Epidemiology of Methicillin-Resistant Staphylococcus aureus in Slovakia, 2020 - Emergence of an Epidemic USA300 Clone in Community and Hospitals.

Methicillin-resistant Staphylococcus aureus (MRSA) is a leading cause of health care-associated infections. Additionally, over the decades, the spread of community-associated (CA-MRSA) clones has become a serious problem. The aim of this study was to gain data on the current epidemiology of MRSA in Slovakia. Between January 2020 and March 2020, single-patient MRSA isolates (invasive and/or colonizing) were collected in Slovakia from hospitalized inpatients (16 hospitals) or outpatients (77 cities). Isolates were characterized via antimicrobial susceptibility testing, spa typing, SCCmec typing, the detection of mecA/mecC, genes coding for Panton-Valentine leukocidin (PVL), and the arcA gene (part of the arginine catabolic mobile element [ACME]). Out of 412 isolates, 167 and 245 originated from hospitalized patients and outpatients, respectively. Inpatients were most likely older (P < 0.001) and carried a strain exhibiting multiple resistance (P = 0.015). Isolates were frequently resistant to erythromycin (n = 320), clindamycin (n = 268), and ciprofloxacin/norfloxacin (n = 261). 55 isolates were resistant to oxacillin/cefoxitin only. By clonal structure, CC5-MRSA-II (n = 106; spa types t003, t014), CC22-MRSA-IV (n = 75; t032), and CC8-MRSA-IV (n = 65; t008) were the most frequent. We identified PVL in 72 isolates (17.48%; 17/412), with the majority belonging to CC8-MRSA-IV (n = 55; arcA+; t008, t622; the USA300 CA-MRSA clone) and CC5-MRSA-IV (n = 13; t311, t323). To the best of our knowledge, this is the first study on the epidemiology of MRSA in Slovakia. The presence of the epidemic HA-MRSA clones CC5-MRSA-II and CC22-MRSA-IV was found, as was, importantly, the emergence of the global epidemic USA300 CA-MRSA clone. The extensive spread of USA300 among inpatients and outpatients across the Slovakian regions warrants further investigation. IMPORTANCE The epidemiology of MRSA is characterized by the rise and fall of epidemic clones. Understanding the spread, as well as the evolution of successful MRSA clones, depends on the knowledge of global MRSA epidemiology. However, basic knowledge about MRSA epidemiology is still fragmented or completely missing in some parts of the world. This is the first study of MRSA epidemiology in Slovakia to identify the presence of the epidemic HA-MRSA clones CC5-MRSA-II and CC22-MRSA-IV and, importantly and unexpectedly, the emergence of the global epidemic USA300 CA-MRSA clone in the Slovakian community and hospitals. So far, USA300 has failed to spread in Europe, and this study documents an extensive spread of this epidemic clone in a European country for the first time.

Clostridioides difficile infections were predominantly driven by fluoroquinolone-resistant Clostridioides difficile ribotypes 176 and 001 in Slovakia in 2018-2019.

AIM: To investigate the epidemiology of Clostridioides difficile infection (CDI) in Slovakian hospitals after the emergence of ribotype 176 (027-like) in 2016. METHODS: Between 2018 and 2019, European Centre for Disease Control and Prevention CDI surveillance protocol v2.3 was applied to 14 hospitals, with additional data collected on recent antimicrobial use and the characterization of C. difficile isolates. RESULTS: The mean hospital incidence of CDI was 4.1 cases per 10,000 patient bed-days. One hundred and five (27.6%) in-hospital deaths were reported among the 381 cases. Antimicrobial treatment within the previous 4 weeks was recorded in 90.5% (333/368) of cases. Ribotype (RT)176 was detected in 50% (n=185/370, 14 hospitals) and RT001 was detected in 34.6% (n=128/370,13/14 hospitals) of cases with RT data. Overall, 86% (n=318/370) of isolates were resistant to moxifloxacin by Thr82Ile in GyrA (99.7%). Multi-locus variable tandem repeat analysis showed clonal relatedness of predominant RTs within and between hospitals. Seven of 14 sequenced RT176 isolates and five of 13 RT001 isolates showed between zero and three allelic differences by whole-genome multi-locus sequence typing. The majority of sequenced isolates (24/27) carried the erm(B) gene and 16/27 also carried the aac(6')-aph(2'') gene with the corresponding antimicrobial susceptibility phenotypes. Nine RT176 strains carried the cfr(E)gene and one RT001 strain carried the cfr(C) gene, but without linezolid resistance. CONCLUSIONS: The newly-predominant RT176 and endemic RT001 are driving the epidemiology of CDI in Slovakia. In addition to fluoroquinolones, the use of macrolide-lincosamide-streptogramin B antibiotics can represent another driving force for the spread of these epidemic lineages. In C. difficile, linezolid resistance should be confirmed phenotypically in strains with detected cfr gene(s).

The evaluation of an automated mariPOC SARS-CoV-2 antigen test compared to RT-qPCR SARS-CoV-2 assay and comparison of its sensitivity in Delta- and Omicron-variant samples.

BACKGROUND: The rapid diagnostics tests for SARS-CoV-2 antigen vary in their sensitivities, and moreover, genomic mutations may further affect the performance of the assays. We aimed to evaluate the analytical performance of an automated antigen assay and compare its sensitivity in Delta- and Omicron-variant positive clinical samples. MATERIAL AND METHODS: The analytical performance of an automated mariPOC SARS-CoV-2 antigen test was evaluated on a population of community-dwelling subjects with mild respiratory symptoms or being asymptomatic investigated by the RT-qPCR Allplex™ SARS-CoV-2 assay. The sensitivity and specificity of the antigen test were evaluated on prospective 621 nasopharyngeal swabs along with oropharyngeal swabs. The sensitivity regarding variants determined by the Allplex™ SARS-CoV-2 Variant assays was analysed in additional, retrospective 158 Delta and 59 Omicron samples. RESULTS: The overall sensitivity of the antigen test in prospective samples was 77.9% (113/145; 95% confidence interval [CI] 70.3-84.4) with the specificity of 99.8% (95% CI 98.8-100). Regarding the variant, the sensitivity was higher in Omicron-variant samples, 93.2% (55/59; 95% CI 83.5-98.1), compared to Delta-variant samples, 71.5% (113/158; 95% CI 63.8-78.4; p = .001). CONCLUSION: In community-dwelling subjects with mild respiratory symptoms or being asymptomatic, the automated mariPOC SARS-CoV-2 antigen test showed high sensitivity over 98.0% in subgroup samples with cycle threshold (Ct) values < 25. Regarding the variant, the antigen test sensitivity was higher in the Omicron-variant samples compared to the Delta-variant samples. The analytical performance of the antigen test can differ between the SARS-CoV-2 variants, and a re-evaluation should be performed for new circulating lineages.

Detection of Plasmid-Mediated Resistance to Metronidazole in Clostridioides difficile from River Water.

Clostridioides difficile is one of the most important human pathogens. The identification of its possible sources is important for the understanding of C. difficile infection (CDI) epidemiology. A total of 16 water samples from wastewater and surface water in South Moravia in the Czech Republic and 82 samples of fish and gulls were collected between May and July 2019. C. difficile isolates were cultured by direct plating and after enrichment on chromogenic media. Susceptibility testing to eight antimicrobials was performed by Etest. C. difficile isolates were characterized by ribotyping, multilocus sequence typing, multilocus tandem repeats analysis, and toxin gene detection. Samples from fish and gulls were C. difficile negative; a total of 15 C. difficile isolates from 8 out of 16 water samples were cultured (6 out of 14 surface water samples yielded 6 isolates, and 2 out of 2 wastewater samples yielded 9 isolates). Direct plating was culture positive in 6 out of 16 samples (12 isolates), and enrichment culture was positive in an additional 2 out of 16 samples (3 isolates). Twelve different ribotyping profiles and 14 sequence types of clades 1, 4, and 5 were identified. Five isolates did not carry genes for toxins, and eight isolates carried genes for toxins A and B; the remaining two isolates (RT078) carried the genes for toxins A, B, and binary. All C. difficile isolates were susceptible to amoxicillin, moxifloxacin, tetracycline, and vancomycin and resistant to ciprofloxacin. A high level of erythromycin resistance (>256 mg/L) was detected in eight isolates. Clindamycin resistance was found in 14 isolates, 6 of which showed a high level of resistance (>256 mg/L) and carried ermB. Surprisingly, one isolate (RT010, ST15) showed resistance to metronidazole (12 mg/L) with the presence of the plasmid pCD-METRO. In conclusion, a diverse spectrum of C. difficile strains was found in wastewater and surface water. A recently discovered plasmid-bound resistance to metronidazole was detected in C. difficile from the surface water sample. IMPORTANCE The combination of direct plating and culture after enrichment was used in order to gain a spectrum of C. difficile ribotypes present in the water samples. Toxigenic C. difficile ribotypes detected in surface water and in wastewater treatment plants overlapped with those derived from patients with CDI and/or animals. Importantly, a recently discovered plasmid-mediated resistance to metronidazole, a drug used for the treatment of CDI, was detected in C. difficile from river water.

Clostridioides difficile epidemiology in the Middle and the Far East.

OBJECTIVES: Clostridioides difficile is an important pathogen of healthcare-associated gastrointestinal infections. Recently, an increased number of C. difficile infection (CDI) surveillance data has been reported from Asia. The aim of this review is to summarize the data on the prevalence, distribution and molecular epidemiology of CDI in the Middle and the Far East. METHODS: Literature was drawn from a search of PubMed up to September 30, 2021. RESULTS: The meta-analysis of data from 111 studies revealed the pooled CDI prevalence rate in the Middle and the Far East of 12.4% (95% CI 11.4-13.3); 48 studies used PCR for CDI laboratory diagnoses. The predominant types (RT)/sequence type (ST) differ between individual countries (24 studies, 14 countries). Frequently found RTs were 001, 002, 012, 017, 018 and 126; RT017 was predominant in the Far East. The epidemic RT027 was detected in 8 countries (22 studies), but its predominance was reported only in three studies (Israel and Iran). The contamination of vegetable and meat or meat products and/or intestinal carriage of C. difficile in food and companion animals have been reported; the C. difficile RTs/STs identified overlapped with those identified in humans. CONCLUSIONS: A large number of studies on CDI prevalence in humans from the Middle and the Far East have been published; countries with no available data were identified. The number of studies on C. difficile from non-human sources is limited. Comparative genomic studies of isolates from different sources are needed.

Motility and the genotype diversity of the flagellin genes fliC and fliD among Clostridioides difficile ribotypes.

OBJECTIVE: The motility and genotype of the flagellin fliC and fliD genes were investigated in 82 Clostridioides difficile isolates belonging to the ribotypes (RTs): 027 (n = 41), 176 (n = 17), 023 (n = 8), 017 (n = 6) and 046 (n = 10). The reference C. difficile strains 630 and M120 were included as controls for the motility assay. METHODS: A Multiple Locus Variable-number Tandem Repeat Analysis (MLVA) was used to exclude the genetic relatedness of C. difficile isolates belonging to the same RT. The variability of the fliC and fliD genes was determined by PCR-restriction fragment length polymorphism (RFLP) analysis and Sanger sequencing. The motility assay was carried out with 0.175% BHI agar tubes and BHI solid media plates with 0.4% agar. RESULTS: The highest motility was observed in C. difficile RT023 isolates (p < 0.01), followed by RTs 027 and 176. C. difficile isolates of RTs 017 and 046 were less motile than RTs 027, 176 and 023 (p < 0.01). The fliC and fliD genes were present in all clinical isolates irrespective of the motility results. In the fliC gene analysis, four different RFLP groups were identified (I, II, VII, X). The fliC group VII was identified in two RTs (027 and 176), whereas the remaining three groups (I, II and X) belonged to a single RT 046, 017 and 023, respectively. The fliD gene analysis identified four new RFLP groups (a, b, c and d). CONCLUSIONS: C. difficile RT023 is highly motile and its motility is comparable to the hypervirulent RT027 and its genetic relative RT176.

Genetic diversity and phylogenetic analysis of the surface layer protein A gene (slpA) among Clostridioides difficile clinical isolates from Tehran, Iran.

Clostridioides difficile is the most common causative agent of healthcare-associated diarrhea. C. difficile strains produce a crystalline surface layer protein (SlpA), encoded by the slpA gene. Previous studies have shown that SlpA varies among C. difficile strains. In this study, we used the SlpA sequence-based typing system (SlpAST) for the molecular genotyping of C. difficile clinical isolates identified in Iran; the PCR ribotypes (RTs) and toxin profiles of the isolates were also characterized. Forty-eight C. difficile isolates were obtained from diarrheal patients, and characterized by capillary electrophoresis (CE) PCR ribotyping and the detection of toxin genes. In addition, the genetic diversity of the slpA gene was investigated by Sanger sequencing. The most common RTs were RT126 (20.8%), followed by RT001 (12.5%) and RT084 (10.4%). The intact PaLoc arrangement representing cdu2+/tcdR+/tcdB+/tcdE+/tcdA+/tcdC+/cdd3+ profile was the predominant pattern and cdtA and cdtB genes were found in one-third of the isolates. Using the SlpA genotyping, 12 main genotypes and 16 subtypes were identified. The SlpA type 078-1 was the most prevalent genotype (20.8%), and identified within the isolates of RT126. The yok-1, gr-1, cr-1 and kr-3 genotypes were detected in 14.5%, 12.5%, 12.5% and 8.3% of isolates, respectively. Almost all the isolates with the same RT were clustered in similar SlpA sequence types. In comparison to PCR ribotyping, SlpAST, as a simple and highly reproducible sequenced-based technique, can discriminate well between C. difficile isolates. This typing method appears to be a valuable tool for the epidemiological study of C. difficile isolates worldwide.

The Intestinal Carriage of Plasmid-Mediated Colistin-Resistant Enterobacteriaceae in Tertiary Care Settings.

Background: In order to estimate the prevalence of plasmid borne colistin resistance and to characterize in detail the mcr-positive isolates, we carried out a sentinel testing survey on the intestinal carriage of plasmid-mediated colistin-resistant Enterobacteriaceae in hospitalized patients. Methods: Between June 2018 and September 2019, 1922 faecal samples from hospitalised patients were analysed by selective culture in presence of colistin (3.5 mg/L), and in parallel by direct detection of the mcr-1 to mcr-8 genes by qPCR. The mcr-positive isolates were characterised by whole-genome sequencing. Results: The prevalence of the mcr-1 gene was 0.21% (n = 4/1922); the mcr-2 to 8 genes were not detected. The mcr-1 gene was found to be localised in the IncX4 (n = 3) and IncHI2 (n = 1) plasmid type. One Escherichia coli isolate was susceptible to colistin due to the inactivation of the mcr-1 gene through the insertion of the IS2 element; however, the colistin resistance was inducible by culture in low concentrations of colistin. One human mcr-1 positive E. coli isolate was related genetically to the mcr-1 E. coli isolate derived from turkey meat of Czech origin. Conclusions:mcr-mediated colistin resistance currently poses little threat to patients hospitalised in Czech healthcare settings. The presence of the mcr-1 gene in the human population has a possible link to domestically produced, retail meat.

The molecular epidemiology of methicillin-resistant Staphylococcus aureus (MRSA) in the Czech Republic.

OBJECTIVES: To gain data on the current molecular epidemiology and resistance of MRSA in the Czech Republic. METHODS: Between September 2017 and January 2018, a total of 441 single-patient MRSA isolates were collected from 11 Czech hospitals and analysed by spa typing, SCCmec typing, antibiotic susceptibility testing, detection of the PVL toxin and the arcA gene. RESULTS: Of all MRSA isolates, 81.41% (n = 359) belonged to the CC5-MRSA clone represented by the spa types t003 (n = 136), t586 (n = 92), t014 (n = 81), t002 (n = 20) and other spa types (n = 30); a majority of the CC5 isolates (n = 348, 96.94%) carried SCCmec type II. The occurrence of CC5-MRSA was more likely in older inpatients and associated with a healthcare origin (P < 0.001). The CC5-MRSA isolates were resistant to more antimicrobial drugs compared with the other MRSAs (P < 0.001). Interestingly, t586 was detected in blood samples more often than the other spa types and, contrary to other spa types belonging to CC5-MRSA, t586 was not associated with patients of advanced age. Other frequently found lineages were CC8 (n = 17), CC398 (n = 11) and CC59 (n = 10). The presence of the PVL was detected in 8.62% (n = 38) of the MRSA isolates. CONCLUSIONS: The healthcare-associated CC5-MRSA-II lineage (t003, t586, t014) was found to be predominant in the Czech Republic. t586 is a newly emerging spa type in the Czech Republic, yet reported rarely in other countries. Our observations stress the need for MRSA surveillance in the Czech Republic in order to monitor changes in MRSA epidemiology.