First Report of Campylobacter jejuni Strains Belonging to ST-21 Clonal Complex Isolated from Human, Poultry and Wild Birds in Croatia: Antimicrobial Resistance and Genetic Distance.

In the era of growing antimicrobial resistance, a threat affecting humans, endangering animals, as well as livelihoods and food security worldwide, we wanted to find possible explanations for its continuous spread from a new perspective. The ubiquity of resistance genes requires a One Health approach to finding the explanations for continuous AMR spread. The natural transformability of Campylobacter jejuni, its high incidence of infections, and emerging resistance worldwide inspired us to choose C. jejuni ST-21CC to be our pathogen for analyzing its contribution and connection to the cycle of AMR dissemination. ST-21CC is known as a generalist among humans and broilers, the most prevalent lineage worldwide, but it is rarely found in wild birds. Emerging in wild birds, genetic relatedness and similar resistance profiles were expected. We analyzed 23 Croatian C. jejuni strains belonging specifically to ST-21CC from humans, broilers, and wild birds. The genomic data obtained through whole genome sequencing and phenotypic susceptibility data of strains were compared. Our findings suggest high fluoroquinolone resistance in ST-21CC strains, with more diverse genetic backgrounds in wild birds. Intriguing were three isolates of ST-822 (from human and storks), sharing a similar genetic fingerprint.

Molecular characterization of colistin resistance genes in a high-risk ST101/KPC-2 clone of Klebsiella pneumoniae in a University Hospital of Split, Croatia / Caracterización molecular de genes de resistencia a la colistina en un clon ST101/KPC-2 de alto riesgo de Klebsiella pneumoniae en un Hospital Universitario de Split, Croacia

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.(AU)

Interspecies transmission of porcine-originated G4P[6] rotavirus A between pigs and humans: a synchronized spatiotemporal approach.

As a leading viral cause of acute gastroenteritis in both humans and pigs, rotavirus A (RVA) poses a potential public health concern. Although zoonotic spillover of porcine RVA strains to humans is sporadic, it has been detected worldwide. The origin of chimeric human-animal strains of RVA is closely linked to the crucial role of mixed genotypes in driving reassortment and homologous recombination, which play a major role in shaping the genetic diversity of RVA. To better understand how genetically intertwined porcine and zoonotic human-derived G4P[6] RVA strains are, the present study employed a spatiotemporal approach to whole-genome characterization of RVA strains collected during three consecutive RVA seasons in Croatia (2018-2021). Notably, sampled children under 2 years of age and weanling piglets with diarrhea were included in the study. In addition to samples tested by real-time RT-PCR, genotyping of VP7 and VP4 gene segments was conducted. The unusual genotype combinations detected in the initial screening, including three human and three porcine G4P[6] strains, were subjected to next-generation sequencing, followed by phylogenetic analysis of all gene segments, and intragenic recombination analysis. Results showed a porcine or porcine-like origin for each of the eleven gene segments in all six RVA strains. The G4P[6] RVA strains detected in children most likely resulted from porcine-to-human interspecies transmission. Furthermore, the genetic diversity of Croatian porcine and porcine-like human G4P[6] strains was propelled by reassortment events between porcine and porcine-like human G4P[6] RVA strains, along with homologous intragenotype and intergenotype recombinations in VP4, NSP1, and NSP3 segments. Described concurrent spatiotemporal approach in investigating autochthonous human and animal RVA strains is essential in drawing relevant conclusions about their phylogeographical relationship. Therefore, continuous surveillance of RVA, following the One Health principles, may provide relevant data for assessing the impact on the protectiveness of currently available vaccines.

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.

Resistome Analysis of Campylobacter jejuni Strains Isolated from Human Stool and Primary Sterile Samples in Croatia.

Campylobacteriosis represents a global health challenge due to continuously increasing trends of antimicrobial resistance in Campylobacter jejuni. C. jejuni can sometimes cause life-threatening and severe systematic infections (bacteremia, meningitis, and other extraintestinal infections) with very few antibiotics left as treatment options. Bearing in mind that C. jejuni is the predominant species in humans, in this paper, we present a study of the C. jejuni differences in antimicrobial resistance and genotype distribution between strains isolated from stool and primary sterile sites. We compared the genomic data obtained through whole genome sequencing (WGS) and phenotypic susceptibility data of C. jejuni strains. Once antimicrobial susceptibility testing of C. jejuni strains was carried out by the broth microdilution method for six of interest, results were compared to the identified genotypic determinants derived from WGS. The high rate of resistance to fluoroquinolones presented in this study is in accordance with national surveillance data. The proportion of strains with acquired resistance was 71% for ciprofloxacin and 20% for tetracycline. When invasive isolates were analysed separately, 40% exhibited MIC values of ciprofloxacin higher than the ECOFFs, suggesting a lower flouroquinolone resistance rate in invasive isolates. All isolates demonstrated wilde-type phenotype for chloramphenicol, erythromycin, gentamicin, and ertapenem. A special focus and review in this study was performed on a group of C.jejuni strains found in primary sterile samples. Apart from demonstrating a lower resistance rate, these isolates seem genetically more uniform, showing epidemiologically more homogenous patterns, which cluster to several clonal complexes, with CC49 being the most represented clonal complex.

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.

Meningitis caused by Campylobacter jejuni: a case presentation and literature review.

Objectives: Campylobacter jejuni is one of the most common causative agents of gastroenteritis; however C. jejuni meningitis is rarely described. Therefore, little is known about its epidemiology, clinical presentation, diagnostic methods, treatment, and outcomes.Methods: In this paper, we report a case of an adult patient with C. jejuni meningitis. In addition, we reviewed 16 cases of C. jejuni published since 1980.Results: We described a 62-year-old immunocompromised patient with meningitis and gastroenteritis in whom C. jejuni was rapidly detected in cerebrospinal fluid (CSF) using 16S rDNA, while blood culture yielded the same pathogen with 48 h delay. Following 21 day-long treatment with meropenem, our patient fully recovered. Literature review revealed that C. jejuni meningitis is mainly described in newborns and adults with central nervous system comorbidities and it is most frequently detected by bacterial cultures.Conclusion: There are no clear recommendations for antimicrobial treatment of C. jejuni meningitis, but meropenem seems to be a safe and effective choice. High hopes are placed in new, broad-range culture-independent molecular methods that enable rapid pathogen detection, even in case of negative cultures.

Occurrence of Campylobacter jejuni in Gulls Feeding on Zagreb Rubbish Tip, Croatia; Their Diversity and Antimicrobial Susceptibility in Perspective with Human and Broiler Isolates.

Campylobacteriosis is the most common gastrointestinal bacterial disease in the European Union (EU). Wild birds are one of the natural reservoirs of these pathogens. In this study we tested cloacal swabs of 643 gulls captured on rubbish tip in Zagreb, Croatia for the presence of Campylobacter spp. and found 168 Campylobacter positive samples. We used multilocus sequence typing (MLST) to genotype 62 random C. jejuni isolates from gulls, 24 isolates from broiler caeca, 27 isolates from broiler neck skins and 23 human isolates. Altogether, we identified 44 different STs, from which 19 were newly described. Most of the new STs (14) originate from gulls. Although humans and broilers share the majority of STs and isolates from gulls are separated from these, there was one ST present in all three hosts: 45. Additionally antimicrobial susceptibility to six antimicrobials was performed on 123 C. jejuni strains isolated from broiler caeca (n = 22), neck skins of broilers (n = 20), gulls cloacal swabs (n = 50) and human faeces (n = 31) by the broth microdilution method. Results show lower resistance of gull isolates to NAL and CIP, while resistance to TET was as high as in human and broiler isolates.

Serum levels of metalloproteinases and their inhibitors during infection with pathogens having integrin receptor-mediated cellular entry.

BACKGROUND: Matrix metalloproteinases (MMPs) are a family of proteolytic enzymes with numerous roles in the normal immune response to infection. However, excess MMP activity following infection may lead to immunopathological processes that cause tissue damage. Their activity in normal tissues is subject to tight control, which is regulated by its specific endogenous tissue inhibitors (TIMPs). It is known that MMPs bind to cell surface proteins (e.g. integrins) and that such interactions can have modulatory effects on MMP functionality. The objective of this study was to determine whether there are differences in MMP and TIMP production during the acute phase of infection with different pathogens that use ß-integrins as their receptors for cell entry. METHODS: We measured the total amounts of soluble MMP-2, MMP-9, TIMP-1, and TIMP-2 in the sera from patients infected with Dobrava virus (DOBV), Coxiella burnetii, or uropathogenic Escherichia coli. Statistical analyses were used to correlate MMP/TIMP serum levels with different clinical laboratory parameters. RESULTS: The results showed that both of the bacterial infections generally manifested the stronger effect on MMP production, while in contrast, viral infection introduced stronger changes to metalloproteinase inhibitors. MMPs and TIMPs were significantly correlated with some of the clinical laboratory parameters in both bacterial infections, but no correlations were found for DOBV infection. CONCLUSIONS: These findings suggest diverse mechanisms by which MMP activity could be implicated in the pathology of these 2 bacterial infections versus the viral DOBV infection, despite the type of their cellular entry receptors.

[Virologic and epidemiological characteristics of non-polio infection in Croatia over a ten-year period (2000-2009)]. / Non-polio enterovirusne infekcije u Hrvatskoj tijekom desetogodisnjeg razdoblja (2000.-2009.): viroloske i epidemioloske znacajke.

AIM: The aim of the present study was to show the virologic and epidemiological characteristics of non-polio enterovirus (NPEV) infection over a ten-year period in Croatia. METHODS: During the 2000-2009 period, the Laboratory for Enteroviruses, Croatian Public Health Institute analyzed 2754 clinical samples collected from 1880 patients with a clinical picture of enteroviral infection. The diagnosis of enteroviral infection was confirmed by virus isolation in cell culture. Viruses were typed using indirect immunofluorescence and/or neutralization assay. RESULTS: NPEV was proven in 394 (21%) of 1880 patients. Males were more commonly infected than females, at a ratio of 1.8:1, while the number of infected cases was highest among preschoolers and schoolchildren. Patients with isolated NPEV were most frequently diagnosed with aseptic meningitis (234/394; 59.4%). Infections were most commonly caused by echoviruses (218/394; 55.3%), followed by Coxsackie B (126/394; 32.0%) and Coxsackie A (31/394; 7.9%) viruses, rarely by echovirus 22 - parechovirus 1 (16/394; 4.1%) and enterovirus 71 (3/394; 0.8%). In most cases, echoviruses of the following serotypes were proven: 6, 9, 11, 13, 14, 18 and 30 (45/218, 21%; 14/218, 6.4%; 18/218, 8.3%; 15/218, 6.9%; 11/218, 5.0%; 55/218, 25.2% and 42/218, 19.3%), while serotypes 2, 3, 4, 5, 7, 12, 20, 21,25 and 26 were evidenced in a minority of patients or individual cases. Coxsackie B5 was the predominant serotype among Coxsackie B viruses (50/126, 40%), while Coxsackie A9 was most common Coxsackie A virus (26/31, 84%). Coxsackie B5 and B4 viruses were continually detected during the study period and appeared more frequently every four to five years. The most common echovirus isolate was echovirus 18, detected continually between 2000 and the outbreak year of 2006. Echovirus 6 and echovirus 30 were also isolated continually with peaks in 2002 and 2008, or 2002, 2006 and 2008. CONCLUSION: The results of this study pointed to a specific pattern of the occurrence of certain NPEV serotypes in Croatia. The epidemic pattern (echovirus 18 and Coxsackie 81) was characterized by peaks with an elevated number of isolations in given years. Contrary to this, endemic viruses were isolated in similar counts every year (Coxsackie B3), or manifested milder epidemic peaks every few years (echoviruses 30 and 6, Coxsackie B4 and B5). Data on NPEV infections, given their serotype and specific pattern of occurrence, contribute significantly to prompt diagnostic, clinical and epidemiological response to NPEV infections.

Zoonoses and vector-borne diseases in Croatia - a multidisciplinary approach.

Emerging and re-emerging infectious diseases create constant and serious concerns for public health. The majority of emerging infectious diseases (EID) are wildlife zoonotic diseases and vector-borne diseases. Croatia has a long tradition in the control, management and research of EID zoonotic diseases and vector-borne diseases. There has also been a long and advantageous tradition in the collaboration of different experts and professionals in EID research in Croatia involving physician clinicians in infectious diseases, microbiologists, pathologists, veterinarians and animal scientists, ecologists, forestry experts, wildlife scientists, public health specialists and epidemiologists and laboratory scientists. The University Hospital for Infectious Diseases in Zagreb established the Centre for Emerging and Re-emerging Infectious Diseases in liaison with national and international partners from Europe and the United States. This Centre is working in line with the 'One Health initiative' which recognises the inter-relationships between human, animal and environmental health.