Zrinka Bošnjak

NDM-1-producing Klebsiella pneumoniae, Croatia.

To the Editor: The novel metallo-β-lactamase named New Delhi metallo-β-lactamase (NDM-1) was identified from Klebsiella pneumoniae and Escherichia coli isolates in Sweden from a patient previously hospitalized in India (1). NDM-1 is spreading rapidly worldwide to nonclonally related isolates, many of which are directly or indirectly tracked to the Indian subcontinent (2). A carbapenem-resistant K. pneumoniae strain, KLZA, was isolated in May 2009 from the culture of a blood sample from of a 40-year-old man on the day after his admission to a surgical intensive care unit of the Clinical Hospital Center in Zagreb, Croatia. The patient had been transferred after 5 days of hospitalization in Bosnia and Herzegovina following a car accident. The clinical history mentioned antimicrobial drug treatment that did not include carbapenems (gentamicin, metronidazole, and ceftriaxone) and no link to the Indian subcontinent. Antimicrobial drug susceptibility testing was performed by Vitek2 (bioMerieux, Marcy-l’Etoile, France) and broth microdilution and interpreted according to the latest documents from the European Committee on Antimicrobial Susceptibility Testing (www.eucast.org/clinical_breakpoints/, version 1.1). The strain proved resistant to imipenem and meropenem, to all broad-spectrum cephalosporins, and to aminoglycosides and susceptible to ciprofloxacin and tigecycline (Table). We checked for blaVIM, blaIMP, blaSPM, blaGIM, blaSIM, and blaNDM resistance genes by using PCR. A PCR product was obtained only with the NDM primers, after being purified (QIAquick PCR Purification Kit, QIAGEN, Hilden, Germany), its sequence showed 100% identity with blaNDM-1. Table MIC of the KLZA strain of Klebsiella pneumoniae and its transconjugant and recipient Strain genotyping was performed by multilocus sequence typing to determine the sequence type (ST) of the isolate and to establish a comparison with previously reported NDM-1–producing isolates. Allelic numbers were obtained on the basis of sequences of 7 housekeeping genes at www.pasteur.fr/recherche/genopole/PF8/mlst/Kpneumoniae.html. Multilocus sequence typing identified K. pneumoniae KLZA as an ST25 strain, which significantly differs from the ST14 type found in the index NDM-1–producing strain and from other isolates originating from India (1) and then in other countries. ST25 K. pneumoniae was also found in K. pneumoniae isolates in Geneva (3). Other K. pneumoniae STs harboring NDM-1 were ST15, ST16, and ST147 (4–7). Resistance was transferred by conjugation to E. coli J53, with selection based on growth on agar in the presence of ceftazidime (10 mg/L) and azide (100 mg/L). The conjugant T1 showed resistance to β-lactams, including all carbapenems, as well as decreased susceptibility to ciprofloxacin. The KLZA strain and its transconjugant harbored other determinant of resistance, namely blaCTX-M-15, blaCMY-16, and qnrA6. Plasmid incompatibility groups, determined by a PCR-based replicon typing method, belonged to the incA/C replicon type. This report of an NDM-1–producing K. pneumoniae in Croatia adds to those of other cases in patients from patients hospitalized in the Balkan area. The patient in this report had no apparent link to the Indian subcontinent. In a survey conducted by the European Centre for Disease Prevention and Control to gather information about the spread of NDM-1–producing Enterobacteriaceae in Europe and reporting cases from 13 countries during 2008–2010, five of the 55 persons with known travel histories had traveled to the Balkan region during the month before diagnosis of their infection: 2 to Kosovo and 1 each to Serbia, Montenegro, and Bosnia and Herzegovina. All had received hospital care in Balkan countries because of an illness or accident that occurred during the journey (7). Two of the latter cases (4,8) and a case from Germany (9) were subsequently published. No patient had any apparent link to the Indian subcontinent. Although the way NDM-1 isolates might have been imported to western Europe not only from the Indian subcontinent but also from Balkan countries (10) has been highlighted, awareness of western Europe as a possible area of endemicity remains limited. The aforementioned report from Germany, although recognizing that the patient had been repatriated after hospitalization in Serbia, declared “no evidence about contact with people from regions where NDM-enterobacteria are endemic” (9). This limited awareness shows the threat of neglecting to screen patients who are transferred from countries thought not to be at risk for NDM-1. Furthermore, it means that specimen are not sent to the local reference laboratories and recognized as positive for NDM-1, thus permitting wide dissemination of NDM-1–producing enterobacteria in the community (4). The accumulating evidence of NDM-1 from the Balkan area could suggest a possible multifocal spread of this enzyme, with the Balkans as a possible second area of endemicity, in addition to the Indian subcontinent, and prompts for widespread epidemiologic surveillance.

Infrequent Finding of Metallo-β-Lactamase VIM-2 in Carbapenem-Resistant Pseudomonas aeruginosa Strains from Croatia

ABSTRACT One hundred sixty-nine nonreplicate imipenem-resistant Pseudomonas aeruginosa strains isolated in a large hospital on the coastal region of Croatia were studied. The most active antibiotics were colistin and amikacin. Most of the isolates were multiresistant. The most prevalent serotype was O12, followed by O11. Six strains carried the blaVIM-2 gene located in a novel class 1 integron composed in its variable part of the blaVIM-2-blaoxa-10-ΔqacF-aacA4 genes. Metallo-β-lactamase-producing strains belonged to sequence types ST235 and ST111.

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.

VIM-2 β-lactamase in Pseudomonas aeruginosa isolates from Zagreb, Croatia

Abstract The aim of this investigation was to characterize metallo-β-lactamases (MBLs) in Pseudomonas aeruginosa isolates from Zagreb, Croatia. One hundred P. aeruginosa isolates with reduced susceptibility to either imipenem or meropenem were tested for the production of MBLs by MBL-Etest. The susceptibility to a wide range of antibiotics was determined by broth microdilution method. The presence of blaMBL genes was detected by polymerase chain reaction (PCR). Hydrolysis of 0.1 mM imipenem by crude enzyme preparations of β-lactamases was monitored by UV spectrophotometer. Outer membrane proteins were prepared and analysed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Six out of 100 isolates were positive for MBLs by Etest. All strains were resistant to gentamicin, ceftazidime and cefotaxime, and all except 1 were resistant to imipenem. Six strains positive for MBLs by Etest were identified as VIM MBL-producers by PCR. Sequencing of blaVIM genes revealed the production of VIM-2 β-lactamase in all 6 strains. This investigation proved the occurrence of VIM-2 β-lactamase among P. aeruginosa strains from Zagreb, Croatia. VIM-2 β-lactamase with similar properties has previously been described in another region of Croatia and in Italy, France, Spain, Greece, Taiwan and South Korea, suggesting that this type of enzyme is widespread in the Mediterranean region of Europe and in the Far East.

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

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

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.

Evaluation of Matrix-Assisted Laser Desorption/ Ionization Time-of-Flight Mass Spectrometry in Identification of Nontuberculous Mycobacteria

Background/Aims: Species-level identification of nontuberculous mycobacteria (NTM) is important in making decisions about the necessity and choice of antimicrobial treatment. The reason is predictable clinical significance and the susceptibility profile of specific NTM species. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is recognized as a diagnostic tool for routine identification of bacteria and yeasts in the clinical laboratory based on protein fingerprint analysis. The aim of the study was to evaluate MALDI-TOF MS in the identification of NTM. Methods: A total of 25 NTM isolates from liquid cultures were identified with both polymerase chain reaction (PCR)-based hybridization assay and MALDI-TOF MS at the University Hospital Center Zagreb. Results: PCR-based hybridization assay identified 96% (24/25) and MALDI-TOF MS 80% (20/25) of tested NTM isolates. Five isolates with no reliable MALDI-TOF MS identification belonged to the Mycobacterium avium-intracellulare complex. Seventy percent (14/20) of NTM isolates successfully identified with MALDI-TOF MS had a score higher than 2.0, indicating reliable species identification. Conclusion: MALDI-TOF MS is a promising tool for the identification of NTM. With a further improvement of the protein extraction protocol, especially regarding the M. avium-intracellulare complex, MALDI-TOF MS could be an additional standard method for identification of NTM.

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 Pearsons 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.

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.