Analysis and Characterization of Staphylococcus aureus Small Colony Variants Isolated From Cystic Fibrosis Patients in Austria.

Cystic fibrosis (CF) is the most common hereditary lung disease in the Caucasian population, characterized by viscous bronchial secretion, consecutive defective mucociliary clearance, and unavoidable colonization with microorganisms. Besides Pseudomonas aeruginosa, Staphylococcus aureus is the most common bacterial species colonizing the CF respiratory tract. Under antibiotic pressure S. aureus is able to switch to small colony variants (SCV). These small colony variants can invade epithelial cells, overcome antibiotic therapy inside the cells and can be the starting point for extracellular recolonization. The aim of the present study was the isolation and characterization of S. aureus small colony variants from Austrian cystic fibrosis patients. Samples collected from 147 patients were screened for the presence of S. aureus wild-type and small colony variants. Antibiotic susceptibility testing and determination of the small colony variants causing auxotrophism were performed. Wild-type isolates were assigned to corresponding small colony variants with spa typing. In total, 17 different small colony variant isolates and 12 corresponding wild-type isolates were obtained. 13 isolates were determined thymidine auxotroph, 2 isolates were auxotroph for hemin, and none of the tested isolates was auxotroph for both, respectively. The presence of SCVs is directly related to a poor clinical outcome, therefore a monitoring of SCV prevalence is recommended. This study revealed rather low SCV ratios in CF patients compared to other countries.

Severe forefoot infection complicated by Fusobacterium russii.

We present the first case of a complicated foot infection caused by Fusobacterium russii in Austria. F. russii is highly associated with mammals such as cats and dogs. Our case underlines the difficulties in isolation and identification of anaerobes and the pitfalls in antimicrobial treatment of polymicrobial infections.

Contaminated handwashing sinks as the source of a clonal outbreak of KPC-2-producing Klebsiella oxytoca on a hematology ward.

We investigated sinks as possible sources of a prolonged Klebsiella pneumonia carbapenemase (KPC)-producing Klebsiella oxytoca outbreak. Seven carbapenem-resistant K. oxytoca isolates were identified in sink drains in 4 patient rooms and in the medication room. Investigations for resistance genes and genetic relatedness of patient and environmental isolates revealed that all the isolates harbored the blaKPC-2 and blaTEM-1 genes and were genetically indistinguishable. We describe here a clonal outbreak caused by KPC-2-producing K. oxytoca, and handwashing sinks were a possible reservoir.

Prevalence of emm types and antimicrobial susceptibility of Streptococcus dysgalactiae subsp. equisimilis in Austria.

OBJECTIVES: An increase of severe infections caused by Streptococcus dysgalactiae subsp. equisimilis (SDSE) similar to infections caused by Streptococcus pyogenes has been reported over the last years. Little is known about infections with SDSE in Austria. Therefore, we investigated a collection of 113 SDSE invasive and non-invasive isolates from different infection sites and type of infections as well as patients' characteristics. METHODS: The isolates were phenotypically identified and emm typed using the enlarged emm database from the Centers for Disease Control and Prevention. Additionally, 13 antimicrobial agents were tested using EUCAST guidelines and virulence genes were investigated. RESULTS: Severe SDSE infections were most common in elderly men with underlying diseases especially diabetes mellitus. With VitekMS identification of SDSE isolates was successful to the species level only. Emm typing revealed 24 different emm types, one new type and one new subtype. StG485, stG6, stC74a, stG643, and stG480 were the predominant types in this study, stC74a and stG652 in invasive infections and stG643, stC74a and stG485 in non-invasive infections. Resistance was observed to tetracycline (62%), macrolides (13%) with one M phenotype, and clindamycin (12%) presenting 6 constitutive MLS(B) phenotypes and 8 inducible MLS(B) phenotypes. Levofloxacin resistance was detected only in one isolate. All isolates tested for virulence genes were positive for scpA, ska, saga and slo. Superantigenic genes were negative except speG(dys) (positive 17/34; 50%). CONCLUSION: This paper presents the first report of SDSE infections in Austria. Severe SDSE infections were found mainly in elderly men with underlying diseases. SDSE isolates demonstrated substantial emm type diversity without association with infections site or invasiveness. Analysis of virulence genes showed no significant difference between invasive and non-invasive infections.

Genetic and Phenotypic Characteristics of Staphylococcus aureus Isolates from Cystic Fibrosis Patients in Austria.

BACKGROUND: Cystic fibrosis (CF) is the most common life-limiting inherited disease in Caucasian populations. While pathological changes can be seen in various organs, morbidity and mortality are mainly related to the respiratory tract, with patients suffering from chronic bronchopulmonary infections with characteristic pathogens including Staphylococcus aureus. OBJECTIVES: To date, there is only very limited data on the genetic and phenotypic characteristics of S. aureus in CF patients. Therefore, in our study, we characterized 58 S. aureus isolates collected from CF patients in Austria by spa typing, DNA microarray profiling, as well as antimicrobial susceptibility testing in order to determine common genomic and antimicrobial resistance features. The tested strain collection exhibited high genomic diversity. RESULTS: The 58 isolates were assigned to 16 clonal complexes and 48 spa types and differed greatly regarding their virulence and resistance gene profiles. The predominant clonal complexes were MLST CC30 (22%), CC15 (16%), CC45 (14%), and CC5 (12%), complexes that are highly prevalent worldwide among S. aureus strains isolated from humans colonized or infected with S. aureus. DNA microarray profiles showed a wide variety of genes encoding antimicrobial resistance and virulence factors such as various leukocidins, haemolysins, enterotoxins, exfoliative toxins, toxic shock syndrome toxin, as well as genes involved in adhesion and immune evasion. CONCLUSIONS: While a large number of strains exhibited resistance to one or several antimicrobial agents, methicillin-resistant S. aureus was found at a low prevalence of 3% (n = 2) only. The two methicillin-resistant S. aureus isolates were assigned to CC152/t355 (SCCmecV) and CC5/t001 (SCCmecI). This is the first study to genetically characterize S. aureus isolates in CF patients in Austria.

Isolation and characterization of multidrug-resistant bacteria from minced meat in Austria.

INTRODUCTION: Resistant bacteria are a well-known public health problem. This study was conducted to investigate the prevalence and genetic characteristics of extended spectrum ß-lactamase (ESBL) producing enterobacteria, methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococci (VRE) in mixed minced meat from pork and beef. METHODS: One hundred samples of mixed minced meat were collected from supermarkets (n = 70) and local butcher shops (n = 30) in the city of Graz (Austria). After enrichment and inoculation on selective media, bacteria were identified with MALDI-TOF MS or Vitek2 systems, tested for antibiotic resistance and further characterized with PCR and sequencing. RESULTS: In 20 of the 100 meat samples 24 ESBL positive Escherichia coli isolates were found. The most common ESBL among the isolates was CTX-M-1. Other detected bla genes contained CTX-M-14, CTX-M-32, SHV-12 and TEM-52 types. Nine samples were tested positive for MRSA and spa-typed. Detected spa-types were hospital-acquired t3928, as well as livestock-associated t011, t034 and t2241. No VRE were found. CONCLUSION: A contamination of meat with ESBL-producing E. coli and MRSA was confirmed in this study. The large diversity of ESBL producing E. coli could indicate a growing dissemination of ESBL genes in E. coli found in meat products from porcine and bovine origin.

First report of Nocardia asiatica olecranon bursitis in an immunocompetent traveler returning to Austria.

Nocardia spp. are rarely isolated in extrapulmonary clinical specimens. We describe the first case of olecranon bursitis caused by Nocardia asiatica. The patient, a traveler returning from Thailand, was successfully treated with linezolid.

Ventilator-associated pneumonia caused by OXA-48-producing Escherichia coli complicated by ciprofloxacin-associated rhabdomyolysis.

We report the emergence of OXA-48 carbapenemase-producing Escherichia coli in Austria causing ventilator-associated pneumonia in a traveler returning from Egypt. Depending on resistance testing, quinolones may remain a therapeutic option for infections caused by these multiple resistant pathogens, as this class of drugs has a favorable safety and tolerability profile when compared to the alternatives. In this patient, however, the clinical course was dramatically complicated by the development of ciprofloxacin-associated rhabdomyolysis.

No Changes in the Occurrence of Methicillin-Resistant Staphylococcus aureus (MRSA) in South-East Austria during the COVID-19 Pandemic.

Methicillin-resistant Staphylococcus aureus (MRSA) is a universal threat. Once being well established in the healthcare setting, MRSA has undergone various epidemiological changes. This includes the emergence of more aggressive community-acquired MRSA (CA-MRSA) and the occurrence of MRSA which have their origin in animal breeding, called livestock-associated MRSA (LA-MRSA). Emergence of new clones as well as changes in the occurrence of some clonal lineages also describes the fluctuating dynamic within the MRSA family. There is paucity of data describing the possible impact of the COVID-19 pandemic on the MRSA dynamics. The aim of the study was the analysis of MRSA isolates in a three-year time period, including the pre-COVID-19 years 2018 and 2019 and the first year of the pandemic 2020. The analysis includes prevalence determination, antibiotic susceptibility testing, spa typing, and detection of genes encoding the PVL toxin. The MRSA rate remained constant throughout the study period. In terms of a dynamic within the MRSA family, only a few significant changes could be observed, but all except one occurred before the start of the COVID-19 pandemic. In summary, there was no significant impact of the COVID-19 pandemic on MRSA in Austria.

Incidental finding of Clostridium perfringens on human corpses used for the anatomy course.

Post-mortem specimens used for anatomy teaching are commonly embalmed using compositions of chemicals, with the objective to maintain tissue quality and to avoid putrefaction. Monitoring for bacterial or fungal contamination is becoming increasingly important especially when measures are taken to minimize exposure by chemicals such as formaldehyde. In this case, random swabs were taken from six corpses embalmed with ethanol-glycerin and Thiel embalming. Cultures and MALDI-TOF analyses yielded four cases of Clostridium perfringens contamination. C. perfringens is of special interest as a human pathogen. A potential source was identified in the containers filled with the moistening solution. Cross contamination with Clostridium species has likely occurred between corpses sharing the moistening solution and soaking the cover linen directly within the containers. To minimize any risk for those exposed, the moistening solutions were discarded and all equipment thoroughly disinfected. The specimens had to be cremated as they formed a potential source of Clostridium spores. Deviating from previous routines it was formalized that the cover linen must not be submerged in the moistening contains rather than moistening the specimens directly with dedicated vessels. Follow-up analyses yielded no further contamination with C. perfringens.

Nosocomial outbreak of Klebsiella pneumoniae carbapenemase-producing Klebsiella oxytoca in Austria.

To date, no outbreak of carbapenemase-producing bacteria has been reported for Austria. While outbreaks of Klebsiella pneumoniae carbapenemase (KPC)-producing K. pneumoniae have been increasingly reported, no outbreak caused by KPC-producing Klebsiella oxytoca has been described yet, to the best of our knowledge. We report an outbreak of KPC-producing K. oxytoca. In 5 months, 31 KPC-producing Klebsiella oxytoca strains were isolated from five patients. All patients were admitted to the same medical intensive care unit in Austria.

Osteoarticular Infections in Pediatric Hospitals in Europe: A Prospective Cohort Study From the EUCLIDS Consortium.

Background: Pediatric osteoarticular infections (POAIs) are serious diseases requiring early diagnosis and treatment. Methods: In this prospective multicenter cohort study, children with POAIs were selected from the European Union Childhood Life-threatening Infectious Diseases Study (EUCLIDS) database to analyze their demographic, clinical, and microbiological data. Results: A cohort of 380 patients with POAIs, 203 with osteomyelitis (OM), 158 with septic arthritis (SA), and 19 with both OM and SA, was analyzed. Thirty-five patients were admitted to the Pediatric Intensive Care Unit; out of these, six suffered from shock, one needed an amputation of the right foot and of four left toes, and two had skin transplantation. According to the Pediatric Overall Performance Score, 36 (10.5%) showed a mild overall disability, 3 (0.8%) a moderate, and 1 (0.2%) a severe overall disability at discharge. A causative organism was detected in 65% (247/380) of patients. Staphylococcus aureus (S. aureus) was identified in 57.1% (141/247) of microbiological confirmed cases, including 1 (0.7%) methicillin-resistant S. aureus (MRSA) and 6 (4.2%) Panton-Valentine leukocidin (PVL)-producing S. aureus, followed by Group A Streptococcus (18.2%) and Kingella kingae (8.9%). K. kingae and PVL production in S. aureus were less frequently reported than expected from the literature. Conclusion: POAIs are associated with a substantial morbidity in European children, with S. aureus being the major detected pathogen. In one-third of patients, no causative organism is identified. Our observations show an urgent need for the development of a vaccine against S. aureus and for the development of new microbiologic diagnostic guidelines for POAIs in European pediatric hospitals.

Surveillance of Antimicrobial Susceptibility of Anaerobe Clinical Isolates in Southeast Austria: Bacteroides fragilis Group Is on the Fast Track to Resistance.

Anaerobic bacteria play an important role in human infections. Bacteroides spp. are some of the 15 most common pathogens causing nosocomial infections. We present antimicrobial susceptibility testing (AST) results of 114 Gram-positive anaerobic isolates and 110 Bacteroides-fragilis-group-isolates (BFGI). Resistance profiles were determined by MIC gradient testing. Furthermore, we performed disk diffusion testing of BFGI and compared the results of the two methods. Within Gram-positive anaerobes, the highest resistance rates were found for clindamycin and moxifloxacin (21.9% and 16.7%, respectively), and resistance for beta-lactams and metronidazole was low (<1%). For BFGI, the highest resistance rates were also detected for clindamycin and moxifloxacin (50.9% and 36.4%, respectively). Resistance rates for piperacillin/tazobactam and amoxicillin/clavulanic acid were 10% and 7.3%, respectively. Two B. fragilis isolates were classified as multi-drug-resistant (MDR), with resistance against all tested beta-lactam antibiotics. The comparative study of 109 BFGI resulted in 130 discrepancies in 763 readings (17%) with a high number of Very Major Errors (VME) and Major Errors (ME). In summary, resistance rates, with the exception of clindamycin and moxifloxacin, are still low, but we are facing increasing resistance rates for BFGI. Surveillance studies on a regular basis are still recommended.

Diversity of Oxacillinases and Sequence Types in Carbapenem-Resistant Acinetobacter baumannii from Austria.

Carbapenem-resistant Acinetobacter baumannii is a significant health problem worldwide. A multicenter study on A. baumannii was performed to investigate the molecular epidemiology and genetic background of carbapenem resistance of A. baumannii isolates collected from 2014-2017 in Austria. In total, 117 non-repetitive Acinetobacter spp. assigned to A. baumannii (n = 114) and A. pittii (n = 3) were collected from four centers in Austria. The isolates were uniformly resistant to piperacillin/tazobactam, ceftazidime, and carbapenems, and resistance to imipenem and meropenem was 97.4% and 98.2%, respectively. The most prominent OXA-types were OXA-58-like (46.5%) and OXA-23-like (41.2%), followed by OXA-24-like (10.5%), with notable regional differences. Carbapenem-hydrolyzing class D carbapenemases (CHDLs) were the only carbapenemases found in A.baumannii isolates in Austria since no metallo-ß-lactamases (MBLs) nor KPC or GES carbapenemases were detected in any of the isolates. One-third of the isolates harbored multiple CHDLs. The population structure of A. baumannii isolates from Austria was found to be very diverse, while a total of twenty-three different sequence types (STs) were identified. The most frequent was ST195 found in 15.8%, followed by ST218 and ST231 equally found in 11.4% of isolates. Two new ST types, ST2025 and ST2026, were detected. In one A. pittii isolate, blaOXA-143-like was detected for the first time in Austria.

CD19+IgD+CD27- Naïve B Cells as Predictors of Humoral Response to COVID 19 mRNA Vaccination in Immunocompromised Patients.

Immunocompromised patients are considered high-risk and prioritized for vaccination against COVID-19. We aimed to analyze B-cell subsets in these patients to identify potential predictors of humoral vaccination response. Patients (n=120) suffering from hematologic malignancies or other causes of immunodeficiency and healthy controls (n=79) received a full vaccination series with an mRNA vaccine. B-cell subsets were analyzed prior to vaccination. Two independent anti-SARS-CoV-2 immunoassays targeting the receptor-binding domain (RBD) or trimeric S protein (TSP) were performed three to four weeks after the second vaccination. Seroconversion occurred in 100% of healthy controls, in contrast to 67% (RBD) and 82% (TSP) of immunocompromised patients, while only 32% (RBD) and 22% (TSP) achieved antibody levels comparable to those of healthy controls. The number of circulating CD19+IgD+CD27- naïve B cells was strongly associated with antibody levels (ρ=0.761, P<0.001) and the only independent predictor for achieving antibody levels comparable to healthy controls (OR 1.07 per 10-µL increase, 95%CI 1.02-1.12, P=0.009). Receiver operating characteristic analysis identified a cut-off at ≥61 naïve B cells per µl to discriminate between patients with and without an optimal antibody response. Consequently, measuring of naïve B cells in immunocompromised hematologic patients could be useful in predicting their humoral vaccination response.

Submasseteric abscess caused by Mycoplasma salivarium infection.

Mycoplasma salivarium preferentially resides in the human oral cavity. Unlike other Mycoplasma species, M. salivarium has not been regarded as a pathogen, although one case of M. salivarium-caused arthritis in a patient with hypogammaglobulinemia has been reported. We describe the first case of submasseteric abscess caused by M. salivarium.

Use of hybridization probes in a real-time PCR assay on the LightCycler for the detection of methicillin-resistant Staphylococcus aureus.

The rapid and accurate identification of methicillin-resistant Staphylococcus aureus (MRSA) is of great importance for the affected patient, the involved ward, and the microbiological laboratory. Resistance to methicillin is encoded by the mecA gene in S. aureus. Because routine laboratory diagnostics may be time consuming and because species differentiation encounters a variety of difficulties, molecular techniques detecting both the mecA and a S. aureus-specific gene are used for rapid and accurate detection and identification of MRSA. Various protocols, including the manual extraction of DNA have been established. In this chapter, the identification of MRSA based on simultaneous detection of the mecA gene and the S. aureus-specific Sa442 DNA fragment using automated DNA extraction and real-time polymerase chain reaction is described. This method is an attractive alternative to labor-intensive manual protocols and can easily be incorporated into the diagnostic microbiology laboratory workflow, with the ability to obtain results within 4 h.

Susceptibility of Austrian Clinical Klebsiella and Enterobacter Isolates Linked to Patient-Related Data.

The aim of the study was to analyze the antimicrobial susceptibility of Austrian clinical Klebsiella sp. and Enterobacter sp. isolates linked to patient-related data over a time period from 1998 to 2014. The main findings of this study were (i) a marked difference of antibiotic susceptibility rates between different infection sites for both Klebsiella sp. and Enterobacter sp., (ii) significantly greater percentages of resistant isolates among both Klebsiella sp. and Enterobacter sp. in male patients compared to female patients and (iii) significantly greater percentages of resistant isolates among both Klebsiella sp. and Enterobacter sp. from hospital-derived samples compared to samples from the community. In conclusion, our statistical data analysis clearly indicated a strong association of patient-related data and Klebsiella sp. and Enterobacter sp. susceptibility profiles.