Emergence and Spread of Enterobacterales with Multiple Carbapenemases after COVID-19 Pandemic.

Resistance to carbapenems in Enterobacterales has become a matter of the highest concern in the last decade. Recently, Enterobacterales harboring multiple carbapenemases were detected in three hospital centers in Croatia and in the outpatient setting, posing a serious therapeutic challenge for clinicians. In this study, we analyzed eight Klebsiella pneumoniae and two Enterobacter cloacae complex isolates with multiple carbapenemases, with regard to antibiotic susceptibility, ß-lactamase production and plasmid content. The isolates demonstrated uniform resistance to amoxicillin/clavulanate, piperacillin/tazobactam, cefuroxime, ceftazidime, cefotaxime, ceftriaxone and ertapenem. Among novel ß-lactam/inhibitor combinations, ceftazidime/avibactam exhibited moderate activity, with 50% of isolates susceptible. All isolates demonstrated resistance to imipenem/cilastatin/relebactam, and all but one to ceftolozane/tazobactam. Four isolates exhibited a multidrug-resistant phenotype (MDR), whereas six were allocated to an extensively drug-resistant phenotype (XDR). OKNV detected three combinations of carbapenemases: OXA-48+NDM (five isolates), OXA-48+VIM (three isolates) and OXA-48+KPC (two isolates). Inter-array testing identified a wide variety of resistance genes for ß-lactam antibiotics: blaCTX-M-15, blaTEM, blaSHV, blaOXA-1, blaOXA-2, blaOXA-9, aminoglycosides: aac6, aad, rmt, arm and aph, fluoroquinolones: qnrA, qnrB and qnrS, sulphonamides: sul1 and sul2 and trimethoprim: dfrA5, dfrA7, dfrA14, dfrA17 and dfrA19. mcr genes were reported for the first time in Croatia. This study demonstrated the ability of K. pneumoniae and E. cloacae to acquire various resistance determinants under the selection pressure of antibiotics widely used during the COVID-19 pandemic. The novel inter-array method showed good correlation with OKNV and PCR, although some discrepancies were found.

BUILDING THE COVID-19 TESTING CAPACITIES IN CROATIA: ESTABLISHING THE INTERDEPARTMENTAL COVID-19 UNIT AT THE SPLIT UNIVERSITY HOSPITAL CENTRE.

This paper presents the chronology, experiences, and challenges in introducing COVID-19 RT-PCR testing in Split, Croatia. We describe the processes from March 12, 2020 to May 26, 2020, starting from the initial knowledge transfer, expert team formation and management, testing implementation, and concluding with the standalone testing facilities, which used automated processes sufficient to meet testing requirements at that time. In the case presented, the COVID-19 unit was organized by joining human and laboratory resources from five clinical departments at the Split University Hospital Centre. Sample preparation procedures and analyses were launched within the restricted time frame while simultaneously training and organizing new laboratory staff and completing equipment requirements. As a result, the process that started with 30 tests per day was constantly improved over time and reached up to 160 tests per day when MagNA Pure was added to automatize RNA extraction at the end of April. At that pace, the cumulative number of samples soon exceeded the first thousand, and by the end of May it exceeded 4000. The case presented provides an example of good practice for crisis response and organization that successfully enabled sufficient COVID-19 testing capacities within the restricted time frame, human and technical resources. Despite limited understanding of COVID-19 at that time, appropriate management, transfer of knowledge, previous experiences in related laboratory and diagnostic work, as well as interdisciplinary and interdepartmental cooperation proved appropriate to overcome the above limitations and ensure adequate healthcare response.

Polyclonal spread of colistin resistant Klebsiella pneumoniae in Croatian hospitals and outpatient setting.

INTRODUCTION: Recently, a marked increase in the rate of colistin resistant Klebsiella pneumoniae was observed in Croatian hospitals and the outpatient setting. This prompted us to analyze the molecular epidemiology of these isolates and the mechanisms of spread. METHODS: In total 46 colistin-resistant K. pneumoniae isolates from five hospitals and the community were analyzed. The presence of genes encoding broad and extended-spectrum ß-lactamases, plasmid-mediated AmpC ß-lactamases and carbapenemases was determined by PCR. Plasmids were characterized by PCR based replicon typing. Isolates were genotyped by pulsed-field gel electrophoresis. Virulence traits such as hemolysins, hyperviscosity and resistance to serum bactericidal activity were determined by phenotypic methods. RESULTS: High resistance rates were observed for cefuroxime, ceftazidime, cefotaxime, ceftriaxone and ertapenem, ciprofloxacin and gentamicin. The majority of OXA-48 producing isolates were resistant to ertapenem but susceptible to imipenem and meropenem. Nine strains transferred ertapenem resistance to E. coli recipient strain. Thirty-nine strains were phenotypically positive for ESBLs and harbored group 1 of CTX-M ß-lactamases. OXA-48 was detected in 39 isolates, KPC-2 in four and NDM-1 in one isolate. The isolates belonged to six PFGE clusters. All isolates were found to be resistant to serum bactericidal activity and all except four strains positive for KPC, produced ß-hemolysins. String test indicating hypermucosity was positive in only one KPC producing organism. CONCLUSIONS: The study demonstrated the ability of K. pneumoniae to accumulate different resistance and virulence determinants. We reported dissemination of colistin resistant K. pneumoniae in five hospitals, located in different geographic regions of Croatia and in the outpatients setting. mcr genes responsible for transferable colistin resistance were not found, indicating that resistance was probably due to chromosomal mutations.

Klebsiella pneumoniae carbapenemase (KPC) in urinary infection isolates.

Recently, emergence of carbapenem-resistance, in particular due to Klebsiella pneumoniae carbapenemase (KPC), was observed among K. pneumoniae causing urinary tract infections in Croatia. The aim of the study was to characterize, antimicrobial susceptibility, carbapenem resistance, virulence traits and plasmid types of the urinary KPC positive isolates of K. pneumoniae. The antimicrobial susceptibility to a wide range of antibiotics was determined by broth microdilution method. The transferability of meropenem resistance was determined by conjugation (broth mating method) employing Escherichia coli J63 strain resistant to sodium azide. Genes encoding broad and extended-spectrum ß-lactamases, plasmid-mediated AmpC ß-lactamases, group A and B carbapenemases, and carbapenem hydrolyzing oxacillinases (blaOXA-48like), respectively, were determined by Polymerase chain reaction (PCR). In total 30 KPC-positive K. pneumoniae urinary isolates collected from different regions of Croatia were analysed. The isolates were uniformly resistant to all tested antibiotics except for variable susceptibility to gentamicin, sulphamethoxazole/trimethoprim, and colistin, respectively. Four isolates were resistant to colistin with MICs values ranging from 4 to 16 mg/L. All tested isolates were susceptible to ceftazidime/avibactam. Sixteen isolates transferred meropenem resistance to E. coli recipient strain by conjugation. Other resistance markers were not co-transferred. PCR was positive for blaKPC and blaSHV genes in all isolates whereas 13 isolates tested positive also for blaTEM genes. PCR based replicon typing (PBRT) revealed the presence of FIIs in 13 and FIA plasmid in two strains. The study showed dissemination of KPC-producing K. pneumoniae in urinary isolates, posing a new epidemiological and treatment challenge. Sulphamethoxazole/trimethoprim, colistin, and ceftazidime/avibactam remain so far, as the therapeutic options.

Distribution of hepatitis C virus genotypes and subtypes in Croatia: 2008-2015.

OBJECTIVE: Hepatitis C virus (HCV) genotyping is an important part of pre-treatment diagnostic algorithms as it guides the choice of therapeutic regimens. The aim of this study was to analyse the distribution of HCV genotypes in patients with chronic hepatitis C from Croatia in the period 2008-2015. METHODS: The study enrolled 3,655 anti-HCV positive patients with available results of HCV genotyping from the three largest national HCV genotyping laboratories. RESULTS: The majority of HCV-infected individuals enrolled in the study were male (70.7%). Analysis of age distribution in a subset of 2,164 individuals showed a mean age of 40.9 years (SD 11.77 years). Croatian patients were mostly infected with HCV genotype 1 (56.6%), followed by genotype 3 (37.3%), genotype 4 (4.2%) and genotype 2 (1.8%). Genotype 1 subtyping in a subset of 1,488 patients showed 54% (803/1,488) of 1b infections and 46% (685/1,488) of 1a infections. Percentages of genotype 1 were the highest in Central/Northwestern and Eastern Croatia and the lowest in the Central/Southern Adriatic Region. Genotype 3 was most frequently found in the Central/Southern Adriatic Region (49.1%) but represented only 17.5% of infections in Eastern Croatia (p < 0.001). CONCLUSIONS: The results of this nine-year retrospective analysis on the distribution of HCV genotypes and subtypes in 3,655 HCV-infected individuals from Croatia showed that the majority of infections can be attributed to genotypes 1 and 3 with absence of major changes in the molecular epidemiology of the two most frequent HCV genotypes infection in Croatia in the past 20 years.

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

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

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.

[Carbapenemases of gram-negative bacteria]. / Karbapenemaze gram-negativnih bakterija.

Carbapenems are often antibiotics of last resort for the treatment of severe infections. They are stable to most beta-lactamases produced by gram-negative bacteria. However, bacterial enzymes named carbapenemases can efficiently hydrolyze carbapenems. They are produced most frequently by Enterobacteriaceae and non-fermentative bacteria such as Pseudomonas aeruginosa and Acinetobacter baumannnii. They belong to group A (KPC, SME, IMI, NMC), B (VIM, IMP, SPM, GIM, NDM, SIM, DIM, AIM) and D (OXA-23, OXA-24, OXA-48, OXA-58, OXA-143). The accurate and rapid laboratory identification of carbapenem-resistant isolates is important to prevent spread of such multidrug resistant strains and to avoid therapeutic failures. Therapeutic options are often limited because carbapenemases are encoded on mobile genetic elements which often harbour resistance genes to other groups of antibiotics. Thus, colistin is often the only therapeutic option.

Microbial ethanol production in postmortem urine sample.

We present a case in which postmortem blood ethanol concentration was 0.02 g/kg and acetone concentration was 0.51 g/kg, while urine ethanol concentration was 6.0 g/kg and acetone concentration was 0.63 g/kg. In the urine sample, sodium fluoride was not added. The urinary ethanol concentration continued to increase without any remarkable increase of isopropanol concentration and external contamination was excluded. Species of bacteria and yeasts, including Candida glabrata, were isolated from urine and blood samples. A few days after the collection of samples, we received the information that the patient was diabetic and did not receive insulin therapy regularly. To prevent postmortem microbial ethanol production and incorrect diagnosis of the cause of death, it is necessary to add sodium fluoride to blood and urine samples collected from diabetic patients.

Spread of CTX-M-15 positive Providencia spp. causing urinary tract infections at the University Hospital Split in Croatia.

AIM: During 2010-2011, six Providencia spp. (five Providencia stuartii and one Providencia rettgeri) urine isolates with unusual resistance phenotype were collected from various hospital units at the University Hospital Split in Croatia. The aim of the study was to analyze the mechanisms of resistance to expanded-spectrum cephalosporins. METHODS: The antimicrobial susceptibility to a wide range of antibiotics was determined by broth microdilution method according to CLSI guidelines. A double-disk-synergy test (DDST) was performed to detect ESBLs. The transferability of cefotaxime resistance was determined by conjugation. The presence of genes encoding ESBLs was determined by PCR while genotyping of the isolates was performed by PFGE. RESULTS: All strains were positive for ESBL production by DDST. They were uniformly resistant to amoxycillin alone and combined with clavulanate, cefazoline, cefuroxime, ceftazidime, cefotaxime, ceftriaxone, gentamicin and ciprofloxacin. P. stuartii strains transferred cefotaxime resistance to E. coli recipient strain with frequency ranging from 10-5 to 5x10-4. Five P. stuartii strains were positive for TEM and CTX-M ß-lactamases while P. rettgeri was positive only for TEM ß-lactamases. Five CTX-M producing isolates were shown to be clonally related. CONCLUSIONS: Continuous surveillance in tracking CTX-M-15- producing P. stuartii in the hospitals is necessary to prevent their spread to other hospitals and community. Global spread of ESBL positive Providencia spp all over the world is of great clinical concern.

Infrequent finding of metallo-ß-lactamase VIM-2 in carbapenem-resistant Pseudomonas aeruginosa strains from Croatia.

One hundred sixty-nine nonreplicate imipenem-resistant Pseudomonas aeruginosa strains isolated in a large hospital on the coastal region of Croatia were studied. The most active antibiotics were colistin and amikacin. Most of the isolates were multiresistant. The most prevalent serotype was O12, followed by O11. Six strains carried the bla(VIM-2) gene located in a novel class 1 integron composed in its variable part of the bla(VIM-2)-bla(oxa-10)-ΔqacF-aacA4 genes. Metallo-ß-lactamase-producing strains belonged to sequence types ST235 and ST111.

[ISKRA guidelines for antimicrobial prophylaxis in surgery--Croatian National Guidelines]. / Smjernice ISKRA za antimikrobnu profilaksu u kirurgiji--Hrvatske Nacionalne Smjernice.

Summary. The development of the Guidelines for perioperative prophylactic use of antimicrobial agents (further on 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 a consensus between all involved professional societies. Guidelines were composed in order to improve antibiotic use in surgical professions. Data obtained from observational studies have shown that the use of antimicrobials in surgical professions is unsatisfactory, and since around 50% of all prescribed drugs in surgical professions refer to perioperative prophylaxis, such guidelines could significantly improve current negative trend and reduce the occurrence of infections in surgical patients as well as slow down the selection of resistant bacteria. In the introductory part of the guidelines, principles of perioperative prophylaxis are presented. The advantages and risks of prophylaxis are listed as well as factors that determine prophylaxis effectiveness. For easier orientation, surgical professions have been divided into basic surgical fields. In each field, the specificity of the field has been described followed by uniform structured tables and with every listed surgical procedure there is the most probable cause of infection, the drug of choice for prophylaxis, alternative drug, remark for particular surgical procedure and finally the grade of recommendation. The Guidelines do not cover perioperative prophylaxis in immunocompromised patients nor perioperative prophylaxis in children. The Guidelines do not cover all possible surgical interventions, but can be used as a basis for most surgical procedures performed in our hospitals. At the very end of these Guidelines, a comprehensive list of references enables all those interested to find further information and details about this topic. The revision of the Guidelines is planned in three years' time.

Emergence of Proteus mirabilis isolates producing TEM-52 extended-spectrum beta-lactamases in Croatia.

BACKGROUND: An increased frequency of extended-spectrum beta-lactamase (ESBL)-positive Proteus mirabilis isolates was observed recently in the Clinical Hospital Center Split in Croatia. The aim of this study was the molecular characterization of ESBLs in P. mirabilis isolates from this hospital. MATERIAL AND METHODS: Seven strains showing reduced susceptibility to ceftazidime were investigated. Antimicrobial susceptibility was determined using the broth microdilution method. ESBLs were characterized by PCR and sequencing of bla(ESBL) genes. Quinolone resistance determinants (qnr genes) were characterized by PCR. Genotyping of strains was performed by pulsed-field gel electrophoresis (PFGE). RESULTS: The presence of an ESBL was confirmed in all strains by a double-disk synergy test. All strains were resistant to amoxicillin, piperacillin, gentamicin, ciprofloxacin, chloramphenicol, sulfamethoxazole and trimethoprim, but susceptible to ceftazidime/clavulanic acid, piperacillin/tazobactam, cefoxitin, imipenem and meropenem; PCR sequencing using primers targeting bla(ESBL) genes revealed TEM-52 beta-lactamase. PFGE genotyping demonstrated the clonal relatedness of TEM-52-producing P. mirabilis strains isolated from different clinical samples and wards within the hospital. Bla(TEM-52) in 3 isolates was carried by a 70-kb conjugative plasmid. CONCLUSIONS: Our findings indicate the emergence of the TEM-52 enzyme among P. mirabilis in Croatia.

Late ventriculoperitoneal shunt infection caused by Shewanella algae.

We present a case of ventriculitis and peritonitis in a child with ventriculoperitoneal shunt, which occurred 5 years after the surgery. The infection developed after contact with seawater and began as otitis. For the first time, Shewanella algae, a marine microorganism, was identified as the cause of ventriculoperitoneal shunt infection.

First isolation of Rickettsia conorii from human blood in Croatia.

AIM: To detect and isolate rickettsial strains from blood samples of patients with presumptive diagnosis of Mediterranean spotted fever (MSF) in the coastal region of south Croatia, and to compare the results with routine serology. METHODS: A "suicide" polymerase chain reaction (PCR), and a shell vial culture were done on samples of ethylenediamine tetra-acetic acid (EDTA) and citrate-anticoagulated blood samples. Indirect immunofluorescence was performed on sera collected from 17 patients clinically diagnosed with MSF during summer in three consecutive years, from 1998 to 2000. RESULTS: The primers used in PCR amplified the expected part of the rickettsia genomic DNA and Rickettsia conorii grew from the shell vial-cultured blood of a single patient. In 13 patients, the diagnosis was confirmed serologically by paired sera, whereas in 4 patients the diagnosis remained presumptive, since no paired sera were available. Analyzing sequences of the ompA and citrate synthase gene, respectively, derived from the shell vial isolate, a 100% similarity with Rickettsia conorii, strain Seven (Malish), was found. CONCLUSION: To the best of our knowledge, this is the first isolation of Rickettsia conorii from a human sample in Croatia, and the first proof of a causative agent of MSF in the country. Beside PCR-based methods and isolation, correct diagnosis of MSF could be still routinely reached by serology.

[Occurrence of Toxoplasma gondii antibodies in the population of Split-Dalmatia County]. / Ucestalost protutijela za Toxoplasmu gondii u populaciji splitsko-dalmatinske zupanije.

The prevalence of antibodies reactive with Toxoplasma gondii in the population of the Split-Dalmatia county (southern Croatia) was investigated by enzyme linked immunosorbent assay (ELISA). Of a total of 1464 serum samples collected from persons aged 2-84 years 36.4% reacted with Toxoplasma gondii. The frequency of positive sera increased significantly with age (p < 0.001). Prevalence of specific antibodies to T. gondii does not vary significantly according to gender, except, among children younger than 10 years (boys 14.0%; girls 28.2%; p < 0.01). Of a total of 398 pregnant women tested 31.4% were seropositive. According to increase in seroprevalence among the pregnant women of different age the theoretical incidence of congenital toxoplasmosis was calculated.