Co-occurrence of triple carbapenemase genes, blaVIM-2, blaNDM-1, and blaOXA-48 in Enterobacter hormaechei clinical isolates -first report from Croatia.

Two Enterobacter hormaechei isolates harbouring three carbapenemase genes each, were isolated from two patients from different ICUs at University Hospital Centre Zagreb, Croatia, which is to our knowledge, the first report of triple carbapenemase (blaVIM-2, blaNDM-1, and blaOXA-48) co-existence in E. hormachei strains and also among Enterobacterales members in Croatia. Antimicrobial susceptibility testing showed susceptibility only to colistin and amikacin. The production of carbapenemases was phenotypically tested by immunochromatographic assay and confirmed by PCR. Detailed analysis by Whole Genome Sequencing (WGS) of short reads by Illumina and long reads by Oxford Nanopore Technologies (ONT) was additionally performed and showed that both isolates belonged to ST200. They were separated by 98 Single Nucleotide Polymorphisms (SNPs) having variations in the number of blaVIM-2 genes on the chromosome, the number of blaNDM-1 genes on the plasmid, non-identical blaNDM-1 plasmids, different plasmid content in general, and only one isolate carried a 94 kb prophage.

Sepsityper® Kit versus In-House Method in Rapid Identification of Bacteria from Positive Blood Cultures by MALDI-TOF Mass Spectrometry.

In order to further accelerate pathogen identification from positive blood cultures (BC), various sample preparation protocols to identify bacteria with MALDI-TOF MS directly from positive BCs have been developed. We evaluated an in-house method in comparison to the Sepsityper® Kit (Bruker Daltonics, Bremen, Germany) as well as the benefit of an on-plate formic acid extraction step following positive signal by the BACTECTM FX system. Confirmation of identification was achieved using subcultured growing biomass used for MALDI-TOF MS analysis. A total of 113 monomicrobial positive BCs were analyzed. The rates of Gram-positive bacteria correctly identified to the genus level using in-house method and Sepsityper® Kit were 63.3% (38/60) and 81.7% (49/60), respectively (p = 0.025). Identification rates at species level for Gram-positive bacteria with in-house method and Sepsityper® kit were 30.0% (18/60) and 66.7% (40/60), respectively (p < 0.001). Identification rates of Gram-negative bacteria were similar with the in-house method and Sepsityper® Kit. Additional on-plate formic acid extraction demonstrated significant improvement in the identification rate of Gram-positive bacteria at both genus and species level for both in-house (p = 0.001, p < 0.001) and Sepsityper® Kit methods (p = 0.007, p < 0.001). Our in-house method is a candidate for laboratory routines with Sepsityper® Kit as a back-up solution when identification of Gram-positive bacteria is unsuccessful.

In vitro susceptibility of carbapenem-resistant Enterobacterales to eravacycline - the first report from Croatia.

The main obstacle in treatment of infections caused by carbapenem-resistant Enterobacterales (CRE) are limited treatment options. The novel antimicrobial agents other than ß-lactams with activity not being dependent on ß-lactamase class are especially important. Eravacycline (ERV) is the first fully synthetic fluorocycline indicated for the treatment of complicated intra-abdominal infections in adults. Eighty CRE isolates at the University Hospital Centre Zagreb, Croatia were examined for susceptibility to ERV by disc diffusion method and minimal inhibitory concentration (MIC). Total of 54 (54/80; 67.5%) isolates were susceptible to ERV with MIC50 of ≤0.5 µg/mL and MIC90 of 4 µg/mL. Susceptibility of OXA-48 positive isolates was not significantly higher in comparison with NDM positive (P = 0.539) and VIM positive (P = 0.7805) isolates. ERV is possible alternative to novel ß-lactamase inhibitor combinations for treatment of CRE infections with antimicrobial susceptibility testing of CRE isolate to ERV in particular patient as condicio sine qua non before administration.

Subgingival Microbiota Profile in Association with Cigarette Smoking in Young Adults: A Cross-Sectional Study.

While smoking is recognized as one of the factors for the development and progression of periodontal diseases, a relation between the composition of the subgingival microbiota and smoking is yet to be elucidated. The aim of this study was to investigate the prevalence of subgingival bacteria in young smokers and non-smokers without clinical signs of periodontal disease. In this cross-sectional study, performed at the Department of Pharmacology, School of Dental Medicine, University of Zagreb, we enrolled 32 periodontally healthy smokers and 32 non-smokers, aged 25-35 years old. The number of oral bacteria and the prevalence of particular bacteria were assessed for each subject. Subgingival plaque samples were collected with sterile paper points from two first molars for microbiological analyses with MALDI-TOF mass spectrometry. In smokers, a significantly higher prevalence of Actinomyces odontolyticus was observed compared to non-smokers, and a significantly lower prevalence of Streptococcus sanguinis was observed compared to non-smokers. Smoking affects the composition of subgingival microbiota, either via depletion of beneficial bacteria or the increase in pathogenic bacteria.

Follow-up of renal transplant recipients after acute COVID-19-A prospective cohort single-center study.

INTRODUCTION: Although most patients recover within several weeks after acute COVID-19, some of them develop long-lasting clinical symptoms. Renal transplant recipients have an increased mortality risk from COVID-19. We aimed to describe complications occurring after COVID-19 in this group of patients. METHODS: A prospective single-center cohort study was conducted at University Hospital Centre Zagreb. Patients with two negative reverse transcriptase-polymerase chain reaction (RT-PCR) tests for SARS-CoV-2 after COVID-19 were eligible for further follow-up at our outpatient clinic. They underwent detailed clinical and laboratory assessments. The primary outcome was the development of complications after COVID-19. RESULTS: Only 11.53% of renal transplant recipients who survived acute COVID-19 were symptomless and free from new-onset laboratory abnormalities during the median follow-up of 64 days (range: 50-76 days). Three patients died from sepsis after discharge from the hospital. In 47 patients (45.2%), clinical complications were present, while 74 patients (71.2%) had one or more laboratory abnormalities. The most common clinical complications included shortness of breath (19.2%), tiredness (11.5%), peripheral neuropathy (7.7%), self-reported cognitive impairments (5.7%), and dry cough (7.7%). Most common laboratory abnormalities included shortened activated partial thromboplastin time (50%), elevated D-dimers (36.5%), elevated fibrinogen (30.16%), and hypogammaglobulinemia (24%). Positive RT-PCR for cytomegalovirus (8.7%), Epstein-Barr virus (26%), or BK virus (16.3%). Multivariate analysis identified the history of diabetes mellitus and eGFR CKD-EPI as predictors for the development of post-COVID clinical complications. Six months after acute COVID-19, elevated D-dimers persisted with normalization of other laboratory parameters. Twenty-nine patients were hospitalized, mostly with several concomitant problems. However, initially reported clinical problems gradually improved in the majority of patients. CONCLUSION: Post-COVID-19 clinical and laboratory complications are frequent in the renal transplant population, in some of them associated with significant morbidity. All patients recovered from acute COVID-19 should undergo long-term monitoring for evaluation and treatment of complications.

Combined association of recipient killer cell immunoglobulin-like haplotype AA and donor HLA-C*07 gene with BK virus associated nephropathy in kidney transplant patients.

Kidney transplant recipient killer cell immunoglobulin-like receptors (KIR) genotype and HLA-C status of their donors have been separately associated with BK virus-associated nephropathy (BKVAN) and BK virus infection. Our aim was to determine whether different combinations of recipients KIR genes and donor HLA-C ligands influence the risk of BKVAN. Retrospective case-control study included 23 recipients with BKVAN and 46 recipients with persistently negative BK virus. Donor HLA-C*07 positivity was associated with lower odds for BKVAN, recipients bearing KIR haplotype AA or lacking any activating KIR genes were more frequent in BKVAN while recipient/donor combination HLA-C*07 negative/KIR AA positive was significantly associated with BKVAN. Our study complements and confirms results from several previously published studies, suggesting potential clinical usefulness.

The photo-activated and photo-thermal effect of the 445/970 nm diode laser on the mixed biofilm inside root canals of human teeth in vitro: A pilot study.

AIMS: 1) Evaluation of the photo-thermal (PT) and photo-activated (PAD) antibacterial effect of the 445/970 nm diode laser on E. faecalis, S. aureus and C. albicans mixed biofilms grown together inside root canals of human teeth. 2) Defining a potentially efficient clinical protocol for safe and predictable usage in endodontic procedures. METHODOLOGY: The root canals of 100 extracted human teeth with single straight canals were prepared with ProTaper NEXT files, sterilized, contaminated with a combination of three cultures (E. faecalis, S. aureus, C. albicans) and incubated for 15 days. The samples were randomly distributed into three groups (n = 20) and treated as follows: Group 1 (G1) - the 445 nm photo-thermal (PT) effect, Group 2 (G2) - a combination of the 445 nm and 970 nm PT effect, Group 3 (G3) - the 445 nm photo-activated (PAD) effect with 0.1% riboflavin, Group 4 (G4) - a combination of 3% sodium hypochlorite (NaOCl) and the 445 nm PAD effect. Four samples were used as positive control (non-treated) and four as a negative control. 12 aditional samples were used as a control for the G4 (3% NaOCl rinse without the laser). The number of viable microbes in each canal was determined by the colony forming unit (CFU) count. RESULTS: A statistically significant reduction in the microbial population after all treatments was observed (P < 0.001). Groups 2 and 3 showed similar results, both better than Group 1. Group 4 produced the best results. CONCLUSIONS: The 445 nm PAD protocol has a stronger antimicrobial effect than the 445 nm PT protocol. Prolonged exposure time to laser light and a combination of wavelengths (445/970 PT protocol) helps in the reduction of microbes. C. albicans appears to be more sensitive to laser irradiation than the other bacteria tested in this study. Following current results, tested laser protocols could be recommended for clinical usage but only as an adjunct to "classic" NaOCl rinse since alone they are not able to completely eradicate all microorganisms.

Susceptibility of Escherichia coli to nitroxoline, an option for uncomplicated urinary tract infections - the first report from Croatia.

Nitroxoline (NTX), 5-nitro-8-hydroxyquinoline is an oral antibiotic with mechanism of bacteriostatic activity that is based on chelation of divalent cations required for bacterial RNA polymerase. Susceptibility to NTX of 100 Escherichia coli urine isolates was determined at the Department of Clinical and Molecular Microbiology, University Hospital Centre Zagreb during September and October 2017. Antimicrobial susceptibility was tested by disc diffusion and the results were interpreted according to the European Committee for Antimicrobial Susceptibility Testing (EUCAST) standards. All E. coli isolates, including ESBL-positive ones, were fully susceptible to imipenem, meropenem, amikacin, fosfomycin and NTX. This is the first report from Croatia about sensitivity of E. coli isolates to NTX. Besides fosfomycin, NTX was the only antimicrobial drug available for peroral administration demonstrating the sensitivity for all tested isolates. The results of the study demonstrated the potential of NTX as an additional therapeutically applicable option for the treatment of uncomplicated UTI.

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.

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.

[MIDDLE EAR INFECTION]. / UPALA SREDNJEG UHA.

Middle ear infection is one of the most common childhood infections and the leading reason for antibiotic prescriptions. Although the etiological diagnosis is rarely discovered, successful identification of pathogens depends on properly collected sample, chosen method and microbiological analysis made on time. The most common bacterial pathogen is Streptococcus pneumoniae. Others include Haemophilus influenzae, Moraxella catarrhalis and Pseudomonas aeruginosa, known as the most common bacterial pathogen of chronic inflamations. Viral or polimicrobial upper respiratory tract infections often precede this infection. Pneumococcal conjugate vaccines given during infancy decrease rates of acute middle ear inflammation. It is a self-limited disease with rare complications. The best treatment is watchful waiting for two days followed by amoxicillin during 7 days, only if it is necessary. If there is resistance, then combination of amoxicillin and beta lactamase inhibitor is second line. The best choice for patients allergic to penicillin are macrolides. Antibiotic treatment has contributed to frequent relapses and increase of multi-drug resistant pathogens by permitting their colonization, which eliminates protective nasopharyngeal flora.

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.

First Report of NDM-1-Producing Acinetobacter guillouiae.

BACKGROUND: Acinetobacter spp. is an opportunistic pathogen that has demonstrated increasing relevance in nosocomial infections. Carbapenem-resistant strains have been reported worldwide. METHODS: Since 2014, screening for metallo-ß-lactamases (MBLs) in all Acinetobacter spp. isolates using phenotypic methods and PCR has been implemented at the University Hospital Center Zagreb. RESULTS: The bacterial strain was isolated from the drain of a child hospitalized in a paediatric intensive care unit and identified as Acinetobacter guillouiae using a MALDI TOF automated system. The strain was resistant to meropenem, ceftazidime, cefotaxime, ceftriaxone, cefepime, sulbactam/ampicillin, gentamicin and ciprofloxacin, intermediately susceptible to piperacillin/tazobactam and imipenem, and susceptible to amikacin and colistin. The Hodge test and combined disk test with EDTA were positive. The MICs of meropenem and imipenem were not reduced by cloxacillin, but a small reduction of two dilutions was observed following the addition of sodium chloride, which indicated that OXA-58 was produced. PCR and sequencing of chromosomal DNA from boiled colonies revealed blaOXA-58 and blaNDM-1 genes. CONCLUSION: This is the first report of NDM-1 in Acinetobacter spp. in Croatia. The early detection of these genes will aid in the prevention and in the achievement of adequate infection control by limiting the spread of these organisms.

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.

First report of KPC-producing Klebsiella pneumoniae in Croatia.

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 bla(KPC), bla(TEM), and bla(SHV) genes. Sequencing of bla(KPC) 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.

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.