Anita Novak

Outbreak in Croatia caused by a new carbapenem-resistant clone of Acinetobacter baumannii producing OXA-72 carbapenemase.

Acinetobacter baumannii is a multidrug-resistant opportunistic pathogen that causes nosocomial infections and outbreaks, particularly in the intensive care unit (ICU) setting.1 Many outbreak strains belong to one of three worldwide lineages, known originally as European clones I, II and III. These correspond to sequence groups 2, 1 and 3, respectively, each of which includes a number of different genotypes defined by pulsed-field gel electrophoresis (PFGE).2 Only two previous reports have analysed carbapenem resistance inmultidrug-resistant isolates of A. baumannii from Croatia.3,4 In Split, the first carbapenem-resistant isolate of A. baumannii [meropenem minimum inhibitory concentration (MIC): 16 mg/mL] was isolated in 2002 at Split University Hospital. Between 2002 and 2008, >100 patient isolates were found to belong to the European clone 1 lineage, with most isolates carrying a blaOXA-107 gene associated with ISAba1.3 We now wish to report a new clone of multidrugresistant A. baumannii causing an outbreak in Split University Hospital following inward transfer of a patient. On 5 January 2009, a female aged 51 years was transferred to the intensive care unit (ICU) of Split University Hospital, Croatia, following brain surgery for glioblastoma at the General Hospital Mostar, Bosnia Herzegovina. According to the transfer letter, multidrug-resistant Acinetobacter sp. was isolated from a bronchial aspirate at Mostar General Hospital. No published data are available concerning the incidence, molecular basis and epidemiology of carbapenem-resistant isolates of Acinetobacter in Bosnia Herzegovina. On the day of transfer, blood cultures, cerebrospinal fluid and bronchial lavage were taken. Two days after transfer, carbapenemresistant A. baumannii was isolated from blood culture, and a day later from the bronchial lavage. Initial identification was made using the ATB 32GN and Vitek 2 systems (bioMérieux, Marcy l’Etoile, France), with A. baumannii confirmed by tRNA spacer fingerprinting and the presence of an OXA-51-type b-lactamase (specific to A. baumannii).2 Susceptibility to b-lactams (ceftazidime, cefepime, imipenem, meropenem), b-lactam/b-lactamase inhibitor combinations (ampicillin/sulbactam, piperacillin/tazobactam), aminoglycosides (amikacin, gentamicin) and fluoroquinolones (ciprofloxacin) was determined by disc-diffusion tests, and susceptibility to colistin by E-tests (AB Biodisk, Solna, Sweden). MICs were determined by broth microdilution according to Clinical and Laboratory Standards Institute recommendations. The A. baumannii isolates were resistant to all antimicrobials tested except colistin (MIC: 0.5 mg/mL) and ampicillin/sulbactam (MIC: 1 mg/mL). The patient was treated with intravenous ampicillin/sulbactam and colistin in contact isolation, but died two weeks following transfer. During the next 6 months (January to July 2009), 32 similar consecutive isolates were obtained from blood cultures, urine samples, catheter tip specimens, cerebrospinal fluid, throat and nasal swabs, and bronchial secretions collected from 23 different patients hospitalised in two ICUs and three different departments at Split University Hospital. PFGE following macrorestriction of genomic DNA with ApaI revealed that all isolates belonged to the European clone 2 lineage. All isolates also displayed the same multidrug resistance pattern (with no inhibition zone around imipenem or meropenem discs), but susceptibility to sulbactam and colistin (Table I). Bacterial DNA was extracted using a DNAze kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. The presence of genes encoding class D carbapenemases was detected by multiplex polymerase chain reaction using primers specific for the OXA-23, OXA-40, OXA-51 and OXA-58 gene families.5 Each isolate possessed a blaOXA-51-like and a blaOXA-40-like gene. Sequencing (both strands) of the blaOXA-51-like amplicons from a randomly selected subset of isolates by a commercial laboratory (Macrogen, Seoul, South Korea) revealed the presence of the OXA-90 gene (a variant of OXA-66), whereas sequencing of the blaOXA-40-like amplicons revealed the presence of a gene encoding OXA-72.2 OXA-40-like carbapenemases were originally described in A. baumannii isolates from the Iberian peninsula, but have now been reported worldwide, with sequence divergences between the members of this family varying from one to five amino acids.6,7 OXA-72was first described in an Acinetobacter isolate fromThailand in 2004, and has since been reported in a single A. baumannii isolate from mainland China and as a major mechanism of carbapenem resistance in a Taiwanese hospital.8 This is the first report of OXA-72 in Southeast Europe. PFGE analysis revealed that this new imported clone belonged to the European clone 2 lineage, in contrast to carbapenem-resistant isolates of A. baumannii from the same hospital during the preceding six years, which belonged to the European clone 1 lineage and encoded only the unusual OXA-51-type enzyme OXA-107, associated with ISAba1.3 These observations reinforce the continued importance of careful epidemiological surveillance and enhanced control measures following international patient transfers. Wherever possible, hospitals should routinely isolate and screen all patients admitted from hospitals in other countries. This is particularly important when the source country is known to have a high prevalence of multidrug-resistant bacteria or when no antimicrobial surveillance data are available. It is mandatory to rapidly identify the source and/or reservoir of colonisation/infection with multidrug-resistant A. baumannii in order to prevent further dissemination inside a hospital.

Occurrence of OXA-107 and ISAba1 in Carbapenem-Resistant Isolates of Acinetobacter baumannii from Croatia

ABSTRACT Carbapenem-resistant isolates of Acinetobacter baumannii from intensive care units at Split University Hospital, Split, Croatia, were studied. Most (100 of 106) had ISAba1 inserted upstream of a blaOXA-107 gene, encoding an unusual OXA-51-type oxacillinase. Pulsed-field gel electrophoresis revealed that the isolates formed three clusters belonging to the sequence group 2 (European clone 1) lineage.

Antimicrobial susceptibility of clinically isolated anaerobic bacteria in a University Hospital Centre Split, Croatia in 2013

Anaerobic bacteria play a significant role in many endogenous polymicrobial infections. Since antimicrobial resistance among anaerobes has increased worldwide, it is useful to provide local susceptibility data to guide empirical therapy. The present study reports recent data on the susceptibility of clinically relevant anaerobes in a University Hospital Centre (UHC) Split, Croatia. A total of 63 Gram-negative and 59 Gram-positive anaerobic clinical isolates from various body sites were consecutively collected from January to December 2013. Antimicrobial susceptibility testing was performed using standardized methods and interpreted using EUCAST criteria. Patients clinical and demographic data were recorded by clinical microbiologist. Among 35 isolates of Bacteroides spp., 97.1% were resistant to penicillin (PCN), 5.7% to amoxicillin/clavulanic acid (AMC), 8.6% to piperacillin/tazobactam (TZP), 29.0% to clindamycin (CLI) and 2.9% to metronidazole (MZ). Percentages of susceptible strains to imipenem (IPM), meropenem (MEM) and ertapenem (ETP) were 94.3. Resistance of other Gram-negative bacilli was 76.0% to PCN, 8.0% to AMC, 12.0% to TZP, 28.0% to CLI and 8% to MZ. All other Gram-negative strains were fully susceptible to MEM and ETP, while 96.0% were susceptible to IPM. Clostridium spp. isolates were 100% susceptible to all tested antibiotics except to CLI (two of four tested isolates were resistant). Propionibacterium spp. showed resistance to CLI in 4.3%, while 100% were resistant to MZ. Among other Gram-positive bacilli, 18.2% were resistant to PCN, 9.1% to CLI and 54.5% to MZ, while 81.8% of isolates were susceptible to carbapenems. Gram-positive cocci were 100% susceptible to all tested antimicrobials except to MZ, where 28.6% of resistant strains were recorded. Abdomen was the most common source of isolates (82.5%). The most prevalent types of infection were abscess (22.1%), sepsis (14.8%), appendicitis (13.9%) and peritonitis (6.6%). Twenty four patients (19.7%) received empiric antimicrobial therapy. One hundred and one patients (82.8%) had polymicrobial aerobic/anaerobic isolates cultivated from the same specimens. Almost all aerobic bacteria were of endogenous origin and showed fully susceptible antimicrobial profile; only 8.7% (9/104) were multiresistant and considered as hospital acquired. Based on our findings, β-lactam/β-lactamase inhibitor combinations and metronidazole remain useful antimicrobials for empiric treatment of anaerobic infections, while carbapenems should be reserved for situations were multidrug resistant, aerobic or facultative Gram-negative bacteria are expected. However, a certain percentage of resistant isolates were observed for each of these agents. Therefore, periodic resistance surveillance in anaerobes is highly recommended in order to guide empirical therapy.

High prevalence and molecular characterization of extended-spectrum β-lactamase-producing Proteus mirabilis strains in southern Croatia.

The aim of this study was to determine the prevalence and antibiotic resistance rates of extended-spectrum β-lactamase (ESBL)-producing Proteus mirabilis strains isolated from inpatients at the Split University Hospital (southern Croatia) during a survey performed between 2005 and 2008. A total of 2152 consecutive isolates of P. mirabilis were isolated. The prevalence was 0.5 % in 2005 and increased significantly to 20.9 % by 2008. Strains were most frequently isolated from urine (36.5 %) and bronchial aspirates and wound swabs (11.3 %). ESBL-producing P. mirabilis isolates showed very high resistance rates to the majority of non-β-lactam antibiotics and were susceptible to a β-lactam/β-lactamase inhibitor and carbapenems. The isolates were genotyped and their ESBLs were molecularly characterized. Strains originating from the intensive care unit and the surgery and neurosurgery wards were clonally related. All P. mirabilis isolates produced the TEM-52 type of ESBL. To the best of our knowledge, our work detailed here and summarized in an earlier communication is the first report of such isolates from southern Croatia. Increased monitoring and screening for ESBL production in this species at our hospital is mandatory.

Antimicrobial Susceptibility and Beta-Lactamase Production of Selected Gram-Negative Bacilli from Two Croatian Hospitals: MYSTIC Study Results

Abstract The Meropenem yearly Susceptibility test information Collection (MYSTIC) programme is a global, longitudinal resistance surveillance network that monitors the activity of meropenem and compares its activity with other broad-spectrum antimicrobial agents. We now report the antimicrobial efficacy of meropenem compared to other broad-spectrum agents within the selective Gram-negative pathogen groups from two Croatian Hospitals investigated between 2002-2007. A total of 1510 Gram-negative pathogens were tested and the minimum-inhibitory concentrations (MICS) were determined by broth microdilution method according to CLSI. There was no resistance to either imipenem or meropenem observed for Escherichia coli, Klebsiella pneumoniae and Proteus mirabilis in both medical centers. High resistance rates of K. pneumoniae to ceftazidime (18%), cefepime (17%) and gentamicin (39%) are raising concern. Acinetobacter baumannii turned out to be the most resistant Gram-negative bacteria with 81% resistant to ceftazidime, 73% to cefepime, 69% to gentamicin and 71% to ciprofloxacin. Almost 20% of Pseudomonas aeruginosa strains were resistant to imipenem, 13% to meropenem, 69% to gentamicin and 38% to ciprofloxacin. The prevalence of extended-spectrum beta-lactamases (ESBLs) in E. coli was 10% and in K. pneumoniae 49%. PCR and sequencing of the amplicons revealed the presence of SHV-5 in nine E. coli strains and additional TEM-1 β-lactamase five strains. Five K. pneumoniae strains were positive for bla SHV-5 gene. Eight ESBL positive Enterobacter spp. Strains were found to produce tem and CTX-M β-lactamases. Plasmid-mediated AmpC β-lactamases were not found among K. pneumoniae, E. coli and Enterobacter spp. Three A. baumannii strains from Zagreb University Center were identified by multiplex PCR as OXA-58 like producers. Six A. baumannii strains from Split University Center were found to possess an ISAba1 insertion sequence upstream of bla OXA-51 gene. According to our results meropenem remains an appropriate antibiotic for the treatment of severe infections caused by Gramnegative bacteria. These data indicate that despite continued use of meropenem, carbapenem resistance is not increasing among species tested, except for A. baumannii, in the two study hospitals and suggest that clinicians can still administer carbapenems as a reliable and effective choice in managing serious nosocomial infections.

First clinical and microbiological characterization of Clostridium difficile infection in a Croatian University Hospital

Clinical background and molecular epidemiology of Clostridium difficile infection (CDI) in the University Hospital Centre Split were investigated from January 2010 to December 2011. In total, 54 patients with first episode of CDI were consecutively included in the study based on the positive EIA test specific for A and B toxins. Demographic and clinical data were prospectively analyzed from medical records. CDI incidence rate was 0.6 per 10,000 patient-days. Thirty six cases (70.6%) were healthcare-associated, twelve cases (23.5%) were community-associated and three (5.9%) were indeterminate. Six patients (11.7%) had suffered one or more recurrences and 37 patients (72.5%) showed severe CDI. Prior therapy with third generation cephalosporin was significantly associated with severe CDI (P<0.021). Fifty four toxigenic C. difficile strains were isolated and 50 of them were available for PCR-ribotyping. Sixteen different PCR-ribotypes were identified. The most prevalent were PCR-ribotype 001 (27.8%) and 014/020 (24.1%). Twenty three strains were resistant to at least one of the antibiotics tested. Among resistant strains, three (13.0%)--all PCR-ribotype 001--were multi-resistant. Resistance to fluoroquinolones was significantly higher in strains that caused infection after previous use of fluoroquinolones (P=0.04).

Campylobacter jejuni strains coresistant to tetracycline and ciprofloxacin in patients with gastroenteritis in Croatia

Abstract Background: Antibiotic-resistant Campylobacter jejuni strains are rapidly emerging worldwide. Here, we aimed to determine the antibiotic-resistance patterns and genetic structure of C. jejuni from stool samples of symptomatic patients in Dalmatia, the largest Croatian county. Methods: In a population-based laboratory surveillance programme for campylobacteriosis in Dalmatia from May 2012 to May 2013, C. jejuni (n = 76) were collected from stool samples of all the patients hospitalized with gastroenteritis and matched positive outpatients (n = 77). Antibiotic susceptibility testing and pulsed-field gel electrophoresis (PFGE) genotyping of isolates were performed. Results: Approximately 60% of the isolates were resistant to ciprofloxacin, whereas 24% of isolates were resistant to tetracycline; of the latter, 89% were also coresistant to ciprofloxacin. Resistance to erythromycin and gentamicin was infrequent (≤ 0.7%). Antibiotic-resistant strains were generally not associated with the need for hospitalization. However, the prevalence of coresistant strains increased sharply after 2010, and these coresistant strains were more prevalent in infections caused by clonal PFGE types, with distinct patterns of temporal occurrence and age distribution in infected patients. Conclusion: A high prevalence of coresistant TcR/CipR C. jejuni strains were detected in patients in Croatia. Strains were significantly associated with several clonal-type PFGE genotypes, shared common patterns of temporal occurrence, and showed distinct age distribution in infected patients, suggestive of newly identified strains. Since a high prevalence of coresistant TcR/CipR strains was also observed in other countries, further in-depth studies are essential to evaluate whether this phenomenon is linked to C. jejuni epidemiology in food animals and agricultural ecosystems.

Evaluation of PNA FISH® Yeast Traffic Light in identification of Candida species from blood and non-blood culture specimens

PNA FISH(®) (peptide nucleic acid fluorescent in situ hybridization) Yeast Traffic Light (PNA FISH(®) YTL) assay is a commercially avaliable method for rapid identification of Candida spp. directly from positive blood cultures. This report provides a one-year experience in identification of yeasts from 25 specimens (15 positive blood cultures and 10 other clinically significant specimens) using PNA FISH(®) YTL and comparing it to VITEK 2 System. Overall, assay identification compatibility with VITEK 2 System was found among 21/25 (84%) isolates tested. Only 3/25 (12%) of the isolates were not identified, and one isolate was misidentified by the PNA FISH(®) YTL assay. Our results show that the assay is a reliable method in identification of Candida spp. not only from blood cultures, but even from other clinically significant specimens (urine cultures, catheter tip cultures, peritoneal fluid cultures) when compared to automated method like VITEK 2 System. This novel application of the PNA FISH(®) YTL assay could therefore contribute to cost savings and significant benefit to patients, as rapid information about isolated yeast species is provided.

Prompt Diagnosis and Effective Treatment of Trichosporon asahii Catheter-Related Infection in Non-immunocompromised Neurosurgical Patient

Trichosporon asahii is a rare but emerging fungal pathogen that causes severe and life-threatening infections with high mortality rate, mostly in immunocompromised patients. It could be easily misdiagnosed due to lack of awareness, especially when invasive or deep-seated infections occur in non-immunocompromised patients, and inadequately treated since the clinical failures and high minimum inhibitory concentrations to some antifungal agents have been described. We present a case of T. asahii catheter-related infection in 66-year-old comatose patient with polytrauma, who was not immunodeficient, but was receiving broad-spectrum antibiotics for a long period. Due to prompt diagnosis and treatment which included catheter replacement and voriconazole, the patient successfully recovered from this infection. The aims of this case report were to highlight the importance of recognizing this otherwise colonizing yeast as potentially dangerous pathogen in non-immunocompromised patients with a long-term antibiotic therapy, and to emphasize the importance of the right therapeutic choice due to its resistance to certain antifungal agents.

No difference in the intention to engage others in academic transgression among medical students from neighboring countries: a cross-national study on medical students from Bosnia and Herzegovina, Croatia, and Macedonia

Aim To asses if the level of intention to engage others in academic transgressions was comparable among medical students from five schools from neighboring Southern-European countries: Croatia, Bosnia and Herzegovina, and Macedonia; and medical students from western EU studying at Split, Croatia. Methods Five medical schools were surveyed in 2011, with ≥87% of the targeted population sampled and a response rate of ≥76%. Students’ intention to engage a family member, friend, colleague, or a stranger in academic transgression was measured using a previously validated the Intention to Engage Others in Academic Transgression (IEOAT) questionnaire and compared with their intention to ask others for a non-academic, material favor. Data on students’ motivation measured by Work Preference Inventory scale, and general data were also collected. Multiple linear regression models of the intention to engage others in a particular behavior were developed. Results The most important determinants of the intention to engage others in academic transgression were psychological factors, such as intention to ask others for a material favor, or students’ motivation (median determinant’s β of 0.18, P ≤ 0.045 for all), whereas social and cultural factors associated with the country of origin were either weak (median β of 0.07, P ≤ 0.031) or not relevant. A significant proportion of students were aware of the ethical violations in academic transgressions (P ≤ 0.004 for all transgressions), but a large proportion of students also perceived academic cheating as a collective effort and were likely to engage people randomly (P ≤ 0.001 for all, but the most severe transgression). This collective effort was more pronounced for academic than non-academic behavior. Conclusion Culture differences among neighboring Southern-European countries were not an important determinant of the intention to engage others in academic cheating.