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, β-lactamase production and plasmid content. The isolates demonstrated uniform resistance to amoxicillin/clavulanate, piperacillin/tazobactam, cefuroxime, ceftazidime, cefotaxime, ceftriaxone and ertapenem. Among novel β-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 β-lactam antibiotics: bla_CTX-M-15, bla_TEM, bla_SHV, bla_OXA-1, bla_OXA-2, bla_OXA-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.

K. pneumoniae harbors various antibiotic resistance determinants like extended-spectrum and plasmid-mediated AmpC β-lactamases and carbapenemases. In the last three years, in the period of intense population aging, migrations and climate changes in Europe and Croatia as well, we observed changes in antibiotic resistance patters of carbapenem-resistant K. pneumoniae (CRKP) isolates obtained routinely in community and inpatient setting. The aim was to compare and subsequently analyze CRKP hospital and community isolates resistance mechanisms, traits and molecular epidemiology, in order to analyze the dynamic of resistance trends, carbapenemase types and plasmid epidemiology. Disk diffusion and broth dilution method were the methods of choice to determine antibiotic susceptibility. β-lactamases were screened by phenotypic methods and confirmed with PCR. In total 113 isolates were analysed. Resistance to amoxicillin-clavulanate and ertapenem was confirmed in all strains. High resistance rates (over 90%) were observed for extended-spectrum cephalosporins, and ciprofloxacin. OKNV (OXA-48, KPC, NDM, VIM) testing and PCR detected OXA-48 in 106, NDM in 7 and KPC in only one isolate. ESBLs accompanied carbapenemases in 103 isolates. IncL, associated with OXA-48, was the dominant plasmid type. No significant differences in the resistance profile and resistance determinants were found between hospital and community isolates plasmid type. The predominance of OXA-48 carbapenemase is in line with the reports from the neigbouring countries.

Bloodstream infections associated with AmpC-producing Enterobacterales are severe medical conditions which, without prompt and effective treatment, may have dire ramifications. This study aimed to assess whether certain comorbidities and previous surgical procedures coincide with resistance determinants of AmpC-producing Enterobacterales associated with bloodstream infections. Antibiotic resistance patterns and therapy outcome were also determined. The patients’ data obtained revealed that the prevalence of recent surgical procedures, solid organ tumors, metabolic diseases, kidney and liver failure, and hematological malignancies do not differ between resistant and susceptible isolates of AmpC-producing Enterobacterales. Furthermore, no difference was reported in mortality rates. Regarding antibiotic resistance, 34.52% of isolates were confirmed to be resistant (AmpC hyperproduction, ESBL, or carbapenemase). More than one in five AmpC hyperproducers were reported amid Providencia spp., K. aerogenes, E. cloacae, and C. freundii. strains. Carbapenemases were mostly noted in Providencia spp. followed by M. morganii and K. aerogenes strains. Serratia marcescens had the highest proportion of ESBLsof ESBLs. Resistance to expanded-spectrum cephalosporins of Providencia spp. and K. aerogenes strains exceeded 50%, and resistance to meropenem over 10% was observed only in C. freundii strains. Enterobacterales’ ever-growing resistance to antibiotics is becoming quite a challenge for clinicians and new treatment options are required.

The COVID-19 pandemic has underscored the intricate interplay between viral infections and opportunistic bacterial pathogens such as Klebsiella pneumoniae. K. pneumoniae harbors various antibiotic resistance determinants like extended-spectrum and plasmid-mediated AmpC β-lactamases and carbapenemases. In the two years following the pandemic, carbapenemase producing K. pneumoniae isolates obtained routinely in outpatient and inpatient setting demonstrated similar resistance patterns which prompted research. The aim was to compare and subsequently analyze carbapenem-resistant hospital and outpatient’s K. pneumoniae isolates’ resistance mechanisms, traits and molecular epidemiology, following the COVID-19 pandemic. Disk diffusion and broth dilution method were the methods of choice to determine antibiotic susceptibility. β-lactamases were screened by phenotypic methods and confirmed with PCR. In total 113 isolates were analysed. Resistance to amoxicillin-clavulanate and ertapenem was confirmed in all strains. High resistance rates (over 90%) were observed for extended-spectrum cephalosporins, and ciprofloxacin. OKNV testing and PCR detected OXA-48 in 106, NDM in 7 and KPC in only one isolate. ESBLs accompanied carbapenemases in 103 isolates. Carbapenem resistance in K. pneumoniae is becoming a global problem, hence careful monitoring of its resistance traits is of utmost importance in order to combat the arising threat. OXA-48 seems to continue predominating in the turbulent period after pandemic.

Background/objectives: A rise in infections associated with carbapenem-resistant Providencia species (CRPS) has been observed worldwide. This study presents a genomic analysis of CRPS isolates from four hospitals in Croatia and the outpatient setting, in order to determine the extent of the spread of CRPS in Croatia. In the present study, we applied a combination of phenotypic characterization and molecular analysis of resistance traits to determine the mechanisms and the routes of spread of CRPS. Material and methods: The antibiotic susceptibility testing was performed using disk-diffusion and broth dilution methods. The nature of extended-spectrum β-lactamases (ESBLs), carbapenemases, and fluoroquinolone resistance determinants was investigated by polymerase chain reaction (PCR). In order to obtain an insight into the whole resistome, selected isolates were subjected to the Interarray Genotyping Kit CarbaResist and whole genome sequencing (WGS). Results: In total, 30 isolates were collected from four centers, located in different geographic regions of Croatia. There was uniform resistance to piperacillin-tazobactam, cefuroxime, expanded-spectrum cephalosporins (ESCs), imipenem, ertapenem, meropenem, and ciprofloxacin. Immunochromatographic testing and PCR revealed OXA-48 and NDM carbapenemase in 15 isolates, respectively. Phenotypic tests for ESBLs were positive in all OXA-48 and one NDM-positive organism (16 isolates). The isolates were categorized as extensively drug-resistant (XDR). OXA-48-producing isolates were susceptible only to ceftazidime-avibactam, whereas NDM producers were susceptible to cefiderocol and, in the majority of cases, also to amikacin. WGS identified a plethora of genes encoding resistance to aminoglycosides, such as aadA1 and aadA2, (aph(3″)-Ib and aph(6)-Id, sulfonamides sul1 and sul2, trimethoprim dfrA1, dfrA10, and dfrA12, tetracyclines tet(A) and tet(B), and chloramphenicol catA3 and catA5. Conclusions: Providencia spp., in spite of being a rare pathogen, should be included in the surveillance studies across the medical centers in Croatia.