Evolution of Beta-Lactamases in Urinary Klebsiella pneumoniae Isolates from Croatia; from Extended-Spectrum Beta-Lactamases to Carbapenemases and Colistin Resistance.

K. pneumoniae isolates often harbor various antibiotic resistance determinants including extended-spectrum ß-lactamases (ESBLs), plasmid-mediated AmpC ß-lactamases (p-Amp-C) and carbapenemases. In this study we analyzed 65 K. pneumoniae isolates obtained from urinary tract infections in the outpatients setting, with regard to antibiotic susceptibility, ß-lactamase production, virulence traits and plasmid content.Antibiotic susceptibility was determined by broth microdilution method. PCR was applied to detect genes encoding ESBLs, p-Amp-C and carbapenemases and plasmid incompatibility groups. Phenotypic methods were applied to characterize virulence determinants. Increasing resistance trend was observed for amoxicillin/clavulanate, imipenem, meropenem and ciprofloxacin. The study showed that ESBLs belonging to the CTX-M family, conferring high level of resistance to expanded-spectrum cephalosporins (ESC) were the dominant resistance trait among early isolates (2013 to 2016) whereas OXA-48 carbapenemase, belonging to class D, emerged in significant numbers after 2017. OXA-48 producing organisms coharbored ESBLs. KPC-2 was dominant among isolates from Dubrovnik in the recent years. Colistin resistance was reported in three isolates. Inc L/M was the dominant plasmid in the later period, encoding OXA-48. Hyperviscosity was linked to KPC positivity and emerged in the later period. This report describes evolution of antibiotic resistance in K. pneumoniae from ESBLs to carbapenemases and colistin resistance. The study demonstrated the ability of K. pneumoniae to acquire various resistance determinants, over time. The striking diversity of the UTI isolates could result from introduction of the isolates from the hospitals, transfer of plasmids and multidirectional evolution.

Management of Cardiac Patients in Epidemic Outbreak.

In times of COVID-19 epidemic/pandemic, cardiac patients are vulnerable group with many specific conditions that could aggravate their condition. In this narrative review, we present possible measures adequate in managing cardiac patients in epidemic outbreak. An overview of the role of cardiologists and Crisis Management Team in management of cardiac patients is given. Protocols and measures implemented in COVID-19 crises are presented in light of risk assessment and disease prevention of cardiac patients and measures that should be taken for each cardiac condition. Specificity of epidemics calls for specific measures in addressing cardiac patients as part of the affected population. Many possible outcomes could be expected in an epidemic outbreak in relation to cardiovascular diseases, but tailored measures will keep cardiac patients safe. Proposed preventive measures for cardiac patients could be implemented in existing protocols for epidemic outbreak.

Management of Measurable Variable Cardiovascular Disease' Risk Factors.

AIM: To summarize the main findings on variable cardiovascular risk factors and their management in everyday practice. METHODS: A narrative review of the relevant literature known to the authors and incorporation of healthy changes tips in defined variable cardiovascular risk factors. RESULTS: There are known variable cardiovascular risk factors to be claimed as those that should be changed in order to achieve a better prevention of cardiovascular disease development. But, most papers are informative and they didn't incorporate exact measures for each variable risk factor. Our paper shows exact measures for each variable cardiovascular risk factor that should be incorporate in everyday practice of family practitioners and cardiologists as well. CONCLUSION: The best cardiovascular disease' prevention should include a multidisciplinary team of experts and the entire community with the support of governmental and non-governmental organizations that will contribute to improving the lifestyle of individuals and the entire community through their activities and legal provisions. The most important factors in cardiovascular disease management are: recognizing individual risk factors, monitoring them, and assisting in changes in life-style habits that directly affect the defined risk factors of a patient. The simplest and most practicable guidelines for CV prevention in accordance with the national, cultural and socioeconomic aspects of their country of work are needed.

COMPARISON OF TWO DIFFERENT METHODS FOR TIGECYCLINE SUSCEPTIBILITY TESTING IN ACINETOBACTER BAUMANNII.

- Tigecycline susceptibility testing (TST) presents a tremendous challenge for clinical microbiologists. Previous studies have shown that the Epsilometer test (E-test) and Vitek 2 automated system significantly overestimate the minimum inhibitory concentrations for tigecycline resistance compared to the broth microdilution method (BMM). This leads to very major errors or false susceptibility (i.e. the isolate is called susceptible when it is actually resistant). The aim of this study was to compare E-test against BMM for TST in carbapenem-resistant and carbapenem-susceptible Acinetobacter (A.) baumannii and to analyze changes in tigecycline susceptibility between two time periods (2009-2012 and 2013-2014), with BMM as the gold standard. Using the EUCAST criteria, the rate of resistance to tigecycline for the OXA-23 MBL-positive, OXA-23 MBL-negative and carbapenemase-negative strains for BMM was 54.5% (6/11), 29.4% (5/17) and 2.7% (1/37), respectively; the OXA-24/40 and OXA-58 producing organisms did not exhibit any resistance. With E-test, all OXA-23 MBL-positive organisms (11/11), 23.5% (4/17) of OXA-23 MBL-negative, and 4.1% of OXA-24/40 (3/74) strains displayed tigecycline resistance; there were no resistant strains among the OXA-58 and carbapenemase-negative isolates. Resistance emerged in the bacterial isolates from 2013 to 2014. Although tigecycline does not display cross-resistance, the highest rates of resistant A. baumannii isolates were observed among those producing VIM MBL, regardless of the testing method. These findings suggest that the commercial E-test does not provide reliable results for TST of A. baumannii. Further confirmation with the dilution method should be recommended, particularly in cases of serious infections.

Molecular characterization of class b carbapenemases in advanced stage of dissemination and emergence of class d carbapenemases in Enterobacteriaceae from Croatia.

Carbapenemases involved in acquired carbapenem resistance in Enterobacteriaceae belong to Ambler class A serin ß-lactamases, class B metallo-ß-lactamases (MBL) or class D OXA-48-like ß-lactamases. The aim of the present study was to analyse the molecular epidemiology and the mechanisms and routes of spread of class B and class D carbapenemases in Croatia. In total 68 isolates were analyzed. Antibiotic susceptibility was determined by broth microdilution method. PCR was used to detect antibiotic-resistance genes. Genotyping was performed by rep-PCR and MLST. Sixty-five isolates were found to harbour VIM-1 carbapenemase, seven of which were positive also for NDM-1, while two strains harboured only NDM-1. OXA-48 was detected in three isolates, two of which coproduced VIM-1. Thirty-six strains possessed additional CTX-M-15 ß-lactamase whereas 64 were positive for TEM-1. CMY was found in 18 Citrobacter freundii isolates and DHA-1 in one Enterobacter cloacae isolate. Four different plasmid-incompatibility groups were found: A/C, L/M, N and FIIAs. Unlike C. freundii and E. cloacae, Klebsiella pneumoniae showed high diversity of rep-PCR patterns. E. cloacae and C. freundii predominantly belonged to one large clone which was allocated to ST105 and ST24, respectively. Three different types of carbapenemases were identified showing the complexity of CRE in Croatia.

[EVOLUTION OF BETA-LACTAM ANTIBIOTIC RESISTANCE IN ENTEROBACTER SPP. IN CROATIA].

Enterobacter spp. develops resistance to expanded-spectrum cephalosporins by induction or derepression of chromosomal AmpC ß-lactamase, or production of extended-spectrum ß-lactamases (ESBLs) or carbapenemases. The aim of the study was to analyze the mechanisms of resistance to expanded-spectrum cephalosporins and the evolution of resistance mechanism during the study period (2008­2011) on a collection of 58 randomly collected Enterobacter spp. strains from three hospital centers in Croatia and Bosnia and Herzegovina during 2008-2010. The antibiotic susceptibility was determined by broth microdilution method according to CLSI. Resistance genes were determined by PCR. Plasmids were characterized by PCR-based replicon typing (PBRT). The hypothesis of the study was that there will be multiple mechanisms of ceftazidime resistance involved, from inducible and derepressed AmpC ß-lactamases to extended-spectrum ß-lactamases and carbapenemases at the end of the study. The isolates from different centers were expected to express different phenotypes and mechanisms of resistance. The study showed the predominance of derepressed AmpC ß-lactamases combined with ESBLs belonging to CTX-M family as a mechanism of resistance to expanded-spectrum cephalosporins. The emergency of MBLs was reported in the last year of the study in University Hospital Center Zagreb. The plasmids encoding ESBLs belonged to different incompatibility groups. This points out to the evolution of ß-lactam resistance in Enterobacter spp. from derepressed AmpC ß-lactamases and ESBL to carbapenemases.

Antibiotic susceptibility of isolates from paediatric intensive care units in Zagreb.

AIM: Meropenem Yearly Susceptibility Test Information Collection (MYSTIC) Program is a longitudinal global surveillance study to monitor in vitro data on microbial susceptibility in centers that prescribe meropenem. Results of the six years period (2002-2007) for the antimicrobial efficacy of meropenem compared to other broad-spectrum agents against Gram-negative and Gram-positive isolates collected at pediatric intensive care units of the University Hospital Center Zagreb in Croatia were reported. METHODS: A total of 110 Gram-negative and 43 Gram-positive pathogens from pediatric specimens were tested. The minimum-inhibitory concentrations (MICs) were determined by broth microdilution method according to CLSI. RESULTS: There was no resistance to either imipenem or meropenem observed for Escherichia coli, Klebsiella pneumoniae and Proteus mirabilis. High resistance rates of K. pneumoniae to ceftazidime and gentamicin (50%) are a raising concern. Pseudomonas aeruginosa was the most resistant Gram-negative species with two (12%) of the strains resistant to meropenem, three (18%) to imipenem, 10 (47%) to gentamicin and six (35%) to piperacillin/tazobactam and ciprofloxacin. According to our results meropenem remains an appropriate antibiotic for the treatment of severe infections caused by Gram-negative bacteria in pediatric population. CONCLUSIONS: The results indicate that meropenem has excellent potency and spectrum of activity despite being prescribed for a long time for the treatment of seriously ill patients, and still appears to be a reliable option for the initial empirical therapy of serious nosocomial infections in children. However, later studies have shown the emergence of carbapenem-resistant Gram-negative bacteria after 2008.