Different sampling strategies for optimal detection of the overall genetic diversity of methicillin-resistant Staphylococcus aureus.

Surveillance schemes for methicillin-resistant Staphylococcus aureus (MRSA) are widely established at the national and international levels. Due to the simple standardization of the protocol, mainly isolates from bloodstream infections are used. However, the limitations of this simple surveillance system are well described. We conducted a comprehensive analysis of MRSA isolates in a large Slovenian region over 5 years to identify the optimal sample group for assessing the overall MRSA diversity. At the same time, this study provides to date non-available molecular characterization of Slovenian MRSA isolates. A total of 306 MRSA isolates from various sources were sequenced and phenotypically tested for resistance. The isolates exhibited significant molecular diversity, encompassing 30 multi locus sequence type (MLST) sequence types (STs), 39 ST-SCCmec genetic lineages, 49 spa types, and 29 antibiotic resistance profiles. Furthermore, the isolate pool comprised 57 resistance genes, representing 22 resistance mechanisms, and 96 virulence genes. While bloodstream isolates, commonly used in surveillance, provided insights into frequently detected clones, they overlooked majority of clones and important virulence and resistance genes. Blood culture isolates detected 21.3% spa types, 24.1% resistance phenotypes, and 28.2% MLST-SCCmec profiles. In contrast, strains from soft tissues demonstrated superior genomic diversity capture, with 65.3% spa types, 58.6% resistance phenotypes, and 71.8% MLST-SCCmec profiles. These strains also encompassed 100.0% of virulence and 82.5% of resistance genes, making them better candidates for inclusion in surveillance programs. This study highlights the limitations of relying solely on bloodstream isolates in MRSA surveillance and suggests incorporating strains from soft tissues to obtain a more comprehensive understanding of the epidemiology of MRSA.IMPORTANCEIn this study, we investigated the diversity of methicillin-resistant Staphylococcus aureus (MRSA), a bacterium that can cause infections that are difficult to treat due to its resistance to antimicrobial agents. Currently, surveillance programs for MRSA mainly rely on isolates from bloodstream infections, employing a standardized protocol. However, this study highlights the limitations of this approach and introduces a more comprehensive method. The main goal was to determine which group of samples is best suited to understand the overall diversity of MRSA and to provide, for the first time, molecular characterization of Slovenian MRSA isolates. Our results suggest that including MRSA strains from soft tissue infections rather than just blood infections provides a more accurate and comprehensive view of bacterial diversity and characteristics. This insight is valuable for improving the effectiveness of surveillance programs and for developing strategies to better manage MRSA infections.

SARS-CoV-2 molecular epidemiology in Slovenia, January to September 2021.

BackgroundSequencing of SARS-CoV-2 PCR-positive samples was introduced in Slovenia in January 2021. Our surveillance programme comprised three complementary schemes: (A) non-targeted sequencing of at least 10% of samples, (B) sequencing of samples positive after PCR screening for variants of concern (VOC) and (C) sequencing as per epidemiological indication.AimWe present the analysis of cumulative data of the non-targeted surveillance of SARS-CoV-2 and variant-dependent growth kinetics for the five most common variants in Slovenia for the first 9 months of 2021.MethodsSARS-CoV-2 PCR-positive samples, from January to September 2021, were selected for sequencing according to the national surveillance plan. Growth kinetics studies were done on Vero E6 cells.ResultsAltogether 15,175 genomes were sequenced and 64 variants were detected, of which three successively prevailed. Variant B.1.258.17 was detected in ca 80% of samples in January and was replaced, within 9 weeks, by the Alpha variant. The number of cases decreased substantially during the summer of 2021. However, the introduction of the Delta variant caused a fourth wave and completely outcompeted other variants. Other VOC were only detected in small numbers. Infection of Vero E6 cells showed higher replication rates for the variants Alpha and Delta, compared with B.1.258.17, B.1.258, and B.1.1.70, which dominated in Slovenia before the introduction of the Alpha and Delta variants.ConclusionInformation on SARS-CoV-2 variant diversity provided context to the epidemiological data of PCR-positive cases, contributed to control of the initial spread of known VOC and influenced epidemiological measures.

Milder outcomes of SARS-CoV-2 genetically confirmed reinfections compared to primary infections with the delta variant: A retrospective case-control study.

Background: SARS-CoV-2 infection does not confer long immunity. However, studies suggest that prior infection is associated with lower risk of reinfection and milder outcomes of recurrent infections. The aims of this retrospective observational case-control study were to describe the clinical and molecular characteristics of genetically confirmed Delta reinfection cases and to assess the potential protective role of preceding infection on the severity of reinfection. Methods: We used next generation sequencing (NGS) to explore if cases with two positive real time RT-PCR tests > 90 days apart were infected with a different SARS-CoV-2 variant. Cases with confirmed reinfection between August 1st and October 31st, 2021 (the Delta wave) in Slovenia were matched 1:4 by age, sex and timeframe (week of positive test) with individuals with primary infection. Sociodemographic and epidemiologic data, vaccination status, and data on hospitalization and outcome of infection were retrieved from several centralized and standardized national databases. Additional epidemiologic surveys were performed on a limited number of cases and controls. Results: We identified 628 cases of genetically confirmed reinfection during the study period and matched them with 2,512 control subjects with Delta primary infection. Primary infections in individuals with reinfection were mainly caused by B.1.258.17 (51.1%), followed by B.1.1.7 (15.1%) and reinfection was detected on average 271 days after primary infection (range 101-477 days). Our results show a substantially lower probability of hospitalization in cases with reinfection compared with controls (OR: 0.21, p = 0.017), but no significant difference was observed in intensive care unit admission and deaths. We observed a significantly lower proportion of vaccinated individuals among cases compared to controls (4.5% vs. 28.2%), suggesting that hybrid immunity leads to lower probability of reinfection. Detailed analysis of the temporal distribution of variants, responsible for reinfections, showed no significant differences in reinfection potential. Conclusion: Reinfection with the SARS-CoV-2 Delta variant resulted in fewer hospitalizations compared to the primary Delta infection, suggesting that primary infection may, to some extent, produce at least short lasting protective immunity. This study provides additional insight into the reinfection dynamics that may allow appropriate public health measures to be taken in subsequent waves of the COVID-19 pandemic.

Prevalence of sexually transmitted infections with Chlamydia trachomatis, Neisseria gonorrhoeae, Mycoplasma genitalium and Trichomonas vaginalis: findings from the National Survey of Sexual Lifestyles, Attitudes and Health, Slovenia, 2016 to 2017.

BackgroundTo inform prevention and control of sexually transmitted infections (STIs), we need reliable prevalence estimates.AimOne objective of the Slovenian National Survey of Sexual Lifestyles, Attitudes and Health was to estimate the prevalence of STIs with Chlamydia trachomatis, Neisseria gonorrhoeae, Mycoplasma genitalium and Trichomonas vaginalis.MethodsData were collected between October 2016 and July 2017 in a probability sample of the general population aged 18-49 years. Computer-assisted face-to-face interviewing and self-completion of questionnaires were used. Respondents were invited to provide urine samples to be tested for STIs.ResultsOf 1,929 survey participants, 1,087 individuals provided urine samples which were tested confidentially for C. trachomatis and a subset (n = 1,023) were tested anonymously for the other STIs. The prevalence of C. trachomatis was 0.5% (95% confidence interval (CI): 0.1-1.8) in men and 1.7% (95% CI: 0.9-3.2) in women. Age-specific prevalence was the highest among individuals aged 18-24 years, 2.8% (95% CI: 0.7-10.6) in men and 4.7% (95% CI: 1.7-12.3) in women. N. gonorrhoea was not detected. Prevalence of M. genitalium was 0.5% (95% CI: 0.1-2.2) in men and 0.3% (95% CI: 0.1-1.1) in women; the highest prevalence was among men aged 25-34 years (1.1%; 95% CI: 0.2-7.5) and women aged 35-49 years (0.5%; 95% CI: 0.1-2.0). T. vaginalis was detected in the sample from one woman (0.2%; 95% CI: 0.1-1.2).ConclusionThe substantial prevalence of C. trachomatis among young adults suggests gaps in testing, diagnosis and treatment.

Lack of correlation between reduced outpatient consumption of macrolides and macrolide resistance of invasive Streptococcus pneumoniae isolates in Slovenia during 1997-2017.

OBJECTIVES: The objective of this study was to investigate the correlation between decreased national consumption of macrolides and resistance of invasive Streptococcus pneumoniae isolates in Slovenia during 1997-2017. METHODS: A total of 4241 invasive S. pneumoniae isolates were collected in Slovenia from 1997 to 2017. The presence of erm(B), mef(E), mef(A) and erm(TR) genes was determined by PCR in 612 erythromycin-resistant isolates. Selected isolates carrying the mef(A) gene were further examined by pulsed-field gel electrophoresis (PFGE). Multilocus sequence typing (MLST) was performed for 161 erythromycin-resistant isolates from 2004 to 2009. RESULTS: Consumption of macrolides decreased by 42.5% between 1997 and 2017, and by 57.0% from the highest consumption during 1999 to 2017. Resistance of S. pneumoniae increased by 120.7% in the same period, from 5.8% in 1997 to 12.8% in 2017. The most prevalent serotypes among macrolide-resistant isolates were 14 (54.9%), 19A (9.0%), 19F (8.3%), 6B (7.2%), 6A (5.2%) and 9V (19; 3.0%). The most prevalent determinant of macrolide resistance in the observed period was erm(B) (43.0%; 263/612), followed by mef(A) (36.3%; 222/612) and mef(E) (14.9%; 91/612). During the study period, an increasing trend in serotype 14, mef(A)-carrying isolates was observed, with a peak in 2011 (P<0.001); 63/71 isolates (88.7%) with the mef(A) gene were clonally related and were related to the international England14-9 clonal cluster. CONCLUSIONS: The reason for the observed increase in macrolide resistance among invasive S. pneumoniae in Slovenia despite decreased macrolide consumption was spread of the England14-9 clonal cluster.

The evaluation of MRSA surveillance cultures by the number and combinations of anatomical sites.

INTRODUCTION: The identification of patients infected and/or colonised by methicillin resistant Staphylococcus aureus (MRSA) is necessary for the timely introduction of measures for infection control. We compared the diagnostic efficacy of combinations of MRSA surveillance swabs routinely taken by health institutions in the country. METHODS: All surveillance samples, which were sent for a microbiological analysis to detect MRSA with the culture method in 2014, in the three departments for medical microbiology of the National Laboratory for Health, Environment and Food, were included in this study. RESULTS: Among 65,251 surveillance cultures from 13,274 persons, 1,233 (2.1%) were positive (490 positive persons). Prevailing positive surveillance cultures were throat swabs (31.3%), followed by nose swab (31.2%), skin swab (18.9%), perineum (16.4%) and wound swabs (1.4%). The contribution of other samples, such as aspirate, urine and excreta, was under 1%. We found no statistically significant differences in the frequency of detection of a positive patient, if the combination of samples NTS (nose, throat, skin) or NTP (nose, throat, perineum) was analysed. However, statistically significant differences were confirmed when any of the anatomic sites would be omitted from the sets of NTP and NTS (chi square; p<0.01). Adding additional samples resulted in only 24 additional positive patients (4.9%). CONCLUSIONS: The results indicate that increasing the number of surveillance cultures above three does not add much to the sensitivity of MRSA surveillance, the exception could be wound. The swabs from the perineum and from the skin are exchangeable.

Analysis of Slovenian MRSA strains with susceptibility patterns suggestive of CA-MRSA.

BACKGROUND: Community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) differs from healthcare-associated MRSA (HA-MRSA) in its molecular and microbiological characteristics. MATERIALS AND METHODS: Six Slovenian regional public health institutes and the National Institute of Public Health took part in monitoring CA-MRSA infections. S. aureus isolates resistant to oxacillin and susceptible to > or = two of the four antibiotics ciprofloxacin, erythromycin, clindamycin or gentamicin were defined as CA-MRSA and further analyzed. The presence of the gene for Panton-Valentine leukocidin (PVL) was confirmed using PCR, the type of staphylococcal cassette chromosome (SCCmec) using multiplex PCR, and macrorestriction analysis of chromosomal DNA using pulsed-field gel electrophoresis (PFGE). RESULTS: A total of 31 strains from 31 patients were analyzed during a period of 21 months: 23 specimens were sent from hospitals, six from primary care, two from a long-term care facility. All 31 isolates contained the gene mecA. Sixteen (51.6%) isolates were identified as SCCmec type IV, three isolates were PVL positive. Using PFGE, the CA-MRSA strains were classified into 15 similarity groups. Results of antibiotic susceptibility showed there were five resistance types among the 31 strains. Simultaneous resistance against ciprofloxacin and gentamicin was often associated with the presence of SCCmec type I, strongly resembling HA-MRSA. CONCLUSIONS: PVL-positive strains of CA-MRSA have been isolated in Slovenia only rarely. We will continue to monitor strains of MRSA in order to obtain the complete microbiological and epidemiological features.

Complex endemic situation regarding extended-spectrum beta-lactamase-producing Klebsiella pneumoniae in a hospital in Slovenia.

The first detailed epidemiological study of extended-spectrum beta-lactamase (ESBL)-producing organisms identified in Slovenia was carried out. It was performed on a group of 40 Klebsiella pneumoniae isolates that were randomly selected from all putative ESBL producers of this species recovered in a large hospital in Celje in 1997-2001. At least three different ESBLs, SHV-2, -5, and -12, were produced by the isolates and these enzymes seem to be common in nosocomial Enterobacteriaceae populations in countries of the region (e.g., Italy, Hungary, Croatia). The analysis revealed a complex epidemiology of the organisms, illustrated mostly by their high clonal variety but also by the diversity of their beta-lactamase and plasmid content, mating capability, and antimicrobial susceptibility. Although some cases of a 'fresh' dissemination of strains or plasmids could be identified, the overall situation should be described rather as endemic, and its complexity may be in part attributed to the late introduction of the ESBL detection procedure to the hospital.