A head-to-head comparison of three MALDI-TOF mass spectrometry systems with 16S rRNA gene sequencing.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has revolutionized diagnostics in culture-based microbiology. Commonly used MALDI-TOF MS systems in clinical microbiology laboratories are MALDI Biotyper (Bruker Daltonics) and Vitek MS (bioMérieux), but recently the new EXS2600 (Zybio) has been launched. This study aimed to evaluate the performance of the three devices by comparing the results to 16S rRNA gene sequencing. A set of 356 previously collected difficult-to-identify bacteria was tested in parallel with the three systems. Only the direct smear method and simple formic acid extraction were applied. Valid results were achieved for 98.6%, 94.4%, and 93.3% of all isolates by MALDI Biotyper, EXS2600, and Vitek MS, respectively. Of all valid results, agreement with sequencing data was achieved in 98.9%, 98.5%, and 99.7% by MALDI Biotyper, EXS2600, and Vitek MS, respectively. Considering only the isolates with valid measurements at the single-species level, misidentification rates were 0%, 2.6%, and 1.1% for MALDI Biotyper, EXS2600, and Vitek MS, respectively. Apart from minor performance differences, our data demonstrate that the three systems provide comparable results and are suitable for use in medical diagnostic laboratories.

Emergence of Carbapenem-Hydrolyzing Oxacillinases in Acinetobacter baumannii in Children from Croatia.

INTRODUCTION: Carbapenem resistance in Acinetobacter baumannii can be mediated by carbapenemases of class A, class B metallo-ß-lactamases (MBLs), and class D carbapenem-hydrolyzing oxacillinases (CHDL). The aim of the study was to investigate the antimicrobial susceptibility and ß-lactamase production of carbapenem-resistant A. baumannii isolates (CRAB) from the Children's Hospital Zagreb, Croatia. METHODS: A total of 12 A. baumannii isolates collected between August 2016 and March 2018 were analyzed. Antibiotic susceptibility was determined by the broth microdilution method. The presence of MBLs was explored by combined disk test with EDTA. The presence of carbapenemases of class A, B, and D was explored by PCR. The occurrence of the ISAba1 upstream of the blaOXA-51-like or blaOXA-23-like was determined by PCR mapping. Epidemiological typing was performed by determination of sequence groups (SG). Genotyping was performed by SG determination, rep-PCR, and MLST. RESULTS: All CRAB were resistant to piperacillin/tazobactam, ceftazidime, cefotaxime, ceftriaxone, cefepime, imipenem, meropenem, gentamicin, and ciprofloxacin. Moderate resistance rates were observed for ampicillin/sulbactam (67%) and tigecycline (42%). The isolates were uniformly susceptible to colistin. PCR revealed the presence of genes encoding OXA-24-like CHDL in nine and OXA-23-like CHDL in three isolates. blaOXA-51 genes were preceded by ISAba1. PCR for the common MBLs in Acinetobacter was negative. All isolates belonged to SG 1 corresponding to ICL (International Clonal Lineage) II. Rep-PCR identified four major clones. CONCLUSIONS: The study found OXA-24-like ß-lactamase to be the dominant CHDL among children'sCRAB. The predominant spread of OXA-24-like is in contrast with the recent global dissemination of OXA-23 reported all over the world. In contrast to the previous studies in which emergency of OXA-24-like positive isolates was monoclonal, we found considerable genetic diversity of the isolates.

Culturable fungi in potting soils and compost.

In the present study the spectrum and the incidence of fungi in potting soils and compost was investigated. Since soil is one of the most important biotopes for fungi, relatively high concentrations of fungal propagules are to be expected. For detection of fungi, samples of commercial soils, compost and soils from potted plants (both surface and sub-surface) were suspended and plated onto several mycological media. The resulting colonies were evaluated qualitatively and quantitatively. The results from the different sampling series vary, but concentrations on the surface of potted plants and in commercial soils are increased tenfold compared to compost and sub-surface soils. Median values range from 9.5 × 10(4) colony forming units (CFU)/g to 5.5 × 10(5) CFU/g. The spectrum of fungi also varies in the soils. However, all sampling series show high proportion of Aspergillus and Penicillium species, including potentially pathogenic species such as Aspergillus fumigatus. Cladosporium, a genus dominant in the ambient air, was found preferably in samples which were in contact with the air. The results show that potentially pathogenic fungi are present in soils. Immunocompromised individuals should avoid handling soils or potted plants in their immediate vicinity.

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.

Detection of central venous catheter-related bloodstream infections in haematooncological patients.

BACKGROUND: Catheter-related bloodstream infections (CRBSIs) are currently detected in patients with clinically suspicion. The aim of our study was to evaluate whether CRBSIs could be anticipated and detected in a subclinical stage by peptide nucleic acid fluorescence in situ hybridization (PNA FISH) using universal hybridization probes or acridine orange leucocyte cytospin (AOLC) tests in haematooncological patients with central venous catheters (CVCs) in situ. MATERIALS AND METHODS: Peptide nucleic acid fluorescence in situ hybridization and AOLC tests using blood samples from one CVC lumen/port chamber in haematooncological patients were continuously performed. These results were compared to those obtained from routinely performed CRBSI diagnostic tests. RESULTS: One hundred and eighty-two patients with 342 catheter periods were investigated. Seventeen CRBSI cases were detected in 6466 CVC days by routine measures resulting in a CRBSI rate of 2.6/1000 catheter days. Two of 17 showed positive PNA FISH tests, and five positive AOLC test results before the diagnosis were established with routine measures. The screening revealed further seven patients with positive universal PNA FISH tests and 10 positive AOLC tests without symptoms indicative for infection and were therefore considered not to have CRBSI. CONCLUSIONS: Sampling of only one CVC lumen/port chamber screening for CRBSI in haematooncological patients seems not to be a useful tool for anticipative diagnosis of CRBSI. Reasons for false-negative results might include origin of CRBSIs from the other CVC lumina not sampled for screening, and false-positive results might origin from catheter colonization without subsequent spread of micro-organisms into the peripheral bloodstream.

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.

Multidrug-Resistant Bacteria in Surgical Intensive Care Units: Antibiotic Susceptibility and ß-Lactamase Characterization.

Multidrug-resistant (MDR) bacteria of the utmost importance are extended-spectrum ß-lactamase (ESBL) and carbapenemase-producing Enterobacterales (CRE), carbapenem-resistant Acinetobacter baumannii (CRAB), carbapenem-resistant Pseudomonas aeruginosa (CRPA), methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus spp. (VRE). In this study, an evaluation of MDR bacteria in surgical intensive care units in a tertiary referral hospital was conducted. The study aimed to characterize ß-lactamases and other resistance traits of Gram-negative bacteria isolated in surgical intensive care units (ICUs). Disk diffusion and the broth dilution method were used for antibiotic susceptibility testing, whereas ESBL screening was performed through a double disk synergy test and an inhibitor-based test with clavulanic acid. A total of 119 MDR bacterial isolates were analysed. ESBL production was observed in half of the Proteus mirabilis, 90% of the Klebsiella pneumoniae and all of the Enterobacter cloacae and Escherichia coli isolates. OXA-48 carbapenemase, carried by the L plasmid, was detected in 34 K. pneumoniae and one E. coli and Enterobacter cloacae complex isolates, whereas NDM occurred sporadically and was identified in three K. pneumoniae isolates. OXA-48 positive isolates coharboured ESBLs belonging to the CTX-M family in all but one isolate. OXA-23 carbapenemase was confirmed in all A. baumannii isolates. The findings of this study provide valuable insight of resistance determinants of Enterobacterales and A. baumannii which will enhance surveillance and intervention strategies that are necessary to curb the ever-growing carbapenem resistance rates.

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.

Emergence and Spread of Enterobacterales with Multiple Carbapenemases after COVID-19 Pandemic.

Resistance to carbapenems in Enterobacterales has become a matter of the highest concern in the last decade. Recently, Enterobacterales harboring multiple carbapenemases were detected in three hospital centers in Croatia and in the outpatient setting, posing a serious therapeutic challenge for clinicians. In this study, we analyzed eight Klebsiella pneumoniae and two Enterobacter cloacae complex isolates with multiple carbapenemases, with regard to antibiotic susceptibility, ß-lactamase production and plasmid content. The isolates demonstrated uniform resistance to amoxicillin/clavulanate, piperacillin/tazobactam, cefuroxime, ceftazidime, cefotaxime, ceftriaxone and ertapenem. Among novel ß-lactam/inhibitor combinations, ceftazidime/avibactam exhibited moderate activity, with 50% of isolates susceptible. All isolates demonstrated resistance to imipenem/cilastatin/relebactam, and all but one to ceftolozane/tazobactam. Four isolates exhibited a multidrug-resistant phenotype (MDR), whereas six were allocated to an extensively drug-resistant phenotype (XDR). OKNV detected three combinations of carbapenemases: OXA-48+NDM (five isolates), OXA-48+VIM (three isolates) and OXA-48+KPC (two isolates). Inter-array testing identified a wide variety of resistance genes for ß-lactam antibiotics: blaCTX-M-15, blaTEM, blaSHV, blaOXA-1, blaOXA-2, blaOXA-9, aminoglycosides: aac6, aad, rmt, arm and aph, fluoroquinolones: qnrA, qnrB and qnrS, sulphonamides: sul1 and sul2 and trimethoprim: dfrA5, dfrA7, dfrA14, dfrA17 and dfrA19. mcr genes were reported for the first time in Croatia. This study demonstrated the ability of K. pneumoniae and E. cloacae to acquire various resistance determinants under the selection pressure of antibiotics widely used during the COVID-19 pandemic. The novel inter-array method showed good correlation with OKNV and PCR, although some discrepancies were found.

Detection and characterisation of extended-spectrum and plasmid-mediated AmpC ß-lactamase produced by Escherichia coli isolates found at poultry farms in Bosnia and Herzegovina.

Extended-spectrum ß-lactamases (ESBLs) hydrolyse extended-spectrum cephalosporins (ESC) and aztreonam. As ESBL-producing organisms have been identified in food producing animals, the aim of our study was to detect and analyse such Escherichia coli isolates from poultry. Antibiotic susceptibility of the isolates was determined with disk-diffusion and broth microdilution methods. ESBLs were detected with the double-disk synergy and inhibitor-based test with clavulanic acid. The transferability of cefotaxime resistance was determined with conjugation experiments, and genes encoding ESBLs, plasmid-mediated AmpC ß-lactamases, and quinolone resistance determinants identified by polymerase chain reaction. The study included 108 faecal samples (cloacal swabs) from 25 different poultry farms in the Zenica-Doboj Canton, Bosnia and Herzegovina. Of these, 75 (69.4 %) were positive for E. coli, of which 27 were resistant to cefotaxime, amoxicillin, cefazoline, and cefriaxone, and susceptible to imipenem, meropenem, ertapenem, and amikacin. All 27 cefotaxime-resistant isolates were positive in double-disk synergy and combined disk tests. Eighteen isolates transferred cefotaxime resistance to E. coli recipient. Twenty-one isolates were positive for the bla CTX-M-1 cluster genes and seven for bla CTX-M-15. Fourteen were positive for the bla TEM genes. The most frequent plasmid incompatibility group was IncFIB, whereas IncFIA and Inc HI1 were present in only a few isolates. Two different sequence types (STs) were identified: ST117 and ST155. The emergence of ESBL-producing E. coli in farm animals presents a public health threat, as they can colonise the intestine and cause infections in humans.

Multiresistant Bacteria Isolated from Intestinal Faeces of Farm Animals in Austria.

In recent years, antibiotic-resistant bacteria with an impact on human health, such as extended spectrum ß-lactamase (ESBL)-containing Enterobacteriaceae, methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococci (VRE), have become more common in food. This is due to the use of antibiotics in animal husbandry, which leads to the promotion of antibiotic resistance and thus also makes food a source of such resistant bacteria. Most studies dealing with this issue usually focus on the animals or processed food products to examine the antibiotic resistant bacteria. This study investigated the intestine as another main habitat besides the skin for multiresistant bacteria. For this purpose, faeces samples were taken directly from the intestines of swine (n = 71) and broiler (n = 100) during the slaughter process and analysed. All samples were from animals fed in Austria and slaughtered in Austrian slaughterhouses for food production. The samples were examined for the presence of ESBL-producing Enterobacteriaceae, MRSA, MRCoNS and VRE. The resistance genes of the isolated bacteria were detected and sequenced by PCR. Phenotypic ESBL-producing Escherichia coli could be isolated in 10% of broiler casings (10 out of 100) and 43.6% of swine casings (31 out of 71). In line with previous studies, the results of this study showed that CTX-M-1 was the dominant ESBL produced by E. coli from swine (n = 25, 83.3%) and SHV-12 from broilers (n = 13, 81.3%). Overall, the frequency of positive samples with multidrug-resistant bacteria was lower than in most comparable studies focusing on meat products.

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.

Proteus mirabilis harboring carbapenemase NDM-5 and ESBL VEB-6 detected in Austria.

We describe a case of carbapenemase-harboring Proteus mirabilis together with detection of NDM-5 in Austria accompanied by other bacterial strains with a wide range of beta-lactamases including OXA-181 and VEB-6. Isolates were obtained from a subphrenic abscess from one patient who was previously treated with broad-spectrum antibiotics in Bangladesh.

Multiresistant Bacteria Isolated from Activated Sludge in Austria.

Wastewater contains different kinds of contaminants, including antibiotics and bacterial isolates with human-generated antibiotic resistances. In industrialized countries most of the wastewater is processed in wastewater treatment plants which do not only include commercial wastewater, but also wastewater from hospitals. Three multiresistant pathogens-extended spectrum ß-lactamase (ESBL)-harbouring Enterobacteriaceae (Gram negative bacilli), methicillin resistant Staphylococcus aureus (MRSA) and vancomycin resistant Enterococci (VRE)-were chosen for screening in a state of the art wastewater treatment plant in Austria. Over an investigation period of six months all three multiresistant pathogens could be isolated from activated sludge. ESBL was the most common resistance mechanism, which was found in different species of Enterobacteriaceae, and in one Aeromonas spp. Sequencing of ESBL genes revealed the dominance of genes encoding members of CTX-M ß-lactamases family and a gene encoding for PER-1 ESBL was detected for the first time in Austria. MRSA and VRE could be isolated sporadically, including one EMRSA-15 isolate. Whereas ESBL is well documented as a surface water contaminant, reports of MRSA and VRE are rare. The results of this study show that these three multiresistant phenotypes were present in activated sludge, as well as species and genes which were not reported before in the region. The ESBL-harbouring Gram negative bacilli were most common.

Low prevalence of colonization with multidrug-resistant gram-negative bacteria in long-term care facilities in Graz, Austria.

BACKGROUND: Residents in long-term care facilities (LTCFs) are increasingly found to be an important reservoir of multidrug-resistant gram-negative (MRGN) bacteria. AIMS: We aimed to determine colonization by MRGN bacteria over 6 months in LTCFs and geriatric wards in Graz, Austria, and to evaluate risk factors for such colonization. METHODS: During August 2015, we conducted a point-prevalence survey at LTCFs and geriatric wards of the Geriatric Health Centers of the City of Graz. Inguinal and perianal swabs were taken from 137 patients and screened for MRGN using standard procedures. Six months after the initial investigation all colonized patients were sampled again and use of antibiotics, hospital admissions, and mortality was registered. Genetic relatedness of MRGN bacteria was evaluated. RESULTS: We detected 12 patients harboring MRGN isolates (prevalence, 8.7%). Overall inguinal colonization was 5.1%. After 6 months, only 2 out of 12 patients were still colonized. Presence of a urinary catheter was associated with a higher risk of MRGN colonization (odds ratio [OR], 17.5; 95% CI, 1.6-192). Chronic wounds and gastrostomy were also risk factors of MRGN colonization (OR, 10.7; 95% CI, 1.6-69.3 and OR, 18.3; 95% CI, 2.4-139.4, respectively). There was no difference in mortality between colonized and noncolonized patients. CONCLUSIONS: Prevalence of colonization with MRGN bacteria was low in patients in LTCFs and geriatric wards in Graz, Austria.

Colonization of long term care facility patients with MDR-Gram-negatives during an Acinetobacter baumannii outbreak.

BACKGROUND: We aimed to determine the prevalence of colonization by multidrug-resistant Gram-negative bacteria including ESBL-producing enterobacteriaceae, carbapenem-resistant enterobacteriaceae, Pseudomonas aeruginosa and Acinetobacter baumannii at two wards caring long term for patients with disorder of consciousness at the Geriatric Health Centers Graz, Austria. During our study we detected two A. baumannii outbreaks. METHODS: In August 2015, we conducted a point-prevalence study. Inguinal and perianal swabs were taken from 38 patients and screened for multidrug-resistant Gram-negative rods using standard procedures. Six months after the initial investigation all patients were sampled again and use of antibiotics during the past 6 months and mortality was registered. Genetic relatedness of bacteria was evaluated by DiversiLab system. RESULTS: Fifty percent of patients were colonized by multidrug-resistant Gram-negative isolates. Five patients harboured ESBL-producing enterobacteriaceae. No carbapenem-resistant enterobacteriaceae were detected. 13/38 patients were colonized by A. baumannii isolates (resistant to ciprofloxacin but susceptible to carbapenems). There was a significant difference in the prevalence of colonization by A. baumannii between ward 2 and ward 1 (60% vs. 5.6%, p < 0.001). Two clusters of A. baumannii isolates were identified including one isolate detected on a chair in a patient's room. CONCLUSIONS: We detected a high prevalence of two multidrug-resistant A. baumannii strains in patients with disorder of consciousness at a LTCF in Graz, Austria. Our findings strongly suggest nosocomial cross-transmission between patients. An active surveillance strategy is warranted to avoid missing newly emerging pathogens.

Increase of genetic diversity and clonal replacement of epidemic methicillin-resistant Staphylococcus aureus strains in South-East Austria.

Spa-typing and microarray techniques were used to study epidemiological changes in methicillin-resistant Staphylococcus aureus (MRSA) in South-East Austria. The population structure of 327 MRSA isolated between 2002 and 2012 was investigated. MRSA was assigned to 58 different spa types and 14 different MLST CC (multilocus sequence type clonal complexes); in particular, between 2007 and 2012, an increasing diversity in MRSA clones could be observed. The most abundant clonal complex was CC5. On the respective SCCmec cassettes, the CC5 isolates differed clearly within this decade and CC5/SCCmecI, the South German MRSA, predominant in 2002, was replaced by CC5/SCCmecII, the Rhine-Hesse MRSA in 2012. Whereas in many European countries MLST CC22-MRSA (EMRSA 15, the Barnim epidemic MRSA) is predominant, this clone occurred in Austria nearly 10 years later than in neighbouring countries. CC45, the Berlin EMRSA, epidemic in Germany, was only sporadically found in South-East Austria. The Irish ST8-MRSA-II represented by spa-type t190 was frequently found in 2002 and 2007, but disappeared in 2012. Our results demonstrate clonal replacement of MRSA clones within the last years in Austria. Ongoing surveillance is warranted for detection of changes within the MRSA population.

Molecular characterization of class b carbapenemases in advanced stage of dissemination and emergence of class d carbapenemases in Enterobacteriaceae from Croatia.

Carbapenemases involved in acquired carbapenem resistance in Enterobacteriaceae belong to Ambler class A serin ß-lactamases, class B metallo-ß-lactamases (MBL) or class D OXA-48-like ß-lactamases. The aim of the present study was to analyse the molecular epidemiology and the mechanisms and routes of spread of class B and class D carbapenemases in Croatia. In total 68 isolates were analyzed. Antibiotic susceptibility was determined by broth microdilution method. PCR was used to detect antibiotic-resistance genes. Genotyping was performed by rep-PCR and MLST. Sixty-five isolates were found to harbour VIM-1 carbapenemase, seven of which were positive also for NDM-1, while two strains harboured only NDM-1. OXA-48 was detected in three isolates, two of which coproduced VIM-1. Thirty-six strains possessed additional CTX-M-15 ß-lactamase whereas 64 were positive for TEM-1. CMY was found in 18 Citrobacter freundii isolates and DHA-1 in one Enterobacter cloacae isolate. Four different plasmid-incompatibility groups were found: A/C, L/M, N and FIIAs. Unlike C. freundii and E. cloacae, Klebsiella pneumoniae showed high diversity of rep-PCR patterns. E. cloacae and C. freundii predominantly belonged to one large clone which was allocated to ST105 and ST24, respectively. Three different types of carbapenemases were identified showing the complexity of CRE in Croatia.

Emergence of multidrug-resistant Proteus mirabilis in a long-term care facility in Croatia.

BACKGROUND: An increased frequency of Proteus mirabilis isolates resistant to expanded-spectrum cephalosporins was observed recently in a long-term care facility in Zagreb (Godan). The aim of this study was the molecular characterization of resistance mechanisms to new cephalosporins in P. mirabilis isolates from this nursing home. METHODS: Thirty-eight isolates collected from 2013-2015 showing reduced susceptibility to ceftazidime were investigated. Antibiotic susceptibilities were determined by broth microdilution method. Inhibitor-based tests were performed to detect extended-spectrum (ESBLs) and AmpC ß-lactamases. AmpC ß-lactamases were characterized by polymerase chain reaction (PCR) followed by sequencing of bla ampC genes. Quinolone resistance determinants (qnr genes) were characterized by PCR. Genotyping of the isolates was performed by repetitive element sequence (rep)-PCR and pulsed-field gel electrophoresis (PFGE). RESULTS: Presence of an AmpC ß-lactamase was confirmed in all isolates by combined-disk test with phenylboronic acid. All isolates were resistant to amoxicillin alone and combined with clavulanate, cefotaxime, ceftriaxone, cefoxitin, and ciprofloxacin; but susceptible to cefepime, imipenem, and meropenem. PCR followed by sequencing using primers targeting bla ampc genes revealed CMY-16 ß-lactamase in all but one strain. Bla cmy-16 was carried by a non-conjugative plasmid which did not belong to any known plasmid-based replicon typing (PBRT) group. Rep-PCR identified one large clone consisting of 15 isolates, three pairs or related isolates, one triplet, and four singletons. PFGE confirmed the clonality of the isolates. CONCLUSIONS: This is the first report of multidrug resistant P. mirabilis in a nursing home in Croatia. Cephalosporin resistance was due to plasmid-mediated AmpC ß-lactamase CMY-16.