First report on PVL-positive methicillin-resistant Staphylococcus aureus of SCCmec type V, spa type T441 in Croatia.

The aim of the study was to investigate the molecular epidemiology of community- associated MRSA in Primorsko-Goranska County of Croatia during a six-year period(2001-2007). In period from 2001 and 2007, 46 MRSA isolates were collected in Rijeka, strains were subjected to susceptibility testing according to CLSI guidelines, mecA gene detection and SCCmec typing as well as detection of PVL. Strains were typed by Pulse Field Gel Electrophoresis (PFGE) and spa typing. All isolates were susceptible to vancomycin, linezolid, mupirocin, nitrofurantoin, only one strain was resistant to fusidic acid and co-trimoxazole. Results of SCCmec typing showed the presence of SCCmec type IV in 26 MRSA strains, SCCmec type V in three strains, and 13 strains comprised SCCmec I. SCCmec type II and III were not observed. Four MRSA strains were non-typeable by applied SCCmec typing methods. PVL was detected in 4 strains, two SCCmec IV and two SCCmec V. PFGE analysis, grouped MRSA strains into six similarity groups and 18 singletons. Dominating spa types in this collection of strains were t015, with 15 strains, followed by t041(N=7), t051,(N=2 ), t2850(N=2), t008(N=2)and single isolates t441, t002, t448, t018, t019, t355, t390, t026, t449, t148. We also detected two new spa types, t3510 and t3509, respectively. This is the first report on SCCmec type V in Croatia, and, to our knowledge, first report of PVL-positive mehicillin-resistant Staphylococcus aureus SCCmec type V and t441(ST59-MRSA-V) in this part of Europe.

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.

Prevalence and molecular characteristics of methicillin-resistant Staphylococcus aureus strains isolated in a multicenter study of nursing home residents in Croatia.

BACKGROUND: Residents of nursing homes (NHs) are often hospitalized and could present a potential reservoir for methicillin-resistant Staphylococcus aureus (MRSA). The aim of the study was to determine the prevalence for MRSA carriage in residents and staff in Croatian NHs and to characterize MRSA strains using genotyping techniques. METHODS: A cross-sectional study was performed among 877 residents and staff of 7 NHs representing 3 major Croatian regions. Nasal swabs from residents and staff and other samples from residents with invasive devices were obtained. Identified isolates were submitted to susceptibility testing and genotyping with SCCmec typing, S aureus protein A (spa) locus typing, and pulsed-field gel electrophoresis (PFGE). RESULTS: The overall prevalence of MRSA colonization was 7.1% (95 confidence interval, 5.4%-8.8%), ranging from 0% to 28.8%. Four MRSA isolates were found in NH staff. All MRSA isolates were negative for Panton-Valentine leukocidin-encoding genes. SCCmec type II was found in 32 MRSA strains; SCCmec IV, in 27 strains; SCCmec I, in 3 strains. The predominant spa type was t008, found in 49 strains; PFGE analysis revealed 2 major clonal groups. CONCLUSIONS: MRSA strains were found to be colonizing residents and staff of 7 NHs in Croatia. Our study demonstrates the spread of 2 clones within and among Croatian NHs. The data presented here provide an important baseline for future surveillance of MRSA in NH.

Antibiotic susceptibility of isolates from paediatric intensive care units in Zagreb.

AIM: Meropenem Yearly Susceptibility Test Information Collection (MYSTIC) Program is a longitudinal global surveillance study to monitor in vitro data on microbial susceptibility in centers that prescribe meropenem. Results of the six years period (2002-2007) for the antimicrobial efficacy of meropenem compared to other broad-spectrum agents against Gram-negative and Gram-positive isolates collected at pediatric intensive care units of the University Hospital Center Zagreb in Croatia were reported. METHODS: A total of 110 Gram-negative and 43 Gram-positive pathogens from pediatric specimens were tested. The minimum-inhibitory concentrations (MICs) were determined by broth microdilution method according to CLSI. RESULTS: There was no resistance to either imipenem or meropenem observed for Escherichia coli, Klebsiella pneumoniae and Proteus mirabilis. High resistance rates of K. pneumoniae to ceftazidime and gentamicin (50%) are a raising concern. Pseudomonas aeruginosa was the most resistant Gram-negative species with two (12%) of the strains resistant to meropenem, three (18%) to imipenem, 10 (47%) to gentamicin and six (35%) to piperacillin/tazobactam and ciprofloxacin. According to our results meropenem remains an appropriate antibiotic for the treatment of severe infections caused by Gram-negative bacteria in pediatric population. CONCLUSIONS: The results indicate that meropenem has excellent potency and spectrum of activity despite being prescribed for a long time for the treatment of seriously ill patients, and still appears to be a reliable option for the initial empirical therapy of serious nosocomial infections in children. However, later studies have shown the emergence of carbapenem-resistant Gram-negative bacteria after 2008.

Influenza and hepatitis B vaccination coverage among healthcare workers in Croatian hospitals: a series of cross-sectional surveys, 2006-2011.

BACKGROUND: Healthcare workers (HCWs) are at an increased risk of exposure to and transmission of infectious diseases. Vaccination lowers morbidity and mortality of HCWs and their patients. To assess vaccination coverage for influenza and hepatitis B virus (HBV) among HCWs in Croatian hospitals, we conducted yearly nationwide surveys. METHODS: From 2006 to 2011, all 66 Croatian public hospitals, representing 43-60% of all the HCWs in Croatia, were included. Statistical analysis was performed using the Kruskal-Wallis analysis of variance, Dunn's multiple comparison analysis and the chi-square test, as appropriate. RESULTS: The median seasonal influenza vaccination coverage rates in pre-pandemic (2006-2008) seasons were 36%, 25% and 29%, respectively. By occupation, influenza vaccination rates among physicians were 33 ± 21%, 33 ± 22% among graduate nurses, 30 ± 34% among other HCWs, 26 ± 21% among housekeeping and the lowest, 23 ± 17%, among practical nurses (p < 0.01). In 2009-2010 season, seasonal influenza vaccination coverage was 30%, while overall vaccination coverage against pandemic influenza was fewer than 5%. Median vaccination coverage in the post-pandemic seasons of 2010-2011 and 2011-2012 decreased to 15% and 14%, respectively (reduction of 24% and 35%, respectively, p < 0.0001). Meanwhile, the median mandatory HBV vaccination coverage was 98%, albeit with considerable differences according to work setting (range 19-100%) and occupation (range 4-100%). CONCLUSIONS: We found substantial year-on-year variations in seasonal influenza vaccination rates, with reduction in post pandemic influenza seasons. HBV vaccination is satisfactory compared to seasonal influenza vaccination coverage, although substantial variations by occupation and work setting were observed. These findings highlight the need for national strategies that optimize vaccination coverage among HCWs in Croatian hospitals. Further studies are needed to establish the potential role of mandatory vaccination for seasonal influenza.

Efficacy of ozone on microorganisms in the tooth root canal.

The aim of this study was to examine the effect of ozone gas on the remaining bacteria after chemomechanical instrumentation of tooth root canal. The study was carried out at the Department of Endodontics and Restorative dentistry, School of Dental Medicine, University of Zagreb. A total of 37 tooth root canals from 23 teeth (10 incisors, 2 canines, 8 premolars and 3 molars) with a diagnosis of chronic apical periodontitis (17 untreated teeth and 6 retreatments) from 20 adult patients (11 females and 9 male) were selected. Endodontic samples consisted of 74 swabs from 37 canals. The first root canal swab was taken following a completed chemomechanical instrumentation by a sterile paper point after rinsing the root canal with a sterile saline solution. The canal was dried and treated with ozone gas for 40 seconds (HealOzone, Kavo, Germany). After the ozone treatment the canal was rinsed with a sterile saline solution a second swab was taken. The swabs were stored in transport media until cultivation. Microbiological identification was performed by macromorphological, micromorphological, commercial biochemical test microbiological analysis and bacteria count. A significant decrease in the number of bacteria (p < 0.001) was found after the ozone treatment: the total number of bacteria was 82%, 67% of aerobic and 93% of anaerobic bacteria. When analysing individually, a significant decrease was found for Streptococcus mitis and Propionibacterium acnes (p < 0.05). The results of this study shows the efficacy of ozone on the bacterial count reduction in the root canal treatment.

Surveillance of surgical site infection after cholecystectomy using the hospital in Europe link for infection control through surveillance protocol.

BACKGROUND: The third most common healthcare-associated infection is surgical site infection (SSI), accounting for 14%-16% of infections. These SSIs are associated with high morbidity, numerous deaths, and greater cost. METHODS: A prospective study was conducted to assess the incidence of SSI in a single university hospital in Croatia. We used the Hospital in Europe Link for Infection Control through Surveillance (HELICS) protocol for surveillance. The SSIs were classified using the standard definition of the National Nosocomial Infections Surveillance (NNIS) system. RESULTS: The overall incidence of SSI was 1.44%. The incidence of infection in the open cholecystectomy group was 6.06%, whereas in the laparoscopic group, it was only 0.60%. The incidence density of in-hospital SSIs per 1,000 post-operative days was 5.76. Patients who underwent a laparoscopic cholecystectomy were significantly younger (53.65±14.65 vs. 64.42±14.17 years; p<0.001), spent roughly one-third as many days in the hospital (2.40±1.72 vs. 8.13±4.78; p<0.001), and had significantly shorter operations by nearly 26 min (60.34±28.34 vs. 85.80±37.17 min; p<0.001). Procedures that started as laparoscopic cholecystectomies and were converted to open procedures (n=28) were reviewed separately. The incidence of SSI in this group was 17.9%. The majority of converted procedures (71.4%) were elective, and the operating time was significantly longer than in other two groups (109.64±85.36 min). CONCLUSION: The HELICS protocol has a good concept for the monitoring of SSI, but in the case of cholecystectomy, additional factors such as antibiotic appropriateness, gallbladder entry, empyema of the gallbladder, and obstructive jaundice must be considered.

The screening of methicillin-resistant staphylococcus aureus in vascular surgery patients: a comparison of molecular testing and broth-enriched culture.

BACKGROUND: Methicillin-resistant Staphylococcus aureus (MRSA) is a major global health care-associated pathogen. This study sought to examine the prevalence of MRSA in patients who were admitted to a vascular surgery ward during a 3-month period. METHODS: MRSA screening was accomplished through the acquisition of nasal, throat and perineal swabs. These swabs were placed in tryptic soy broth that had been supplemented with 6.5% NaCl and incubated for 24 h. The resulting isolates were subcultured on agar plates containing 5% sheep blood. The BD GeneOhm MRSA assay for screening swabs was performed in accordance with the manufacturer's instructions. RESULTS: A total of 58 patients were included in the study and swabs from 232 sites were obtained during the sampling period. MRSA was detected in 33 samples of 12 patients during the study period; thus, there was a 20.6% prevalence of patients who were recognized as MRSA carriers. There were discrepancies between the results of classical bacteriological screening and molecular MRSA detection methods in 8 of the patients. CONCLUSIONS: Nasal, throat and perineal MRSA screening can detect the carriage of this pathogen and allow for the timely use of appropriate infection control measures. The choice of screening techniques poses a challenge; it has been demonstrated that molecular detection methods should be performed with great sensitivity, specificity and, most importantly, speed. The cost of the PCR screening method is the only disadvantage of this approach.

Value of rapid aetiological diagnosis in optimization of antimicrobial treatment in bacterial community acquired pneumonia.

In 80 adult patients with community acquired pneumonia (CAP) conventional microbiological methods, polymerase chain reaction (PCR) and serum C-reactive protein (CRP) levels were performed and the appropriateness of the empirical antimicrobial treatment was evaluated according to bacterial pathogen detected. The aetiology was determined in 42 (52.5%) patients, with Streptococcus pneumoniae as the most common pathogen. PCR applied to bronchoalveolar lavage (BAL) provided 2 and PCR on sputum samples 1 additional aetiological diagnosis of CAP The mean CRP values in the S. pneumoniae group were not significantly higher than in the group with other aetiological diagnoses (166.89 mg/L vs. 160.11 mg/L, p = 0.457). In 23.8% (10/42) of patients with determined aetiology, the empirical antimicrobial treatment was inappropriate. PCR tests need further investigation, particularly those for the atypical pathogens, as they are predominant in inappropriately treated patients. Our results do not support the use of CRP as a rapid test to guide the antimicrobial treatment in patients with CAP.

[Comparative urinary bactericidal activity of oral antibiotics against gram-positive pathogens]. / Usporedna urinarna baktericidna aktivnost oralnih antibiotika prema gram-pozitivnim urinarnim patogenima.

In routine bacteriological laboratories the antibacterial activity of antibiotics is determined by in vitro testing, usually by disk-diffusion test. However, in vitro testing does not always reflect antibacterial efficiency of antibiotics in vivo. In this investigation, the urine samples obtained in a single oral dose pharmacokinetic study were examined for their bactericidal activity against a range of relevant Gram-positive urinary tract pathogens. Urinary bactericidal activity of linezolid had been previously compared with ciprofloxacin but not with other oral antibiotics such as beta-lactams. Linezolid showed satisfactory urinary bactericidal titres throughout the whole testing period against all Gram-positive cocci. Fluoroquinolones displayed high and persisting levels of urinary bactericidal activity against staphylococci, but their activity against enterococci was weaker. According to the results of ex-vivo testing amoxycillin could be recommended only for infections caused by E. faecalis. Amoxycillin combined with clavulanic acid can be considered as a therapeutic option for infections caused by S. saprophyticus and E. faecalis. Older cephalosporins had high titres only against S. saprophyticus. Their drawback is a short elimination half-time in urine resulting in rapid decrease of urinary bactericidal titers during dosing interval. Furthermore, they do not show activity against enterococci due to their intrinsic resistance to cephalosporins.

[Guidelines on hand hygiene in health care institutions]. / Smjernice za higijenu ruku u zdravstvenim ustanovama.

Healthcare associated infections (HCAI) are huge problem all over the world, and 5-10% of all hospitalized patients will develop infection during hospitalization. From the times of I. P. Semelweiss we know that clean hands are the most important single factor that can decrease the number of HCAI. World Health Organization (WHO) has recognised this problem and developed Guidelines for hand hygiene in healthcare institutions. This also was the reason of developing Croatian national Guidelines. The main goal of the Guidelines was to decrease number of HCAI associated with the hands of healthcare workers. These Guidelines are meant for all healthcare workers and other hospital staff who come to the direct contact with patients. An interdisciplinary team of experts developed these Guidelines using WHO Guidelines, other existing guidelines and literature reviews for hand hygiene. Grades of evidence for specific recommendations were determined using CDC/HICPAC grading system. Categorization is based on existing data, theoretical basis, applicability and economic impact. After a broad discussion in different professional societies, Guidelines were accepted. Guidelines include recommendations for hand hygiene indications, hand hygiene technique, surgical hand preparation, choosing hand hygiene preparations, skin care, nails, glove use, patients and visitors hand hygiene, role of education, as well as role of healthcare institution and role of government. Furthermore, in the Guidelines the concept of "Five moments for hand hygiene" is explained in detail, and main literature data are presented.

Distributed lags time series analysis versus linear correlation analysis (Pearson's r) in identifying the relationship between antipseudomonal antibiotic consumption and the susceptibility of Pseudomonas aeruginosa isolates in a single Intensive Care Unit of a tertiary hospital.

The relationship between antibiotic consumption and selection of resistant strains has been studied mainly by employing conventional statistical methods. A time delay in effect must be anticipated and this has rarely been taken into account in previous studies. Therefore, distributed lags time series analysis and simple linear correlation were compared in their ability to evaluate this relationship. Data on monthly antibiotic consumption for ciprofloxacin, piperacillin/tazobactam, carbapenems and cefepime as well as Pseudomonas aeruginosa susceptibility were retrospectively collected for the period April 2006 to July 2007. Using distributed lags analysis, a significant temporal relationship was identified between ciprofloxacin, meropenem and cefepime consumption and the resistance rates of P. aeruginosa isolates to these antibiotics. This effect was lagged for ciprofloxacin and cefepime [1 month (R=0.827, P=0.039) and 2 months (R=0.962, P=0.001), respectively] and was simultaneous for meropenem (lag 0, R=0.876, P=0.002). Furthermore, a significant concomitant effect of meropenem consumption on the appearance of multidrug-resistant P. aeruginosa strains (resistant to three or more representatives of classes of antibiotics) was identified (lag 0, R=0.992, P<0.001). This effect was not delayed and it was therefore identified both by distributed lags analysis and the Pearson's correlation coefficient. Correlation coefficient analysis was not able to identify relationships between antibiotic consumption and bacterial resistance when the effect was delayed. These results indicate that the use of diverse statistical methods can yield significantly different results, thus leading to the introduction of possibly inappropriate infection control measures.

Cotton textiles modified with citric acid as efficient anti-bacterial agent for prevention of nosocomial infections.

AIM: To study the antimicrobial activity of citric acid (CA) and sodium hypophosphite monohydrate (SHP) against gram-positive and gram-negative bacteria, and to determine the influence of conventional and microwave thermal treatments, on the effectiveness of antimicrobial treatment of cotton textiles. METHOD: Textile material was impregnated with CA and SHP solution and thermally treated by either conventional or microwave drying/curing treatment. Antibacterial effectiveness was tested according to ISO 20743:2009 standard, using absorption method. The surfaces were morphologically observed by scanning electron microscopy, while physical characteristics were determined by wrinkle recovery angles method (DIN 53 891), tensile strength (DIN 53 837), and whiteness degree method (AATCC 110-2000). RESULTS: Cotton fabric treated with CA and SHP showed significant antibacterial activity against MRSA (6.38 log10 treated by conventional drying and 6.46 log10 treated by microwave drying before washing, and 6.90 log10 and 7.86 log10, respectively, after 1 cycle of home domestic laundering washing [HDLW]). Antibacterial activity was also remarkable against S. aureus (4.25 log10 by conventional drying, 4.58 log10 by microwave drying) and against P. aeruginosa (1.93 log10 by conventional drying and 4.66 log10 by microwave drying). Antibacterial activity against P. aeruginosa was higher in samples subjected to microwave than in conventional drying. Antibacterial activity was reduced after 10 HDLW cycles but the compound was still effective. The surface of the untreated cotton polymer was smooth, while minor erosion stripes appeared on the surfaces treated with antimicrobial agent, and long and deep stripes were found on the surface of the washed sample. CONCLUSION: CA can be used both for the disposable (non-durable) materials (gowns, masks, and cuffs for blood pressure measurement) and the materials that require durability to laundering. The current protocols and initiatives in infection control could be improved by the use of antimicrobial agents applied on cotton carbohydrate polymer.

[Antibacterial effect of the materials for the root canal filling, bases and material for the cavity fillings]. / Antibakterijski ucinak materijala za punjenje korijenskih kanala, podloge i ispune kaviteta.

AIM: To assess antimicrobial effects of the materials used in the endodontic treatment of the teeth. METHODS: The following root-canal fillings were examined: Ketac Endo, AH Plus, Diaket, and gutta-percha, by means of the agar diffusion test. As for the base materials, Zink Oxide/Eugenol cement, glass-ionomere cements Fuji II LC Improved, Ketac Cem, and phosphate cement and Harvard cement were investigated. Finally, of the materials for final cavity filling, amalgam, Ketac Molar and Fuji II LC Improved were tested. In the present research, the following bacteria were applied: Streptococcus mutans, Streptotoccus mitis, Lactobacillus species, Staphylococcus aureus, Enterococcusfaecalis, and polymicrobial suspention. For each individual bacterium tested, material samples were placed on the inoculated plates of blood-agar. RESULTS: Antibacterial effects were confirmed in the following materials: root-canal fillings--Diaket, AH Plus, Ketac Endo; bases--Zink Oxide/Eugenol cement, phosphat cement and Harvard cement. No antibacterial effect was established in: amalgam, gutta-percha, Fuji II LC Improved, Ketac Cem and Ketac Molar. Diaket showed a statistically more significant antibacterial effect in comparison with AH Plus, Ketac Endo, and gutta-percha (P(s.mitis) < 0.05; P(s.mutans) < 0.05; p(lactobacillus) < 0.05; p(enterococcus) < 0.05; P(staphilococcus) < 0.05; p(polymicrobial) < 0.05); of the base materials, however, a somewhat stronger antimicrobial effect was found in phosphate cement, Harvard cement, and Zink Oxide/Eugenol (ZnOE) (p < 0.05) as compared with Fuji II LC Improved and Ketac Cem. CONCLUSION: According to the results of this study, and considering the fact that all cavity-filling materials failed to show any antimicrobial effect at all, when choosing the materials for root-canal fillings, and for bases, advantage should be given to those with the manifested strongest antibacterial effect, namely Diaket and phosphate cement.

Clinical impact of antimicrobial resistance in European hospitals: excess mortality and length of hospital stay related to methicillin-resistant Staphylococcus aureus bloodstream infections.

Antimicrobial resistance is threatening the successful management of nosocomial infections worldwide. Despite the therapeutic limitations imposed by methicillin-resistant Staphylococcus aureus (MRSA), its clinical impact is still debated. The objective of this study was to estimate the excess mortality and length of hospital stay (LOS) associated with MRSA bloodstream infections (BSI) in European hospitals. Between July 2007 and June 2008, a multicenter, prospective, parallel matched-cohort study was carried out in 13 tertiary care hospitals in as many European countries. Cohort I consisted of patients with MRSA BSI and cohort II of patients with methicillin-susceptible S. aureus (MSSA) BSI. The patients in both cohorts were matched for LOS prior to the onset of BSI with patients free of the respective BSI. Cohort I consisted of 248 MRSA patients and 453 controls and cohort II of 618 MSSA patients and 1,170 controls. Compared to the controls, MRSA patients had higher 30-day mortality (adjusted odds ratio [aOR] = 4.4) and higher hospital mortality (adjusted hazard ratio [aHR] = 3.5). Their excess LOS was 9.2 days. MSSA patients also had higher 30-day (aOR = 2.4) and hospital (aHR = 3.1) mortality and an excess LOS of 8.6 days. When the outcomes from the two cohorts were compared, an effect attributable to methicillin resistance was found for 30-day mortality (OR = 1.8; P = 0.04), but not for hospital mortality (HR = 1.1; P = 0.63) or LOS (difference = 0.6 days; P = 0.96). Irrespective of methicillin susceptibility, S. aureus BSI has a significant impact on morbidity and mortality. In addition, MRSA BSI leads to a fatal outcome more frequently than MSSA BSI. Infection control efforts in hospitals should aim to contain infections caused by both resistant and susceptible S. aureus.

[Diagnostic approach and therapy for cytomegalovirus (CMV) infection following allogeneic stem cell transplantation]. / Dijagnosticki pristup, pracenje i lijecenje citomegalovirusne (CMV) infekcije nakon alogene transplantacije krvotvornih maticnih stanica.

In spite of improvements in diagnostics and prevention of CMV disease in recent decades, CMV infection still remains major concern in terms of diagnosis and therapy in recipients of allogeneic stem cells. Besides considerable morbidity with direct effects of CMV infection (hepatitis, gastrointestinal disease, pneumonia, retinitis), there are also indirect effects such as increased susceptibility to opportunistic infections and an increased risk of graft rejection and transplant-related mortality. Also, myelosuppression, nephrotoxicity and emergence of drug-resistant CMV strains may limit the use of antiviral agents for the control of CMV infection. The aim of this paper is to show the problems associated with CMV infection in recipients of allogeneic stem cells with special emphasis on diagnostic procedures and treatment or prophylaxis of CMV disease.

Clonal spread of CTX-M-15-producing Klebsiella pneumoniae in a Croatian hospital.

This study was conducted to detect and analyse the presence of extended-spectrum beta-lactamase (ESBL)-producing Klebsiella pneumoniae associated with a nosocomial outbreak at a Croatian hospital. During 2007, 162 K. pneumoniae isolates with reduced susceptibility to third-generation cephalosporins were collected from various hospital units and patient specimens. Most of the strains were isolated from urine (61 %), followed by blood cultures (13 %), wound swabs (13 %), tracheal aspirates (5 %), intra-abdominal abscess aspirates (4 %), intravascular catheters (3 %) and cerebrospinal fluid (1 %). Medical wards were the most important source of the isolates (46 %); 21 % of the isolates originated from surgical intensive-care units. All patients had infections acquired during their stay in hospital. No community-acquired infections were reported. Sixty of these isolates were chosen for further analysis. A double-disc synergy test (DDST) was used to detect ESBLs. MICs were determined by the broth microdilution method according to CLSI guidelines. The transferability of ceftazidime resistance was tested by conjugation (broth mating method). PCR was used to detect alleles encoding ESBL enzymes. Plasmids encoding ESBLs were extracted with the Macherey Nagel Mini kit according to the manufacturer's recommendations. The genotypes of the strains were compared by analysis of banding patterns generated by PFGE of XbaI-digested genomic DNA. ESBLs were found by DDST in all isolates. All strains were resistant to cefuroxime, ceftazidime, cefotaxime, ceftriaxone, aztreonam, piperacillin/tazobactam and ciprofloxacin. There was variable susceptibility/resistance to cefepime and gentamicin. No resistance to ceftazidime/clavulanate and carbapenems was observed. Only six strains transferred resistance to an Escherichia coli recipient strain, with low frequency. All isolates yielded an amplicon of 545 bp with consensus MA primers. Multiplex PCR was positive for group 1 CTX-M beta-lactamases. Sequencing of selected amplicons revealed the presence of bla(CTX-M-15), with coding regions containing identical nucleotide sequences. Similarly to isolates from India, our isolates contained the ISEcpI insertion sequence located upstream of the bla(CTX-M-15) gene, which has recently been demonstrated to mobilize 3'-adjacent genes to transfer between DNA replicons. The isolates contained a large plasmid of approximately 150 kb. The isolates were assigned to five clusters (>85 % similarity), which contained subclusters. The results of this work provided insights into the molecular epidemiology of the spread of ESBLs in K. pneumoniae involved in an outbreak at a Croatian hospital. The hospital antibiotic policy resulted in ceftriaxone being the most heavily prescribed third-generation cephalosporin, which might be expected to select for cefotaximases such as CTX-M-15.

Emergence of Proteus mirabilis isolates producing TEM-52 extended-spectrum beta-lactamases in Croatia.

BACKGROUND: An increased frequency of extended-spectrum beta-lactamase (ESBL)-positive Proteus mirabilis isolates was observed recently in the Clinical Hospital Center Split in Croatia. The aim of this study was the molecular characterization of ESBLs in P. mirabilis isolates from this hospital. MATERIAL AND METHODS: Seven strains showing reduced susceptibility to ceftazidime were investigated. Antimicrobial susceptibility was determined using the broth microdilution method. ESBLs were characterized by PCR and sequencing of bla(ESBL) genes. Quinolone resistance determinants (qnr genes) were characterized by PCR. Genotyping of strains was performed by pulsed-field gel electrophoresis (PFGE). RESULTS: The presence of an ESBL was confirmed in all strains by a double-disk synergy test. All strains were resistant to amoxicillin, piperacillin, gentamicin, ciprofloxacin, chloramphenicol, sulfamethoxazole and trimethoprim, but susceptible to ceftazidime/clavulanic acid, piperacillin/tazobactam, cefoxitin, imipenem and meropenem; PCR sequencing using primers targeting bla(ESBL) genes revealed TEM-52 beta-lactamase. PFGE genotyping demonstrated the clonal relatedness of TEM-52-producing P. mirabilis strains isolated from different clinical samples and wards within the hospital. Bla(TEM-52) in 3 isolates was carried by a 70-kb conjugative plasmid. CONCLUSIONS: Our findings indicate the emergence of the TEM-52 enzyme among P. mirabilis in Croatia.

Emergence of CTX-M group 1 extended-spectrum beta-lactamase-producing Klebsiella pneumoniae strains in the community.

AIM: Molecular characterization of ESBL-producing K. pneumoniae strains isolated from urine of outpatients in Zagreb region during the last five years. METHODS: During the five-year study period a total of 2, 651 K. pneumoniae strains were isolated from urine of nonhospitalized patients with significant bacteriuria. ESBL production was detected by double-disk diffusion technique and by > or = 3-dilution reduction in the minimal inhibitory concentration of ceftazidime in the presence of clavulanate. A total of 441 ESBL-producing K. pneumoniae strains (15.5%) were collected and 17 strains were further characterised. Double-disk synergy test was used to detect ESBLs. Minimum inhibitory concentrations (MICs) were determined by broth microdilution method according to CLSI. The transferability of cefotaxime resistance was tested by conjugation (broth mating method). PCR was used to detect alleles encoding ESBL enzymes. The genotypes of the strains were compared by pulsed-field gel electrophoresis (PFGE) of Xba I-digested genomic DNA. RESULTS: A significant difference in frequencies of ESBL isolates was observed. In the first year of study only 4.9% of isolated strains were ESBL producers, while in the second year 17.% ESBL-positive strains were detected (p < 0.01), and the frequency remained stabile within following years. All strains yielded an amplicon with primers specific for SHV beta-lactamases and CTX-M beta-lactamases. Based on sequencing of bla(CTX-M) genes enzymes of nine strains were identified as CTX-M 15 beta -lactamase and three as CTX-M-14. Isolates were not clonally related. CONCLUSION: The study demonstrated community-associated emergence of CTX-M 1 beta-lactamase-producing K. pneumoniae strains.