Molecular characterization of colistin resistance genes in a high-risk ST101/KPC-2 clone of Klebsiella pneumoniae in a University Hospital of Split, Croatia.

Klebsiella pneumoniae carbapenemase-producing K. pneumoniae (KPC-KP) has become a major concern worldwide due to multidrug resistance and the ability to spread locally and globally. Infections caused by KPC-KP are great challenge in the healthcare systems because these are associated with longer hospitalization and high mortality. The emergence of colistin resistance has significantly reduced already limited treatment options. This study describes the molecular background of colistin-resistant KPC-KP isolates in the largest hospital in southern Croatia. Thirty-four non-duplicate colistin-resistant KPC-KP isolates were collected during routine work from April 2019 to January 2020 and from February to May 2021. Antimicrobial susceptibility was determined using disk diffusion, broth microdilution, and the gradient strip method. Carbapenemase was detected with an immunochromatographic test. Identification of blaKPC and mcr genes or mutations in pmrA, pmrB, mgrB, phoP, and phoQ genes were performed by polymerase chain reaction (PCR) and positive products were sequenced. Pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) were used for epidemiological analysis. All isolates were multidrug-resistant, with colistin minimum inhibitory concentrations (MICs) from 4 to >16 mg/L, and all harbored blaKPC-2 and had a single point mutation in the mgrB gene resulting in a premature stop codon, with the exception of one isolate with four point mutations corresponding to stop codons. All isolates were negative for mcr genes. PFGE analysis identified a single genetic cluster, and MLST revealed that all isolates belonged to sequence type 101 (ST101). These results show emergence of the high-risk ST101/KPC-2 clone of K. pneumoniae in Croatia as well as appearance of colistin resistance due to mutations in the mgrB gene. Molecular analysis of epidemiology and possible resistance mechanisms are important to develop further strategies to combat such threats.

Molecular Characterization and Mutational Analysis of Clarithromycin- and Levofloxacin-Resistance Genes in Helicobacter pylori from Gastric Biopsies in Southern Croatia.

Point mutations in the 23S rRNA, gyrA, and gyrB genes can confer resistance to clarithromycin (CAM) and levofloxacin (LVX) by altering target sites or protein structure, thereby reducing the efficacy of standard antibiotics in the treatment of Helicobacter pylori infections. Considering the confirmed primary CAM and LVX resistance in H. pylori infected patients from southern Croatia, we performed a molecular genetic analysis of three target genes (23S rRNA, gyrA, and gyrB) by PCR and sequencing, together with computational molecular docking analysis. In the CAM-resistant isolates, the mutation sites in the 23S rRNA gene were A2142C, A2142G, and A2143G. In addition, the mutations D91G and D91N in GyrA and N481E and R484K in GyrB were associated with resistance to LVX. Molecular docking analyses revealed that mutant H. pylori strains with resistance-related mutations exhibited a lower susceptibility to CAM and LVX compared with wild-type strains due to significant differences in non-covalent interactions (e.g., hydrogen bonds, ionic interactions) leading to destabilized antibiotic-protein binding, ultimately resulting in antibiotic resistance. Dual resistance to CAM and LVX was found, indicating the successful evolution of H. pylori resistance to unrelated antimicrobials and thus an increased risk to human health.

Measles cases in Split-Dalmatia County (a Croatian tourist region), in May-July 2019: outbreak report and lessons learnt.

BACKGROUND: Measles elimination was accomplished in Croatia in 2016. Split-Dalmatia County, with population of ca. 425 000 inhabitants, is among the most important Croatian tourist areas with numerous seasonal workers coming during summer months. In both 2018 and 2019, more than 3 million tourists visited this county. In 2000-2018, there were no measles cases in this county, or their number was low (1-3 cases per year). METHODS: After measles was clinically suspected, all contacts were traced and contacted. Detection of specific IgM/IgG antibodies and real-time reverse transcription-polymerase chain reaction detection of viral RNA were used for laboratory confirmation. Sequencing and genotyping were performed for strains' molecular epidemiology analysis. RESULTS: Six epidemiologically unlinked measles virus occurrences happened in Split-Dalmatia County in 15 May-19 July 2019. Causative viral strains belonged to genotypes B3 and D8. Four were single imported cases. Ten patients belonged to two separate clusters within domicile population. Multiple individual and public health measures were implemented. In total, 483 contacts were identified, 64.2% within healthcare system where two persons contracted the disease. CONCLUSIONS: Besides the importance of timely vaccination of children, the lessons learned from this outbreak point to the need of stricter implementation of other aspects of Croatian measles prevention programme, such as checking of vaccination status in early adulthood. Despite the fact that measles elimination within domicile population in this tourist region has been accomplished and maintained for years, continuous public health workers' efforts are still necessary for identification and diminishment of populational pockets of susceptibility.

A RARE PRESENTATION OF GENITOURINARY TUBERCULOSIS MIMICKING ABDOMINAL TUMOR.

Genital tuberculosis is a rare and unexpected disease in European countries including Croatia. Diagnosis of female genital tract tuberculosis is challenging and is rarely pin-pointed by clinical symptoms because of their low specificity. The authors decided to present a case of genitourinary tuberculosis in a young, immunocompetent fertile woman with high clinical suspicion of abdominal tumor mass. Although considered a desease of the past, rare clinical presentation of genital tuberculosis should be expected and taken into account.

Vancomycin-resistant Enterococcus faecium COLONIZATION and Clostridium difficile infection in a HEMATOLOGIC patient.

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.

A six-year epidemiological surveillance study in Split-Dalmatia County, Croatia: urban versus rural differences in human campylobacteriosis incidence.

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.

PGLa-H tandem-repeat peptides active against multidrug resistant clinical bacterial isolates.

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.

Evaluation of PNA FISH® Yeast Traffic Light in identification of Candida species from blood and non-blood culture specimens.

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.

Carbapenem-resistant isolates of Acinetobacter baumannii in a municipal wastewater treatment plant, Croatia, 2014.

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 blaOXA genes, all harboured the constitutive blaOXA-51-like gene, while the acquired blaOXA-23-like and blaOXA-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.

Prompt diagnosis and effective treatment of Trichosporon asahii catheter-related infection in non-immunocompromised neurosurgical patient.

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.

Antimicrobial susceptibility of clinically isolated anaerobic bacteria in a University Hospital Centre Split, Croatia in 2013.

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, ß-lactam/ß-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.

[Characteristics of children tuberculosis in University Hospital Centre Split from 1990 to 2012]. / Osobitosti djecje tuberkuloze u Klinickome Bolnickom Centru Split u Razdoblju od 1990. Do 2012.

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.

First clinical and microbiological characterization of Clostridium difficile infection in a Croatian University Hospital.

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).

Prolonged watery diarrhea and malnutrition caused by Aliarcobacter butzleri (formerly Arcobacter butzleri): the first pediatric case in Croatia and a literature review.

Aliarcobacter butzleri (formerly Arcobacter butzleri), is a newly recognized Campylobacter-like emerging foodborne pathogen worldwide, usually causing gastrointestinal symptoms in young children. A 4-year-old boy was admitted to the Department of Pediatrics, University Hospital of Split, Croatia, because of malnutrition, lost appetite and prolonged watery diarrhea. A comprehensive diagnostics, including biochemistry, haematology, allergology, microbiology and radiology, were performed. The only positive microbiology result was unexpected isolation of Aliarcobacter butzleri on selective media for Campylobacter, after 48 hours of incubation on 42°C, among microaerophilic atmosphere. Clinical course was favorable and after symptomatic therapy child was discharged in good clinical condition and normal peristalsis to home care, with the recommendation of taking high-protein preparations to improve nutritional status. In addition, we performed a literature review of clinical cases caused by Aliarcobacter butzleri infection. The first report of Aliarcobacter butzleri isolated from stool sample in a 4-year old boy in Croatia, along with other clinical reports in literature, highlights the importance of standardisation and improvement of microbiological analysis, especially implementation of new methods for the identification of emerging pathogens.

Phenotypic and Molecular Characterization of a Hospital Outbreak Clonal Lineage of Salmonella enterica Subspecies enterica serovar Mikawasima Containing blaTEM-1B and blaSHV-2 That Emerged on a Neonatal Ward, During the COVID-19 Pandemic.

Nontyphoid salmonella can cause severe infections in newborns and is therefore declared a pathogen of major health significance at this age. The aim of the study was molecular and antimicrobial characterization of ß-lactamase-producing Salmonella Mikawasima outbreak clone on a Neonatal ward, University Hospital of Split (UHS), Croatia during the COVID-19 pandemic. From April 2020, until April 2023, 75 nonrepetitive strains of Salmonella Mikawasima were isolated from stool specimens and tested for antimicrobial resistance. All 75 isolates were resistant to ampicillin and gentamicin, while 98% of isolates were resistant to amoxicillin/clavulanic acid. A high level of resistance was observed to third-generation cephalosporins (36% to ceftriaxone and 47% to ceftazidime). Extended-spectrum ß-lactamase production was phenotypically detected by double-disk synergy test in 40% of isolates. Moderate resistance to quinolones was detected; 7% of isolates were resistant to pefloxacin and ciprofloxacin. All isolates were susceptible to carbapenems, chloramphenicol, and co-trimoxazole. Fourteen representative isolates, from 2020, 2021, 2022, and 2023, were analyzed with PFGE and all of them belong to the same clone. Whole-genome sequencing (WGS) analysis of three outbreak-related strains (SM1 and SM2 from 2020 and SM3 from 2023) confirmed that these strains share the same serotype (Mikawasima), multilocus sequence typing profile (ST2030), resistance genes [blaTEM-1B, aac(6')-Iaa, aac(6')-Im, and aph(2'')-Ib)] and carry incompatibility group C (IncC) plasmid. Furthermore, the gene blaSHV-2 was detected in SM1 and SM2. In summary, WGS analysis of three representative strains clearly demonstrates the persistence of ß-lactamase-producing Salmonella Mikawasima in UHS during the 4-year period.

Microbiome profiling and characterization of virulent and vancomycin-resistant Enterococcus faecium from treated and untreated wastewater, beach water and clinical sources.

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 genes, 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 gene. 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.

Molecular characterisation of colistin and carbapenem-resistant clinical isolates of Acinetobacter baumannii from Southeast Europe.

OBJECTIVES: To characterise 11 colistin- and carbapenem-resistant Acinetobacter baumannii isolates recently emerging in hospital settings. METHODS: A. baumannii isolates were collected from hospitalised patients under colistin treatment in three countries of Southeast Europe: Turkey, Croatia, and Bosnia and Herzegovina. Isolates were identified using molecular methods. RESULTS: Isolates from Turkey and Croatia belong to the sequence types ST195 or ST281 of the clone lineage 2, while the single isolate from Bosnia and Herzegovina belongs to the ST231 of clone lineage 1. All isolates turned out to be highly resistant to colistin (MIC ≥ 16 mg/L) and have point mutations in pmrCAB operon genes. The colistin-resistant isolate from Bosnia and Herzegovina had a unique P170L point mutation in the pmrB gene and the R125H point mutation in the pmrC gene. The L20S mutation in the pmrA gene was detected only in isolates from Croatia and has never been reported before in isolates from this country. CONCLUSION: Colistin resistance in A. baumannii in hospitalised patients receiving colistin treatment is a result of chromosomal mutations. The pattern of point mutations in pmrCAB genes suggests a spread of specific colistin-resistant isolates within the hospital.

Epidemiology and diagnosis of Helicobacter pylori infection.

Medline, PubMed and the Cochrane databases were searched on epidemiology and diagnosis of Helicobacter pylori for the period of April 2011-March 2012. Several studies have shown that the prevalence of H. pylori infection is decreasing in adults and children in many countries. Various diagnostic tests are available, and most of them have high sensitivity and specificity. The Maastricht IV/Florence consensus report states that the urea breath test using (13)C urea remains the best test to diagnose H. pylori infection. Among the stool antigen tests, the ELISA monoclonal antibody test is recommended. All these tests were used, either as a single diagnostic test or in combination, to investigate H. pylori infection among different populations throughout the world. Of particular interest, current improvements in high-resolution endoscopic technologies enable increased diagnostic accuracy for the detection of H. pylori infection, but none of these techniques, at present, are specific enough for obtaining a real-time diagnosis of H. pylori infection.

Submarine Outfalls of Treated Wastewater Effluents are Sources of Extensively- and Multidrug-Resistant KPC- and OXA-48-Producing Enterobacteriaceae in Coastal Marine Environment.

The rapid and ongoing spread of carbapenemase-producing Enterobacteriaceae has led to a global health threat. However, a limited number of studies have addressed this problem in the marine environment. We investigated their emergence in the coastal waters of the central Adriatic Sea (Croatia), which are recipients of submarine effluents from two wastewater treatment plants. Fifteen KPC-producing Enterobacteriaceae (nine Escherichia coli, four Klebsiella pneumoniae and two Citrobacter freundii) were recovered, and susceptibility testing to 14 antimicrobials from 10 classes showed that four isolates were extensively drug resistant (XDR) and two were resistant to colistin. After ERIC and BOX-PCR typing, eight isolates were selected for whole genome sequencing. The E. coli isolates belonged to serotype O21:H27 and sequence type (ST) 2795, while K. pneumoniae isolates were assigned to STs 37 and 534. Large-scale genome analysis revealed an arsenal of 137 genes conferring resistance to 19 antimicrobial drug classes, 35 genes associated with virulence, and 20 plasmid replicons. The isolates simultaneously carried 43-90 genes encoding for antibiotic resistance, while four isolates co-harbored carbapenemase genes bla KPC-2 and bla OXA-48. The bla OXA-48 was associated with IncL-type plasmids in E. coli and K. pneumoniae. Importantly, the bla KPC-2 in four E. coli isolates was located on ~40 kb IncP6 broad-host-range plasmids which recently emerged as bla KPC-2 vesicles, providing first report of these bla KPC-2-bearing resistance plasmids circulating in E. coli in Europe. This study also represents the first evidence of XDR and potentially virulent strains of KPC-producing E. coli in coastal waters and the co-occurrence of bla KPC-2 and bla OXA-48 carbapenemase genes in this species. The leakage of these strains through submarine effluents into coastal waters is of concern, indicating a reservoir of this infectious threat in the marine environment.

Molecular Characterization of ß-Lactam Resistance and Antimicrobial Susceptibility to Possible Therapeutic Options of AmpC-Producing Multidrug-Resistant Proteus mirabilis in a University Hospital of Split, Croatia.

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.