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.

A comparative study on antibiotic resistant Escherichia coli isolates from Austrian patients and wastewater-influenced Danube River water and biofilms.

Antimicrobial resistance (AMR) poses a major threat to human health worldwide. AMR can be introduced into natural aquatic ecosystems, for example, from clinical facilities via wastewater emissions. Understanding AMR patterns in environmental populations of bacterial pathogens is important to elucidate propagation routes and develop mitigation strategies. In this study, AMR patterns of Escherichia coli isolates from urinary tract infections and colonised urinary catheters of inpatients and outpatients were compared to isolates from the Danube River within the same catchment in Austria to potentially link environmental with clinical resistance patterns. Susceptibility to 20 antibiotics was tested for 697 patient, 489 water and 440 biofilm isolates. The resistance ratios in patient isolates were significantly higher than in the environmental isolates and higher resistance ratios were found in biofilm in comparison to water isolates. The role of the biofilm as potential sink of resistances was reflected by two extended-spectrum beta-lactamase (ESBL) producing isolates in the biofilm while none were found in water, and by higher amoxicillin/clavulanic acid resistance ratios in biofilm compared to patient isolates. Although, resistances to last-line antibiotics such as carbapenems and tigecycline were found in the patient and in the environmental isolates, they still occurred at low frequency.

Comparison of the Antibiotic Resistance of Escherichia coli Populations from Water and Biofilm in River Environments.

Antibiotic-resistant, facultative pathogenic bacteria are commonly found in surface water; however, the factors influencing the spread and stabilization of antibiotic resistance in this habitat, particularly the role of biofilms, are not fully understood. The extent to which bacterial populations in biofilms or sediments exacerbate the problem for specific antibiotic classes or more broadly remains unanswered. In this study, we investigated the differences between the bacterial populations found in the surface water and sediment/biofilm of the Mur River and the Drava River in Austria. Samples of Escherichia coli were collected from both the water and sediment at two locations per river: upstream and downstream of urban areas that included a sewage treatment plant. The isolates were subjected to antimicrobial susceptibility testing against 21 antibiotics belonging to seven distinct classes. Additionally, isolates exhibiting either extended-spectrum beta-lactamase (ESBL) or carbapenemase phenotypes were further analyzed for specific antimicrobial resistance genes. E. coli isolates collected from all locations exhibited resistance to at least one of the tested antibiotics; on average, isolates from the Mur and Drava rivers showed 25.85% and 23.66% resistance, respectively. The most prevalent resistance observed was to ampicillin, amoxicillin-clavulanic acid, tetracycline, and nalidixic acid. Surprisingly, there was a similar proportion of resistant bacteria observed in both open water and sediment samples. The difference in resistance levels between the samples collected upstream and downstream of the cities was minimal. Out of all 831 isolates examined, 13 were identified as carrying ESBL genes, with 1 of these isolates also containing the gene for the KPC-2 carbapenemase. There were no significant differences between the biofilm (sediment) and open water samples in the occurrence of antibiotic resistance. For the E. coli populations in the examined rivers, the different factors in water and the sediment do not appear to influence the stability of resistance. No significant differences in antimicrobial resistance were observed between the bacterial populations collected from the biofilm (sediment) and open-water samples in either river. The different factors in water and the sediment do not appear to influence the stability of resistance. The minimal differences observed upstream and downstream of the cities could indicate that the river population already exhibits generalized resistance.

Linking antibiotic resistance gene patterns with advanced faecal pollution assessment and environmental key parameters along 2300 km of the Danube River.

The global spread of antimicrobial resistance (AMR) in the environment is a growing health threat. Large rivers are of particular concern as they are highly impacted by wastewater discharge while being vital lifelines serving various human needs. A comprehensive understanding of occurrence, spread and key drivers of AMR along whole river courses is largely lacking. We provide a holistic approach by studying spatiotemporal patterns and hotspots of antibiotic resistance genes (ARGs) along 2311 km of the navigable Danube River, combining a longitudinal and temporal monitoring campaign. The integration of advanced faecal pollution diagnostics and environmental and chemical key parameters allowed linking ARG concentrations to the major pollution sources and explaining the observed patterns. Nine AMR markers, including genes conferring resistance to five different antibiotic classes of clinical and environmental relevance, and one integrase gene were determined by probe-based qPCR. All AMR targets could be quantified in Danube River water, with intI1 and sul1 being ubiquitously abundant, qnrS, tetM, blaTEM with intermediate abundance and blaOXA-48like, blaCTX-M-1 group, blaCTX-M-9 group and blaKPC genes with rare occurrence. Human faecal pollution from municipal wastewater discharges was the dominant factor shaping ARG patterns along the Danube River. Other significant correlations of specific ARGs were observed with discharge, certain metals and pesticides. In contrast, intI1 was not associated with wastewater but was already established in the water microbiome. Animal contamination was detected only sporadically and was correlated with ARGs only in the temporal sampling set. During temporal monitoring, an extraordinary hotspot was identified emphasizing the variability within natural waters. This study provides the first comprehensive baseline concentrations of ARGs in the Danube River and lays the foundation for monitoring future trends and evaluating potential reduction measures. The applided holistic approach proved to be a valuable methodological contribution towards a better understanding of the environmental occurrence of AMR.

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.

Low antimicrobial resistance in Escherichia coli isolates from two large Austrian alpine karstic spring catchments.

The increasing occurrence of antibiotic resistant bacteria poses a threat to global public health. Clinically relevant resistances also spread through the environment. Aquatic ecosystems in particular represent important dispersal pathways. In the past, pristine water resources have not been a study focus, although ingestion of resistant bacteria through water consumption constitutes a potentially important transmission route. This study assessed antibiotic resistances in Escherichia coli populations in two large well-protected and well-managed Austrian karstic spring catchments representing essential groundwater resources for water supply. E. coli were detected seasonally only during the summer period. By screening a representative number of 551 E. coli isolates from 13 sites in two catchments, it could be shown that the prevalence of antibiotic resistance in this study area is low. 3.4 % of the isolates showed resistances to one or two antibiotic classes, 0.5 % were resistant to three antibiotic classes. No resistances to critical and last-line antibiotics were detected. By integrating fecal pollution assessment and microbial source tracking, we could infer that ruminants were the main hosts for antibiotic resistant bacteria in the studied catchment areas. A comparison with other studies on antibiotic resistances in karstic or mountainous springs highlighted the low contamination status of the model catchments studied here, most likely due to the high protection and careful management while other, less pristine catchments showed much higher antibiotic resistances. We demonstrate that studying easily accessible karstic springs allows a holistic view on large catchments concerning the extent and origin of fecal pollution as well as antibiotic resistance. This representative monitoring approach is also in line with the proposed update of the EU Groundwater Directive (GWD).

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.

Multidrug-Resistant Bacteria in a COVID-19 Hospital in Zagreb.

During November to December 2020, a high rate of COVID-19-associated pneumonia with bacterial superinfections due to multidrug-resistant (MDR) pathogens was recorded in a COVID-19 hospital in Zagreb. This study analyzed the causative agents of bacterial superinfections among patients with serious forms of COVID-19. In total, 118 patients were hospitalized in the intensive care unit (ICU) of the COVID-19 hospital. Forty-six out of 118 patients (39%) developed serious bacterial infection (VAP or BSI or both) during their stay in ICU. The total mortality rate was 83/118 (70%). The mortality rate due to bacterial infection or a combination of ARDS with bacterial superinfection was 33% (40/118). Six patients had MDR organisms and 34 had XDR (extensively drug-resistant). The dominant species was Acinetobacter baumannii with all isolates (34) being carbapenem-resistant (CRAB) and positive for carbapenem-hydrolyzing oxacillinases (CHDL). One Escherichia coli causing pneumonia harboured the blaCTX-M-15 gene. It appears that the dominant resistance determinants of causative agents depend on the local epidemiology in the particular COVID center. Acinetobacter baumannii seems to easily spread in overcrowded ICUs. Croatia belongs to the 15 countries in the world with the highest mortality rate among COVID-19 patients, which could be in part attributable to the high prevalence of bacterial infections in local ICUs.

Diffusion of OXA-48 carbapenemase among urinary isolates of Klebsiella pneumoniae in non-hospitalized elderly patients.

BACKGROUND: Recently, a dramatic increase of Klebsiella pneumoniae positive for OXA-48 ß-lactamases was observed first in the hospital setting and later in the long-term care facilities (LTCFs) and community in the Zagreb County, particularly, in urinary isolates. The aim of the study was to analyse the epidemiology and the mechanisms of antibiotic resistance of OXA-48 carbapenemase producing K. pneumoniae strains isolated from urine of non-hospitalized elderly patients. RESULTS: The isolates were classified into two groups: one originated from the LTCFs and the other from the community. Extended-spectrum ß-lactamases (ESBLs) were detected by double disk-synergy (DDST) and combined disk tests in 55% of the isolates (51/92). The ESBL-positive isolates exhibited resistance to expanded-spectrum cephalosporins (ESC) and in majority of cases to gentamicin. LTCFs isolates showed a significantly lower rate of additional ESBLs and consequential resistance to ESC and a lower gentamicin resistance rate compared to the community isolates, similarly to hospital isolates in Zagreb, pointing out to the possible transmission from hospitals.ESBL production was associated with group 1 of CTX-M or SHV-12 ß-lactamases. Ertapenem resistance was transferable from only 12 isolates. blaOXA-48 genes were carried by IncL plasmid in 42 isolates. In addition IncFII and IncFIB were identified in 18 and 2 isolates, respectively. Two new sequence types were reported: ST4870 and ST4781. CONCLUSIONS: This study showed eruptive and extensive diffusion of OXA-48 carbapenemase to LTCFs and community population in Zagreb County, particularly affecting patients with UTIs and urinary catheters. On the basis of susceptibility testing, ß-lactamase production, conjugation experiments, MLST and plasmid characterization it can be concluded that there was horizontal gene transfer between unrelated isolates, responsible for epidemic spread of OXA-48 carbapenemase in the LTCFs and the community The rapid spread of OXA-48 producing K. pneumoniae points out to the shortcomings in the infection control measures.

Analysis of Extended Spectrum Beta Lactamase (ESBL) Genes of Non-Invasive ESBL Enterobacterales in Southeast Austria in 2017.

Extended spectrum beta lactamases producing Enterobacteriaceae are a major player in the antibiotic resistance challenge. In general, the situation regarding antibiotic resistance in Austria is very good compared to many other countries. Perhaps this is why there is a lack of data on the distribution of ESBL genes in the clinical setting. The aim of this study was to collect data on ESBL genes from a larger sample of human non-invasive clinical isolates from one region in Austria. In total, 468 isolates from different sample materials isolated at the Medical University of Graz from 2017 were examined. The most frequent organisms were Escherichia coli and Klebsiella pneumoniae. Among the enzymes produced, CTX-M-15 was clearly dominant, exotic ESBLs were only represented by three Proteus mirabilis isolates harboring genes for VEB-6 and one P. mirabilis for CTX-M-2, respectively. Compared to other countries, the results are in line with the expectations. The data help to better classify the many studies from the non-clinical field in Austria and to shift the focus slightly away from the exotic results and sample sites.

Effects of Ribosomal Protein S10 Flexible Loop Mutations on Tetracycline and Tigecycline Susceptibility of Escherichia coli.

Tigecycline is a tetracycline derivative that is being used as an antibiotic of last resort. Both tigecycline and tetracycline bind to the small (30S) ribosomal subunit and inhibit translation. Target mutations leading to resistance to these antibiotics have been identified both in the 16S ribosomal RNA and in ribosomal proteins S3 and S10 (encoded by the rpsJ gene). Several different mutations in the S10 flexible loop tip residue valine 57 (V57) have been observed in tigecycline-resistant Escherichia coli isolates. However, the role of these mutations in E. coli has not yet been characterized in a defined genetic background. In this study, we chromosomally integrated 10 different rpsJ mutations into E. coli, resulting in different exchanges or a deletion of S10 V57, and investigated the effects of the mutations on growth and tigecycline/tetracycline resistance. While one exchange, V57K, decreased the minimal inhibitory concentration (MIC) (Etest) to tetracycline to 0.75 µg/ml (compared to 2 µg/ml in the parent strain) and hence resulted in hypersensitivity to tetracycline, most exchanges, including the ones reported previously in resistant isolates (V57L, V57D, and V57I) resulted in slightly increased MICs to tigecycline and tetracycline. The strongest increase was observed for the V57L mutant, with a MIC (Etest) to tigecycline of 0.5 µg/ml (compared to 0.125 µg/ml in the parent strain) and a MIC to tetracycline of 4.0 µg/ml. Nevertheless, none of these exchanges increased the MIC to the extent observed in previously described clinical tigecycline-resistant isolates. We conclude that, next to S10 mutations, additional mutations are necessary in order to reach high-level tigecycline resistance in E. coli. In addition, our data reveal that mutants carrying S10 V57 exchanges or deletion display growth defects and, in most cases, also thermosensitivity. The defects are particularly strong in the V57 deletion mutant, which is additionally cold-sensitive. We hypothesize that the S10 loop tip residue is critical for the correct functioning of S10. Both the S10 flexible loop and tigecycline are in contact with helix h31 of the 16S rRNA. We speculate that exchanges or deletion of V57 alter the positioning of h31, thereby influencing both tigecycline binding and S10 function.

Density-dependent microbial calcium carbonate precipitation by drinking water bacteria via amino acid metabolism and biosorption.

Drinking water plumbing systems appear to be a unique environment for microorganisms as they contain few nutrients but a high mineral concentration. Interactions between mineral content and bacteria, such as microbial calcium carbonate precipitation (MCP) however, has not yet attracted too much attention in drinking water sector. This study aims to carefully examine MCP behavior of two drinking water bacteria species, which may potentially link scaling and biofouling processes in drinking water distribution systems. Evidence from cell density evolution, chemical parameters, and microscopy suggest that drinking water isolates can mediate CaCO3 precipitation through previously overlooked MCP mechanisms like ammonification or biosorption. The results also illustrate the active control of bacteria on the MCP process, as the calcium starts to concentrate onto cell surfaces only after reaching a certain cell density, even though the cell surfaces are shown to be the ideal location for the CaCO3 nucleation.

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.

Klebsiella pneumoniae carbapenemase (KPC) in urinary infection isolates.

Recently, emergence of carbapenem-resistance, in particular due to Klebsiella pneumoniae carbapenemase (KPC), was observed among K. pneumoniae causing urinary tract infections in Croatia. The aim of the study was to characterize, antimicrobial susceptibility, carbapenem resistance, virulence traits and plasmid types of the urinary KPC positive isolates of K. pneumoniae. The antimicrobial susceptibility to a wide range of antibiotics was determined by broth microdilution method. The transferability of meropenem resistance was determined by conjugation (broth mating method) employing Escherichia coli J63 strain resistant to sodium azide. Genes encoding broad and extended-spectrum ß-lactamases, plasmid-mediated AmpC ß-lactamases, group A and B carbapenemases, and carbapenem hydrolyzing oxacillinases (blaOXA-48like), respectively, were determined by Polymerase chain reaction (PCR). In total 30 KPC-positive K. pneumoniae urinary isolates collected from different regions of Croatia were analysed. The isolates were uniformly resistant to all tested antibiotics except for variable susceptibility to gentamicin, sulphamethoxazole/trimethoprim, and colistin, respectively. Four isolates were resistant to colistin with MICs values ranging from 4 to 16 mg/L. All tested isolates were susceptible to ceftazidime/avibactam. Sixteen isolates transferred meropenem resistance to E. coli recipient strain by conjugation. Other resistance markers were not co-transferred. PCR was positive for blaKPC and blaSHV genes in all isolates whereas 13 isolates tested positive also for blaTEM genes. PCR based replicon typing (PBRT) revealed the presence of FIIs in 13 and FIA plasmid in two strains. The study showed dissemination of KPC-producing K. pneumoniae in urinary isolates, posing a new epidemiological and treatment challenge. Sulphamethoxazole/trimethoprim, colistin, and ceftazidime/avibactam remain so far, as the therapeutic options.

Pseudomonas Species Diversity Along the Danube River Assessed by rpoD Gene Sequence and MALDI-TOF MS Analyses of Cultivated Strains.

A collection of 611 Pseudomonas isolated from 14 sampling sites along the Danube River were identified previously by MALDI-TOF MS with the VITEK MS system and were grouped in 53 clusters by their main protein profiles. The strains were identified in the present study at the phylospecies level by rpoD gene sequencing. Partial sequences of the rpoD gene of 190 isolates representatives of all clusters were analyzed. Strains in the same MALDI-TOF cluster were grouped in the same phylospecies when they shared a minimum 95% similarity in their rpoD sequences. The sequenced strains were assigned to 34 known species (108 strains) and to 32 possible new species (82 strains). The 611 strains were identified at the phylospecies level combining both methods. Most strains were assigned to phylospecies in the Pseudomonas putida phylogenetic group of species. Special attention was given to 14 multidrug resistant strains that could not be assigned to any known Pseudomonas species and were considered environmental reservoir of antibiotic resistance genes. Coverage indices and rarefaction curves demonstrated that at least 50% of the Pseudomonas species in the Danube River able to grow in the isolation conditions have been identified at the species level. Main objectives were the confirmation of the correlation between the protein profile clusters detected by MALDI-TOF MS and the phylogeny of Pseudomonas strains based on the rpoD gene sequence, the assessment of the higher species discriminative power of the rpoD gene sequence, as well as the estimation of the high diversity of Pseudomonas ssp. along the Danube river. This study highlights the Pseudomonas species diversity in freshwater ecosystems and the usefulness of the combination of MALDI-TOF mass spectrometry for the dereplication of large sets of strains and the rpoD gene sequences for rapid and accurate identifications at the species level.

First Report of Kosakonia radicincitans Bacteraemia from Europe (Austria) - Identification and Whole-Genome Sequencing of Strain DSM 107547.

Kosakonia radicincitans is a species within the new genus Kosakonia. Many strains of this genus have been isolated from plants, but some strains are assumed to act as facultative human pathogens. In this study, an in-depth analysis of a Kosakonia isolate from human blood was performed. The strain was originally isolated from blood and identified as a member of the Enterobacter cloacae complex, exhibiting an atypical result in susceptibility testing. Therefore, the genetic background was examined, including phylogenetic classification and screening for virulence factors. Using whole-genome sequencing, the isolate was identified as a K. radicincitans strain, revealing a virulence gene cluster for yersiniabactin biosynthesis in contrast to all other strains of the species. Whole-genome sequencing was the perfect method for identifying putative virulence factors of a particular Kosakonia strain and will help distinguish beneficial strains from pathogenic strains in the future. To our knowledge, this is the first report of Kosakonia-related bacteraemia from Europe.

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.

Phenotypic Stability of Staphylococcus Aureus Small Colony Variants (SCV) Isolates from Cystic Fibrosis (CF) Patients.

One of the most interesting features of Staphylococcus aureus is its ability to switch to a small colony variant (SCV). This switch allows the pathogen to survive periods of antibiotic treatment or pressure from the immune system of the host and further enables it to start the infection once again after the environmental stress declines. However, so far only little is known about this reversion back to the more virulent wild type phenotype. Therefore, this study aimed to analyze the frequency of reversion to the wild type phenotype of thymidine auxotroph S. aureus SCV isolates (TD-SCVs) obtained from patients with cystic fibrosis (CF). With the use of single cell starting cultures, the occurrence of the thymidine prototroph revertants was monitored. The underlying mutational cause of the SCVs and subsequent revertants were analyzed by sequencing the gene coding for thymidylate synthase (ThyA), whose mutations are known to produce thymidine auxotroph S. aureus SCV. In our study, the underlying mutational cause for the switch to the TD-SCV phenotype was primarily point mutations. Out of twelve isolates, seven isolates showed an occurrence of revertants with a frequency ranging from 90.06% to 0.16%. This high variability in the frequency of reversion to the wild type was not expected. However, this variability in the frequency of reversion may also be the key to successful re-infection of the host. Sometimes quick reversion to the wild type proves necessary for survival, whereas other times, staying hidden for a bit longer leads to success in re-colonization of the host.

Hidden Carbapenem Resistance in OXA-48 and Extended-Spectrum ß-Lactamase-Positive Escherichia coli.

The purpose of this study was to report the identification OXA-48 carbapenemase in seven extended-spectrum ß-lactamase (ESBL)-positive Escherichia coli clinical isolates, fully susceptible to all carbapenems by disk diffusion and E-test methods, but with borderline minimal inhibitory concentration (MIC) values of ertapenem. This report points to the necessity for determination of carbapenem MICs in ESBL-positive E. coli isolates and additional phenotypic testing for carbapenemases in all isolates with borderline ertapenem MIC defined by EUCAST. The isolates showed a high level of resistance to expanded-spectrum cephalosporins because of the production of an additional ESBL belonging to CTX-M family. All isolates and their respective tranconjugants were found to possess L plasmid. Pulsed-field gel electrophoresis analysis revealed two clusters containing highly related isolates. The global spread of multidrug-resistant E. coli should be monitored closely because of the ability of isolates to rapidly obtain additional antibiotic resistance traits such as plasmid-mediated OXA-48 genes.