Methicillin-resistant Staphylococcus Aureus Typing Research Articles

MOLECULAR IDENTIFICATION AND GENOTYPING OF METHICILLIN-RESISTANT STAPHYLOCOCCUS AUREUS (MRSA) IN DIFFERENT CLINICAL SAMPLES

Methicillin-resistant Staphylococcus aureus (MRSA) is a serious human pathogen that can spread in healthcare facilities and among the general public. This study was aimed to evaluate the prevalence and diversity of SCCmec types of this superbug among hospitalized patients. This study involved phenotypic identification and molecular confirmation of S. aureus based on the nuc gene, molecular detection of MRSA, SCCmec typing, and virulence factor profiling of MRSA clinical isolates obtained from hospitalized patients in Duhok province. Out of the 310 enrolled patients, 33 isolates (10.64%) were identified and confirmed as Staphylococcus aureus, of which 51.5% were identified as MRSA based on phenotypic and molecular targeting of the mecA gene. There were no discernible variations between the prevalence rates of this pathogen in different clinical sources, sexes, or age groups (p-values: 0.71, 0.39, and 0.15 respectively). The isolates had elevated rates of resistance to most antibiotic classes. They were classified as extensive drug-resistant (30.3%), multidrug-resistant (57.5%), and non-multidrug-resistant (12.1%). Additionally, SCCmec typing of MRSA by multiplex PCR identified three different SCCmec types and subtypes, including SCCmec type II (35.5%), followed by 17.64% of SCCmec type IV subtype d (IVd), and SCCmec type III (11.76%). However, 35.3% of the MRSA isolates were found to be non-typeable. Molecular profiling of major virulence factors and toxin genes revealed that 57.5% of the isolates were positive for the exfolitative toxin (ETA), 45.4% of the isolates carried TSST-1 (Toxic Shock Syndrome Toxin-1), the PVL (Panton-Valentine Leukocidin) cytotoxin was identified in 15% of the isolates, and 18.1% of the identified S. aureus isolates were positive for the ACME (arginine catabolic mobile element). The findings of the current investigation pointed out the circulating of highly virulent and extensively resistant MRSA strains among hospitalized patients, which may require active surveillance and better control policies

The molecular epidemiology and clinical implication of methicillin-resistant Staphylococcus aureus (MRSA) sequence types in pediatric bacteremia: a restrospective observational study, 2016-2021.

While there is a high burden of methicillin-resistant Staphylococcus aureus (MRSA) infections among pediatric patients, studies on the molecular epidemiology of MRSA infections in Korean children since the 2010s are lacking. This study aimed to investigate the molecular genotypes and clinical characteristics of MRSA isolates from children with MRSA bacteremia at Asan Medical Center Children's Hospital from 2016 to 2021. Clinical data were retrospectively reviewed, and the molecular types of MRSA were determined using multilocus sequence typing (MLST) and Staphylococcal cassette chromosome mec (SCCmec) typing. The overall methicillin resistance rate of S. aureus bacteremia was 44.8% (77/172); 49.5% in the period 2016-2018 (period 1) and 37.3% in the period 2019-2021 (period 2) (P = 0.116). Community-acquired infections accounted for only 3.9% of cases. The predominant ST group was ST72 group (67.6%), followed by ST5 group (18.9%) and ST1 group (5.4%). The proportion of ST5 was significantly lower in period 2 compared to period 1 (P = 0.02). Compared to the ST5 and ST1 groups, the ST72 group exhibited lower overall antibiotic resistance and multidrug-resistant (MDR) rates (12.0% [6/50] in ST72 group vs. 100.0% [14/14] in ST5 group vs. 50.0% [2/4] in ST1 group; P < 0.001). In the multivariate analysis, the ST1 group was an independent risk factor for 30-day all-cause mortality (aOR, 44.12; 95% CI, 3.46-562.19). The ST72-MRSA strain remained the most frequently isolated genotype in Korean children, while the ST1 group emerged as an independent risk factor for 30-day all-cause mortality in pediatric MRSA bacteremia. Ongoing efforts to uncover the evolving epidemiology of MRSA are essential for developing effective strategies for prevention and treatment.

Emergence and Genomic Characterization of a spa Type t4407 ST6-SCCmec Type IVa Methicillin-Resistant Staphylococcus aureus Strain Isolated from Al-Karak Hospital, Jordan.

Background and Objectives: Methicillin-resistant Staphylococcus aureus (MRSA) is a major concern in Jordanian hospitals in terms of infection control. The purpose of this study was to identify the resistance patterns of Staphylococcus aureus strains isolated from surfaces of critical locations within the Al-Karak Governmental Hospital in 2019. Additionally, the study aimed to conduct whole-genome sequencing on the isolates. Materials and Methods: In February 2019, fourteen S. aureus strains were isolated from surfaces in critical sites in the Al-Karak Governmental Hospital. These isolates underwent antibiogram testing to determine their resistance profile. Genome sequencing using the Illumina MiSeq platform was applied to the extracted DNA from these isolates. The genomic data, including coding sequences, were analyzed to identify lineage, resistance genes, and plasmids. Results: The antibiogram results revealed that 11 of the 14 isolates were resistant to oxacillin, 6 to linezolid, and 1 to rifampicin, while none showed resistance to chloramphenicol. Eleven isolates were identified as MRSA, with a novel spa type (t4407) not previously reported in Jordan. High-quality sequencing data were obtained for only one isolate, i.e., A29, the genome showed 2,789,641 bp with a 32.7% GC content and contained 2650 coding sequences. Genomic analysis indicated the ST6 lineage, mecA gene (SCCmec type IVa(2B)), and a hybrid plasmid (pJOR_blaZ) carrying the blaZ gene for β-lactam resistance. Genomic data were deposited in NCBI (CP104989). The A29 genome closely resembled an MRSA genome isolated from a Danish hospital in 2011. The SNP analysis revealed identical antimicrobial resistance genes in these two genomes. Conclusions: This study unveils the first genomic sequence of an MRSA isolate from Jordan, marked by distinctive genotypic traits. The findings enhance our understanding of the MRSA types circulating in Jordan and the region and substantiate the phenomenon of intercontinental MRSA transmission.

In Vitro Antibacterial Activity of Ceftobiprole and Comparator Compounds against Nation-Wide Bloodstream Isolates and Different Sequence Types of MRSA.

Bloodstream infections by bacteria, especially multidrug-resistant bacteria, remain a worldwide public health concern. We evaluated the antibacterial activity of ceftobiprole and comparable drugs against different bloodstream isolates and different sequence types of methicillin-resistant Staphylococcus aureus (MRSA) in China. We found that MRSA, methicillin-susceptible Staphylococcus aureus (MSSA), and methicillin-susceptible coagulase-negative Staphylococcus (MSCNS) displayed ceftobiprole sensitivity rates of >95%, which are similar to the rates for linezolid, daptomycin, and vancomycin. Of the tested MRCNS strains, 90.4% were sensitive to ceftobiprole. The sensitivities of ST59, ST398, and ST22 MRSA to ceftobiprole were higher than that of ST239. Ceftobiprole's MIC50/90 value against Enterococcus faecalis was 0.25/2 mg/L, whereas Enterococcus faecium was completely resistant to this drug. Ceftobiprole exhibited no activity against ESBL-positive Enterobacterales, with resistance rates between 78.6% and 100%. For ESBL-negative Enterobacterales, excluding Klebsiella oxytoca, the sensitivity to ceftobiprole was comparable to that of ceftazidime, ceftriaxone, and cefepime. The MIC50/90 value of ceftobiprole against Pseudomonas aeruginosa was 2/16 mg/L, and for Acinetobacter baumannii, it was 32/>32 mg/L. Thus, ceftobiprole shows excellent antimicrobial activity against ESBL-negative Enterobacterales and Pseudomonas aeruginosa (comparable to that of ceftazidime, ceftriaxone, and cefepime); however, it is not effective against ESBL-positive Enterobacterales and Acinetobacter baumannii. These results provide important information to clinicians.

The Prevalence, Epidemiological, and Molecular Characterization of Methicillin-Resistant Staphylococcus aureus (MRSA) in Macau (2017-2022).

Macau, recognized as a global tourism hub and the world's most densely populated region, provides a unique environment conducive to methicillin-resistant Staphylococcus aureus (MRSA) transmission in healthcare and community settings, posing a significant public health concern both locally and globally. The epidemiology and molecular characteristics of MRSA in the distinct city of Macau remain largely unelucidated. This five-year longitudinal study (2017-2022) examined the local prevalence and molecular typing of MRSA in Macau, with future MRSA type distribution predicted through ARIMA modeling. We subsequently analyzed the epidemiological characteristics of MRSA, including specimen source, clinical department, collection year, season, patient age, sex, and the annual number of tourists. Comprehensive antibiotic resistance profiles of the strains were also assessed. Of 504 clinically isolated S. aureus strains, 183 (36.3%) were identified as MRSA by the cefoxitin disk diffusion method and validated through multi-locus sequence typing (MLST). The MRSA detection rate showed an upward trend, increasing from 30.1% in 2017 to 45.7% in 2022. SCCmec type IV was predominant (28.9%), followed by types II (25.4%), III (22.1%), and V (22.1%). The primary sources of MRSA isolates were sputum (39.2%) and secretions (25.6%). Older age emerged as a risk factor for MRSA infection, whereas no significant associations were found with seasonal variations, gender, or the annual number of tourists. Despite displaying universal resistance to cefoxitin, oxacillin, and benzylpenicillin, MRSA isolates in Macau remained fully sensitive to vancomycin, tigecycline, quinupristin, nitrofurantoin, and linezolid. Continuous surveillance and analysis of MRSA distribution in Macau could provide invaluable insights for the effective management of MRSA prevention and control measures within healthcare settings.

Within-host genomic evolution of methicillin-resistant Staphylococcus aureus in long-term carriers

Assessing the genomic evolution of Staphylococcus aureus can help us understand how the bacteria adapt to its environment. In this study, we aimed to assess the mutation rate within 144 methicillin-resistant Staphylococcus aureus (MRSA) carriers with a carriage time from 4 to 11 years, including some carriers who belonged to the same households. We found that 23 of the 144 individuals had completely different MRSA types over time and were therefore not long-term carriers of the same MRSA. From the remaining 121 individuals, we performed whole-genome sequencing (WGS) on 424 isolates and then compared these pairwise using core genome multilocus sequence typing (cgMLST) and single-nucleotide polymorphism (SNP) analyses. We found a median within-host mutation rate in long-term MRSA carriers of 4.9 (3.4–6.9) SNPs/genome/year and 2.7 (1.8–4.2) allelic differences/genome/year, when excluding presumed recombination. Furthermore, we stratified the cohort into subgroups and found no significant difference between the median mutation rate of members of households, individuals with presumed continued exposure, e.g., from travel and persons without known continued exposure. Finally, we found that SNPs occurred at random within the genes in our cohort.Key points• Median mutation rate within long-term MRSA carriers of 4.9 (3.4–6.9) SNPs/genome/year• Similar median mutation rates in subgroups (households, travelers)• No hotspots for SNPs within the genome

Molecular epidemiology and change trend of methicillin-resistant Staphylococcus aureus from invasive infections of a hospital in Hangzhou from 2012 to 2018.

The disease caused by methicillin-resistant Staphylococcus aureus (MRSA) is a global public health challenge that threatens society and patients seriously. Therefore, the molecular epidemiology and change trend of MRSA is essential for the control and treatment of diseases caused by the pathogen in their regions. To explore molecular epidemiology of MRSA in Hangzhou, we collected 162 MRSA isolates from 2012 to 2018, conducted the antimicrobial susceptibility and used polymerase chain reaction(PCR) to test the molecular typing including multilocus sequence typing (MLST), staphylococcal chromosome cassette mec (SCCmec), staphylococcal protein A (spa A) and Panton-Valentine leucocidin (PVL). All the strains was divided into community-associated MRSA (CA-MRSA) or hospital-associated MRSA (HA-MRSA). 162 MRSA isolates were divided into 16 STs and 30 spa types. The major ST type was ST5 (96/162, 59.3%) and the predominant spa type was t311 (83/162, 51.2%). Five SCCmec types were found and the most common SCCmec type was type II (101/162, 61.7%). ST5-II-t311 was the predominant MRSA clone. And the prevalence of ST5 MRSA gradually declined from 2014 to 2018 but the prevalence of ST59 MRSA significantly increased. At the same time, livestock-associated methicillin-resistant Staphylococcus aureus(LA-MRSA) ST398 and ST9 were detected. Twenty-eight isolates were PVL gene positive (28/162, 17.3%). The most prevalent PVL-positive clone was ST59-IVa-t437. Comparing with HA-MRSA, CA-MRSA had a lower probability of ST5 (9.1% vs 67.1%, P=0.000) but a higher probability of ST59 (63.6% vs 11.4%, P=0.000), not only that, it was more likely to carrying PVL-positive gene (36.4% vs 14.3%, P=0.028). In summary, the molecular types of MRSA were getting complex over time. ST5-II-t311 was the predominant clone of MRSA isolate with a downward incidence from 2014 to 2018. ST59 MRSA strains, which is thought community related strain are spreading into hospitals and has an upward incidence from 2014 to 2018.

Comparative Genomic Analysis of Staphylococcal Cassette Chromosome mec Type V Staphylococcus aureus Strains and Estimation of the Emergence of SCCmec V Clinical Isolates in Korea.

Staphylococcal cassette chromosome mec type V (SCCmec V) methicillin-resistant Staphylococcus aureus (MRSA) has been recovered from patients and livestock. Using comparative genomic analyses, we evaluated the phylogenetic emergence of SCCmec V after transmission from overseas donor strains to Korean recipient strains. Sixty-three complete MRSA SCCmec V genomes (including six Korean clinical isolates) were used to construct a phylogenetic tree. Single-nucleotide polymorphisms were identified using Snippy, and a maximum-likelihood-based phylogenetic tree was constructed using RAxML. The possible emergence of the most common ancestor was estimated using BactDating. To estimate mecA horizontal gene transfer (HGT) events, Ranger-dtl was applied to 818 SCCmec V strains using publicly available whole-genome data. The phylogenetic tree showed five major clades. German strains formed a major clade; their possible origin was traced to the 1980s. The emergence of Korean SCCmec V clinical isolates was traced to 2000-2010. mecA HGT events in Staphylococcus spp. were identified in seven strains. P7 (Hong Kong outbreak strain) served as the donor strain for two Korean sequence type (ST) 59 strains, whereas the other five recipient strains emerged from different SCCmec V donors. Most Korean SCCmec V strains may have emerged during 2000-2010. A unique MRSA SCCmec V strain, ST72 (a Korean common type of community-associated MRSA), was also identified. The genomic dynamics of this clone with a zoonotic background should be monitored to accurately understand MRSA evolution.

Comparing the Phylogenetic Distribution of Multilocus Sequence Typing, Staphylococcal Protein A, and Staphylococcal Cassette Chromosome Mec Types in Methicillin-Resistant Staphylococcus Aureus (MRSA) in Korea from 1994 to 2020.

Staphylococcus aureus (S. aureus) bacteremia is one of the most frequent and severe bacterial infections worldwide. Methicillin-resistant Staphylococcus aureus (MRSA) is a serious human pathogen that can cause a wide variety of infections. Comparative genetic analyses have shown that despite the existence of a vast number of genotypes, genotypes are restricted to certain geographical locations. By comparing multilocus sequence typing (MLST) and SCCmec types from 1994 to 2020, the present study intended to discover which genotype genes were related to MRSA infections. MLST, Staphylococcus aureus protein A (spa), and SCCmec typings were performed to determine their relationship during those years. Results revealed that MRSA isolates in the Republic of Korea were distributed among all major staphylococcal cassette chromosome mec (SCCmec) types. The majority of SCCmec isolates belonged to SCCmec type II and type IV. The majority of MLST had the sequence type (ST) 72, 239, 8, or 188. By contrast, minorities belonged to ST22 (SCCmec IV), ST772 (SCCmec V), and ST672 (SCCmec V) genotypes. The SCCmec type was determined for various types. The spa type was dispersed, seemingly regardless of its multidrug resistance property. The MLST type was found to be similar to the existing typical type. These results showed some correlations between resistance characteristics and types according to the characteristics of the MLST types distributed, compared to previous papers. Reports on genotype distribution of MLST and SCCmec types in MRSA are rare. These results show a clear distribution of MLST and SCCmec types of MRSA from 1994 to 2020 in the Republic of Korea.

Molecular characterization and epidemiology of methicillin-resistant Staphylococcus aureus isolated from clinical samples in Sokoto, Nigeria

Objectives: Methicillin-resistant Staphylococcus aureus (MRSA) is a major public health threat and a cause of hospital-acquired and community-acquired infections. This study was undertaken to investigate antimicrobial resistance pattern, the genetic lineage, presence of S. aureus protein A (SPA) gene, and staphylococcal chromosomal cassette mec (SCC mec) types of MRSA isolated from clinical samples sent for microbiological test in major government hospitals in Sokoto. Material and Methods: A total of 90 S. aureus MRSA isolates were collected and confirmed using standard microbiological techniques. Antibiotic susceptibility testing was done using the disk diffusion method; mecA detection and sequencing were carried out. Phylogenetic reconstruction was also done using the molecular evolutionary genetics analysis X software and phylogeny tree constructed by Neighbor-Joining method. SCC mec typing and SPA detection were also done. Results: Of the 90 S. aureus isolates, 42 were found to be MRSA using the cefoxitin disk diffusion, the most potent antibiotic against them was quinupristin/dalfopristin with 83.3% followed by rifampicin with 81.0% and 6 clindamycin with a 71.4%. With 78.6% of the isolates showing resistance to the fluoroquinolone antibiotic ciprofloxacin, tetracycline and gentamicin came in second and third, with 64.3% and 61.9% of isolates showing resistance, respectively. Most of the MRSA isolates were resistant to more than three antibiotics. Polymerase chain reaction showed 36 (85.7%) harbored the mecA gene and of the 36 mecA positive isolates, phylogenetic reconstruction of representative MRSA sequences showed that MRSA sequences in this study clustered in two closest clades suggesting a possible horizontal transfer. Of the 36 isolates, 23 were SCC mec type I, ten were type IV, and three were non-typeable, while the SPA gene was detected in all the isolates amplified. Conclusion: The use of phenotypic and molecular methods in this study provided useful information on antibiotic resistance profile, epidemiology, and molecular characteristics of MRSA isolates in Sokoto Nigeria. The information provided could help in monitoring the evolution of MRSA strains in Nigeria over time.

Molecular epidemiological characteristics and genetic evolutionary relationships of methicillin-resistant Staphylococcus aureus of different avian origins in Qingdao, China, using whole-genome sequencing.

To understand the prevalence of avian methicillin-resistant Staphylococcus aureus (MRSA) and the current status of drug resistance in Qingdao, a comprehensive molecular epidemiological investigation and analysis of evolutionary relationships of MRSA isolates from broiler and layer chickens and waterfowl was conducted. One hundred and two avian MRSA strains were identified by multi-locus sequence typing, staphylococcal protein A (spa) and staphylococcal cassette chromosome mec (SCCmec) typing, and whole-genome sequencing. The sequence type (ST) 9-t899-SCCmec IVb type represented the highest proportion of avian-derived MRSA strains (71.57%), with ST398 type strains occasionally observed in broilers and waterfowl. The poultry-derived MRSA strains were all resistant to eight or more antimicrobials. Avian-derived MRSA strains carried 20 resistance genes, 109 virulence genes and 10 plasmids. Strains carrying the cfr oxazolidinone resistance gene were occasionally seen in broiler- and layer-derived MRSA. Single nucleotide polymorphism (SNP) core genome evolution and locus difference analysis showed that the closest strains were all of ST9-t899 type (to which also affiliated the highest number of strains) and this type occurred on all three kinds of poultry farm, but the SNP difference loci between strains of the same type ranged from 0 to 1472. The dominant type of MRSA from different poultry sources in Qingdao is ST9-t899-SCCmec IVb, which is commonly resistant to a variety of antimicrobial drugs and carries a variety of resistance genes and a large number of virulence genes. Sequence type 9-t899 type is widely spread among the three kinds of poultry investigated, but there are differences in affiliations.

Repurposing CD5789 as an Antimicrobial Agent Against MRSA and Its High Resistant Phonotypes.

Methicillin-resistant Staphylococcus aureus (MRSA) poses a great threat to human health, and the formation of biofilm and persister cells make the situation even worse. Drug repurposing is an effective way to solve this problem by shortening the drug development times and reducing the research costs. In this study, CD5789 (trifarotene), a fourth-generation retinoid to be approved by the FDA in 2019 for the topical acne vulgaris regimens, was exhibited antimicrobial activity against MRSA type strains and its clinical isolates with the minimal concentration (MIC) of 2-4μg/mL and 4-16μg/mL, respectively, in a dose-dependent manner. By crystal violet staining, we found that CD5789 could inhibit the biofilm formation by MRSA and could further eradicate the pre-formed biofilm at the concentration of 8μg/mL. By checkerboard dilution assay, sub-MIC of CD5789 showed synergistic antimicrobial effects with sub-MIC of gentamycin against MRSA type strains as well as clinical isolates. In addition, CD5789 also exhibited effective bactericidal activity against MRSA persister cells at the concentration of 8 ~ 16μg/mL. Extremely low cytotoxicity of CD5789 was observed by CCK-8 assay indicated the well tolerability to human body. In all, CD5789 has the potential to be an alternative for the treatment of refractory MRSA-related infections.

Spa Typing of Methicillin-Resistant Staphylococcus aureus Based on Whole-Genome Sequencing: the Impact of the Assembler.

Sequencing of the spa gene of methicillin-resistant Staphylococcus aureus (MRSA) is used for assigning spa types to e.g., detect transmission and control outbreaks. Traditionally, spa typing is performed by Sanger sequencing but has in recent years been replaced by whole-genome sequencing (WGS) in some laboratories. Spa typing by WGS involves de novo assembly of millions of short sequencing reads into larger contiguous sequences, from which the spa type is then determined. The choice of assembly program therefore potentially impacts the spa typing result. In this study, WGS of 1,754 MRSA isolates was followed by de novo assembly using the assembly programs SPAdes (with two different sets of parameters) and SKESA. The spa types were assigned and compared to the spa types obtained by Sanger sequencing, regarding the latter as the correct spa types. SPAdes with the two different settings resulted in assembly of the correct spa type for 84.8% and 97.6% of the isolates, respectively, while SKESA assembled the correct spa type in 98.6% of cases. The misassembled spa types were generally two spa repeats shorter than the correct spa type and mainly included spa types with repetition of the same repeats. WGS-based spa typing is thus very accurate compared to Sanger sequencing, when the best assembly program for this purpose is used. IMPORTANCE spa typing of methicillin-resistant Staphylococcus aureus (MRSA) is widely used by clinicians, infection control workers, and researchers both in local outbreak investigations and as an easy way to communicate and compare MRSA types between laboratories and countries. Traditionally, spa types are determined by Sanger sequencing, but in recent years a whole-genome sequencing (WGS)-based approach has become increasingly used. In this study, we compared spa typing by WGS using different methods for assembling the genome from short sequencing reads and compared to Sanger sequencing as the gold standard. We find substantial differences in correct assembly of spa types between the assembly methods. Our findings are therefore important for the quality of WGS based spa typing data being exchanged by clinical microbiology laboratories.

Performance of a Machine Learning-Based Methicillin Resistance of Staphylococcus aureus Identification System Using MALDI-TOF MS and Comparison of the Accuracy according to SCCmec Types.

The prompt presumptive identification of methicillin-resistant Staphylococcus aureus (MRSA) using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) can aid in early clinical management and infection control during routine bacterial identification procedures. This study applied a machine learning approach to MALDI-TOF peaks for the presumptive identification of MRSA and compared the accuracy according to staphylococcal cassette chromosome mec (SCCmec) types. We analyzed 194 S. aureus clinical isolates to evaluate the machine learning-based identification system (AMRQuest software, v.2.1, ASTA: Suwon, Korea), which was constructed with 359 S. aureus clinical isolates for the learning dataset. This system showed a sensitivity of 91.8%, specificity of 83.3%, and accuracy of 87.6% in distinguishing MRSA. For SCCmec II and IVA types, common MRSA types in a hospital context, the accuracy was 95.4% and 96.1%, respectively, while for the SCCmec IV type, it was 21.4%. The accuracy was 90.9% for methicillin-susceptible S. aureus. This presumptive MRSA identification system may be helpful for the management of patients before the performance of routine antimicrobial resistance testing. Further optimization of the machine learning model with more datasets could help achieve rapid identification of MRSA with less effort in routine clinical procedures using MALDI-TOF MS as an identification method.

Characterization of Methicillin-Resistant Staphylococcus aureus Isolates from Periprosthetic Joint Infections.

Periprosthetic joint infection (PJI) is a troublesome clinical issue in total joint arthroplasty (TJA). Although methicillin-resistant Staphylococcus aureus (MRSA) is considered to be the most serious pathogen in PJIs, little is known about the genotypic and phenotypic characteristics of MRSA clones isolated from PJI patients. A total of 36 MRSA isolates from PJI patients were collected at the Chang-Gung Memorial Hospital in Taiwan from May 2016 to October 2019. All MRSA isolates were subjected to genome typing. The prevalence of Panton–Valentine leucocidin (PVL), the antibiotic susceptibility profile, and the biofilm formation ability were compared among different MRSA genogroups. Additionally, demographics and clinical manifestations of patients infected with different MRSA genogroups were investigated. Eight sequence types (STs) were identified among 36 isolated from PJIs. According to the incidence of MRSA genotypes in PJIs, in this study, we divided them into four groups, including ST8 (n = 10), ST59 (n = 8), ST239 (n = 11), and other STs (n = 7). For the antibiotic susceptibility testing, we found that all MRSA isolates in the ST239 group were highly resistant to ciprofloxacin, gentamicin trimethoprim-sulfamethoxazole, and levofloxacin. Additionally, ST239 MRSA also had a higher ability to form biofilm than other groups. Importantly, patients with ST239 infection typically had a fever and exhibited higher levels of inflammatory markers, including C-reactive protein (CRP) and white blood cell count (WBC). Epidemiological investigations revealed that knee PJIs were mainly attributed to infection with ST59 MRSA and increasing trends for infection with ST8 and other ST types of MRSAs in PJI patients were observed from 2016 to 2019. The identification of MRSA genotypes in PJIs may be helpful for the management of PJIs.

Predominance of ST8 and CC1/spa-t1784 methicillin-resistant Staphylococcus aureus isolates in Japan and their genomic characteristics

Methicillin-resistant Staphylococcus aureus (MRSA) has become a serious epidemiologic problem worldwide. In this study, we aimed to investigate recently isolated MRSA types and determine their characteristics. We collected 164 strains isolated from 13 hospitals located in Tokyo and surrounding prefectures. In addition to drug resistance tests, we sequenced whole genomes of the prevalent MRSA clones and analysed their genomic characteristics, such as drug resistance genes, virulence factor genes, and genome arrangements. Multilocus sequencing typing showed that 51% of the SCCmecⅣ MRSA isolates belonged to clonal complex 1 (CC1). Staphylococcus protein A gene (spa) typing showed that 91% of these CC1 isolates could be categorised as t1784 type. These CC1/t1784 isolates possessed genes encoding erythromycin resistance protein, spectinomycin 9-adenylyltransferase, and staphylococcal enterotoxins (SEA, SEI, SEM), but not the pvl gene encoding Panton-Valentine leukocidin. Complete genomic analysis of nine CC1/t1784 isolates showed that they shared an intact phage, which carried no annotated virulence factor genes except for two encoding a hypothetical membrane protein and a teichoic acid biosynthesis protein. No significant genomic rearrangements were observed among the CC1/t1784 isolates. These data and previous reports indicate that this CC1/t1784 clone has been expanding rapidly in Japan without genomic changes.

Molecular Typing of Methicillin Resistant Staphylococcus aureus using Coa Gene Polymerase Chain Reaction-Restriction Fragment Length Polymorphism: A Cross-sectional Study

Introduction: Methicillin Resistant Staphylococcus aureus (MRSA) are the most important multidrug resistant pathogen of humans causing a wide array of infections. Polymorphic Coagulase gene (coa) could be targeted for specific typing of Staphylococcus aureus isolates. ‘Possible source’ can be identified and discriminated rapidly for control and prevention of Staphylococcus aureus infections especially in case of suspected outbreaks. Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) have high discriminatory power and enhanced reproducibility. Mutation and variation in number of short sequence repeats lead to distinct amplification of this region of coa gene by PCR. Deoxyribonucleic Acid (DNA) fragments of different sizes obtained by digestion with specific restriction enzymes like Arthrobacter luteus I (AluI) help to discriminate various types based on the patterns produced as a result of PCR-RFLP. Aim: To determine molecular typing of MRSA by coa gene PCR-RFLP. Materials and Methods: A cross-sectional study was done in the Department of Microbiology involving 150 clinical isolates of Staphylococcus aureus obtained from various clinical specimens. Genotypic identification of MRSA was done by detecting coa gene and mecA gene by PCR. Molecular typing of MRSA was done by coa gene PCR-RFLP. Results: Coa gene PCR identified six Genotypic codes (Code I to Code VI) ranging from 300 bp-800 bp size. Restriction digestion of the amplicons of the coa gene by PCR-RFLP using the enzyme AluI provided 17 unique restriction patterns among MRSA isolates in toto. The predominant coa gene PCR Genotype code was “Type IV” which was 600 bp size and predominant RFLP fragment for Genotype code “Type IV” was RFLP Pattern ‘b’ (500 bp, 240 bp and 140 bp fragments) among the study population. Conclusion: Genotype code ‘IV’ and RFLP pattern ‘b’ was found to be predominant coa gene type among MRSA in this study

Comparison of the Staphylococcal Chromosome Cassette (SCC) mec in Methicillin-Resistant Staphylococcus aureus (MRSA) and Non-aureus Staphylococci (MRNAS) from Animals and Humans.

Methicillin-resistant Staphylococcus aureus (MRSA) and non-aureus staphylococci (MRNAS) cause different infections in animals, including mastitis, in livestock and humans. This study aimed to identify and compare the staphylococcal chromosome cassette mec (SCCmec) types of MRSA or MRNAS isolated from several animal species and humans in different countries. Of 1462 S. aureus and non-aureus staphylococci, 68 grew on Chrom MRSA ID® agar, were phenotypically resistant to cefoxitin and tested positive with the PCR for the mecA gene. These 60 MRSA and 8 MRNAS were isolated in Belgium mainly from cows (livestock-associated (LA) MRS) and humans (community-acquired (CA) MRS) and in Japan from dogs and cats. The SCCmec cassettes were identified by multiplex PCR in 52 MRSA and 7 MRNAS and by whole genome sequencing (WGS) in 8 additional MRSA. The SCCmec types IV and V were the most frequent in Belgian LA-MRS and CA-MRS, while the SCCmec type II was identified in four of the five Japanese MRSA. The remaining isolate was a bovine S. haemolyticus in which no SCCmec was identified. These results confirm the high prevalence of the SCCmec types IV and V in LA-MRS and CA-MRS in Belgium, emphasizing the possible public health hazard of the former, and the absence of SCCmec in some MRNAS.

Emergence of HA-MRSA Associated Staphylococcal Cassette Chromosome mec (SCCmec) Elements Spillover to LA-MRSA

Methicillin-resistant Staphylococcus aureus (MRSA) once confined to hospitals (HA-MRSA), in the last two decades, have emerged in the community in people with no exposure to hospital and also in livestock animals, referred to as community-associated (CA-) and livestock-associated (LA-) MRSA, respectively. Prevention of the dissemination of MRSA requires better knowledge of its distribution and transmission. The study aims to investigate samples from different animal farms and veterinary clinics for identification and typing of MRSA circulating among different farm animals. Phenotypic and genetic characteristics were evaluated based on morpho-cultural and biochemical properties and PCR amplification of nuc genes. Methicillin resistance was assessed on ORSAB media and PCR amplification of methicillin resistance mecA gene. SCCmec typing was performed by multiplex PCR to assess the epidemiological relationship between the isolates. Of the 406 samples, 72 confirmed the presence of S. aureus. Among S. aureus isolates, based on PCR amplification of mecA gene, 15 were identified as LA-MRSA. Majority of the LA-MRSA were found to be SCCmec type III with two isolates each from cattle and pig showed presence of both type I and III. The finding suggests the spillover of the SCCmec type III into livestock and emergence of new clones possessing both type I and III elements.

Molecular analysis and the toxin, MSCRAMM, and biofilm genes of methicillin-resistant Staphylococcus aureus strains isolated from pemphigus wounds: A study based on SCCmec and dru typing.

Pemphigus is a chronic autoimmune blistering disease. Pemphigus blisters can damage the natural skin barrier and increase the risk of life-threatening conditions. Colonization of pemphigus wounds with methicillin-resistant Staphylococcus aureus (MRSA) prolongs wound healing and increases mortality rate. Assessing MRSA prevalence, types, and toxin and adhesion genes can facilitate the detection of MRSA strains which cause infections, selection of appropriate treatments, and healing of pemphigus wounds. This study aimed to determine the SCCmec, the direct repeat unit (dru) types (dts), and the toxin, MSCRAMM, and biofilm genes of MRSA strains isolated from pemphigus wounds. In this cross-sectional study, 118S. aureus isolates were gathered from 118 patients with pemphigus. MRSA detection was performed using the mecA gene. Using the polymerase chain reaction method, all MRSA isolates were assessed for the presence of the sea, seb, sec, tst, eta, pvl, hla, hlb, MSCRAMM, and ica genes. Typing and subtyping were performed through respectively SCCmec typing and dru typing methods. The Bionumerics software was used for analyzing the data and drawing the minimum spanning tree. From 118S. aureus isolates, 51 were MRSA. SCCmec typing revealed the prevalence of SCCmec II with a prevalence of 64.7% (33 out of 51 isolates) and SCCmec III with a prevalence of 35.3% (18 out of 51 isolates). Dru typing indicated seven dts, namely dts 10a, 10g, 10m, 13i, 8h, 8i, and 9ca in two main clusters. The dt9ca was a new dru type and was registered in the dru-typing database (www.dru-typing.org). The prevalence rates of the hla, sea, and sec genes in MRSA isolates were respectively 54.9%, 27.4%, and 1.9%, while the hlb, seb, eta, and pvl genes were not detected at all. Only one MRSA with SCCmec III and dt10a carried the tst encoding gene. MSCRAMM gene analysis revealed the high prevalence of the eno (31.3%) and the fib (21.5%) genes. The prevalence rates of the icaA and icaD biofilm formation genes were 3.9% and 5.8%, respectively. There were no significant differences between the two detected SCCmec types and between the two detected dts clusters respecting the prevalence of the encoding genes of virulence factors and MSCRAMMs. The toxin genes hla and sea are prevalent among MRSA strains with SCCmec II and III isolated from pemphigus wounds. The most prevalent dts are dt10a and dt10g among MRSA with SCCmec III and dt8h and dt8i among MRSA with SCCmec II.