Detection of Macrolide and/or Fluoroquinolone Resistance Genes in Mycoplasma genitalium Strains Isolated from Men in the Northwest Region of Croatia in 2018-2023.

Mycoplasma genitalium (M. genitalium) poses a significant public health challenge due to its association with non-gonococcal urethritis (particularly in men) and antimicrobial resistance. However, despite the prevalence of M. genitalium infections and the rise in resistance rates, routine testing and surveillance remain limited. This is the first study from Croatia that aimed to assess the prevalence and trends of resistance in M. genitalium strains isolated from male individuals by detecting macrolide and fluoroquinolone resistance genes. The study also aimed to explore the factors associated with resistance and changes in resistance patterns over time. Urine samples collected from male individuals in the Zagreb County and northwest region of Croatia between 2018 and 2023 were tested for M. genitalium with the use of molecular methods. Positive samples were subjected to DNA extraction and multiplex tandem polymerase chain reaction (MT-PCR) targeting genetic mutations associated with macrolide (23S rRNA gene) and fluoroquinolone (parC gene) resistance. Of the 8073 urine samples tested from 6480 male individuals (and following the exclusion of repeated specimens), we found that the prevalence of M. genitalium infection was 2.2%. Macrolide resistance was observed in 60.4% of strains, while fluoroquinolone resistance was found in 19.2%. Co-resistance to both antibiotics was present in 18.2% of cases. A statistically significant increase in fluoroquinolone resistance was noted over the study period (p = 0.010), but this was not evident for azithromycin resistance (p = 0.165). There were no statistically significant differences in resistance patterns between age groups, whereas re-testing of patients revealed dynamic changes in resistance profiles over time. The high burden of macrolide resistance and increasing fluoroquinolone resistance underscore the urgent need for comprehensive resistance testing and surveillance programs. The implementation of resistance-guided treatment strategies, along with enhanced access to molecular diagnostics, is pivotal for effectively managing M. genitalium infections.

Knowledge about and prevalence of Chlamydia trachomatis in a population-based sample of emerging Croatian adults.

To determine the prevalence of genital Chlamydia trachomatis (chlamydia) infection, knowledge about chlamydia and experience of previous testing for chlamydia, we carried out a national probability-based survey in emerging adults aged 18-25 years in Croatia in 2021-2022. Participants (n = 1197), members of a national online panel, completed a web-based questionnaire that collected information on socio-demographics, sexual behaviours and knowledge about sexually transmitted infections (STIs). Urine specimens from a sample of sexually experienced participants were self-collected and tested for chlamydia using Cobas 4800 CT/NG test. To achieve broad representativeness of the emerging adult population in the country, we applied post-hoc weighting for gender and age. Multivariable ordinary least squares linear regression was used to determine correlates of knowledge about chlamydia infection and binomial logistic regression to assess correlates of the willingness to test for chlamydia. Among 448 participants who sent in their urine specimens chlamydia prevalence was 2.5% (95% CI 1.2-5.1) in women and 1.0% (0.3-3.2%) in men. A total of 8.0% of women and 4.7% men reported testing for chlamydia prior to the survey. About a quarter of the sample was characterized by not answering correctly any of the six questions related to knowledge about chlamydia, while only 9.6% had five or six correct answers. In the multivariable analysis, significantly higher odds of willingness to test for chlamydia were found in females compared to males (OR = 1.34, p = 0.024), those with better knowledge about the infection (OR = 1.11, p = 0.005), and those with lower religiosity (OR = 0.91, p = 0.017). In conclusion, prevalence of chlamydia in emerging adults in Croatia is considerable. Efforts to control this infection should focus on primary prevention and targeted testing combined with effective case management strategies.

Assessing the Need for Routine Screening for Mycoplasma genitalium in the Low-risk Female Population: A Prevalence and Co-infection Study on Women from Croatia.

BACKGROUND: There is an ongoing debate regarding possible cost and benefits, but also harm of universal screening for the emerging sexually transmitted pathogen Mycoplasma genitalium. METHODS: From the initial pool of 8665 samples that were tested, a subset of Chlamydia trachomatis-positive and randomly selected C. trachomatis-negative cervical swabs were further interrogated for M. genitalium by real-time polymerase chain reaction, using a 224 bp long fragment of the glyceraldehyde-3-phosphate dehydrogenase gene. RESULTS: M. genitalium was detected in 4.8% of C. trachomatis-positive samples and none of C. trachomatis-negative samples. Accordingly, a significant association was shown between M. genitalium and C. trachomatis (P < 0.01), but also between M. genitalium and Mycoplasma hominis infection (P < 0.01). CONCLUSIONS: Based on the results, routine screening is recommended only for women with one or more identified risk factors. Moreover, younger age does not represent an appropriate inclusion/exclusion criterion for M. genitalium testing in the low-risk female population.

Etiology and Clinical Characteristics of Single and Multiple Respiratory Virus Infections Diagnosed in Croatian Children in Two Respiratory Seasons.

The aim of this study was to determine the causative agent of acute respiratory infection (ARI) in hospitalized children, as well as investigate the characteristics of ARIs with single and multiple virus detection in two respiratory seasons. In 2010 and 2015, nasopharyngeal and pharyngeal swabs from a total of 134 children, admitted to the hospital due to ARI, were tested using multiplex PCR. Viral etiology was established in 81.3% of the patients. Coinfection with two viruses was diagnosed in 27.6% of the patients, and concurrent detection of three or more viruses was diagnosed in 12.8% of the patients. The most commonly diagnosed virus in both seasons combined was respiratory syncytial virus (RSV) (28.6%), followed by parainfluenza viruses (PIVs) types 1-3 (18.4%), rhinovirus (HRV) (14.3%), human metapneumovirus (10.1%), adenovirus (AdV) (7.1%), influenza viruses types A and B (4.8%), and coronaviruses (4.2%). In 2015, additional pathogens were investigated with the following detection rate: enterovirus (13.2%), bocavirus (HBoV) (10.5%), PIV-4 (2.6%), and parechovirus (1.3%). There were no statistical differences between single and multiple virus infection regarding patients age, localization of infection, and severity of disease (P > 0.05). AdV, HRV, HBoV, and PIVs were significantly more often detected in multiple virus infections compared to the other respiratory viruses (P < 0.001).

Emergence of CTX-M group 1 extended-spectrum beta-lactamase-producing Klebsiella pneumoniae strains in the community.

AIM: Molecular characterization of ESBL-producing K. pneumoniae strains isolated from urine of outpatients in Zagreb region during the last five years. METHODS: During the five-year study period a total of 2, 651 K. pneumoniae strains were isolated from urine of nonhospitalized patients with significant bacteriuria. ESBL production was detected by double-disk diffusion technique and by > or = 3-dilution reduction in the minimal inhibitory concentration of ceftazidime in the presence of clavulanate. A total of 441 ESBL-producing K. pneumoniae strains (15.5%) were collected and 17 strains were further characterised. Double-disk synergy test was used to detect ESBLs. Minimum inhibitory concentrations (MICs) were determined by broth microdilution method according to CLSI. The transferability of cefotaxime resistance was tested by conjugation (broth mating method). PCR was used to detect alleles encoding ESBL enzymes. The genotypes of the strains were compared by pulsed-field gel electrophoresis (PFGE) of Xba I-digested genomic DNA. RESULTS: A significant difference in frequencies of ESBL isolates was observed. In the first year of study only 4.9% of isolated strains were ESBL producers, while in the second year 17.% ESBL-positive strains were detected (p < 0.01), and the frequency remained stabile within following years. All strains yielded an amplicon with primers specific for SHV beta-lactamases and CTX-M beta-lactamases. Based on sequencing of bla(CTX-M) genes enzymes of nine strains were identified as CTX-M 15 beta -lactamase and three as CTX-M-14. Isolates were not clonally related. CONCLUSION: The study demonstrated community-associated emergence of CTX-M 1 beta-lactamase-producing K. pneumoniae strains.

[Characterization of ESBL-producing Escherichia coli and Klebsiella pneumoniae strains isolated from urine of nonhospitalized patients in the Zagreb region]. / Karakterizacija ESBL-producirajucih sojeva bakterija Escherichia coli i Klebsiella pneumoniae izoliranih iz mokrace izvanbolnickih pacijenata zagrebacke regije.

AIM: To determine the prevalence of ESBL-producing Escherichia coli and Klebsiella pneumoniae strains isolated from urine of nonhospitalized patients during a three-year period, to determine their antibiotic susceptibility, investigate the transfer of ESBL genes with cotransfer of resistance and to characterize isolated beta-lactamases. METHODS: Antimicrobial susceptibility was determined by disk diffusion and broth microdilution methods. The double-disk test was used for ESBL detection. Transfer of resistance was performed by broth mating method and characterization of isolated beta-lactamases by polymerase chain reaction. RESULTS: The prevalence of ESBL-producing E. coli was 1.5% and of K. pneumoniae 4.1% with its different distribution according to patients' age and gender. ESBL-producing K. pneumoniae showed high resistance rates to aminoglycosides, cotrimoxazole, nitrofurantoin and quinolones while ESBL-producing E. coli isolates, with exception of high aminoglycoside resistance, showed low resistance rates to other antibiotics. Successful conjugation of ESBL genes was obtained with 25% E. coli and 76.2% K. pneumoniae strains. Comparing to E. coli, K. pneumoniae strains showed higher rates of aminoglycoside and cotrimoxazole resistance cotransfer. Beta-lactamases of investigated strains belonged to TEM, SHV and CTX-M families. CONCLUSION: The existence of multiple-resistant ESBL-producing E. coli and K. pneumoniae strains was confirmed in observed outpatient population. ESBL-producing K. pneumoniae isolates, in contrast to ESBL-producing E. coli, showed higher resistance rates to non-beta-lactam antibiotics, probably caused by cotransfer of resistance genes located on the same plasmid as ESBL genes. It is important to monitor the prevalence of such strains and their possible spreading in the outpatient population of the Zagreb region.

[Frequency of Haemophilus spp. in urinary and and genital tract samples]. / Zastupljenost bakterija roda Haemophilus u uzorcima iz mokracnog i genitalnog sustava.

AIM: To determine the prevalence and antibiotic susceptibility of Haemophilus influenzae and H. parainfluenzae isolated from the urinary and genital tracts. METHODS: Identification of strains bacteria Haemophilus spp. was carried out by using API NH identification system, and antibiotic susceptibility was performed by Kirby-Bauer disk diffusion method. RESULTS: A total number of 50 (0.03%) H. influenzae and 14 (0.01%) H. parainfluenzae (out of 180, 415 samples) were isolated from genitourinary tract. From urine samples of the girls under 15 years of age these bacteria were isolated in 13 (0.88%) and two (0.13%) cases, respectively, and only in one case (0.11%) of the UTI in boys (H. influenzae). In persons of fertile age, it was only H. influenzae bacteria that was found in urine samples of the five women (0.04%) and in three men (0.22%). As a cause of vulvovaginitis, H. influenzae was isolated in four (5.63%), and H. parainfluenzae in two (2.82%) girls. In persons of fertile age, H. influenzae was isolated from 10 (0.49%) smears of the cervix, and in nine (1.74%) male samples. H. parainfluenzae was isolated from seven (1.36%) male samples. (p < 0.01). Susceptibility testing ofH. influenzae and H. parainfluenzae revealed that both pathogens were significantly resistant to cotrimoxasol only (26.0% and 42.9%, respectively). CONCLUSION: In the etiology of genitourinary infections of girls during childhood, genital infections of women in fertile age (especially in pregnant women), and men with cases of epididimytis and/or orchitis, it is important to think about this rare and demanding bacteria in terms of cultivation.

Clonal dissemination of highly virulent extended-spectrum beta-lactamase-producing Escherichia coli strains isolated from the urine of non-hospitalised patients in Zagreb region.

Recent data suggest that extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli is an emergent cause of urinary tract infections in non-hospitalised patients in different countries. The aim of this study was to characterise ESBL-producing E. coli strains isolated from the urine of outpatients in the Zagreb region of Croatia. During the 5-month study period, a total of 2451 E. coli strains were isolated from the urine of non-hospitalised patients with significant bacteriuria. A total of 39 ESBL-producing E. coli strains (1.59%) were collected and characterised.

Emergence of uropathogenic extended-spectrum beta lactamases-producing Escherichia coli strains in the community.

The aim of this study was to determine the virulence characteristics and resistance pattern of the extended-spectrum/lactamases (ESBLs)-producing Escherichia coli strains isolated from urine of outpatients in the Zagreb region during a five-month period, and to compare them with the non ESBLs-producing E. coli strains isolated in the same period. Out of 2451 E. coli strains isolated from urine of nonhospitalized patients with significant bacteriuria, a total of 39 ESBLs-producing strains (1.59%) were detected by a double-disk diffusion technique and by the broth-dilution minimal inhibitory concentration reduction method. The 45 non ESBLs-producing strains were randomly chosen, and phenotype of the two groups of strains was characterized and compared. Serogroup O4, hemolysin production, expression of P- and type 1 fimbriae as well as resistance to gentamicin and amikacin were significantly more prevalent characteristics among the ESBLs-producing strains than among non ESBLs-producing strains (p < 0.01), while higher prevalence of trimethoprim-sulfamethoxazole resistance among ESBLs-producing strains was not statistically significant (p > 0.05). Chromosomal DNA analysis by pulsed-field gel electrophoresis exhibited a great genomic similarity among ESBLs-producing strains and revealed that those highly virulent and resistant E. coli strains isolated from urine of outpatients in the Zagreb region had a clonal propagation.

Genital human papillomavirus infection in women from the Zagreb region.

Human papillomavirus (HPV) infection is the most common sexually transmitted infection, especially among young, sexually active individuals. As persistent infection with oncogenic types may lead to cervical cancer, HPV testing is a useful tool to screen for women at risk for subsequent development of cervical cancer. The aim of the study was to determine the prevalence of high-risk HPV (hrHPV) infection in different age groups of cytologically selected women from the Zagreb region, and to evaluate the frequency and results of repeat hrHPV testing. During a one-year study period (November 2005 to November 2006), a total of 3,440 cervical samples from women attending gynecological services of public and private health care systems were received. They were tested for 13 hrHPV genotypes by the polymerase chain reaction based AMPLICOR HPV test (Roche Molecular Systems). The overall prevalence of hrHPV was 34.6%. Most samples were obtained from women aged 21-30 years (44.2%), followed by the 31-40 (27.6%), 41-50 (15.7%), 51-60 (5.3%) and 261 (2.4%) age groups. Out of 3,227 cervical samples obtained from women of known age, 4.9% were obtained from the group of girls younger than 21, in which the highest prevalence of hrHPV (49.4%) was found. A similar prevalence was observed in women aged 21-30 (45.1%). The prevalence gradually decreased with age. During the study period, repeat hrHPV testing was performed in samples from 66 women at different intervals. Out of 28 women that were hrHPV negative on initial testing, only five women turned positive on repeat testing. Out of 38 women that were positive on initial testing, in one-third hrHPV could not be detected on repeat testing. As expected, hrHPV infection was highly prevalent in female adolescents and young women. Further investigation on repeat hrHPV testing is needed to assess virus clearance and rate of newly acquired infection.