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CILJ ISTRAŽIVANJA: Cilj ovoga rada je utvrditi osjetljivost bakterija koje najčešće izazivaju lokalizirane infekcije u KBC Split tijekom 2020. godine na pojedine topikalne antimikrobne lijekove kako bi se ustanovilo koji od testiranih antibiotika je najbolji izbor za empirijsko liječenje ovih infekcija. MATERIJALI I METODE: Retrospektivnim istraživanjem analizirani su podatci o osjetljivosti na topikalne antimikrobne lijekove (aminoglikozide, fluorokinolone, kloramfenikol, kolistin te mupirocin) pojedinih vrsta bakterija koje su najčešći uzročnici lokaliziranih infekcija: Escherichia coli i ESBL E. coli, Klebsiella pneumoniae (uključujući i ESBL sojeve), Pseudomonas aeruginosa, Acinetobacter baumannii te Staphylococcus aureus (uključujući MRSA). Osjetljivost je ispitivana metodom disk difuzije, a za kolistin metodom dilucije u bujonu. REZULTATI: Rezultati pokazuju visoku rezistenciju bakterije Escherichia coli na antibiotike ciprofloksacin i levofloksacin, a nisku na kloramfenikol i kolistin. U usporedbi s Escherichia coli, ESBL producirajući soj Escherichia coli pokazuje znatno visoku rezistenciju na sve ispitivane antibiotike osim na kolistin i kloramfenikol. Sojevi Klebsiella penumoniae su bili visoko rezistentni na antibiotik tobramicin, a nisko rezistentni na kloramfenikol i kolistin. ESBL producirajući sojevi ove bakterije su imali znatno visoku rezistenciju na sve ispitivane antibiotike osim na kloramfenikol i kolistin. Pseudomonas aeruginosa je bio visoko rezistentan na ciprofloksacin i levofloksacin, a nisko na kolistin. Sojevi Acinetobacter baumanni su bili visoko rezistentni na levofloksacin, a nisko na kolistin. Izolati Staphylococcus aureus su bili visoko rezistentni na levofloksacin, a dobro osjetljivi na kloramfenikol i norfloksacin. MRSA sojevi su bili visoko rezistentni na levofloksacin, u značajno većim postotcima od MSSA sojeva, a dobru osjetljivost pokazali su na mupirocin. ZAKLJUČAK: Podatci ove studije pokazuju visoke stope rezistencije ispitivanih bakterija na pojedine topikalne antimikrobne lijekove, što je u skladu s trendovima porasta rezistencije bakterija u svijetu, te možemo pretpostaviti da će se ta rezistencija u budućnosti još povećavati., AIM OF THE RESEARCH: The main aim of this research was to determine the susceptibility of bacterial isolates which are that most frueqent causative agents of localized infections in UHC Split in 2020 to certain topical antimicrobial drugs and to determine which of the antibiotics tested is the best choice for empirical treatment of these infections. MATERIAL AND METHODS: In this retrospective study the data of susceptibility to topical antimicrobial drugs (aminoglycosides, fluoroquinolones, chloramphenicol, colistin and mupirocin) of certain types of bacteria that are the most common causes of localized infections were analyzed. Bacterial isolates included were Escherichia coli and ESBL E. coli, Klebsiella pneumoniae (including Klebsiella pneumoniae ESBL strains), Pseudomonas aeruginosa, Acinetobacter baumannii, and Staphylococcus aureus (including MRSA). Sensitivity was examined by disk diffusion method, and for colistin by broth dilution method. RESULTS: The results showed high resistance rates of Escherichia coli to the antibiotics ciprofloxacin and levofloxacin, and the low rates to chloramphenicol and colistin. Compared with Escherichia coli, the ESBL-producing strain of Escherichia coli showed significantly high resistance to all tested antibiotics except colistin and chloramphenicol. Strains of Klebsiella penumoniae were high resistant to the antibiotic tobramycin, and low resistant to chloramphenicol and colistin. ESBL-producing strains of this bacterium were significantly high resistant to all tested antibiotics except chloramphenicol and colistin. Pseudomonas aeruginosa was high resistant to ciprofloxacin and levofloxacin, and low to colistin. Acinetobacter baumanni strains were high resistant to levofloxacin and low to colistin. Staphylococcus aureus isolates were high resistant to levofloxacin and sensitive to chloramphenicol and norfloxacin. MRSA strains were high resistant to levofloxacin in significantly higher percentages than MSSA strains, and showed the good sensitivity to mupirocin. CONCLUSION: The data from this study follow the global trends of increasing resistance, and can be assumed that this number will increase in the future.

Vancomycin-resistant Enterococcus faecium (VREfm) is an opportunistic pathogen among the highest global priorities regarding public and environmental health. Following One Health approach, we determined for the first time the antibiotic resistance and virulence features, and sequence types (STs) affiliation of VREfm recovered simultaneously from marine beach waters, submarine outfall of a wastewater treatment plant and an offshore discharge of untreated sewage, and compared them with the surveillance VREfm from regional university hospital in Croatia to assess the hazard of their transmission and routes of introduction into the natural environment. Importantly, VREfm recovered from wastewater, coastal bathing waters and hospital shared similar virulence, multidrug resistance, and ST profiles, posing a major public health threat. All isolates carried the vanA gene, while one clinical isolate also possessed the vanC2/C3 gene. The hospital strains largely carried the aminoglycoside- resistance genes aac(6')-Ie-aph(2'')-Ia, and aph(2'')-Ib and aph(2'')-Id, which were also predominant in the environmental isolates. The hyl gene was the most prevalent virulence marker. The isolates belonged to 10 STs of the clonal complex CC17, a major epidemic lineage associated with hospital infections and outbreaks, with ST117 and ST889 common to waterborne and hospital isolates, pointing to their sewage-driven dissemination. To gain better insight into the diversity of accompanying taxons in the surveyed water matrices, microbiome taxonomic profiling was carried out using Illumina-based 16S rDNA sequencing and their resistome features predicted using the PICRUSt2 bioinformatics tool. An additional 60 pathogenic bacterial genera were identified, among which Arcobacter, Acinetobacter, Escherichia-Shigella, Bacteroides and Pseudomonas were the most abundant and associated with a plethora of antibiotic resistance genes and modules, providing further evidence of the hazardous effects of wastewater discharges, including the treated ones, on the natural aquatic environment that should be adequately addressed from a sanitary and technological perspective.

Background. Infections caused by multidrug-resistant organisms (MDRO) are difficult to treat and associated with poor outcomes for patients. Therefore, early identification and management of colonization are essential as first steps in infection prevention. Culture-based methods have been widely used for MDRO screening. The turnaround time (TAT) for the identification of carriers varies between 48-72 h with this method. The aim of our study was to compare the performance of the new rapid semiautomatic method for detection of MDRO (HB&L Uroquattro, Alifax) with standard cultivation on selective media. Methods. Twenty-one axillary, 20 nose and 19 rectal swabs were taken in duplicate on two selected days at the University Hospital Centre Split, Croatia. Swabs were cultivated and MDRO isolates were identified on selective media (Chromagar MRSA and Chromagar ESBL) according to the standard operating procedure. Novel Alifax method was performed according to manufacturer’s instructions. Results. TAT for the new method was significantly lower (6.5 h) in comparison to the classical method. With classical method, 10 extended spectrum ß-lactamases (ESBL) producing strains from 10 different rectal specimens were isolated on Chromagar ESBL media. Exactly the same specimens were positive for the presence of ESBL-producing bacteria by rapid Alifax method. On selective Chromagar MRSA media, 11 MDRO were isolated, while rapid method detected 11 MDRO from the same specimens. Conclusions. High concordance of positive and negative results obtained with classical and rapid method is encouraging. However, our study was performed on a small sample size and further research with larger sample size is needed.

SUMMARY Vancomycin-resistant enterococci (VRE), especially Enterococcus faecium, have emerged as significant nosocomial pathogens and patients with impaired host defenses are at a particular risk of VRE infection. The most common occurrence is asymptomatic colonization of the gastrointestinal tract that can persist for a long time and serve as a reservoir for transmission of VRE to other patients. We present a case of a patient who was diagnosed with acute myelogenous leukemia and suffered from bone marrow aplasia following induction therapy. The patient received prolonged broad-spectrum antimicrobial therapy. During hospital stay, the patient developed Clostridium difficile infection (CDI) and was found to be colonized with a strain of Enterococcus faecium resistant to vancomycin during therapy for CDI. This case also highlights the role of risk factors that could contribute to development of resistance, particularly CDI. Early detection of VRE colonization or infection is a crucial component in hospital program designed to prevent transmission of nosocomial infections. Surveillance cultures of such patients should be mandatory.

As high clarithromycin resistance (&gt, 20%) in the Split-Dalmatia region of Croatia hinders the treatment of H. pylori infection, the primary objective of this study was to compare concomitant quadruple with the tailored, personalized therapy as first-line eradication treatment of H. pylori. In an open-label, randomized clinical trial, 80 patients with H. pylori infection were randomly assigned to either concomitant (esomeprazole 40 mg, amoxicillin 1 gr, metronidazole 500 mg, clarithromycin 500 mg, twice daily for 14 days) or tailored therapy in accordance with the results of the antimicrobial susceptibility testing. Eradication status was assessed 4 weeks after treatment. Eradication rates were significantly higher in tailored group than in concomitant group both in intention-to-treat (70 vs. 92.5%, p = 0.010) and per-protocol (87.5 vs. 100%, p = 0.030) analysis in the setting of increasing antibiotic resistance (clarithromycin 37.5%, metronidazole 17.5%, dual resistance 10%). Adverse effects were more frequent in the concomitant group (32.5 vs. 7.5%, p = 0.006). Tailored therapy achieves higher eradication with a lower adverse events rate. With the increasing resistance of H. pylori strains to antibiotic treatment, eradication regimes with such characteristics should be strongly considered as a reasonable choice for first-line treatment.

This study was performed to elucidate genetic relatedness and molecular resistance mechanisms of AmpC-producing multidrug-resistant Proteus mirabilis isolates in University Hospital of Split (UHS), and define efficient antibiotics in vitro. A total of 100 nonrepeated, consecutive, amoxicillin/clavulanate- and cefoxitin-resistant P. mirabilis isolates were collected, mostly from urine (44%) and skin and soft-tissue samples (30%). They were all positive in cefoxitin Hodge test and negative for extended spectrum beta- lactamase production. Pulsed field gel electrophoresis identified four clusters and two singletons, with 79% of isolates in dominant cluster. Molecular characterization and I-CeuI analysis of representatives revealed blaCMY-16 gene located on chromosome, and insertion element ISEcp1 positioned 110 pb upstream of blaCMY-16 starting codon. They also harbored blaTEM-1, except one with blaTEM-2. They were all resistant to trimethoprim-sulfamethoxazole, all but one to quinolones, and 81% to all aminoglycosides, while 77% were susceptible (S) and 22% intermediate (I) to piperacillin/tazobactam, and 4% were S and 68% I to cefepime. Only 15% were S to ceftolozane/tazobactam. Meropenem, ertapenem, ceftazidime/avibactam, temocillin, and fosfomycin were 100% efficient in vitro. This is the first report of blaCMY-16 gene in P. mirabilis from hospital samples in Croatia. The findings are in accordance with Italian and Greek reports. The clonal nature of outbreak suggests the high potential of clonal spread. Alternative agents should be considered to spare carbapenem usage.

Although antimicrobial properties of wine have been extensively studied, antimicrobial effects of wine in relation to bacterial resistance to medicinal antibiotics have not been examined. Therefore, our aim was to determine whether bacterial resistance to antibiotics can be related to their resistance to red wine as an unspecific antimicrobial medium. The organisms studied were Salmonella enteritidis (ATCC 13076), Escherichia coli (ATCC 25922), and two clinical isolates which exhibited different resistance to antibiotics, ESBL - producing Escherichia coli UR 3612 and Salmonella enteritidis KK 962. The time-kill curves method was used. The minimal incubation time of the bacterial suspension with wine, necessary for prevention of bacterial growth, was 3 and 20 min for E. coli ATCC and ESBL E. coli respectively. This was associated with susceptibility testing in which E. coli ATCC proved highly sensitive in contrast to ESBL-producing E. coli, which exhibited resistance to a spectrum of antimicrobial drugs of different classes regarding their principal mechanism of action. In the case of S. enteritidis strains, they were similar in their susceptibility against test antibiotics and time-kill curves following exposure to wine. Bacterial resistance to wine is closely associated with bacterial resistance to antimicrobial drugs. The exact mechanisms of antimicrobial activity of wine are still a matter of debate. However, wine might be less susceptible to bacterial resistance development and may include mechanisms different from those of medicinal antibiotics. The present study represents an initial contribution to this important subject which has been practically unexplored.

According to the World Health Organization, bacterium Acinetobacter baumannii is the first on the critical priority list of pathogens in urgent need for new antibiotics. The increasing resistance of A. baumannii to the last-line treatment options, including carbapenems, is a global problem. We report the molecular epidemiology of 12 carbapenem-resistant clinical isolates of A. baumannii collected from hospitalised patients in three neighbouring countries in Southeast Europe: Croatia, Serbia, and Bosnia and Herzegovina, giving an insight into the molecular characterisation and evolutionary history of the acquisition of resistance genes. Besides the blaOXA-23 gene, the endemic presence of OXA-72 oxacillinase of the same origin for more than a decade as the leading mechanism of carbapenem resistance in Southeast Europe was confirmed. To the best of our knowledge, this is the first paper that investigates and analyses the phylogenetic association of the most common mechanisms of resistance to carbapenems in clinical isolates of A. baumannii originating from three neighbouring countries in Southeast Europe.

Increasingly difficult treatment of multidrug-resistant (MDR) bacteria has become a global problem of the 21st century. Within a group of multiresistant bacteria, the Acinetobacter baumannii convincingly occupies the position at the top of the group designated as ESKAPE pathogens. In this study, 61 isolates of A. baumannii were recovered from different samples originating from various departments of the University Clinical Hospital Mostar during 2018. All of the isolates were identified using conventional phenotypic methods and the VITEK® 2 Compact System, and were confirmed by MALDI-TOF mass spectrometry. The minimum inhibitory concentrations (MICs) were determined by the microbroth dilution method using MICRONAUT-S MDR MRGN-Screening and VITEK 2 Compact System. All strains were resistant to carbapenems and classified in eight different resistotypes according to their antibiotic resistance and macrorestriction pulsed-field gel electrophoresis profiles, with all belonging to IC II. One isolate displayed resistance to colistin (MIC ≥16 mg/L). The presence of blaOXA genes encoding OXA-type carbapenemases was investigated by multiplex PCR and the Eazyplex® SuperBugAcineto system and showed 100% compatibility with the detection of acquired oxacillinases. Molecular characterization of the isolates tested in this study revealed the OXA-23- and OXA-40-like groups of acquired oxacillinases. Sequencing of two PCR products of the OXA-40-like group confirmed the presence of OXA-72. Survival assays with two selected isolates of A. baumannii encoding different mechanisms of carbapenem resistance revealed that one isolate was able to survive on a fragment of white laboratory coat during 90 days of monitoring. To the best of our knowledge, this is the first article to present the results of a comprehensive phenotypic, genotypic, and molecular analysis of A. baumannii isolates from the leading clinical hospital center in the southwestern part of Bosnia and Herzegovina, including data for the survival of this pathogen on the white laboratory coats used as compulsory medical clothing.

Increasingly difficult treatment of multidrug-resistant (MDR) bacteria has become a global problem of the 21st century. Within a group of multiresistant bacteria, the

Uvod: Infekcije uzrokovane višestruko otpornim organizmima (MDRO) se teško liječe i često imaju nepovoljan ishod za bolesnika. Budući da infekciji najčešće prethodi kolonizacija, rana identifikacija koloniziranih bolesnika je neophodna u prevenciji nastanka infekcije. Za probir MDRO najčešće se koriste kultivacijske metode kojima vrijeme potrebno za izdavanje nalaza (TAT) iznosi od 48 do 72 h. Cilj ove studije bio je usporediti rezultate nove, brze, poluautomatske metode za detekciju MDRO (HB&L Uroquattro, Alifax) sa standardnom kultivacijom na selektivnim podlogama. Metode/materijali: Tijekom dva nasumično odabrana dana, bolesnicima hospitaliziranima u Kliničkom bolničkom centru Split, uzorkovano je ukupno 21 aksilarnih, 19 rektalnih te 20 briseva vestibuluma nosa. Svaki obrisak je prikupljen u duplikatu, jedan za kultivacijsku, a drugi za brzu poluatomatsku metodu. Brisevi su kultivirani i MDRO izolati identificirani na selektivnim podlogama (Chromagar MRSA i Chromagar ESBL) prema standardnom laboratorijskom protokolu. Nova Alifaxova metoda je provedena prema uputama proizvođača. Rezultati: U usporedbi s klasičnom metodom, TAT nove metode je bio značajno kraći (6,5 h). Klasičnom metodom je na Chromagar ESBL selektivnom mediju izolirano 10 sojeva koji stvaraju ß-laktamaze proširenog spektra (ESBL) iz 10 različitih rektalnih uzoraka. Isti uzorci su bili pozitivni na prisustvo ESBL- producirajućih bakterija brzom Alifaxovom metodom. Kultivacijom na selektivnom Chromagar MRSA mediju i brzom metodom otkriveno je 11 uzoraka pozitivnih na prisustvo MDRO. Zaključak: Visoki stupanj podudarnosti otkrivanja pozitivnih i negativnih uzoraka dobiven klasičnom i novom metodom, kao i značajno skraćivanje TAT-a novom metodom je ohrabrujući. Budući je studija provedena na relativno malom uzorku, potrebno je istraživanje proširiti na veći broj ispitanika., Background. Infections caused by multidrug-resistant organisms (MDRO) are difficult to treat and associated with poor outcomes for patients. Therefore, early identification and management of colonization are essential as first steps in infection prevention. Culture-based methods have been widely used for MDRO screening. The turnaround time (TAT) for the identification of carriers varies between 48-72 h with this method. The aim of our study was to compare the performance of the new rapid semiautomatic method for detection of MDRO (HB&L Uroquattro, Alifax) with standard cultivation on selective media. Methods. Twenty-one axillary, 20 nose and 19 rectal swabs were taken in duplicate on two selected days at the University Hospital Centre Split, Croatia. Swabs were cultivated and MDRO isolates were identified on selective media (Chromagar MRSA and Chromagar ESBL) according to the standard operating procedure. Novel Alifax method was performed according to manufacturer’s instructions. Results. TAT for the new method was significantly lower (6.5 h) in comparison to the classical method. With classical method, 10 extended spectrum ß-lactamases (ESBL) producing strains from 10 different rectal specimens were isolated on Chromagar ESBL media. Exactly the same specimens were positive for the presence of ESBL-producing bacteria by rapid Alifax method. On selective Chromagar MRSA media, 11 MDRO were isolated, while rapid method detected 11 MDRO from the same specimens. Conclusions. High concordance of positive and negative results obtained with classical and rapid method is encouraging. However, our study was performed on a small sample size and further research with larger sample size is needed.

Helicobacter pylori infections represent an important factor in the pathogenesis of chronic gastritis, peptic ulcer, MALT lymphoma and gastric adenocarcinoma. The recently published Maastricht V/Florence consensus report indicated that the urea breath test using 13 C urea still remains the best non-invasive test to diagnose H. pylori infections with high sensitivity and specificity. Among the stool antigen tests, the ELISA monoclonal antibody test is a rational option. Effective therapy should be based only on susceptibility testing in regions with documented high clarithromycin resistance (>15%). Advanced high-resolution endoscopic technologies enable increased diagnostic accuracy for detection of H. pylori infections.

We report a case of cavitary pulmonary disease caused by Mycobacterium shimoidei in 67-year- old female with history of asthma. Even though susceptibility testing was not available, choice of treatment regimen (streptomycin, rifampicin, ethambutol, and clarithromycin), based on a few cases with favorable outcome reported in the literature, resulted with an excellent clinical, microbiological, and radiological response. This is the first report of pulmonary disease caused by M. shimoidei, but also the first ever isolation of M. shimoidei in Croatia.

Objectives: Rotavirus is the major cause of severe diarrhea in young children worldwide. In countries like Croatia, where rotavirus vaccine has not been introduced in the national immunization program, prospective surveillance is necessary to establish the diversity of rotavirus strains. The aim of this study was to describe the prevalence and geographical distribution of rotavirus strains in Croatia and to detect the possible emergence of novel strains. Methods: The study was conducted among children ≤5 years of age with acute gastroenteritis at three hospitals located in different geographical regions of Croatia, during the years 2012 to 2014. Rotavirus was detected in stools using an immunochromatographic assay and then sent for further molecular analysis. Results: Genotyping of 822 rotaviruses showed that the predominant circulating strain was G1P[8] (61.9%), followed by G2P[4] (19.5%), G1P[4] (3.9%), and G3P[8] (2.9%). A high prevalence of reassortants among common human rotavirus genotypes was detected (7.7%). Possible zoonotic reassortants were found, including G8 and G6 strains. The latter is described for the first time in Croatia. Conclusions: This study represents pre-vaccination data that are important for decisions regarding immunization strategies in Croatia. The high prevalence of ‘common’ rotavirus strains circulating in Croatia may advocate for rotavirus vaccine introduction, but further surveillance is necessary to monitor the possible emergence of novel genotypes. Keywords: Rotavirus, Diarrhea, Acute gastroenteritis, Children, Genotypes

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.

The long-standing goal in the field of peptide antibiotics has been to design lead compounds that have a wide spectrum of excellent antibacterial activity but are nontoxic to human cells. Gram-negative and Gram-positive bacteria have very different membranes, which are additionally modified in some drug- resistant species, presenting a challenge for the design of a single membrane-active peptide able to adapt its conformation to various physical properties of membrane microenvironments. In this paper, we describe how a peptide sequence can be constructed starting from an adaptable dynamic turn tandem motif in a central location. The peptide, named flexampin, has been examined firstly by molecular dynamics simulations. It uses a flexible central motif and designed helix- forming cationic amphipathic arms to form a boomerang-like, L-shape, V-shape, and hairpin, super-secondary structures, whichever is the best in matching amphipathic and hydrophobic microenvironments it encounters. Secondly, activity measurements showed that flexampin is bactericidal at low micromolar concentrations against Gram-positive and Gram-negative strains including some multidrug resistant clinical isolates, while it is nontoxic for human circulating blood cells, does not cause DNA damage, and has good selectivity for bacterial cells in comparison to human cells. It is the first membrane-active peptide designed with the ability to self-adjust the orientation of its two cationic helical arms, 3D-hydrophobic moment, and dipole moment for obtaining a better grasp of anionic polar head groups at bacterial membrane surfaces.

Data from population-based laboratory surveillance were used to examine the epidemiological pattern of campylobacteriosis in a sentinel site, Split-Dalmatia County (SDC), Croatia, from 2007 to 2012, and to evaluate the association between disease incidence and demographic, geographical, climatic, agricultural, and microbiological factors. A total of 2658 laboratory-confirmed Campylobacter infections were recorded. Overall mean incidence was 96/100, 000, ranging from 61/100, 000 in rural to 131/100, 000 in urban areas ; rates were highest in the age group 0–4 years. Overall mean and age- and sex-specific incidences were significantly higher in urban versus rural areas (p < 0.01). The number of infections peaked in early summer, and was correlated with higher average monthly temperature (r = 0.58) and lower humidity (r = − 0.27), but not with precipitation. Incidence was not associated with agricultural activities. A distinct campylobacteriosis pattern with consistently higher urban versus rural incidence was observed, which may help formulate further preventive measures.

Antimicrobial peptides often show broad- spectrum activity due to a mechanism based on bacterial membrane disruption, which also reduces development of permanent resistance, a desirable characteristic in view of the escalating multidrug resistance problem. Host cell toxicity however requires design of artificial variants of natural AMPs to increase selectivity and reduce side effects. Kiadins were designed using rules obtained from natural peptides active against E. coli and a validated computational algorithm based on a training set of such peptides, followed by rational conformational alterations. In vitro activity, tested against ESKAPE strains (ATCC and clinical isolates), revealed a varied activity spectrum and cytotoxicity that only in part correlated with conformational flexibility. Peptides with a higher proportion of Gly were generally less potent and caused less bacterial membrane alteration, as observed by flow cytometry and AFM, which correlate to structural characteristics as observed by circular dichroism spectroscopy and predicted by molecular dynamics calculations.

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. Patient's 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.

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.

Antimicrobial peptides (AMPs) are promising candidates for new antibiotic classes but often display an unacceptably high toxicity towards human cells. A naturally produced C-terminal fragment of PGLa, named PGLa-H, has been reported to have a very low haemolytic activity while maintaining a moderate antibacterial activity. A sequential tandem repeat of this fragment, diPGLa-H, was designed, as well as an analogue with a Val to Gly substitution at a key position. These peptides showed markedly improved in vitro bacteriostatic and bactericidal activity against both reference strains and multidrug resistant clinical isolates of Gram-negative and Gram-positive pathogens, with generally low toxicity for human cells as assessed by haemolysis, cell viability, and DNA damage assays. The glycine substitution analogue, kiadin, had a slightly better antibacterial activity and reduced haemolytic activity, which may correlate with an increased flexibility of its helical structure, as deduced using molecular dynamics simulations. These peptides may serve as useful lead compounds for developing anti-infective agents against resistant Gram-negative and Gram-positive species. Copyright A 2016 Elsevier B.V. All rights reserved.

Geotrichum capitatum can cause infections in humans and its importance has recently been recognized in patients with immunosuppressive conditions. In this report we present the first Croatian pediatric patient with pneumonia in whom G. capitatum was isolated in three sequential bronchoalveolar lavage and tracheal aspiration specimens., Geotrichum capitatum može uzrokovati infekcije u ljudi i njegov značaj je nedavno prepoznat u imunosuprimiranih bolesnika. U ovom radu prikazujemo prvog hrvatskog pedijatrijskog bolesnika s pneumonijom u kojeg je G. capitatum izoliran u tri uzastopna bronhoalveolarna lavata i aspirata traheje.

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.

Acinetobacter baumannii is an emerging nosocomial pathogen with also emerging resistance to different antibiotics. Multidrug and pan drug-resistant clinical isolates were reported worldwide. Here we report the first evidence of pan drug-resistant environmental isolate of A. baumannii. The isolate was recovered from the effluent of secondary treated municipal wastewater of the City of Zagreb, Croatia. The isolate was resistant to penicillins/b-lactamase inhibitors, carbapenems, fluoroquinolones, aminoglycosides, folate pathway inhibitors, and polymyxins, except intermediately susceptible to minocycline and tigecycline. Intrinsic chromosomally located blaOXA-51-like gene and acquired plasmid-located blaOXA-23-like gene were related to clinical isolates. Pan drug-resistant A. baumannii can occur in natural environments outside of the hospital. Secondary treated municipal wastewater represents a potential epidemiological reservoir of pan drug-resistant A. baumannii and carbapenem resistance gene.

Aim To evaluate, in vitro antimicrobial ability of two probiotic toothpastes (one containing Lactobacillus paracasei, other containing Lactobacillus acidophilus) and one toothpaste without probiotic separately, and in a combination with two different mouthrinses (one containing essential oils and the other containing hexitidine). Methods Antimicrobial susceptibility was checked by using the ditch method and Clinical Laboratory Standard Institute (CLSI) guidelines. Two different toothpastes with probiotic, toothpaste without probiotic and two different mouthrinses were tested against the following selected microorganisms: Candida albicans, Candida tropicalis, Enterococcus faecalis, Streptococcus salivarius and Staphylococcus aureus. Kruskal-Wallis test and MannWhitney U test were used for the statistical analysis (p≤ 0.05). Results Probiotic toothpastes had better inhibitory effect than toothpaste without probiotic in the case of Candida albicans (p=0.043) and Streptococcus salivarius (p=0.043). In all cases, toothpastes had stronger inhibition capacity than mouthrinses (p≤0.05). Conclusion Probiotic toothpastes, as a relatively new concept in the prevention of oral infectious diseases such as caries and periodontal disease, can contribute to the prevention of oral infectious diseases.

The aim of the study was to characterize ESBL-producing uropathogenic Escherichia coli (UPEC) strains isolated in children. That included the investigation of virulence factors and the analysis of the types of β-lactamases at the molecular genetic level.During the 2-year study period, 77 ESBL-producing E. coli strains were recovered from urine samples of febrile children with significant bacteriuria hospitalized at one Croatian hospital. Susceptibility of isolates to bactericidal serum activity was tested by Shiller and Hatch method, while adhesin expression was determined by agglutination methods. Characterization of ESBLs was performed by PCR with specific primers for ESBLs and by sequencing of bla (ESBL) genes. Genotyping of the E. coli isolates was performed by pulsed-field gel electrophoresis (PFGE).Twenty-seven (35.1 %) and 50 (64.9 %) ESBL-producing UPEC strains were isolated in neonates and infants, respectively. Of 70 strains investigated for the presence of virulence factors, adhesins were detected in 48.6 % strains (8.6 % in the neonate and 40 % in the infants group) giving a statistically significant difference in adhesin expression between the two groups (p 0.01). Hemolysin was produced by 84.3 %, whereas 70 % of strains were serum-resistant. The bla (TEM) gene was detected in 22 (28 %) and bla (SHV) gene in 57 strains (74 %), whereas bla (CTX-M) gene was detected in only two isolates (2.5%). In ten isolates, bla (TEM) and bla (SHV) were simultaneously detected. Sequencing of bla (SHV) genes revealed that SHV-5 β-lactamase was by far the most prevalent and was found in 51 strains (66 %). The strains were clonally related as demonstrated by PFGE and assigned into ten clusters.Infection control measures should be employed and the consumption of expanded-spectrum cephalosporins in the hospital should be restricted.

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.

Although cooking with wine and consumption of wine as a warm beverage is widespread, antibacterial effects of thermally treated wine have not been studied. We examined in vitro antibacterial activity of wine heated at 75 and 125 °C for 45 min against Salmonella enterica serovar Enteritidis and Escherichia coli. Their effects were compared with intact red wine, dealcoholized wine (DW) and dealcoholized wine reconstituted (RDW) with water to the initial volume. Samples were also analysed for their phenolics content, antioxidant capacity, resveratrol and ethanol content and pH. Total phenolics concentration and related antioxidative activity followed changes in samples volume, regardless of treatment type, while pH of all samples remained stable and ranged from 3.09 to 3.24. The order of the antibacterial activity of wine samples was: intact wine > heated at 75 °C > heated at 125 °C > DW > RDW. Antibacterial activity of the samples could not be related to their content of resveratrol as a single phenolics compound, antioxidative capacity or pH. Thermally treated wine under conditions applicable to food processing in everyday life, may be effective antibacterials in spite of significant heat-induced changes in their physical–chemical composition.

CTX-M-producing Escherichia coli isolates from three Croatian hospitals were analyzed. All bla CTX-M-15 genes and one bla CTX-M-3a gene resided in widely spread IS Ecp1 transposition modules, but other bla CTX-M-3a genes were in a new configuration with two IS 26 copies, indicating a new event of gene mobilization from a Kluyvera ascorbata genome. The study confirmed the role of the E. coli ST131 clonal group with IncFII-type plasmids in the spread of bla CTX-M-15 and of IncL/M pCTX-M3-type plasmids in the dissemination of bla CTX-M-3a .

The aim of this study was to compare the sensitivity and specificity of six different beta-lactam antibiotics using five phenotypical tests for detection of extended spectrum beta-lactamases (ESBLs) based on synergism of beta-lactam antibiotics and clavulanate. Experiments were performed on a set of 80 Klebsiella pneumoniae strains and 105 Escherichia coli strains with previously characterized ESBLs (SHV, TEM and CTX-M). ESBLs were detected by five different phenotypical methods: MIC (minimum inhibitory concentration) determination of beta-lactam antibiotics with and without clavulanate, double-disk synergy test (DDST), inhibitor-potentiated disk-diffusion test (IPDDT), CLSI-Clinical and Laboratory Standard Institution (former NCCLS) combined-disk-test, and modified MAST-disk-diffusion test (MAST-DD-test). Seven antibiotics were tested as indicators of ESBL production: ceftazidime, cefotaxime, ceftriaxone, aztreonam, ceftibuten, cefpodoxime and cefepime. Ceftazidime and aztreonam were the best indicators for SHV-5, SHV-12 and TEM beta-lactamases whereas cefotaxime and ceftriaxone were the most sensitive in detection of SHV-2 and CTX-M beta-lactamases in DDST, IPDDT and CLSI test. MIC determination of beta-lactam antibiotics with and without clavulanate was the most sensitive method. DDST was the least sensitive test. Double-disk synergy test, which is the most frequently used test for detection of ESBLs in routine laboratories, was the least sensitive independently of the indicator antibiotic. Since MIC determination is a very laborious and time consuming method, we would recommend the NCCLS combined disk test or IPDD test for detection of ESBLs in routine laboratories with 5 mm zone augmentation breakpoint.

Five clinical Escherichia coli isolates from University Hospital Split, Croatia, with a CTX-M resistance phenotype (high level of resistance to cefotaxime and ceftriaxone) were screened for blaCTX-M alleles by PCR and confirmed to produce a group 1 CTX-M enzyme. ESBL phenotype was transferred by conjugation from 3 isolates to a rifampicin-resistant recipient strain. PFGE proved that two of these five strains were clonally related. This is the first report from Croatia of CTX-M type enzyme. Carbapenems could be recommended as antibiotics of choice for the treatment of infections caused by our CTX-M producing E. coli isolates.

Childhood tuberculosis is a unique disease regarding epidemiological and clinical features. The most effective measures to combat the spread of the disease are rapid detection and prompt and effective treatment. As most of the children have negative sputum microscopy, rapid diagnosis can be challenging. In this paper, the data on incidence, treatment, diagnostic and clinical characteristics of children treated for tuberculosis in the University Hospital Centre Split during 22 years are presented.

Acinetobacter baumannii is an emerging hospital pathogen. Whereas A. baumannii isolated from patients or hospitals has been reported, there are few data regarding propagation of viable A. baumannii in the natural environment. This study investigates the occurrence and antimicrobial susceptibility of viable A. baumannii in municipal wastewater and its persistence through the wastewater treatment process. A total of 21 A. baumannii isolates were recovered at a secondary type of municipal wastewater treatment plant in Zagreb, Croatia: 15 from raw influent wastewater and six from final effluent. All isolates were carbapenem- and multidrug-resistant. Among 14 isolates tested for bla OXA genes, all harboured the constitutive bla OXA-51-like gene, while the acquired bla OXA-23-like and bla OXA-40-like genes were found in 10 and three isolates respectively. Six A. baumannii isolates recovered from effluent wastewater multiplied and survived in sterilised effluent wastewater up to 50 days. These findings support the idea that multidrug-resistant A. baumannii can occur and have the ability to survive in the environment.

Tuberkuloza u dječjoj dobi posebna je bolest po svojim epidemiološkim i kliničkim osobitostima. Najučinkovitija mjera u suzbijanju širenja bolesti jest brzo otkrivanje te neodgodivo i učinkovito liječenje oboljelih. Kako najveći dio djece ima mikroskopski negativne iskašljaje, brzo postavljanje dijagnoze može biti problem. U ovom radu prikazali smo učestalost, liječenje, dijagnostiku i kliničke osobitosti djece liječene u KBC-u Split tijekom 22 godine., Childhood tuberculosis is a unique disease regarding epidemiological and clinical features. The most effective measures to combat the spread of the disease are rapid detection and prompt and effective treatment. As most of the children have negative sputum microscopy, rapid diagnosis can be challenging. In this paper, the data on incidence, treatment, diagnostic and clinical characteristics of children treated for tuberculosis in the University Hospital Centre Split during 22 years are presented.

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).

Acinetobacter baumannii is an important cause of nosocomial infections such as pneumonia, septicaemia, urinary tract infections, and wound infections 1. It may become resistant to a wide range of antibiotics, thus complicating the treatment of nosocomial infections 1. The emergence of carbapenem resistance in Acinetobacter baumannii has become a global concern since these β -lactams are often the only effective treatment left against many multiresistant strains . According to a recent report, the overall resistance rate to imipenem in A. baumannii isolated from 17 representative laboratories in Croatia was 1 % (range 0%-8%) . However, molecular basis of carbapenem resistance was not investigated yet. We would like to report the molecular epidemiology of carbapenem resistant strains obtained from clinical samples of several patients hospitalised on different intensive care units inside University Hospital Split, Croatia. Eleven isolates of A. baumannii with an unusual resistance profile were isolated during 2003 and 2004, from patients hospitalised in three different intensive care units (two adults and one children ICU) on different locations inside UHS. Routine susceptibility testing was done by standard disc diffusion method according to the National Committee for Clinical Laboratory Standards guidelines. All collected isolates of A. baumannii displayed intermediate (MICs>8 mg/L) or resistant (MICs>16 mg/L) profile to imipenem and/or meropenem. Minimum inhibitory concentrations were also determined for ceftazidime, cefepime, ceftriaxone, amikacin, gentamicin, ciprofloxacin and piperacillin-tazobactam by broth microdilution method according to CLSI recommendation.. All isolates were multidrug-resistant exhibiting highly resistance to ceftazidime, cefepime, piperacillin/tazobactam, amikacin, gentamicin, netilmicin and ciprofloxacin. No resistance to ampicillin/sulbactam was observed which is not in use in our hospital. The isolates were genetically characterized with pulsed-field gel electrophoresis (PFGE). The preparation of genomic DNA of A. baumannii isolates was performed as previously described. Oxacillinases were detected by using a standard PCR technique with primers specific for blaOXA-23, blaOXA-40, blaOXA-58, and blaOXA-69 genes. Production of metallo-β -lactamases was screened by E test-MBL and PCR with primers specific for VIM and IMP β -lactamases. Β -lactamases were prepared from overnight broth cultures, released by sonication and cell debris was removed by centrifugation. PCRs were positive for the OXA-69 like enzyme in ten Acinetobacter strains belonging to unique PFGE profile characterised to pulsotype A, while strain characterised to pusotype B was the only strain isolated from children intensive care unit and carbapenemase gene was not found with tested primers. No significant hydrolysis of imipenem was detected. Acccording to the bibliographical data OXA-69 is a naturally occurring oxacillinase in A. baumannii and it does not confer on the producing isolates significant resistance to carbapenems, thus it can be concluded that resistance to imipenem and meropenem in our isolates is due to other mechanisms such as porin loss or efflux. Spread of the pulsotype A with OXA-69-type oxacillinases was due to clonal dissemination of multi-drug resistant A. baumannii between two different adult Intensive Care Units, probably by hospital staff during medical procedures. The infection control team of the hospital implemented restriction of carbapenem usage and strict antiseptic techniques, which included the rigorous use of alcohol-clorhexidine solutions before and between patient and equipment contact and before leaving the unit. To our knowledge, this is the first report on the molecular characterization carbapenem resistance in A. baumannii in Croatia. A similar finding underscores the progressive emergence of these determinants in different geographic areas. Consequently, incidence and spread of multidrug-resistant A. baumannii nosocomial infections suggest the necessity of a surveillance program and enforcing adequate control measures in different hospital settings

Antibiotic resistance of Helicobacter pylori to clarithromycin and azithromycin in Croatia is an emerging problem for eradication treatment. The present study emphasizes the need for susceptibility determination of Helicobacter pylori isolates before beginning eradication treatment especially in women. The continuous monitoring of H. pylori antimicrobial susceptibility is also necessary.

Although most ingested foreign bodies usually pass through the gastrointestinal tract asymptomatically, toothpick injury to the gastrointestinal tract is often associated with significant morbidity and mortality. Toothpick perforation of the gastrointestinal tract is frequently reported but, to the best of our knowledge, bacteremia caused by an impacted toothpick within the gastric mucosa has not yet been described. Here, we report the case of bacteremia caused by an accidentally swallowed toothpick. The toothpick was impacted deeply in the gastric mucosa and was first seen and localized on contrast-enhanced computed tomography (CT). CT scan is a very useful imaging technique in such situations since we lack typical and relevant physical findings or laboratory studies that go with accidentally swallowed objects, in this case a toothpick. Flexible endoscopy was successful in extracting the whole toothpick. In cases without free perforation, flexible endoscopy is the treatment of choice in toothpick removal from the upper gastrointestinal tract.

While the antimicrobial effectiveness of wine is well documented, relative contributions of the wine components to its antimicrobial activity is controversial. To separate the role of wine phenolics, ethanol, and pH from other wine constituents, the antimicrobial effects of intact wine were compared to that of phenols-stripped wine, dealcoholized wine, ethanol, and low pH applied separately and in combination, against 2 common foodborne pathogens, Salmonella enterica serovar Enteritidis and Escherichia coli. All samples were biochemically characterized with respect to their total phenolics and resveratrol content, antioxidant capacity, ethanol content, and pH. Antioxidative activity of the samples corresponded to their total phenolics content. Except for respective controls, pH and ethanol content were similar in all samples. The order of antibacterial activity of the samples was: intact wine > phenols-stripped wine > dealcoholized wine > combination of ethanol and low pH > low pH > ethanol. Separate application of ethanol or low pH showed negligible antibacterial activity while their combination showed synergistic effect. Antibacterial activity of the samples could not be related to their total phenolics and resveratrol content, antioxidant capacity, ethanol content, or pH. Our study indicates that antimicrobial activity of complex solutions such as intact wine cannot be exclusively attributed to its phenolic or nonphenolic constituents, nor can the antimicrobial activity of wine be predicted on the basis of its particular components.

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 broadspectrum 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 beta-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 beta-lactamases. Plasmid-mediated AmpC beta-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 Gram-negative 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.

The prevalence of IgG antibodies reactive with Toxoplasma gondii in the female population of the County of Split Dalmatia was investigated by enzyme linked immunosorbent assay. Of a total of 1109 serum samples collected from female subjects, 423 (38.1%) reacted with T. gondii. The frequency of positive sera increased with age. Theoretical incidence of congenital toxoplasmosis was calculated from the annual increase in cumulative prevalence of antibodies between different age groups among the women of childbearing age. The estimated theoretical incidence of congenital toxoplasmosis was 1.4 per 100 pregnancies of adolescents (16-20 years) and decreased to 0.1% in seronegative pregnant women aged 41-45.