Jasmina Vraneš

First report of KPC-producing Klebsiella pneumoniae in Croatia

Abstract In February 2011, a 78-year-old male patient was admitted to Clinical Hospital Center Zagreb with subdural haematoma. Klebsiella pneumoniae with reduced susceptibility to carbapenems was isolated. PCR revealed the presence of blaKPC, blaTEM, and blaSHV genes. Sequencing of blaKPC gene identified K. pneumoniae carbapenemase (KPC)-2 beta-lactamase. The strain belonged to ST37 clone by multilocus sequence typing. Infection control efforts limited the spread of KPC-producing clone of K. pneumoniae in our hospital so far. To our knowledge, this is the first report of a KPC-producing K. pneumoniae in Croatia.

Clonal spread of carbapenem-resistant OXA-72-positive Acinetobacter baumannii in a Croatian university hospital

BACKGROUND From July to October 2008, 34 Acinetobacter baumannii isolates were involved in an outbreak at the Clinical Hospital Center, Zagreb. The aim of this study was to characterize the mechanisms of carbapenem resistance in our A. baumannii isolates and determine their epidemiology. METHODS Antibiotic susceptibilities were determined by broth microdilution. PCR was used to detect the presence of carbapenemases. Genotyping of the isolates was performed by random amplification of polymorphic DNA (RAPD), pulsed-field gel electrophoresis (PFGE), and repetitive sequence-based PCR (rep-PCR). RESULTS Thirty-three carbapenem-resistant isolates were positive for the acquired bla(OXA-72) and one unrelated isolate was positive for bla(OXA-58). The bla(OXA-72)-positive isolates were shown to be clonally related by RAPD, rep-PCR, and PFGE. CONCLUSIONS On the basis of susceptibility testing, β-lactamase characterization, and genotyping of the isolates we can conclude that clonal spread of endemic isolates was responsible for the high frequency of OXA-72-positive multidrug-resistant A. baumannii in this setting. Most of the isolates originated from the intensive care unit indicating local dissemination within the hospital and pointing to the potential source of isolates.

Effect of Subinhibitory Concentrations of Ceftazidime, Ciprofloxacin, and Azithromycin on the Hemagglutination and Adherence of Uropathogenic Escherichia coli Strains

The effect of subinhibitory concentrations (sub-MICs) of ceftazidime, ciprofloxacin, and azithromycin on the hemagglutination (HA) and adherence ability of 29 P-fimbriated Escherichia coli strains to the buffalo green monkey kidney (BGMK) cell line was investigated. Comparisons were made between the values of HA titer before and those after exposure of strains to 1/2, 1/4, 1/8, 1/16 and 1/32 MIC of antibiotics, as well as between the number of bacteria attached to the BGMK cells before and the number after their exposure to the same concentrations of antibiotics. Azithromycin at concentrations of 1/2 and 1/4 MIC damaged the HA capacity of the studied strains, while ceftazidime at concentrations of 1/2, 1/4, 1/8 and 1/16 MIC and ciprofloxacin at concentrations of 1/2 and 1/4 MIC increased the HA capacity of P-fimbriated E. coli. All three antibiotics decreased the adhesive capacity of E. coli to the BGMK cells. Comparing the number of adhered bacteria before and after exposure to sub-MICs of antibiotics, statistically significant differences were determined (p < 0.01) after exposure of the strains to all the concentrations of ceftazidime used after exposure to 1/2, 1/4, 1/8 and 1/16 MIC of ciprofloxacin, and after exposure to 1/2, 1/4 and 1/8 MIC of azithromycin. Filaments formed by sub-MICs of ceftazidime and ciprofloxacin in a static experimental system caused HA, but in an experimental system imitating in vivo conditions, the strains adhered poorly to the cells.

Sensitivity and Specificity of Various β-Lactam Antibiotics and Phenotypical Methods for Detection of TEM, SHV and CTX-M Extended-Spectrum β-Lactamases

Abstract The aim of this study was to compare the sensitivity and specificity of six different β-lactam antibiotics using five phenotypical tests for detection of extended spectrum beta-lactamases (ESBLs) based on synergism of β-lactam antibiotics and clavulanate. Experiments were performed on a set of 80 Klebsiella pneumoniae strains and 105 Escherichia coli strains with previously characterized ESBLs (SHV, TEM and CTX-M). ESBLs were detected by five different phenotypical methods: MIC (minimum inhibitory concentration) determination of β-lactam antibiotics with and without clavulanate, double-disk synergy test (DDST), inhibitor-potentiated disk-diffusion test (IPDDT), CLSI-Clinical and Laboratory Standard Institution (former NCCLS) combined- disk-test, and modified MAST-disk-diffusion test (MAST-DD-test). Seven antibiotics were tested as indicators of ESBL production: ceftazidime, cefotaxime, ceftriaxone, aztreonam, ceftibuten, cefpodoxime and cefepime. Ceftazidime and aztreonam were the best indicators for SHV-5, SHV-12 and TEM β-lactamases whereas cefotaxime and ceftriaxone were the most sensitive in detection of SHV-2 and CTX-M β-lactamases in DDST, IPDDT and CLSI test. MIC determination of β-lactam antibiotics with and without clavulanate was the most sensitive method. DDST was the least sensitive test. Double-disk synergy test, which is the most frequently used test for detection of ESBLs in routine laboratories, was the least sensitive independently of the indicator antibiotic. Since MIC determination is a very laborious and time consuming method, we would recommend the NCCLS combined disk test or IPDD test for detection of ESBLs in routine laboratories with 5 mm zone augmentation breakpoint.

Postantibiotic and Post-Beta-Lactamase Inhibitor Effect of Carbapenems Combined with EDTA against Pseudomonas aeruginosa Strains Producing VIM-Metallo Beta-Lactamases

Background and Aim: Postantibiotic effect (PAE) is a delay of bacterial growth after short exposure to antibiotics. The phenomenon of continuing suppression of bacterial growth after removal of β-lactamase inhibitors is termed post-β-lactamase inhibitor effect (PLIE). Recently, Pseudomonas aeruginosa strains producing metallo-β-lactamases were described in many countries of the world. The aim of the study was to investigate the PLIE of carbapenems in combinations with EDTA against VIM-MBL-positive strains of P. aeruginosa. Methods: The experiments were performed on two Pseudomonas aeruginosa isolates, one producing VIM-1 and the other producing VIM-2 metallo-β-lactamase. Minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBC) of imipenem and meropenem alone and combined with EDTA, time-kill curves, PAE and PLIE were performed as described previously. Results: The duration of PAE with meropenem combined with EDTA at 8 × MIC was longer against both VIM-1 and VIM-2 producer than that of imipenem with EDTA on VIM-1- and VIM-2-positive strains. The duration of PLIE was similar on both strains of P. aeruginosa regardless of the sort of carbapenem. At lower concentrations, meropenem with EDTA induced slightly longer PAE and PLIE than imipenem with EDTA. Conclusions: This study has shown that EDTA combined with carbapenems produced a significant PLIE on VIM-MBL-positive P. aeruginosa strains. The results do not have any clinical relevance so far since metal chelators such as EDTA are not used as therapeutic agents due to their toxicity.

Hemagglutination ability and adherence to the Buffalo green monkey kidney cell line of uropathogenic Escherichia coli

The hemagglutination ability and adherence capacity to the Buffalo green monkey (BGM) kidney cell line of 160 wild‐type strains of Escherichia coli isolated from bacteriuric patients were investigated. It was found that P‐fimbriated E. coli strains adhered significantly better to BGM cells than did strains in which P‐fimbriae were not detected, which is in accordance with the capacity of P‐fimbriated strains to cause unobstructive pyelonephritis and with receptor distribution for P‐fimbriae in the urinary tract. The strains which exhibited other adhesions, alone or simultaneously, showed reduced adherence to BGM cells, while non‐agglutinating strains, mostly isolated from urine of patients with asymptomatic bacteriuria, did not adhere at all or adhered poorly to the utilized cell line. The BGM cells served as a good experimental model for investigation of uropathogenic E. coli adherence; because these cells originate from the upper urinary tract, they are viable and not coated with Tamm‐Horsfall protein.

Etiology and clinical characteristics of single and multiple respiratory virus infections diagnosed in Croatian children in two respiratory seasons.

The aim of this study was to determine the causative agent of acute respiratory infection (ARI) in hospitalized children, as well as investigate the characteristics of ARIs with single and multiple virus detection in two respiratory seasons. In 2010 and 2015, nasopharyngeal and pharyngeal swabs from a total of 134 children, admitted to the hospital due to ARI, were tested using multiplex PCR. Viral etiology was established in 81.3% of the patients. Coinfection with two viruses was diagnosed in 27.6% of the patients, and concurrent detection of three or more viruses was diagnosed in 12.8% of the patients. The most commonly diagnosed virus in both seasons combined was respiratory syncytial virus (RSV) (28.6%), followed by parainfluenza viruses (PIVs) types 1–3 (18.4%), rhinovirus (HRV) (14.3%), human metapneumovirus (10.1%), adenovirus (AdV) (7.1%), influenza viruses types A and B (4.8%), and coronaviruses (4.2%). In 2015, additional pathogens were investigated with the following detection rate: enterovirus (13.2%), bocavirus (HBoV) (10.5%), PIV-4 (2.6%), and parechovirus (1.3%). There were no statistical differences between single and multiple virus infection regarding patients age, localization of infection, and severity of disease (P > 0.05). AdV, HRV, HBoV, and PIVs were significantly more often detected in multiple virus infections compared to the other respiratory viruses (P < 0.001).

Clonal spread of Klebsiella pneumoniae producing KPC-2 beta-lactamase in Croatian University Hospital.

In this study we report for the first time clonal spread of KPC-2 producing K. pneumoniae at neurosurgery ward of a Croatian University Hospital. Three patients were involved and colonized with KPC-2 producing strain but without any symptoms of infection. The strains were clonally related and harboured additional blaCTX-M-15 gene. Due to prompt and effective infection control measures the strain did not spread to other hospital units

Postantibiotic effect of colistin alone and combined with vancomycin or meropenem against Acinetobacter spp. with well defined resistance mechanisms

Previous studies found short postantibiotic effect of colistin on Acinetobacter baumannii. Many studies have evaluated the potential for synergy between colistin and other antibiotics against A. baumannii. The aim of this study was to determine in vitro synergy and postantibiotic effect (PAE) of colistin alone and combined with other antibiotics (vancomycin or meropenem) against eight carbapenem-non-susceptible Acinetobacter spp. strains with defined resistance mechanisms. It was hypothesised that vancomycin or meropenem would prologue the PAE of colistin since it was previously found that they exert synergism with colistin in time-kill kinetics and chequerboard analysis. After exposure of 1 hour colistin alone exhibited the negative ( − 0.07 hour) (OXA-143), short (0.2–1.82 hours) (OXA-24, OXA-58, OXA-72, VIM-1+OXA-23, OXA-58+NDM-1, ISAba1/OXA-69) or moderate PAE (3.2 hours) for OXA-23 positive strain. When combined with vancomycin, the PAE was moderate (1.7–4 hours) with OXA-23, OXA-23+VIM-1, OXA-72 and OXA-24 positive strains while with OXA-58, OXA-143, OXA-58/NDM-1 and ISAba1/OXA-69 positive strains, it was not possible to calculate mean duration of PAE because there was no regrowth after exposure to antibiotics or it was longer than 5 hours. The combination with meropenem resulted in short (0.2 hours) (OXA-143), moderate (2.4–3.73 hours) (OXA-24, OXA-58, OXA-23, OXA-23+VIM-1), long PAE of 5 hours (OXA-23) or longer than 5 hours (OXA-58+VIM-1, ISAba1/OXA-69). From the clinical point of view, the prolongation of colistin PAE when combined with other antibiotics could provide a rationale for the modification of the dosing interval and could be important for the optimization of the treatment regimen and the minimization of drug-induced side effects.

Assessing the Need for Routine Screening for Mycoplasma genitalium in the Low‑risk Female Population: A Prevalence and Co‑infection Study on Women from Croatia

Background: There is an ongoing debate regarding possible cost and benefits, but also harm of universal screening for the emerging sexually transmitted pathogen Mycoplasma genitalium. Methods: From the initial pool of 8665 samples that were tested, a subset of Chlamydia trachomatis-positive and randomly selected C. trachomatis-negative cervical swabs were further interrogated for M. genitalium by real-time polymerase chain reaction, using a 224 bp long fragment of the glyceraldehyde-3-phosphate dehydrogenase gene. Results: M. genitalium was detected in 4.8% of C. trachomatis-positive samples and none of C. trachomatis-negative samples. Accordingly, a significant association was shown between M. genitalium and C. trachomatis (P < 0.01), but also between M. genitalium and Mycoplasma hominis infection (P < 0.01). Conclusions: Based on the results, routine screening is recommended only for women with one or more identified risk factors. Moreover, younger age does not represent an appropriate inclusion/exclusion criterion for M. genitalium testing in the low-risk female population.