Serotype distribution and antimicrobial resistance of invasive Streptococcus pneumoniae isolates among Croatian adults during a fifteen-year period (2005-2019).

AIM: To assess serotype distribution, antibiotic resistance, and vaccine coverage against Streptococcus pneumoniae causing invasive infections in Croatian adults from 2005 to 2019. METHODS: In this retrospective study, invasive pneumococcal strains were collected through a microbiological laboratory network with country coverage >95%. Capsular typing was performed with the Quellung reaction. In vitro susceptibility testing was carried out according to the European Committee on Antimicrobial Susceptibility Twating guidelines. In macrolide-resistant isolates, the presence of ermB and mefA genes was evaluated. RESULTS: During the fifteen-year study period, 1123 invasive pneumococcal isolates were obtained. The most prevalent serotypes were 3, 14, 19A, 9V, 7F, and 23F, comprising 60% of all invasive pneumococcal isolates. Serotype 3 was the dominant serotype, with the highest prevalence in patients ≥65 years of age. Penicillin susceptibility, increased exposure was 18.6%, mostly associated with serotypes 14 and 19A. Resistance to penicillin was low (<1%). Macrolide resistance was 23%, mostly associated with serotypes 14, 19A, and 19F. The coverage with 13-valent conjugate vaccine (PCV13) and 23-valent polysaccharide vaccine (PPV23) was 80.2% and 93.6%, respectively. CONCLUSIONS: The incidence of invasive pneumococcal disease in adults is highest in patients ≥65 years of age. Penicillin susceptibility, increased exposure and macrolide resistance were mostly associated with serotypes 14 and 19A. PCV13 and PPV23 provide very high serotype coverage. Future studies should evaluate the effects of the 10-valent vaccine, introduced in the Croatian National Immunization Program in June 2019, on serotype distribution and antibiotic resistance rates.

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.

Characterization of macrolide resistance in bacteria isolated from macrolide-polluted and unpolluted river sediments and clinical sources in Croatia.

Environments polluted with excessively high levels of antibiotics released from manufacturing sites can act as a source of transferable antibiotic resistance (AR) genes to human commensal and pathogenic bacteria. The aim of this study was to evaluate AR of bacteria isolated from the Sava river sediments (Croatia) at the discharge site of effluents from azithromycin production compared to those from the upstream site and isolates collected in Croatian hospitals. A total of 228 environmental strains of azithromycin-resistant bacteria were isolated and identified, with 124 from the discharge site and 104 from the upstream site. In addition, a total of 90 clinical, azithromycin-resistant streptococcal and staphylococcal isolates obtained from the Croatian Reference Center for Antibiotic Resistance Surveillance were analyzed. PCR screening of isolates on 11 relevant macrolide-resistance genes (MRGs) showed that discharge isolates had greater detection frequencies for 4 gene targets (ermB, msrE, mphE and ermF) compared to upstream isolates. Among clinical isolates, the most frequently detected gene was ermB, followed by msrD, mefE and mefC. The discharge site demonstrated a greater abundance of isolates with co-occurrence of two different MRGs (predominantly msrE-mphE) than the upstream site, but a lower abundance than the clinical sources (most commonly msrD-mefE). The simultaneous presence of three or even four MRGs was specific for the discharge and clinical isolates, but not for the upstream isolates. When MRG results were sorted by gene mechanism, the ribosomal methylation (erm) and protection genes (msr) were the most frequently detected among both the discharge and the clinical isolates. Following sequencing, high nucleotide sequence similarity was observed between ermB in the discharge isolates and the clinical streptococcal isolates, suggesting a possible transfer of the ermB gene between bacteria of clinical and environmental origin. Our study highlights the importance of environmental bacterial populations as reservoirs for clinically relevant macrolide-resistance genes.

Distribution of rotavirus genotypes in three Croatian regions among children ≤5 years of age (2012-2014).

OBJECTIVES: Rotavirus is the major cause of severe diarrhea in young children worldwide. In countries like Croatia, where rotavirus vaccine has not been introduced in the national immunization program, prospective surveillance is necessary to establish the diversity of rotavirus strains. The aim of this study was to describe the prevalence and geographical distribution of rotavirus strains in Croatia and to detect the possible emergence of novel strains. METHODS: The study was conducted among children ≤5 years of age with acute gastroenteritis at three hospitals located in different geographical regions of Croatia, during the years 2012 to 2014. Rotavirus was detected in stools using an immunochromatographic assay and then sent for further molecular analysis. RESULTS: Genotyping of 822 rotaviruses showed that the predominant circulating strain was G1P[8] (61.9%), followed by G2P[4] (19.5%), G1P[4] (3.9%), and G3P[8] (2.9%). A high prevalence of reassortants among common human rotavirus genotypes was detected (7.7%). Possible zoonotic reassortants were found, including G8 and G6 strains. The latter is described for the first time in Croatia. CONCLUSIONS: This study represents pre-vaccination data that are important for decisions regarding immunization strategies in Croatia. The high prevalence of 'common' rotavirus strains circulating in Croatia may advocate for rotavirus vaccine introduction, but further surveillance is necessary to monitor the possible emergence of novel genotypes.

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.

Imported brucellosis and Q-fever coinfection in Croatia: a case report.

The brucellosis and Q-fever coinfection is very rarely reported. To our knowledge, this is the first case report of concomitant brucellosis and Q-fever, most likely imported in Croatia. A 30-year-old male agricultural worker was hospitalized on 22 April 2017 after a ten days fever up to 40°C with chills, shivering, excessive sweating, general weakness, loss of appetite and headache. A month and a half prior to the hospitalization he lost 18 kg of body weight. Three weeks before hospitalization the patient returned from Kupres (Bosnia and Herzegovina) where he was working for the past year on a sheep farm and consumed unpasteurized dairy products of sheep origin. At admission, his condition was moderately severe due to pronounced dehydration. Routine laboratory tests showed slightly elevated erythrocyte sedimentation rate, anemia, thrombocytopenia and elevated liver transaminases. The chest X-ray showed an inhomogeneous infiltrate of the lower right lung. Three sets of blood culture were cultivated. After 48 hours incubation, bacterial growth was detected in aerobic bottles. Gram-stained smear revealed small, gram-negative coccobacilli. Specimens were subcultured on blood and chocolate agar plates. Using a Vitek GN identification card, the isolated organism was identified as Brucella melitensis. 16S rRNA gene sequencing of the isolate confirmed it as a Brucella sp. Rose-Bengal test was positive, while Wright agglutination test showed a significant increase in antibody titer from 80 to 640 in paired sera. Using indirect immunofluorescence assay (IFA), Coxiella burnetii phase II IgM/IgG titers were 50 and 1024, respectively indicating acute Q-fever. The patient was treated with doxycycline and rifampicin. So far, there has been no relapse or signs of chronic infection.

Characterization of Isolates Associated with Emergence of OXA-48-Producing Klebsiella pneumoniae in Croatia.

Here, we report a retrospective study conducted to elucidate emergence, epidemiology, and molecular mechanisms of resistance underlying the early spread of OXA-48 carbapenemase-producing Enterobacteriaceae in Croatia. Retrospective screening for OXA-48 producers was performed on a collection of 296 nonrepetitive, carbapenem-nonsusceptible enterobacterial isolates collected from January 2011 to December 2012 from 40 participating centers in Croatia. Antimicrobial susceptibility profiles and production of carbapenemases were assessed phenotypically. Pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing were used for epidemiological analysis. Resistance genes were characterized by polymerase chain reaction (PCR) and sequencing. Plasmid localization of blaOXA-48 in isolates and transconjugants was investigated by S1-PFGE and Southern hybridization. PCR mapping was used for identification of genetic platform surrounding blaOXA-48. Out of 296 carbapenem-nonsusceptible isolates, blaOXA-48 gene was detected in 12 Klebsiella pneumoniae isolates. All OXA-48-producing isolates showed varying resistance to carbapenems and 11 were multidrug resistant. All coproduced additional beta-lactamases, including CTX-M-15, which was detected in eight isolates. Isolates were delineated in five clonal types by PFGE corresponding to five sequence types (STs) assigned ST15, ST16, ST37, ST528, and ST1418. All OXA-48 isolates conjugated successfully and other resistance determinants were not cotransferred. blaOXA-48 was carried on a ∼60 kb IncL/M plasmid and was detected within Tn1999.2 composite transposon. OXA-48, a class D carbapenemase, is emerging as a potentially significant contributor among carbapenem-resistant Enterobacteriaceae in Croatia, alongside class A and B carbapenemases. Polyclonal genetic background of K. pneumoniae isolates carrying ∼60 kb incL/M plasmid indicates that dissemination of the blaOXA48 gene is not driven exclusively by the spread of a single clone.

Emergence of colistin resistance in Enterobacter aerogenes from Croatia.

A colistin-resistant Enterobacter aerogenes [study code 12264] was isolated from the tracheal aspirate of a 71-year-old male patient in the General Hospital [GH] in Pula, Croatia. The patient was previously treated in University Hospital Centre in Rijeka with colistin in order to eradicate Acinetobacter baumannii isolate, susceptible only to colistin and tigecycline. Genes encoding ESBLs [blaTEM, blaSHV, blaCTX-M, blaPER-1] were screened by PCR. The strain was shown to possess blaCTX-M-15 and blaTEM-1 genes. To asses genes possibly involved in resistance to colistin the chromosomal enconding mgrB gene and the plasmid-mediated mcr-1 and mcr-2 genes were screened as described previously. Mcr-1 and mcr-2 genes were not detected and mgrB gene presented a wild-type sequence. PCR-based Replicon typing method [PBRT] conducted on an E. aerogenes isolate, showed that the strain carried an IncN plasmid. Adaptive mechanisms such as changes of the bacterial cell outer membrane that cause porin decrease or presence of an efflux pump, due to selection pressure exerted by the therapeutic administration of colistin, could be responsible for the development of colistin resistance in our strain, as recently reported in E. aerogenes from France. Due to effective infection control measures, the colistin-resistant strain did not spread to other patients or hospital wards. This is the first report of an ESBL-producing, colistin-resistant E. aerogenes in clinically relevant samples such as endotracheal aspirate and blood culture, showing the presence of this rare resistance profile among Gram-negative bacteria.

blaNDM-1 Carriage on IncR Plasmid in Enterobacteriaceae Strains.

Four NDM-1-producing Enterobacteriaceae strains (three Klebsiella pneumoniae and one Citrobacter koseri) were isolated between 2009 and 2011 through a nationwide surveillance for carbapenem-resistant Enterobacteriaceae in Croatia to study the molecular genetic background of blaNDM and the responsible plasmid types. Phenotypically, the clinical strains proved to be multidrug resistant. All strains remained susceptible to tigecycline and colistin. The clinical strains harbored variable antibiotic resistance determinants, notably, blaNDM-1, blaTEM-1, blaSHV-1, blaSHV-12, blaOXA-1, blaOXA-9, blaCTX-M-15, blaCMY-4, qnrB1, and aac(6')Ib-cr in different combinations. Two K. pneumoniae belonged to sequence type ST15 and one strain to ST16. As for the plasmid types, C. koseri and one of the ST15 K. pneumoniae carried IncR, and the second ST15 K. pneumoniae carried IncR and colE. The K. pneumoniae ST16 strain hosted A/C and colE plasmids. The blaNDM-1 gene was detected on conjugative high-molecular-weight plasmids, namely, A/C and IncR types. It is noteworthy that this is the first description of K. pneumoniae ST16 expressing NDM-1 in Europe. Remarkably, our study underscores the importance of the IncR plasmid as a reservoir of multidrug resistance. To the best of our knowledge, the IncR plasmid carrying blaNDM-1 in C. koseri is reported for the first time.

Relapsing Bacteraemia due to Corynebacterium striatum in a Patient with Peripheral Arterial Disease.

We describe the first reported case of Corynebacterium striatum (C. striatum) relapsing bacteraemia in a patient with peripheral arterial disease and proven Corynebacterium species colonization of a chronic foot ulcer, focusing on the difficulties in the management of the patient. We conclude that the optimal duration of the antibiotic treatment for relapsing C. striatum bacteraemia from a chronic ulcer should be 6 weeks together with surgical treatment.

Sequence types 235, 111, and 132 predominate among multidrug-resistant pseudomonas aeruginosa clinical isolates in Croatia.

A population analysis of 103 multidrug-resistant Pseudomonas aeruginosa isolates from Croatian hospitals was performed. Twelve sequence types (STs) were identified, with a predominance of international clones ST235 (serotype O11 [41%]), ST111 (serotype O12 [15%]), and ST132 (serotype O6 [11%]). Overexpression of the natural AmpC cephalosporinase was common (42%), but only a few ST235 or ST111 isolates produced VIM-1 or VIM-2 metallo-ß-lactamases or PER-1 or GES-7 extended-spectrum ß-lactamases.

[A report of rarely observed resistance pattern to carbapenems in a clinical isolate of Enterobacter cloacae]. / Prikaz rijetko videnog oblika otpornosti na karbapeneme u vrste Enterobacter cloacae.

The paper reports on the emergence of strain in which Enterobacter cloacae has demonstrated an unusual form of resistance to carbapenems mediated by enzyme IMI-1, class A beta-lactamase. The strain was isolated from a wound swab in the patient who had a surgical wound infection previously treated with meropenem. Limited choice of antibiotics that can treat infections caused by these pathogens indicates the necessity of accurate identification of multiple resistant gram-negative microorganisms and mechanisms of their resistance. Recognition of multiresistant gram-negative microorganisms emphasizes the importance of continuous microbiological monitoring of patients, especially in intensive care units. In the investigated institution there was no secondary spread of this strain. Identifying new mechanisms of resistance will be helpful to clinicians in selection of targeted therapy, while important for efficient prevention of spreading infections caused by multiple resistant microorganisms.

Mycoplasma pneumoniae in adult community-acquired pneumonia increases matrix metalloproteinase-9 serum level and induces its gene expression in peripheral blood mononuclear cells.

BACKGROUND: The objective of this study was to assess the concentration of metalloproteinase-2 (MMP-2) and metalloproteinase-9 (MMP-9) in peripheral circulation and their mRNA expression in peripheral blood mononuclear cells (PBMCs) in patients with CAP caused by M. pneumoniae. MATERIAL/METHODS: We prospectively analyzed MMPs in 40 hospitalized patients with M. pneumoniae CAP on admission, and in the convalescent phase. Twenty healthy men were used as controls. Quantitative real-time PCR and ELISA tests were used. RESULTS: MMP-9 mRNA expression in PBMCs was increased in the acute phase of illness compared to the control group as well as in convalescent phase in which case it was statistically significant (Mann-Whitney; p=0.028). The same was found for MMP-9 plasma levels (Mann-Whitney test; p<0.001; p=0.001). Circulating MMP-2 concentration in acute patients was significantly lower than in the control group and convalescent phase (Mann-Whitney test; p=0.012; p=0.001), while no MMP-2 mRNA expression was found in PBMCs. The plasma level of MMP-9 correlated with leukocyte count in peripheral circulation (r=0.67, p<0.001). CONCLUSIONS: We conclude that M. pneumoniae in adult CAP induces activity of MMP-9 in peripheral blood circulation.

The epidemiology of invasive Streptococcus pneumoniae disease in Croatian children.

BACKGROUND: Invasive pneumococcal disease (IPD) is a major medical problem in childhood. Pneumococcal conjugate vaccines (PCVs) offer a new possibility to reduce the incidence of pneumococcal infections, especially IPD. The aim of this study was to describe the characteristics of IPD among Croatian children and examine the possibilities of introducing different PCVs in our population. MATERIAL/METHODS: Streptococcus pneumoniae isolates causing IPD during three years (2001, 2005, 2006) in Croatian children younger than 14 years old were collected prospectively. Epidemiological and clinical parameters, results of serotyping, and antimicrobial susceptibility data were evaluated. RESULTS: One hundred strains were collected during the study period. The ages of the patients ranged from 30 days to 13 years (median: 25.5 months). Seventy-nine percent of the isolates were from patients younger than five years old. The incidence of IPD was highest among children younger than two years of age (33.9/100,000). Serotypes 14, 6B, 18C, and 23F accounted for 67% of all serotypes. The overall coverage rates of PCV7, PCV10, and PCV13 were 72%, 80%, and 90%, respectively. Low-level resistance to penicillin was found in 20% of the isolates and high resistance to erythromycin in 33.8%. PCV7 covered 85% of the penicillin-resistant strains and 80% of the erythromycin-resistant strains. CONCLUSIONS: The inclusion of a PCV in the immunization program could have a considerable effect on IPD-associated morbidity among Croatian children.