types; rather, a wide range in all strain collections regarding cytotoxicity and invasiveness was observed. Interestingly, all strains were able to persist and to form small colony variants (SCVs). However, the low-cytotoxicity strains survived in higher numbers, and were less efficiently cleared by the host than the highly cytotoxic strains. In summary, our results indicate that not only destructive, but also low-cytotoxicity strains are able to induce infections. The low-cytotoxicity strains can successfully survive, and are less efficiently cleared from the host than the highly cytotoxic strains, which represent a source for chronic infections. The understanding of this interplay/evolution between the host and the pathogen during infection, with specific attention towards low-cytotoxicity isolates, will help to optimize treatment strategies for invasive and therapy-refractory infection courses.
- ItemCharacterization of Staphylococci and Streptococci Isolated from Milk of Bovides with Mastitis in Egypt(Basel : MDPI, 2020) Ahmed, Wedad; Neubauer, Heinrich; Tomaso, Herbert; El Hofy, Fatma Ibrahim; Monecke, Stefan; Abdeltawab, Ashraf Awad; Hotzel, HelmutThe aim of this study was to characterize staphylococci and streptococci in milk from Egyptian bovides. In total, 50 milk samples were collected from localities in the Nile Delta region of Egypt. Isolates were cultivated, identified using matrix-assisted laser desorption/ionization time-offlight mass spectrometry (MALDI-TOF MS), and antibiotic susceptibility testing was performed by the broth microdilution method. PCR amplifications were carried out, targeting resistanceassociated genes. Thirty-eight Staphylococcus isolates and six Streptococcus isolates could be cultivated. Staphylococcus aureus isolates revealed a high resistance rate to penicillin, ampicillin, clindamycin, and erythromycin. The mecA gene defining methicillin-resistant Staphylococcus aureus, erm(C) and aac-aphD genes was found in 87.5% of each. Coagulase-negative staphylococci showed a high prevalence of mecA, blaZ and tetK genes. Other resistance-associated genes were found. All Streptococcus dysgalactiae isolates carried blaZ, erm(A), erm(B), erm(C) and lnuA genes, while Streptococcus suis harbored erm(C), aphA-3, tetL and tetM genes, additionally. In Streptococcus gallolyticus, most of these genes were found. The Streptococcus agalactiae isolate harbored blaZ, erm(B), erm(C), lnuA, tetK, tetL and tetM genes. Streptococcus agalactiae isolate was analyzed by DNA microarray analysis. It was determined as sequence type 14, belonging to clonal complex 19 and represented capsule type VI. Pilus and cell wall protein genes, pavA, cadD and emrB/qacA genes were identified by microarray analysis. © 2020 by the authors.
- ItemCharacterization of Enterococci- and ESBL-Producing Escherichia coli Isolated from Milk of Bovides with Mastitis in Egypt(Basel : MDPI, 2021) Ahmed, Wedad; Neubauer, Heinrich; Tomaso, Herbert; El Hofy, Fatma Ibrahim; Monecke, Stefan; Abd El-Tawab, Ashraf Awad; Hotzel, HelmutThis study aimed to investigate the prevalence and antimicrobial resistance of enterococci- and ESBL-producing E. coli isolated from milk of bovine mastitis cases in Egypt. Fifty milk samples of dairy animals were collected from localities in the Nile Delta region of Egypt. Isolates were identified using MALDI-TOF MS, and antibiotic susceptibility testing was performed by the broth microdilution method. PCR amplifications were carried out, targeting resistance-associated genes. Seventeen Enterococcus isolates and eight coliform isolates could be cultivated. Vancomycin resistance rate was high in Ent. faecalis. The VITEK 2 system confirmed all E. coli isolates as ESBL-producing. All Ent. faecalis isolates harbored erm(B), tetL and aac-aphD genes. The vanA gene was detected in Ent. faecalis isolate, vanB was found in other Enterococcus, while one isolate of E. casseliflavus exhibited the vanA gene. E. coli isolates exhibited high prevalence of erm(B) and tetL. E. coli isolates were analyzed by DNA microarray analysis. Four isolates were determined by O-serotyping as O8 (n = 1), O86 (n = 2) and O157 (n = 1). H-serotyping resulted in H11, H12, H21 (two isolates each) and one was of H16 type. Different virulence-associated genes were detected in E. coli isolates including lpfA, astA, celB, cmahemL, intI1 and intI2, and the iroN gene was identified by DNA microarray analysis.
- 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.