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Introduction: The aims of study were to assess: 1) performance specifications of Atellica 1500, 2) comparability of Atellica 1500 and Iris, 3) the accuracy of both analysers in their ability to detect bacteria., Materials and Methods: Carryover, linearity, precision, reproducibility, and limit of blank (LoB) verification were evaluated for erythrocyte and leukocyte counts. ICSH 2014 protocol was used for estimation of carryover, CLSI EP15-A3 for precision, and CLSI EP17 for LoB verification. Comparison for quantitative parameters was evaluated by Bland-Altman plot and Passing-Bablok regression. Qualitative parameters were evaluated by Weighted kappa analysis. Sixty-five urine samples were randomly selected and sent for urine culture which was used as reference method to determine the accuracy of bacteria detection by analysers., Results: Analytical specifications of Atellica 1500 were successfully verified. Total of 393 samples were used for qualitative comparison, while 269 for sediment urinalysis. Bland-Altman analysis showed statistically significant proportional bias for erythrocytes and leukocytes. Passing-Bablok analysis for leukocytes pointed to significant constant and minor proportional difference, while it was not performed for erythrocytes due to significant data deviation from linearity. Kappa analysis resulted in the strongest agreements for pH, ketones, glucose concentrations and leukocytes, while the poorest agreement for bacteria. The sensitivity and specificity of bacteria detection were: 91 (59-100)% and 76 (66-87)% for Atellica 1500 and 46 (17-77)% and 96 (87-100)% for Iris., Conclusion: There are large differences between Atellica 1500 and Iris analysers, due to which they are not comparable and can not be used interchangeably. While there was no difference in specificity of bacteria detection, Iris analyser had greater sensitivity., Competing Interests: Potential conflict of interest None declared., (Croatian Society of Medical Biochemistry and Laboratory Medicine.)

Here, we report a retrospective study conducted to elucidate emergence, epidemiology, and molecular mechanisms of resistance underlying the early spread of OXA-48 carbapenemase-producing Enterobacteriaceae in Croatia. Retrospective screening for OXA-48 producers was performed on a collection of 296 nonrepetitive, carbapenem-nonsusceptible enterobacterial isolates collected from January 2011 to December 2012 from 40 participating centers in Croatia. Antimicrobial susceptibility profiles and production of carbapenemases were assessed phenotypically. Pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing were used for epidemiological analysis. Resistance genes were characterized by polymerase chain reaction (PCR) and sequencing. Plasmid localization of bla OXA-48 in isolates and transconjugants was investigated by S1-PFGE and Southern hybridization. PCR mapping was used for identification of genetic platform surrounding bla OXA-48 . Out of 296 carbapenem-nonsusceptible isolates, bla OXA-48 gene was detected in 12 Klebsiella pneumoniae isolates. All OXA-48-producing isolates showed varying resistance to carbapenems and 11 were multidrug resistant. All coproduced additional beta-lactamases, including CTX-M-15, which was detected in eight isolates. Isolates were delineated in five clonal types by PFGE corresponding to five sequence types (STs) assigned ST15, ST16, ST37, ST528, and ST1418. All OXA-48 isolates conjugated successfully and other resistance determinants were not cotransferred. bla OXA-48 was carried on a ∼60 kb IncL/M plasmid and was detected within Tn1999.2 composite transposon. OXA-48, a class D carbapenemase, is emerging as a potentially significant contributor among carbapenem-resistant Enterobacteriaceae in Croatia, alongside class A and B carbapenemases. Polyclonal genetic background of K. pneumoniae isolates carrying ∼60 kb incL/M plasmid indicates that dissemination of the bla OXA48 gene is not driven exclusively by the spread of a single clone.

Aim: To determine in vitro susceptibility of multiresistant bacterial isolates to fosfomycin., Methods: In this prospective in vitro study (local non-random sample, level of evidence 3), 288 consecutively collected multiresistant bacterial isolates from seven medical centers in Croatia were tested from February 2014 until October 2016 for susceptibility to fosfomycin and other antibiotics according to Clinical and Laboratory Standards Institute methodology. Susceptibility to fosfomycin was determined by agar dilution method, while disc diffusion was performed for in vitro testing of other antibiotics. Polymerase chain reaction and sequencing were performed for the majority of extended spectrum β-lactamase (ESBL)-producing Klebsiella pneumoniae (K. pneumoniae) and carbapenem-resistant isolates., Results: The majority of 288 multiresistant bacterial isolates (82.6%) were susceptible to fosfomycin. The 236 multiresistant Gram-negative isolates showed excellent susceptibility to fosfomycin. Susceptibility rates were as follows: Escherichia coli ESBL 97%, K. pneumoniae ESBL 80%, Enterobacter species 85.7%, Citrobacter freundii 100%, Proteus mirabilis 93%, and Pseudomonas aeruginosa 60%. Of the 52 multiresistant Gram-positive isolates, methicillin-resistant Staphylococcus aureus showed excellent susceptibility to fosfomycin (94.4%) and vancomycin-resistant enterococcus showed low susceptibility to fosfomycin (31%). Polymerase chain reaction analysis of 36/50 ESBL-producing K. pneumoniae isolates showed that majority of isolates had CTX-M-15 beta lactamase (27/36) preceded by ISEcp insertion sequence. All carbapenem-resistant Enterobacter and Citrobacter isolates had blaVIM-1 metallo-beta-lactamase gene., Conclusion: With the best in vitro activity among the tested antibiotics, fosfomycin could be an effective treatment option for infections caused by multiresistant Gram-negative and Gram-positive bacterial strains in the hospital setting.

Background: In the last few years, Klebsiella pneumoniae strains producing K. pneumoniae carbapenemase (KPC) enzymes have emerged as important multidrug-resistant pathogens in hospitalized patients. This report describes KPC-producing isolates collected through the Croatian antimicrobial resistance surveillance program in the early stage of their dissemination in Croatia., Materials and Methods: Forty-eight KPC-producing K. pneumoniae isolates, collected during a period from February 2011 to August 2013, were analyzed in this study. Antimicrobial susceptibility profiles were determined using disk diffusion and E-test. Pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) were used for epidemiological analysis. Identification of β-lactamase genes and associated antibiotic resistance mechanisms was performed by polymerase chain reaction and positive products were sequenced. Localization of bla KPC was investigated by S1 PFGE and Southern hybridization., Results: Of 40 participating centers in Croatia, KPC isolates were recorded in 9 of them. They all had multidrug-resistant phenotype, but showed varying levels of resistance to carbapenems. All isolates displayed ST258, and PFGE showed that all but one were closely related. All isolates harbored bla KPC-2 . Isolate with a unique PFGE pattern produced TEM-1, while others produced TEM-116. All isolates harbored bla SHV-11 , but were negative for bla CTX-M and bla AmpC genes. All isolates contain one KPC-harboring plasmid, ranging in size from ∼60 to ∼210 kb, characterized as FIIs and IncR., Conclusion: This report describes that the early stage of KPC-producing K. pneumoniae dissemination in Croatia is associated with a prolific PFGE type belonging to ST258. So far, the spread of an outbreak strain is limited to the northwest region of the country.

The paper reports on the emergence of strain in which Enterobacter cloacae has demonstrated an unusual form of resistance to carbapenems mediated by enzyme IMI-1, class A beta-lactamase. The strain was isolated from a wound swab in the patient who had a surgical wound infection previously treated with meropenem. Limited choice of antibiotics that can treat infections caused by these pathogens indicates the necessity of accurate identification of multiple resistant gram-negative microorganisms and mechanisms of their resistance. Recognition of multiresistant gram-negative microorganisms emphasizes the importance of continuous microbiological monitoring of patients, especially in intensive care units. In the investigated institution there was no secondary spread of this strain. Identifying new mechanisms of resistance will be helpful to clinicians in selection of targeted therapy, while important for efficient prevention of spreading infections caused by multiple resistant microorganisms.

Sore throat is most commonly caused by viruses, but when caused by bacteria, the most important is group A streptococcus (GAS). The aim of these guidelines is to determine optimal treatment for streptococcal sore throat and reasonable indications for tonsillectomy, as well as recommend how to differentiate streptococcal infection for which antibiotics are justified, from numerous other sore throats where antibiotics wont have a significant effect on disease course, but might contribute to bacterial resistance to antibiotics. The development of the guidelines was initiated by the Interdisciplinary Section for Antibiotic Resistance Control (ISKRA) of the Croatian Ministry of Health and Social Welfare in accordance with the principles of AGREE (Appraisal of Guidelines for Research and Evaluation) methodology which means that the guidelines are the result of consensus between all interested professional societies and institutions. For streptococcal sore throat diagnostics, the Working Group recommends evaluation of clinical presentation according to Centor criteria and for patients with Centor score 0-1, antibiotic therapy is not recommended nor bacteriological testing, while for patients with Centor score 2-4 bacteriological testing is recommended (rapid test or culture) as well as antibiotic therapy in case of positive result. The drug of choice for the treatment of streptococcal tonsillopharyngitis is oral penicillin taken for ten days (penicillin V) or in case of poor patient compliance benzathine penicillin G can be administered parenterally in a single dose. Other antibiotics (macrolides, clindamycin, cephalosporins, co-amoxiclav) are administered only in case of hypersensitivity to penicillin or in recurrent infections. Tonsillectomy is a widely accepted surgical procedure that decreases the number of sore throats in children and should be performed only if indications for this procedure are established. Absolute indications include five or more streptococcal infections per year, tonsillitis complications, permanent respiratory tract obstruction, obstructive sleep apnea syndrome and suspected tonsillar malignancy. Relative indications include chronic tonsillitis and occlusion disturbances.

War wounds are the most complex type of non-targeted injuries due to uncontrolled tissue damage of varied and multifold localizations, exposing sterile body areas to contamination with a huge amount of bacteria. Wound contamination is caused by both the host microflora and exogenous agents from the environment (bullets, cloth fragments, dust, dirt, water) due to destruction of the host protective barriers. War wounds are the consequence of destructive effects of various types of projectiles, which result in massive tissue devitalization, hematomas, and compromised circulation with tissue ischemia or anoxia. This environment is highly favorable for proliferation of bacteria and their invasion in the surrounding tissue over a relatively short period of time. War wounds are associated with a high risk of local and systemic infection. The infection will develop unless a timely combined treatment is undertaken, including surgical intervention within 6 hours of wounding and antibiotic therapy administered immediately or at latest in 3 hours of wound infliction. Time is a crucial factor in this type of targeted combined treatment consisting of surgical debridement, appropriate empirical antimicrobial therapy, and specific antitetanic prophylaxis. Apart from exposure factors, there are a number of predisposing factors that favor the development of polymicrobial aerobic-anaerobic infection. These are shock, pain, blood loss, hypoxia, hematomas, type and amount of traumatized tissue, age, and comorbidity factors in the wounded. The determinants that define the spectrum of etiologic agents in contaminated war wounds are: wound type, body region involved, time interval between wounding and primary surgical treatment, climate factors, season, geographical area, hygienic conditions, and patient habits. The etiologic agents of infection include gram-positive aerobic cocci, i. e. Staphylococcus spp, Streptococcus spp and Enterococcus spp, which belong to the physiological flora of the human skin and mucosa; gram-negative facultative aerobic rods; members of the family Enterobacteriacea (Escherichia coil, Proteus mirabilis, Klebsiella pneumoniae, Enterobacter cloacae), which predominate in the physiological flora of the intestines, transitory flora of the skin and environment; gram-negative bacteria, i. e. Pseudomonas aeruginosa, Serratia marcescens, Acinetobacter calcoaceticus - A. baumanii complex; environmental bacteria associated with humid environment and dust; anaerobic gram-positive sporogeneous rods Clostridium spp, gram-negative asporogeneous rods Bacteroides spp and gram-positive anaerobic cocci; Peptostreptococcus spp and Peptococcus spp. The latter usually colonize the intestine, primarily the colon, and the skin, while clostridium spores are also found in the environment. Early empirical antibiotic therapy is used instead of standard antibiotic prophylaxis. Empirical antimicrobial therapy is administered to prevent the development of systemic infection, gas gangrene, necrotizing infection of soft tissue, intoxication and death. The choice of antibiotics is determined by the presumed infective agents and localization of the wound. It is used in all types of war wounds over 5-7-10 days. The characteristics of antibiotics used in war wounds are the following: broad spectrum of activity, ability to penetrate deep into the tissue, low toxicity, long half-life, easy storage and application, and cost effectiveness. The use of antibiotics is not a substitution for surgical treatment. The expected incidence of infection, according to literature data, is 35%-40%. If the time elapsed until surgical debridement exceeds 12 hours, or the administration of antibiotics exceeds 6 hours of wound infliction, primary infection of the war wound occurs (early infection) in more than 50% of cases. The keys for the prevention of infection are prompt and thorough surgical exploration of the wound, administration of antibiotics and antitetanic prophylaxis, awareness of the probable pathogens with respect to localization of the wound, and optimal choice of antibiotics and length of their administration.

Although macrolide antibiotics have proved to be a valuable alternative to beta-lactam antibiotics in the treatment of respiratory tract infections, resistance to these agents is now becoming established in streptococci, especially among Streptococcus pneumoniae isolates. Of particular concern is the emergence of cross-resistance to 14-, 15- and 16-membered macrolides, licosamides and group B streptogramins (MLSb phenotype). MLS resistance can be expressed either constitutively (cMLS phenotype) or inducibly (iMLS phenotype). MLS resistance is mediated by two classes of methylase genes--the conventional erm(B) and recently described erm(A) determinants. A new macrolide efflux mechanism has been described for streptococci, in which it is associated with a new resistance pattern (M phenotype) characterized by resistance to 14- and 15-membered macrolides, and susceptibility to 16-membered macrolides, lincosamides and streptogramin B. The recognition of the prevalence of M phenotype in streptococci has implications for sensitivity testing and may have an impact on the choice of antibiotic therapy in clinical practice. While M resistance is similar in S. pyogenes and S. pneumoniae being mediated by mef(A) and mef(E), respectively, MLS resistance in both species appears to be genotypically and phenotypically more varied. Differentiation of M and MLS phenotypes of erythromycin-resistant strains can be performed using the erythromycin-clindamycin double-disc method (ECDD). Distinguishing not only M resistance but also constitutively or inducibly MLS phenotype by ECDD in S. pyogenes is easily and reliably achieved. Inducible MLS phenotype S. pyogenes strain is genotypically and phenotypically heterogeneous and is further subdivided into three recently described subtypes, iMLS-A, iMLS-B and iMLS-C, by a triple-disk test with erythromycin plus clindamycin and josamycin. While distinguishing M from MLS resistance in S. pneumoniae by ECDD test is easily and reliably achieved, the differentiation between constitutive and inducible MLS resistance is by far more uncertain. The meaning of inducible MLS resistance appears to be different in S. pneumoniae from that in S. pyogenes. In order to easily differentiate, within erythromycin-resistant pneumococci, not only the strains of the M phenotype from those with MLS resistance but also among the latter, cMLS from iMcLS strains, a triple-disk test has been set up by adding a rokitamycin disk to the conventional

Aim: The aim of this study was to estimate the changes in the appearance and distribution of M types of Streptococcus pyogenes in different cultures from 78 patients treated during the 1990-1996 period at the Dr. Fran Mihaljević University Hospital for Infectious Diseases in Zagreb., Materials and Methods: Isolates were characterized by the T-agglutination pattern and M type and/or opacity factor type using the standards recommended by two World Health Organization Collaborating Centers for Reference and Research on streptococci from Minneapolis and Prague., Results: In this study, 19% (15/79) of isolates were recovered from normally sterile sites, 26.6% (21/79) came from skin and 54.4% (43/79) from throat swabs. In one patient isolates from the skin and blood culture were analyzed. Of all, 92.4% (73/79) of the isolates were typed by T-agglutination pattern and 73.5% (58/79) by M protein and/or OF typing. The results of M typing showed 14 M types: M1, M3, M4, M5, M6, M11, M12, M28, M57, M58, M60, M75, M76 and M78. The most commonly isolated types were M1 and M3 (13.8%, 8/58 each), followed by M28 found in 12.1% (7/58), and M6 and M12 in 10.3% (6/58) each. These five M types accounted for 60.3% (35/58) of all isolates. Analysis to changes in the distribution of M1 and M3 types during the 1990-1991 and 1992-1993 periods revealed a significantly greater proportion of M1 and M3 isolates in the former (Fisher's two-tailed exact test, p = 0.018). A significantly greater proportion of M1 and M3 isolates was also recorded in the 1990-1991, than in 1994-1996 period. (Fisher's two-tailed exact test, P = 0.021). It was investigated whether Streptococcus pyogenes M1 and M3 types were associated with toxic and invasive infection. There were 28.2% (22/78) of patients with toxic and invasive infection: 31.9% (7/22) of them with the diagnosis of scarlet fever, whereas 68.1% (15/22) of the strains were obtained from normally sterile sites. There were 45.5% (10/22) of M1 and M3 types from patients with toxic and invasive infections. Types M6, M28 and M76 were found in an equal proportion of 9.1% (2/22), and M4, M12 and M 60 of 4.5% (1/22) each. In three strains, M type could not be identified (T8/25 SOR+, T25 SOR+ and T11 SOR+). M1 and M3 types were isolated from 10.3% (6/56) of patients with other streptococcal infections. A significantly greater proportion of M1 and M3 types was recorded in patients with toxic and invasive infections than in those with other streptococcal infections (Fisher's two-tailed exact test, p = 0.004). A multivariate logistic regression model was used to assess whether the increased proportion of streptococcal M types 1 and 3 was associated with the 1990-1991 period, or with the infection characteristics., Conclusions: The changes in the distribution of M types 1 and 3 were found to be significantly associated only with toxic and invasive infections (odds ratio 4.35, p = 0.025). Odds ratio suggests that patients with toxic and invasive infections had a 4.35--fold increased risk of infections caused by types M1 and M3 found in patients with other streptococcal infections. The increased proportion of M types 1 and 3 during the 1990-1991 period was more significantly associated with the characteristics of infection than with the study period.

The aim of the study was to reduce to key tests the 4 extensive polyvalent diagnostic biochemical tables most widely used in Croatia and to adapt them for the demonstration of Vibrio cholerae and its differentiation from the 3 Vibrios (V. alvinolyticus, V. mentschikovii, V. fluvialis) important in differential diagnosis. The fourth table has now been adapted to differentiate among all 12 Vibrio species known to be human pathogens (V. mimicus, V. cincinatiensis, V. holisae, V. damsela, V. furnisi, V. parahaemolyticus, V. vulnificus, V. carchariae). Using the inductive Learning by Logic Minimization Method (ILLM), we analyzed 2 tables (i.e. identification matrices) that were a part of bioMérieux's commercial packaged polyvalent identification systems widely used in Croatia (API 20E and ATB 32E), as well as 2 compilation tables by M. T. Kelly et al. The tables contained 27, 32, 59 and 8 tests, respectively. Cutting these solely to the key tests involved rationalizing them from 59 to the 5 necessary to differentiate Vibrio cholerae from 3 related Vibrios. Further rationalizations were from 32 to 2 and from 27 to the 3 necessary to differentiate Vibrio cholerae from 2 related Vibrios. By reducing the table of 8 tests to 7, and adding 4 new ones to these we achieved an optimization permitting mutual differentiation of all 12 known human Vibrio pathogens. Use of the selective TCBS plating medium was the only precondition for making these tables effective.

In this paper a novel computer example-based learning system (Inductive learning by logic minimization) was used to determine the sufficient set of biochemical reactions and necessary conditions that have to be fulfilled for the correct identification of enterococci isolated from human specimens. Several combinations for accurate identification of Enterococcus faecalis and Enterococcus feacium from other enterococci were found. The simplest combination set for E. faecalis identification consists of 3 reactions: growth in 0.04% K-tellurite, mannitol acidification, failure to acidify raffinose. The simplest set for E. faecium differentiation from other enterococcal species is performed by the following 3 reactions: failure to acidify D-xylose and Na-pyruvate, and acidification of L-arabinose. Only 4 tests are sufficient (acid from D-xylose, mannitol, L-arabinose and Na-pyruvate) for the correct identification of these two, the most frequently isolated species. If it is necessary to analyze all enterococcal species, then even three combinations of 9 tests for the correct differentiation of any of them, are available.

A total number of 836 episodes of bacteremia and fungemia were examined in 823 hospitalized patients in the University Hospital of Infectious Diseases "Dr Fran Mihaljević" Zagreb from the beginning of 1987 to the end of 1991. Twenty-five percent of them were nosocomial bacteremias and 5% were polymicrobial bacteremias. The most frequently isolated causative agents were Salmonella spp. (26%), Escherichia coli (17%), Streptococcus pneumoniae (11%) and Staphylococcus aureus (8%). There were 34% of gram-positive bacteremias. The increased frequency of nosocomial bacteremias caused by coagulase-negative staphylococci was recorded. The frequency of coagulase-negative staphylococci strains resistant to gentamicin and Klebsiella spp. strains resistant to cefotaxime was increased. Shock was present in 19% of episodes. Relation between septic shock occurrence and causative agent of bacteremia was not proved. Mortality in patients with bacteremia was 13%, and total mortality was 20%. The outcome of the disease was in direct relation with causative agent of bacteremia. The initial empiric antimicrobial therapy was prolonged in 91% of episodes of bacteremia after blood culture results were known.

Microorganisms referred to as Streptococcus anginosus (Streptococcus milleri) group which consist of S. anginosus, S. intermedius and S. constellatus is very difficult to identify to the species level, because of their diversity of biochemical, hemolytic and serological characteristics and because of confusion surrounding their taxonomy. Identification procedures on three isolates from primary sterile sites of three different patients, using API 20A system are described. Streptococcus intermedius was established in two and Streptococcus anginosus in one case. The importance of these organisms as human pathogens should enhance efforts of clinical microbiologist towards their accurate identification.

The results of hospital infection surveillance over an eight-month period in the Intensive Care Unit (ICU) of the University Hospital of Infectious Diseases "Dr Fran Mihaljević", Zagreb, are presented together with the results of the antibiotic resistance of isolated gram-negative bacteria in relation to the clinical material taken for culture. Of 110 strains of gram-negative bacteria isolated, 103 (93.6%) were resistant to ampicillin, 80 (72%) to gentamicin, 74 (67%) to cefotaxime, 50 (45.5%) to pefloxacin. Imipenem, ciprofloxacin, ceftazidime and amikacin were the most active representatives of their respective groups with 5, 25, 49 and 50% of resistant strains. Strains isolated in urinary tract infections were significantly less resistant to amikacin and ceftazidime (p < 0.05) than strains isolated from other sources. Our findings stress the need for close surveillance of antibiotic resistance in these selected groups of patients, and are particularly relevant for planning empirical anmtibiotic therapy of hospital infection in the ICU.