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.

Evolution of Beta-Lactamases in Urinary Klebsiella pneumoniae Isolates from Croatia; from Extended-Spectrum Beta-Lactamases to Carbapenemases and Colistin Resistance.

K. pneumoniae isolates often harbor various antibiotic resistance determinants including extended-spectrum ß-lactamases (ESBLs), plasmid-mediated AmpC ß-lactamases (p-Amp-C) and carbapenemases. In this study we analyzed 65 K. pneumoniae isolates obtained from urinary tract infections in the outpatients setting, with regard to antibiotic susceptibility, ß-lactamase production, virulence traits and plasmid content.Antibiotic susceptibility was determined by broth microdilution method. PCR was applied to detect genes encoding ESBLs, p-Amp-C and carbapenemases and plasmid incompatibility groups. Phenotypic methods were applied to characterize virulence determinants. Increasing resistance trend was observed for amoxicillin/clavulanate, imipenem, meropenem and ciprofloxacin. The study showed that ESBLs belonging to the CTX-M family, conferring high level of resistance to expanded-spectrum cephalosporins (ESC) were the dominant resistance trait among early isolates (2013 to 2016) whereas OXA-48 carbapenemase, belonging to class D, emerged in significant numbers after 2017. OXA-48 producing organisms coharbored ESBLs. KPC-2 was dominant among isolates from Dubrovnik in the recent years. Colistin resistance was reported in three isolates. Inc L/M was the dominant plasmid in the later period, encoding OXA-48. Hyperviscosity was linked to KPC positivity and emerged in the later period. This report describes evolution of antibiotic resistance in K. pneumoniae from ESBLs to carbapenemases and colistin resistance. The study demonstrated the ability of K. pneumoniae to acquire various resistance determinants, over time. The striking diversity of the UTI isolates could result from introduction of the isolates from the hospitals, transfer of plasmids and multidirectional evolution.

Characterization of Burkholderia cepacia complex from environment influenced by human waste.

The natural environment is a primary source of infections caused by members of Burkholderia cepacia complex (BCC), but the release of human waste may in return enrich the natural environment with clinically relevant BCC. Seven BCC isolates from environment influenced by human liquid or solid waste across Croatia, and one clinical isolate was characterised. B. multivorans recovered from the soil at illegal dumpsite belonged to sequence type (ST)19; B. ambifaria from the agricultural soil fertilized with swine or poultry manure to ST927 or new ST; B. cenocepacia from creek sediment, river water and wound swab to new STs. Antimicrobial susceptibility of isolates ranged from sensitive to multidrug-resistant. A variety of blaTEM genes was confirmed in isolates. Isolates expressed the virulence factors and survived in river water during 50 days. The BCC present natural environments influenced by the human waste are of clinical relevance and a potential source of sporadic infections.

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.

Oral Cavity Colonization with Multidrug-Resistant Gram-Negative Bacteria after Preoperative Prophylactic Use of Antibiotics as a Risk Factor for Ventilator-Associated Pneumonia.

BACKGROUND: Although it was previously shown that prolonged prophylactic antibiotic exposure and multiple inadequate antibiotic therapies are independent risk factors for multidrug-resistant ventilator associated pneumonia there were no studies investigating whether pre-operative prophylactic dose of antibiotics changes oral microbiome and increases the risk of ventilator associated pneumonia. The aim of the study was to determine if pre-operative prophylactic dose of antibiotics affects the oral microbiome, increases the colonization with Gram-negative bacteria and subsequent risk of ventilator associated pneumonia. SUBJECTS AND METHODS: Mechanically ventilated adult patients receiving surgical antibiotic prophylaxis were included in the study. The presence of Gram negative microorganisms in the pre-prophylactic and post-prophylactic oral swabs and tracheal aspirates, as well as the occurrence of ventilator associated pneumonia, were analyzed. RESULTS: Number of patients colonized with Gram negative bacteria in post- prophylactic oral swab was significantly higher compared to oral swab taken before prophylactic antibiotic. On the other hand, the number of patients with Gram- negative bacteria in tracheal aspirates remained similar as in post- prophylactic oral swabs. Moreover, we found that presence of Gram- negative bacteria in both pre- and post- prophylactic oral swabs was in the positive correlation with the presence of Gram- negative bacteria in tracheal aspirates. CONCLUSIONS: This study showed increased colonization of oral cavity with Gram- negative bacteria after preoperative prophylactic antibiotics. Furthermore, receiving two prophylactic antibiotics from WHO Watch list increased the incidence of Gram- negative bacteria in oral swabs and tracheal aspirates, and the risk of ventilator associated pneumonia development.

Comparison of clinical and sewage isolates of Acinetobacter baumannii from two long-term care facilities in Zagreb; mechanisms and routes of spread.

In the previous studies OXA-23-like and OXA-24-like ß-lactamase were reported among Acinetobacter baumannii in both hospitals and long-term care facilities (LTCF) in Croatia. The aim of this study was to analyze clinical and sewage A. baumannii isolates from two nursing homes in Zagreb, with regard to antibiotic susceptibility and resistance mechanisms, to determine the route of spread of carbapenem-resistant isolates. Nine clinical isolates were collected from February to May 2017 whereas in April 2017, ten A. baumannii isolates were collected from sewage of two nursing homes in Zagreb. Antibiotics susceptibility was determined by broth microdilution method. The presence of carbapenemase and extended spectrum ß-lactamases (ESBL) encoding genes was explored by PCR. Conjugation and transformation experiments were performed as previously described. Genotyping was performed by SG determination, PFGE and MLST. Seven clinical isolates were positive for blaOXA24-like whereas two clinical and environmental carbapenem-resistant isolates, respectively, were found to possess blaOXA-23-like genes. Attempts to transfer imipenem resistance were unsuccessful indicating chromosomal location of blaOXA-23 gene. All carbapenem-resistant isolates belonged to SG- 1 (IC-2) whereas the rest of the isolates susceptible to carbapenems were allocated to SG- 2 (IC-1). PFGE analysis revealed low degree of genetic variability within both IC- I and IC- II. MLST corroborated that two environmental OXA-23 isolates belong to the ST-195. This study showed dissemination of OXA-23 producing A. baumannii from the nursing home into the urban sewage. Disinfection of nursing home sewage should be recommended in order to prevent the spread of resistance genes into the community sewage.

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.

FALSE POSITIVE PHENOTYPIC DETECTION OF METALLO-BETA-LACTAMASES IN ACINETOBACTER BAUMANNII.

Phenotypic detection of metallo-ß-lactamases (MBLs) in Acinetobacter (A.) baumannii is a serious challenge to clinical microbiologists. MBLs are inhibited by metal chelators such as ethylenediaminetetraacetic acid) (EDTA). Production of MBLs cannot be recognized based on resistance phenotype. Therefore, phenotypic tests using EDTA are recommended. The aim of this study was to investigate the sensitivity and specificity of inhibitor based tests (EDTA) for detection of MBL. A total of 172 A. baumannii strains (123 carbapenemase positive and 49 carbapenemase negative) were analyzed. Phenotypic detection of MBLs was performed by the combined disk test with EDTA (CDT-EDTA) and EPI-dilution test (EPI-DT). Both tests were positive in all 11 isolates possessing VIM-1 MBL, showing 100% sensitivity. However, false positive results were observed in strains with class D carbapenemases using both tests, i.e. all OXA-23 and OXA-24/40 producing organisms and most OXA-58 positive strains (77% with CDT-EDTA vs. 65% with EPI-DT). False positive results can occur because oxacillinases are converted to a less active state in the presence of EDTA, leading to augmentation of the inhibition zone around the carbapenem disk or reduction of carbapenem minimum inhibitory concentrations. This study showed high sensitivity but low specificity of phenotypic methods in the detection of MBLs.

Antimicrobial susceptibility and characterization of metallo-ß-lactamases, extended-spectrum ß-lactamases, and carbapenemases of Bacillus cereus isolates.

The susceptibility of 66 clinical and environmental B. cereus isolates were tested to selected antimicrobials by a broth microdilution method. All strains were resistant to ß-lactams and susceptible to gentamicin and imipenem. Sixty-five (98.5%) isolates were susceptible to meropenem and ciprofloxacin and 74.2% to azithromycin. Significant differences in MIC values between environmental and clinical isolates were not demonstrated (p > 0.05). According to the disc diffusion method, 80.3%-98.5% of the strains were resistant to one or more of four cephalosporins. The presence of genes for B. cereus ß-lactamases BCI, BCII, BCIII, extended-spectrum ß-lactamases from the CTX and TEM family and the carbapenemases belonging to IMP and VIM family was studied. BlaII genes were expressed in all isolates; the PCR products for blaIII were also detected in two strains, but none of them was positive for blaI. The amplicon of the family blaCTX-M, mostly M-1 and M-15, was confirmed among 68.2% of the isolates, while were blaVIM-like genes determined in 21.2% of the samples.

In vitro synergy and postantibiotic effect of colistin combinations with meropenem and vancomycin against Enterobacteriaceae with multiple carbapenem resistance mechanisms.

AIM: The aim of the study was to determine in vitro synergy and postantibiotic effect of colistin alone and combined with meropenem or vancomycin against Enterobacteriaceae producing multiple carbapenemases; combinations of two metallo-ß-lactamases (MBL) or MBL with OXA-48. Colistin-resistant strain positive for OXA-48 was also included in the study. METHODS: The antibiotic susceptibility was tested by broth microdilution method. Synergy was tested by chequerboard, time-kill and 2-well method. PAE was determined by viable counting. RESULTS: The chequerboard analysis revealed synergy for colistin combination with meropenem in all isolates with FICI values ranging from 0.12 to 0.24. FICI values for combinations with vancomycin were below 0.5 indicating synergy in two out of four isolates. K. pneumoniae 609815 positive for OXA-48 and colistin resistant showed the most pronounced and consistent synergy effect with meropenem in both chequerboard and time-kill method. Synergy effect in time-kill curves, was observed for K pneumoniae 145846 with two MBLs and colistin resistant K. pneumoniae 609815 positive for OXA-48, with both combinations including meropenem and vancomycin. Colistin alone exhibited short postantibiotic effect (PAE) against all tested isolates. Meropenem markedly prolonged the PAE in two isolates in contrast to vancomycin which did not demonstrate significant effect on the duration of PAE. CONCLUSIONS: The synergy effect and the duration of PAE was strain and antibiotic dependent but not related to the resistance gene content.

Activity of fosfomycin against nosocomial multiresistant bacterial pathogens from Croatia: a multicentric study.

AIM: To determine in vitro susceptibility of multiresistant bacterial isolates to fosfomycin. METHODS: In this prospective in vitro study (local non-random sample, level of evidence 3), 288 consecutively collected multiresistant bacterial isolates from seven medical centers in Croatia were tested from February 2014 until October 2016 for susceptibility to fosfomycin and other antibiotics according to Clinical and Laboratory Standards Institute methodology. Susceptibility to fosfomycin was determined by agar dilution method, while disc diffusion was performed for in vitro testing of other antibiotics. Polymerase chain reaction and sequencing were performed for the majority of extended spectrum ß-lactamase (ESBL)-producing Klebsiella pneumoniae (K. pneumoniae) and carbapenem-resistant isolates. RESULTS: The majority of 288 multiresistant bacterial isolates (82.6%) were susceptible to fosfomycin. The 236 multiresistant Gram-negative isolates showed excellent susceptibility to fosfomycin. Susceptibility rates were as follows: Escherichia coli ESBL 97%, K. pneumoniae ESBL 80%, Enterobacter species 85.7%, Citrobacter freundii 100%, Proteus mirabilis 93%, and Pseudomonas aeruginosa 60%. Of the 52 multiresistant Gram-positive isolates, methicillin-resistant Staphylococcus aureus showed excellent susceptibility to fosfomycin (94.4%) and vancomycin-resistant enterococcus showed low susceptibility to fosfomycin (31%). Polymerase chain reaction analysis of 36/50 ESBL-producing K. pneumoniae isolates showed that majority of isolates had CTX-M-15 beta lactamase (27/36) preceded by ISEcp insertion sequence. All carbapenem-resistant Enterobacter and Citrobacter isolates had blaVIM-1 metallo-beta-lactamase gene. CONCLUSION: With the best in vitro activity among the tested antibiotics, fosfomycin could be an effective treatment option for infections caused by multiresistant Gram-negative and Gram-positive bacterial strains in the hospital setting.

COMPARISON OF TWO DIFFERENT METHODS FOR TIGECYCLINE SUSCEPTIBILITY TESTING IN ACINETOBACTER BAUMANNII.

- Tigecycline susceptibility testing (TST) presents a tremendous challenge for clinical microbiologists. Previous studies have shown that the Epsilometer test (E-test) and Vitek 2 automated system significantly overestimate the minimum inhibitory concentrations for tigecycline resistance compared to the broth microdilution method (BMM). This leads to very major errors or false susceptibility (i.e. the isolate is called susceptible when it is actually resistant). The aim of this study was to compare E-test against BMM for TST in carbapenem-resistant and carbapenem-susceptible Acinetobacter (A.) baumannii and to analyze changes in tigecycline susceptibility between two time periods (2009-2012 and 2013-2014), with BMM as the gold standard. Using the EUCAST criteria, the rate of resistance to tigecycline for the OXA-23 MBL-positive, OXA-23 MBL-negative and carbapenemase-negative strains for BMM was 54.5% (6/11), 29.4% (5/17) and 2.7% (1/37), respectively; the OXA-24/40 and OXA-58 producing organisms did not exhibit any resistance. With E-test, all OXA-23 MBL-positive organisms (11/11), 23.5% (4/17) of OXA-23 MBL-negative, and 4.1% of OXA-24/40 (3/74) strains displayed tigecycline resistance; there were no resistant strains among the OXA-58 and carbapenemase-negative isolates. Resistance emerged in the bacterial isolates from 2013 to 2014. Although tigecycline does not display cross-resistance, the highest rates of resistant A. baumannii isolates were observed among those producing VIM MBL, regardless of the testing method. These findings suggest that the commercial E-test does not provide reliable results for TST of A. baumannii. Further confirmation with the dilution method should be recommended, particularly in cases of serious infections.

Klebsiella Pneumoniaeoxa-48 in a Urology Patient: Case Report

We present an isolate of Klebsiella pneumoniae OXA-48 isolated in a 68-year-old man who underwent radical prostatectomy due to prostate cancer. The antibiotic susceptibility testing to a wide range of antibiotics was performed by disk diffusion method and determination of minimal inhibitory concentrations. The isolate was classified as multidrug-resistant. It showed intermediate susceptibility to imipenem and meropenem, resistance to ertapenem, and sensitivity only to colistin, amikacin, and trimethoprim-sulfamethoxazole. The isolate was positive for ESBLs, negative for AmpC. Polymerase chain reaction and sequencing revealed bla(OXA-48)', bla(CTX-M-15) and bla(SHV-11). The plasmid encoding OXA-48 ß-lactamase did not belong to any known PCR-based replicon typing. According to genotyping, the isolate belonged to ST37.

Antimicrobial Sensitivity Profile of Chlamydia trachomatis isolates from Croatia in McCoy Cell Culture System and Comparison with the Literature.

BACKGROUND: Chlamydia trachomatis (C. trachomatis) is the most common bacterial agent of sexually transmitted infections around the world, but susceptibility testing of this pathogen is rarely pursued due to its intracellular niche. The principal aims of this research were to determine in vitro sensitivity profile of urogenital chlamydial strains isolated from Croatian patients and to compare obtained concentration values of different antimicrobial drugs mutually and with the literature. METHODS: Forty strains of C. trachomatis isolated during 2010-2012 at the National Reference Laboratory for Chlamydia and two reference strains were subjected to susceptibility testing in 96-well microtiter plates containing McCoy cell monolayers. Minimal inhibitory concentration (MIC) and minimal chlamydicidal concentration (MCC) were determined for azithromycin, doxycycline, and levofloxacin. Briefly, strains were inoculated on McCoy cells, followed by addition of serially diluted antimicrobial drugs. Upon incubation, growth of C. trachomatis was detected using fluorescein-conjugated antibody to the lipopolysaccharide genus antigen under the inverted fluorescent microscope. RESULTS: All chlamydial strains were susceptible to the antibiotics tested (MIC < 4 pg/mL), thus the pattern of homotypic or heterotypic resistance has not been found. MCC values were equal or 1-5 dilutions higher than MIC values. Statistically significant differences in the effectiveness of antimicrobial agents in vitro have been proven. Significant correlation has been found for MCCs in the case of two antimicrobial pairs: azithromycin and levofloxacin, and doxycycline and levofloxacin. Comparison of medians for different clinical samples did not reveal any significant difference. CONCLUSIONS: Although resistant strains have not been found in this study, several literature reports of unsuccessfully treated genitourinary infections caused by C. trachomatis require our alertness for possible discovery of resistant strains. Considering the overall antibiotic burden worldwide, pursuing this kind of research is crucial in order to detect possible decreased susceptibility (or even resistance) of chlamydial strains, despite the laborious and time-consuming methodology.

[EVOLUTION OF BETA-LACTAM ANTIBIOTIC RESISTANCE IN ENTEROBACTER SPP. IN CROATIA].

Enterobacter spp. develops resistance to expanded-spectrum cephalosporins by induction or derepression of chromosomal AmpC ß-lactamase, or production of extended-spectrum ß-lactamases (ESBLs) or carbapenemases. The aim of the study was to analyze the mechanisms of resistance to expanded-spectrum cephalosporins and the evolution of resistance mechanism during the study period (2008­2011) on a collection of 58 randomly collected Enterobacter spp. strains from three hospital centers in Croatia and Bosnia and Herzegovina during 2008-2010. The antibiotic susceptibility was determined by broth microdilution method according to CLSI. Resistance genes were determined by PCR. Plasmids were characterized by PCR-based replicon typing (PBRT). The hypothesis of the study was that there will be multiple mechanisms of ceftazidime resistance involved, from inducible and derepressed AmpC ß-lactamases to extended-spectrum ß-lactamases and carbapenemases at the end of the study. The isolates from different centers were expected to express different phenotypes and mechanisms of resistance. The study showed the predominance of derepressed AmpC ß-lactamases combined with ESBLs belonging to CTX-M family as a mechanism of resistance to expanded-spectrum cephalosporins. The emergency of MBLs was reported in the last year of the study in University Hospital Center Zagreb. The plasmids encoding ESBLs belonged to different incompatibility groups. This points out to the evolution of ß-lactam resistance in Enterobacter spp. from derepressed AmpC ß-lactamases and ESBL to carbapenemases.

High prevalence of CTX-M-15 and first report of CTX-M-3, CTX-M-22, CTX-M-28 and plasmid-mediated AmpC beta-lactamase producing Enterobacteriaceae causing urinary tract infections in Bosnia and Herzegovina in hospital and community settings.

AIM: To investigate molecular epidemiology of extended-spectrum ß-lactamase/ESBL and plasmid-mediated AmpC ß-lactamase/pAmpC producing Gram-negative bacteria causing urinary tract infections (UTIs) in Zenica-Doboj Canton, Bosnia and Herzegovina, in the period Decembar 2009-May 2010. METHODS: Antibiotic susceptibility was determined by disc diffusion and broth microdilution according to CLSI guidelines. Double-disk synergy test was performed in order to screen for ESBLs/pAmpC beta-lactamases. PCR was used to detect bla(ESBL)/bla(ampC)/bla(carb) genes. Genetic relatedness of the strains was determined by pulsed-field-gel-electrophoresis (PFGE). RESULTS: Among 85 patients with UTIs caused by ESBL producing isolates, 44 (51.8%) were from in-patients and 41 (48.2%) from outpatients. Klebsiella spp. was the most frequently isolated from in-patients, in 28 (63.6%) cases. Among outpatients, Klebsiella spp./Escherichia coli were the most frequently isolated, in 19 (46.3%)/16 (39.0%) cases. Twenty-one (75.0%) from hospital and nine (47.4%) from outpatient Klebsiella spp. isolates were positive for blaTEM, whereas 27 (96.4%) from in-patients and 6 (31.6%) from outpatient were bla(CTX-M) positive (18 hospital and five outpatient isolates were encoding bla(CTX-M-15)). One Klebsiella oxytoca and one Enterobacter cloacae inpatient isolates were positive for blaCTX-M-28. One Klebsiella pneumoniae outpatient isolate were positive for bla(CTX-M-22) and one E. coli for bla(CTX-M-3). One hospital Proteus mirabilis strain was positive for bla(CMY-2) and two Klebsiella spp. strains for blaDHA-1, whereas two E. coli, one K. oxytoca and one Proteus vulgaris outpatient strains were positive for bla(CMY-2). CONCLUSION: Identification of bla(CTX-M-3), bla(CTX-M-22) and bla(CTX-M-28) among Enterobacteriaceae is uncommon. In this study we report the emergency of CMY-2 and DHA-1 plasmid-mediated beta-lactamases.

Nationwide Survey of Klebsiella Pneumoniae Strains Producing CTX-M Extended-spectrum ß-lactamases in Croatia.

Extended-spectrum ß-lactamases (ESBL) producing bacteria have been increasingly reported in both hospital and community patients. Production of ESBLs is the major mechanism of resistance to oxymino-cephalosporins and aztreonam in Gram-negative bacteria. Recently a new family of ESBLs with predominant activity against cefotaxime (CTX-M ß-lactamases) has been reported. Over 80 CTX-M enzymes have been described so far, which can be grouped into five main subgroups according to amino acid sequence identity (CTX-M-1, CTX-M-2, CTX-M-8, CTX-M-9 and CTX-M-25). In some countries, CTX-M ß-lactamases are the most prevalent types of ESBLs, for instance in Russia, Greece, Spain, Switzerland, Japan, Taiwan, China and Argentina. These enzymes have been identified in countries near Croatia such is Italy, Hungary and Austria. The aim of this study was to determine the prevalence and the types of CTX-M ß lactamases produced by Klebsiella pneumoniae clinical isolates collected from October 2006 to January 2007 from both community- and hospital-based isolates were included (Figure 1.). 128 ESBL isolates were subjected to further analysis: screening with double disc diffusion test and confirmed by ESBL E test.

[First report of carbapenemases in Osijek-Baranja County in imported Enterobacter cloacae isolate in vitro susceptible to carbapenems]. / Prvi opis karbapenemaze u osjecko-baranjskoj zupaniji u unesenom izolatu Enterobactera cloacae in vitro osjetljivom na karbapeneme.

Carbapenems are often the only therapeutic option to treat infections caused by multidrug-resistant Gram-negative bacteria. Emergence of carbapenemases in the isolates of Enterobacteriaceae limits therapeutic options. Carbapenem-resistant Enterobacteriaceae have emerged since 2008 throughout Croatia. In Osijek-Baranja County carbapenem-resistant strains of Enterobacteriaceae were not reported until 2013. The first carbapenem-resistant strain (Enterobacter cloacae) was identified in August 2013 in a patient previously hospitalized at University Hospital Center Zagreb for the treatment of acute lymphoblastic leukemia. Molecular analysis revealed the production of VIM-1 metallo-beta-lactamase (MBL). In spite of the metallo-beta-lactamase production the strain was not resistant to imipenem and meropenem in disk-diffusion and microdilution test. This report shows that routine susceptibility testing carried out in most laboratories does not necessarily detect carbapenemase production in Enterobacteriaceae. Since these enzymes are encoded on mobile genetic elements there is a risk of horizontal spread to other enterobacterial isolates and the development of hospital outbreaks.

First Report of NDM-1-Producing Acinetobacter guillouiae.

BACKGROUND: Acinetobacter spp. is an opportunistic pathogen that has demonstrated increasing relevance in nosocomial infections. Carbapenem-resistant strains have been reported worldwide. METHODS: Since 2014, screening for metallo-ß-lactamases (MBLs) in all Acinetobacter spp. isolates using phenotypic methods and PCR has been implemented at the University Hospital Center Zagreb. RESULTS: The bacterial strain was isolated from the drain of a child hospitalized in a paediatric intensive care unit and identified as Acinetobacter guillouiae using a MALDI TOF automated system. The strain was resistant to meropenem, ceftazidime, cefotaxime, ceftriaxone, cefepime, sulbactam/ampicillin, gentamicin and ciprofloxacin, intermediately susceptible to piperacillin/tazobactam and imipenem, and susceptible to amikacin and colistin. The Hodge test and combined disk test with EDTA were positive. The MICs of meropenem and imipenem were not reduced by cloxacillin, but a small reduction of two dilutions was observed following the addition of sodium chloride, which indicated that OXA-58 was produced. PCR and sequencing of chromosomal DNA from boiled colonies revealed blaOXA-58 and blaNDM-1 genes. CONCLUSION: This is the first report of NDM-1 in Acinetobacter spp. in Croatia. The early detection of these genes will aid in the prevention and in the achievement of adequate infection control by limiting the spread of these organisms.

[Carbapenemases of gram-negative bacteria]. / Karbapenemaze gram-negativnih bakterija.

Carbapenems are often antibiotics of last resort for the treatment of severe infections. They are stable to most beta-lactamases produced by gram-negative bacteria. However, bacterial enzymes named carbapenemases can efficiently hydrolyze carbapenems. They are produced most frequently by Enterobacteriaceae and non-fermentative bacteria such as Pseudomonas aeruginosa and Acinetobacter baumannnii. They belong to group A (KPC, SME, IMI, NMC), B (VIM, IMP, SPM, GIM, NDM, SIM, DIM, AIM) and D (OXA-23, OXA-24, OXA-48, OXA-58, OXA-143). The accurate and rapid laboratory identification of carbapenem-resistant isolates is important to prevent spread of such multidrug resistant strains and to avoid therapeutic failures. Therapeutic options are often limited because carbapenemases are encoded on mobile genetic elements which often harbour resistance genes to other groups of antibiotics. Thus, colistin is often the only therapeutic option.