Outbreak of healthcare-associated bacteremia caused by Burkholderia gladioli due to contaminated multidose vials with saline solutions in three Croatian hospitals.

OBJECTIVES: Burkholderia gladioli has been associated with infections in patients with cystic fibrosis, chronic granulomatous disease, and other immunocompromising conditions. The aim of this study was to better depict the outbreak of healthcare-associated bacteremia caused by B. gladioli due to exposure to contaminated multidose vials with saline solutions. METHODS: An environmental and epidemiologic investigation was conducted by the Infection Prevention and Control Team (IPCT) to identify the source of the outbreak in three Croatian hospitals. RESULTS: During a 3-month period, 13 B. gladioli bacteremia episodes were identified in 10 patients in three Croatian hospitals. At the time of the outbreak, all three hospitals used saline products from the same manufacturer. Two 100-ml multidose vials with saline solutions and needleless dispensing pins were positive for B. gladioli. All 13 bacteremia isolates and two isolates from the saline showed the same antimicrobial susceptibility patterns and pulsed-field gel electrophoresis profile, demonstrating clonal relatedness. CONCLUSION: When an environmental pathogen causes an outbreak, contamination of intravenous products must be considered. Close communication between the local IPCT and the National Hospital Infection Control Advisory Committee is essential to conduct a prompt and thorough investigation and find the source of the outbreak.

The effect of standard-dose wintertime vitamin D supplementation on influenza infection in immunized nursing home elderly residents.

AIM: To investigate whether three-month oral vitamin D supplementation (800 IU in drops) reduces the risk of influenza infection in elderly nursing home residents vaccinated against influenza. METHODS: This cross-sectional observational study enrolled 97 participants (73.2% women) who received one dose of seasonal trivalent 2016-2017 influenza vaccine. The patients were randomized into an experimental group, which received vitamin D supplementation for three months starting on the day of vaccination, and a control group, which did not receive vitamin D supplementation. The primary outcome was the number of influenza infections laboratory-confirmed using a rapid point-of-care test based on nasal swabs collected during vitamin D supplementation. The secondary outcome was serum 25-hydroxyvitamin D level at the end of the study. RESULTS: The mean age ±standard deviation was 78.5± 8.8 years. All participants had vitamin D deficiency at baseline. Twenty-three participants who developed signs of respiratory infections during the study were tested for influenza virus. Although the number of influenza-positive participants was lower in the group receiving vitamin D supplementation as compared with the control group (5 vs 12), this difference was not significant. Vitamin D supplementation failed to increase 25(OH)D levels after three months of supplementation. CONCLUSION: Elderly nursing home residents in Zagreb County have a significant vitamin D deficiency. The recommended national supplementation of 800 IU daily failed to lead to vitamin D sufficiency and did not reduce the risk of influenza infection among the vaccinated elderly.

Seroprevalence of SARS-CoV-2 infection among children in Children's Hospital Zagreb during the initial and second wave of COVID-19 pandemic in Croatia.

INTRODUCTION: The study aimed to investigate the prevalence and titres of anti-SARS-CoV-2 antibodies in children treated at the Children's Hospital Zagreb in the first and the second wave of the COVID-19 pandemic. Statistical significance of difference at two time points was done to determine how restrictive epidemiological measures and exposure of children to COVID-19 infection affect this prevalence in different age groups. MATERIALS AND METHODS: At the first time point (13th to 29th May 2020), 240 samples and in second time point (24th October to 23rd November 2020), 308 serum samples were tested for anti-SARS-CoV-2 antibodies by enzyme-linked immunosorbent assay (ELISA) and electrochemiluminescence immunoassay (ECLIA). Confirmation of results and titre determination was done using virus micro-neutralization test. Subjects were divided according to gender, age and epidemiological history. RESULTS: Seroprevalence of anti-SARS-CoV-2 antibodies differs significantly in two time points (P = 0.010). In first time point 2.9% of seropositive children were determined and in second time point 8.4%. Statistically significant difference (P = 0.007) of seroprevalence between two time points was found only in a group of children aged 11-19 years. At the first time point, all seropositive children were asymptomatic with titre < 8. At the second time point, 69.2% seropositive children were asymptomatic with titre ≥ 8. CONCLUSIONS: The prevalence of anti-SARS-CoV-2 antibodies was significantly lower at the first time point than at the second time point. Values of virus micro-neutralization test showed that low titre in asymptomatic children was not protective at the first time point but in second time point all seropositive children had protective titre of anti-SARS-CoV-2 antibodies.

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.

NDM-1-producing Enterobacter aerogenes isolated from a patient with a JJ ureteric stent in situ.

Urinary tract infections after JJ stent insertion are among the most common complications, and the associated microorganisms carry more antibiotic resistance determinants than those found in urine prior to stent insertion. In line with the trends in healthcare epidemiology which implicate multi-resistant microorganisms in a plethora of healthcare-associated infections, prosthetic stent material also represents an ideal milieu for biofilm formation and subsequent infection development with resistant bacterial agents. Here we describe a case of a 73-year-old Caucasian woman presenting with urinary tract infection after JJ ureteric stent insertion due to ureteric obstruction and hydronephrosis of her left kidney. Extensive microbiological work-up and comprehensive molecular analysis identified the putative microorganism as carbapenem-resistant Enterobacter aerogenes carrying New Delhi metallo-beta-lactamase 1 (NDM-1). This is a first literature report implicating such extensively resistant strain of this species in early indwelling ureteric stent complications, and also the first report of NDM-1 in Enterobacter aerogenes in Croatia and Europe.

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.

Epidemic spread of OXA-48 beta-lactamase in Croatia.

PURPOSE: A dramatic increase in OXA-48 ß-lactamase was observed recently not only in large hospital centres, but also in smaller suburban hospital centres in geographic areas bordering Croatia. The aim of the study was to analyse the epidemiology, the mechanisms of antibiotic resistance and the routes of spread of OXA-48 carbapenemase in Croatia. METHODS: Carbapenemase and other ß-lactamase and fluoroquinolone resistance genes were detected by PCR and sequencing. Whole-genome sequencing (WGS) was performed on five representative isolates. The isolates were genotyped by PFGE. RESULTS: Forty-eight isolates positive for OXA-48, collected from seven hospital centres in Croatia from May 2016 to May 2017, were analysed (40 Klebsiella pneumoniae, 5 Enterobacter cloacae, 2 Escherichia coli and one Citrobacter freundii). Thirty-three isolates were ESBL positive and harboured group 1 CTX-M 1 ß-lactamases. In addition to the ß-lactam resistance genes detected by PCR (blaSHV-1, blaOXA-48 and blaOXA-1), WGS of five representative isolates revealed the presence of genes encoding aminoglycoside resistance, aadA2 and aph3-Ia, fluoroquinolone resistance determinants aac(6)Ib-c, oqxA and oqxB, the sulfonamide resistance gene sul1, and fosA (fosfomycin resistance). IncL plasmid was found in all isolates. Two K. pneumoniae isolates belonged to ST16, two E. cloacae to ST66 and E. coli to ST354. K. pneumoniae isolates were allocated to five clusters by PFGE which occured in different hospitals, indicating epidemic spread. CONCLUSIONS: The OXA-48-positive organisms found in this study showed wide variability in antibiotic susceptibility, ß-lactamase content and PFGE banding patterns. This study revealed a switch from the predominance of VIM-1 in 2012-2013 to that of OXA-48 in the 2015 to 2017.

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.

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 ß-lactamases, class B metallo-ß-lactamases (MBL) or class D OXA-48-like ß-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 ß-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.

[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.

Full-genome characterization of a G8P[8] rotavirus that emerged among children with diarrhea in Croatia in 2006.

The whole genome of a G8P[8] rotavirus from the 2006 epidemic in Croatia was sequenced and showed a Wa-like genotype constellation. Its VP7 gene clustered with DS-1-like G8 African rotaviruses and a G8P[4] German strain. Remaining genes clustered with contemporary Belgian G1P[8] rotaviruses, suggesting reassortment between human G8 and G1P[8] rotaviruses in Croatia or other European countries.

Incidence of Clostridium difficile infection in children with inflammatory bowel disease compared to oncology and immunocompetent patients.

AIMS: The aim of this study was to determine the incidence of Clostridium difficile infection in hospitalized children with inflammatory bowel disease (IBD) and to compare it to other immunosuppressed patients at risk (oncology patients) as well as to immunocompetent patients. METHODS: We analyzed data from all hospitalized children who underwent stool detection of C. difficile toxins A and B (n = 757) in a 5.5-year study period. RESULTS: The number of positive tests was significantly increased in the oncology group compared to the IBD group (12.45 vs. 6.02%, p = 0.03) and immunocompetent group (12.45 vs. 5.7%, p = 0.01). Patients who had C. difficile infection used antibiotics prior to the test more often than patients who did not (12.69 vs. 1.73%, p = 0.03). Pearson's correlation was positive for C. difficile infection and both antibiotics and immunosuppressants, while no correlation was found regarding age and gender. There were no significant differences regarding either IBD diagnosis (Crohn's disease vs. ulcerative colitis, p = 0.71) or treatment used for IBD (p = 0.53) and C. difficile infection. CONCLUSION: In our setting, the incidence of C. difficile infection among hospitalized children with active IBD was found to be low. Children at increased risk for C. difficile infection were oncology patients receiving immunosuppressants and antibiotics.

Antibiotic resistance of Helicobacter pylori in pediatric patients -- 10 years' experience.

The aim of this study was to assess the pattern of evolution of resistance to antibiotics in Helicobacter pylori isolated from children who underwent upper endoscopy with antral biopsy during a 10-year period (2001-2010). We retrospectively analyzed data of all children (n = 3,008) who underwent upper endoscopy during the observed period at the Children's Hospital Zagreb, a university tertiary medical center. We calculated the rate, antibiotic susceptibility and risk factors for the H. pylori infection in our cohort. Antral biopsy was performed in 2,313 (76.89%) patients. Altogether, 382 (16.51%) children had positive biopsy for H. pylori (histology and/or culture). There was no significant difference in the incidence of H. pylori during 10 years of observation (p = 0.21). Infected children compared to non-infected group were older (p = 0.005), and had more often antral nodularity (p < 0.0001), and duodenal ulcer (p = 0.002). Altogether, 22.4% of treatment-naïve patients had strains resistant to tested antibiotics: majority to azithromycin (17.9%), followed by clarithromycin (11.9%), metronidazole (10.1%) and amoxicillin (0.6%). In the eradication failure group, 9/11 of children had strains resistant to tested antibiotics, mostly to metronidazole (7/11), followed by azithromycin (3/11) and clarithromycin (1/11). No correlation was found between age or gender and antibiotic resistance (p = 0.32, for both). In conclusion, our data strongly support current guidelines which recommend antibiotic susceptibility testing prior to eradication therapy. Based on our results we recommend the use of amoxicillin-metronidazole-based regimen as the first-line therapy in our study population.

Candida utilis candidaemia in neonatal patients.

In recent years, an evident rise in the frequency of candidaemia caused by non-albicans Candida species has been reported. In this paper we present three cases of clinically manifested candidaemia caused by Candida utilis in neonatal patients hospitalized in the same neonatal intensive care unit within a 6 month period. To the authors' knowledge, only two cases of C. utilis candidaemia have been reported in the literature to date, but neither of these involved newborns. Clinical resolution and elimination of C. utilis from the blood were achieved using liposomal amphotericin B or caspofungin in all patients.

Microbiological findings in prepubertal girls with vulvovaginitis.

The aim of the study was to define the most common causes, symptoms and clinical features of vulvovaginitis in prepubertal girls, and to evaluate treatment success depending on the causative agent involved. The study included 115 girls aged 2-8 (mean 4.8) years, presenting with vulvovaginitis to the Outpatient Clinic for Pediatric and Adolescent Gynecology, Zagreb Children's Hospital, between September 2006 and July 2007. Medical history data were obtained from parents. Vaginal samples were collected for microbiological culture by using cotton-tipped swabs moistened with saline. All samples were referred to microbiology laboratory, where standard microbiological diagnostic procedures were performed. Selective and non-selective media were used. Of 115 study patients, 43 (37.4%) had received antibiotic therapy more than one month prior to their visit to the Clinic, mainly for upper respiratory tract infection. The most common presenting symptom was increased vaginal discharge usually noticed on the pants or diaper, found in 26 of 115 (22.6%) patients, followed by vulvar redness in 16 (13.9%), burning in seven (6.1%), itching in the vulvovaginal area in seven (6.1%), soreness in six (5.2%), odor in three (2.6%) patients, and two or more of these symptoms in another 50 (43.5%) patients. Fifty-nine of 115 children had normal clinical finding on gynecologic examination. Among the remaining 56 children, the most common finding was erythema observed in 19, vaginal discharge in ten, and a combination of discharge and erythema in 13 patients. Of 115 study patients, causative agents were isolated from vaginal culture in 38 (33%) cases. Of these, 21 grew group A beta hemolytic streptococcus, five patients Haemophilus influenzae, three Escherichia coli, two Enterococcus spp., and one each Staphylococcus aureus, Proteus mirabilis, and Streptococcus pneumoniae. Antibiotic therapy was administered in 31 of these 38 patients, except for those cases where intestinal bacteria and Staphylococcus aureus were isolated and topical therapy and hygienic measures were applied alone. Accordingly, vulvovaginitis in girls was most commonly caused by pathogens originating from the patient upper respiratory tract, accompanied by the symptoms of redness and vaginal discharge. In these cases, antibiotic treatment was administered. In the majority of prepubertal girls with either vulvitis or normal genital finding, simple measures to improve hygiene will lead to resolution of all symptoms.