Mec Gene Complex Research Articles

Optical Mapping: Detecting Genomic Resistance Cassettes in MRSA.

Methicillin-resistant Staphylococcus aureus (MRSA) is a multidrug-resistant bacterium with a global presence in healthcare facilities as well as community settings. The resistance of MRSA to beta-lactam antibiotics can be attributed to a mobile genetic element called the staphylococcal cassette chromosome mec (SCCmec), ranging from 23 to 68 kilobase pairs in length. The mec gene complex contained in SCCmec allows MRSA to survive in the presence of penicillin and other beta-lactam antibiotics. We demonstrate that optical mapping (OM) is able to identify the bacterium as S. aureus, followed by an investigation of the presence of kilobase pair range SCCmec elements by examining the associated OM-generated barcode patterns. By employing OM as an alternative to traditional DNA sequencing, we showcase its potential for the detection of complex genetic elements such as SCCmec in MRSA. This approach holds promise for enhancing our understanding of antibiotic resistance mechanisms and facilitating the development of targeted interventions against MRSA infections.

Unearthing New ccr Genes and Staphylococcal Cassette Chromosome Elements in Staphylococci Through Genome Mining.

Staphylococcal cassette chromosome mec (SCCmec) typing is crucial for investigating methicillin-resistant Staphylococcus aureus (MRSA), relying primarily on the combination of ccr and mec gene complexes. To date, 19 ccr genes and 10 ccr gene complexes have been identified, forming 15 SCCmec types. With the vast release of bacterial genome sequences, mining the database for novel ccr gene complexes and SCC/SCCmec elements could enhance MRSA epidemiological studies. In this study, we identified 12 novel ccr genes (6 ccrA, 3 ccrB, and 3 ccrC) through mining of the National Center for Biotechnology Information (NCBI) database, forming 12 novel ccr gene complexes and 10 novel SCC elements. Overexpression of 5 groups of novel Ccr recombinases (CcrA9B3, CcrA10B1, CcrC3, CcrC4, and CcrC5) in a mutant MRSA strain lacking the ccr gene and extrachromosomal circular intermediate (ciSCC) production significantly promoted ciSCC production, demonstrating their biological activity. This discovery provides an opportunity to advance MRSA epidemiological research and develop database-based bacterial typing methods.

Molecular characterization of typing and subtyping of Staphylococcal cassette chromosome SCCmec types I to V in methicillin-resistant Staphylococcus aureus from clinical isolates from COVID-19 patients.

Methicillin resistance is acquired by the bacterium due to mecA gene which codes for penicillin-binding protein (PBP2a) having low affinity for β-lactam antibiotics. mecA gene is located on a mobile genetic element called staphylococcal cassette chromosome mec (SCCmec). SCCmec genomic island comprises two site-specific recombinase genes namely ccrA and ccrB [cassette chromosome recombinase] accountable for mobility. Currently, SCCmec elements are classified into types I, II, III, IV and V based on the nature of the mec and ccr gene complexes and are further classified into subtypes according to variances in their J region DNA. SSCmec type IV has been found in community-acquired isolates with various genetic backgrounds. The present study was undertaken to categorize the types of SCCmec types and subtypes I, II, III, IVa, b, c, d, and V and PVL genes among clinical MRSA isolates from COVID-19 confirmed cases. Based on the Microbiological and Molecular (mecA gene PCR amplification) confirmation of MRSA isolated from 500 MRSA SCCmec clinical samples, 144 cultures were selected for multiplex analysis. The multiplex PCR method developed by Zhang et al. was adapted with some experimental alterations to determine the specific type of these isolates. Of the total 500 MRSA, 144 MRSA (60 were CA-MRSA and 84 were HA-MRSA) were selected for characterization of novel multiplex PCR assay for SSCmec Types I to V in MRSA. Molecular characterization of multiplex PCR analysis revealed results compare to the phenotypic results. Of the 60 CA-MRSA; in 56 MRSA strains type IVa was found and significantly defined as CA-MRSA while 4 strains showed mixed gens subtypes. Type II, III, IA, and V were present in overall 84 HA-MRSA. Molecular subtyping was significantly correlated to define molecularly as CA-MRSA and HA-MRSA however 15 (10%) strains showed mixed genes which indicates the alarming finding of changing epidemiology of CA-MRSA and HA-MRSA as well. We have all witnessed of COVID-19 pandemic, and its mortality was mostly associated with co-morbid conditions and secondary infections of MDR pathogens. Rapid detections of causative agents of these superbugs with their changing epidemiology by investing in typing and subtyping clones are obligatory. We have described an assay designed for targeting SSCmec types and subtypes I, II, III, IVa,V according to the current updated SCCmec typing system. Changing patterns of molecular epidemiology has been observed by this newly described assay.

Novel SCCmec variants in clonal complex 398 and lineage-specific pseudo-SCCmec identified in ST88 MRSA from invasive bloodstream infections in China.

Methicillin resistance in Staphylococcus aureus is primarily due to the mecA gene found in highly diverse staphylococcal cassette chromosome mec (SCCmec) elements, with an increasing number of variants being continually discovered. To characterize two novel SCCmec variants identified in clonal complex (CC) 398 strains and lineage-specific pseudo-SCCmec elements in the ST88 clone. WGS and comparative genomic analysis were used to elucidate the SCCmec element diversity of representative isolates. The non-typeable 47 kb SCCmec found in the CC398 strain SKLX55795 represents a novel subtype of XIV, showing significant differences in structural organization and genetic content within the joining regions compared with the XIV element from the prototype strain SC792. This unique subtype comprised remnants from various mobile genetic elements that encode antimicrobial resistance genes, ultimately forming a large MDR region. Genome analysis of CC398 strain SKLX61416 revealed the presence of a novel 50 kb composite SCCmec with two distinct domains, carrying the ccr gene complexes 5/8 and containing genes for the detoxification of arsenic and sulphide. Further sequence analysis disclosed that 44.23% (23/52) of ST88 strains in our collection carried a lineage-specific pseudo-SCCmec, termed ΨSCCmecST88. This ΨSCCmecST88 harboured the mec gene complex C2, along with a series of genes associated with heavy metal resistance, but lacked an approximately 28 kb region encompassing the ccr gene complex. Our findings provide evidence for the ongoing evolution of SCCmec elements within the CC398 and ST88 clones, underscoring the need for further surveillance to understand the biological significance of these elements.

Genomic features of mecA-positive methicillin-resistant Mammaliicoccus sciuri causing fatal infections in pets admitted to a veterinary intensive care unit

Methicillin-resistant staphylococci have become leading cause of infectious diseases in humans and animals, being categorized as high priority pathogens by the World Health Organization. Although methicillin-resistant Staphylococcus sciuri (recently moved to Mammaliicoccus sciuri) has been widely reported in companion animals, there is scarce information regarding their clinical impact and genomic features. Herein, we reported the occurrence and genomic characteristics of methicillin-resistant M. sciuri recovered from fatal infections in pets admitted to an intensive care unit of a veterinary hospital, in Brazil. Two M. sciuri strains were isolated from bronchoalveolar lavage samples collected from dog (strain SS01) and cat (strain SS02) presenting with sepsis and acute respiratory distress syndrome. Both isolates displayed a multidrug-resistant profile, whereas whole-genome sequencing analysis confirmed the presence of the mecA gene, along to genetic determinant conferring resistance to macrolides, streptogramins, aminoglycosides, and trimethoprim. For both strains, the mec and crr gene complex shared high identity (≥97%) with analogue sequences from a M. sciuri isolated from a human wound infection, in the Czech Republic. Strains were assigned to the sequence type ST52 and the novel ST74. Phylogenomic analysis revealed a broad host range association of these strains with several hosts and sources, including humans, animals, food, and the environment through different years and geographic locations. Our findings demonstrate that infections caused by mecA-positive M. sciuri strains can be a serious threat for veterinary intensive care patients and the medical staff, with additional implications for One Health approaches.

Investigating the relation between resistance pattern and type of Staphylococcal cassette chromosome mec (SCCmec) in methicillin-resistant Staphylococcus aureus.

Background and Objectives:MRSA became a widely recognized cause of mortality worldwide with necessity of its epidemiological pattern study. Typing of MRSA isolates was performed molecularly based on SCCmec type and relation to resistance pattern was investigated.Materials and Methods:Out of 200 clinical specimens, S. aureus was detected phenotypically and confirmed as MRSA by PCR in 124 isolates obtained from associated laboratories of different hospitals of Zagazig, during 2018–2019. Antimicrobial resistance pattern was detected and MRSA SCCmec was typed by two methods.Results:S. aureus rate was high in wounds, sputum, blood, and urine isolates. Antimicrobial resistance rates against cefotaxime, tetracycline, gentamicin, ciprofloxacin, erythromycin, azithromycin, clindamycin, chloramphenicol, sulfamethoxazole-trimethoprim, linezolid and vancomycin were 82.3%, 65.3%, 56.4%, 45.1%, 37.1%, 32.3%, 32.3%, 25%, 7.3%, 2.4% and 0%, respectively. Multiplex-PCR(M-PCR) was able to detect SCCmec element among 57% of isolates classified as SCCmec II (n=40), III (n=21), IVa (n=3), IVd (n=2), V(n=1), and four isolates contain both SCCmec II and SCCmec IV. Traditional typing by PCR for mec and ccr gene complexes was almost concordant with M-PCR. Furthermore, it was able to identify SCCmec types VI, IX, and XIV among 1, 3 and 18 isolates, respectively. No Statistical correlation was established between type of cassette and rate of antimicrobial resistance. Phylogenetic analysis reveals that all ccr types were related to each other and no significant variation in the same ccr type of different SCCmec cassettes.Conclusion:Most MRSA isolates were MDR reflecting antimicrobials misuse. Detection of various SCCmec types among MRSA isolates indictae the complexity of MRSA epidemiology and increase chance for gene sharing creating new types. The presented investigation was important in understanding MRSA epidemiology and diversity in Egypt providing advice for improving therapeutic protocols.

Novel SCCmec type XV (7A) and two pseudo-SCCmec variants in foodborne MRSA in China.

Staphylococcal cassette chromosome mec (SCCmec) elements are highly diverse and have been classified into 14 types. Novel SCCmec variants have been frequently detected from humans and animals but rarely from food. To characterize a novel SCCmec type and two SCCmec variants identified from food-associated MRSA in China. Three MRSA (NV_1, NT_611 and NT_8) collected from retail foods in China were subjected to WGS and the SCCmec elements were determined. The novel SCCmecXV identified in NV_1 carried the mec gene complex class A (mecI-mecR1-mecA-IS431) and the ccr gene complex 7 (ccrA1B6), and a Tn558-mediated phenicol exporter gene fexA was detected in this SCCmecXV cassette. The pseudo-SCCmec elements ΨSCCmecNT_611 and ΨSCCmecNT_8 showed a truncated SCCmec pattern, carrying the class C2 mec gene complex but missing the ccr genes. The ΨSCCmecNT_611 element shared more similarities with those of Staphylococcus haemolyticus (AB478934.1) and carried a heavy metal resistance gene cluster cadD-cadX-arsC-arsB-arsR-copA. The ΨSCCmecNT_8 MRSA exhibited a highly resistant phenotype, showing the absence of a 19.3 kb segment compared with the reference SCCmecXII element (CP019945.1). Notably, a 46 kb region containing multiple transposons encoding antimicrobial or metal resistance genes flanked by IS431 or IS256 was identified ∼30 kb downstream from the mec gene complex in ΨSCCmecNT_8, which might explain such high resistance in MRSA NT_8. Our finding of novel and pseudo-SCCmec elements reflected the ongoing intra/interspecies genetic rearrangements in staphylococci. Further study will be needed to investigate the biological significance and prevalence of those SCCmec variants along the food chain.

Staphylococcal cassette chromosome mec containing a novel mec gene complex, B4.

ObjectivesTo describe a new subclass of mec class B complex identified in Staphylococcus epidermidis.MethodsFour S. epidermidis isolates obtained from bloodstream infections in patients at University Medical Center Groningen (UMCG) were analysed by phenotypic antibiotic susceptibility testing and WGS.ResultsSequence analysis revealed a new staphylococcal cassette chromosome mec (SCCmec) structure in isolate UMCG335. In this structure, plasmid pUB110 was found to be integrated into SCCmec IVc, creating a new SCCmec subtype, IVUMCG335. SCCmec IVc and a copy of plasmid pUB110 were found in other isolates, UMCG364 and UMCG341, respectively, indicating a probability that SCCmec IVUMCG335 could have evolved at the UMCG. SCCmec of UMCG337 contained a new genetic organization of the mec complex (IS431-ΔmecR1-mecA-IS431-pUB110-IS431-ψIS1272) that we have named B4. This new subclass of mec class B complex originated by IS431-mediated inversion of the DNA segment encompassing the plasmid and most of the genes of the mec complex with the exception of IS1272. As the SCCmec organization in UMCG337 differed by the inversion of an ∼10 kb sequence compared with SCCmec IVUMCG335, we have named it SCCmec subtype IVUMCG337. Isolates UMCG335 and UMCG337 carrying SCCmec IVUMCG335 and IVUMCG337, respectively, were associated with a restriction-modification system and a CRISPR-Cas system, creating a composite island of almost 70 kb.ConclusionsOur findings highlight the importance of IS431 in the evolution of the SCCmec region. The increasing genetic diversity identified in the SCCmec elements imposes a great challenge for SCCmec typing methods and highlights possible difficulties with the SCCmec nomenclature.

Staphylococcal cassette chromosome mec elements from Staphylococcus intermedius group (SIG) isolates from dogs in a center for veterinary diagnostics in Brazil

ABSTRACT: Methicillin resistance in the Staphylococcus intermedius group (SIG) has emerged in small animal practice. Methicillin-resistant SIG (MRSIG) members have been implicated as causes of infections in both companion animals and humans. Staphylococcal cassette chromosome mec (SCCmec) elements carry the mecA/C genes, which encode for the transpeptidase PBP2a (PBP2’) responsible for β-lactam antibiotic resistance in staphylococci. This study examined the SCCmec types of MRSIG isolates from different clinical specimens of dogs that exhibited methicillin MIC ≥ 0.5 μg/mL by an automated identification and susceptibility system in a Center for Veterinary Diagnostics in São Paulo, Brazil. Susceptibility to methicillin was determined by broth microdilution testing, and Oxoid® M.I.C.Evaluator® strips. PBP2a production was detected using a latex agglutination assay. SCCmec typing was performed according to the International Working Group on the Classification of Staphylococcal Cassette Chromosome Elements (IWG-SCC) guidelines. SCCmec type II (2A), SCCmec type III (3A), composite SCC structures consisting of a class A mec gene complex in addition to multiple ccr gene complexes, and non-typable SCCmec elements were reported in these MRSIG isolates. SCCmec type variants differing from those so far acknowledged by IWG-SCC were found, indicating new rearrangements in the genetic context of mecA in these canine MRSIG isolates.

Insufficient repeatability and reproducibility of MALDI-TOF MS-based identification of MRSA.

Rapid identification of methicillin-resistant Staphylococcus aureus (MRSA) is essential for proper initial antibiotic therapy and timely set up of hygienic measures. Recently, detection of MRSA using MALDI-TOF mass spectrometer mediated by the peptide-phenol-soluble modulin (PSM-mec)-linked to the class A mec gene complex present in SCCmec cassettes types II, III, and VIII of MRSA strains, has been commercially available. We present here a multicentre study on MALDI-TOF MS detection of MRSA evincing a poor repeatability and reproducibility of the assay. The sensitivity of the assay varies between 50 and 90% in strains carrying psmMEC and psmδ genes encoding for PSM-mec and δ-toxin (a member of the PSM peptide family), respectively. No false positive results were found. The very major error calculation was 30% and the major error achieved 0%. Interlaboratory repeatability varies between 0 and 100%. No significant difference was observed with the use of different cultivation media. Our data showed a poor sensitivity of the method excluding it from the use in routine laboratory testing.

Prevalence, molecular characteristics and antimicrobial susceptibility patterns of MRSA in hospitalized and nonhospitalized patients in Barbados.

The epidemiology and clonality of methicillin-resistant Staphylococcus aureus (MRSA) has not been investigated, as not much research or surveillance has been undertaken to identify and characterize the circulating MRSA strains in Barbados. Prevalence rates, molecular characteristics and antimicrobial susceptibility pattern of MRSA infections in hospitalized and nonhospitalized patients were investigated. A total of 293 isolates were included in the study, with 100 from the hospital and 193 from the public health laboratory. Isolates were collected over a period of 1 (2015–2016) and 3 years (2013–2016) respectively. MRSA was identified using standard microbiologic techniques and was further analysed by multiplex PCR for the presence of the spa, mec gene complex typing and PVL genes (lukS-PV and lukF-PV). A prevalence rate of 19.7% was calculated for those hospitalized. All hospital isolates were sensitive to vancomycin, rifampin, linezolid and cotrimoxazole (trimethoprim/sulfamethoxazole), whilst 82% were sensitive to clindamycin. The PVL gene was detected in 76% of hospital isolates. In the community isolates, resistance was observed in erythromycin (100%), ciprofloxacin (97.4%), clindamycin (13%) and cotrimoxazole (5.7%). There was no resistance to vancomycin. The PVL gene was detected in 97.9% of the isolates, the mecA gene in only 2.1% and the mecC gene in 0%. Most MRSA isolates were community acquired in both settings, and the antimicrobial susceptibility profile was similar, suggesting transmission of community-associated MRSA into the hospital environment. Further harmonization of antimicrobial policy for the treatment of MRSA (and by extension other pathogens) should be implemented to quell ongoing transmission. We found that 93.4% of MRSA in Barbados treated in the primary healthcare system were sensitive to cotrimoxazole. By typing MRSA isolates and drawing interferences on transmission on the basis of genetic relatedness, transmission pathways may be tracked. Further studies must be performed for this high level of comprehensiveness so that with the surveillance of MRSA, effective strategies may be developed to prevent or limit transmission.

Prevalence and characterisation of methicillin-resistant staphylococci from bovine bulk tank milk in England and Wales

ObjectivesTo investigate the prevalence and characteristics of methicillin-resistant staphylococci on dairy farms in England and Wales including zoonotic MRSA. MethodsBulk tank milk was sampled from 363 dairy farms in 2015-2016 and methicillin-resistant staphylococci were isolated by salt broth enrichment and plating on MRSA Brilliance selective agar. Isolates were characterised through antimicrobial susceptibility testing and whole-genome sequencing. ResultsMethicillin-resistant staphylococci were isolated from ∼5% of dairy farms and belonged to six different species, Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus lentus, Staphylococcus saprophyticus, Staphylococcus fleurettii and Staphylococcus sciuri. Whole-genome sequencing revealed a large variety of antimicrobial resistance genes and SCCmec elements were present, including mecA and mecC alleles. Potentially zoonotic methicillin-resistance S. aureus were found at a low prevalence (0.83% of sampled dairy farms). Whole-genome sequencing also provided evidence for the mobility of a primordial mec gene complex, independently of a SCCmec element, which appears to have been acquired by S. saprophyticus from S. fleurettii. ConclusionsThese data give new insight into the epidemiology of veterinary methicillin-resistant staphylococci to inform future surveillance and zoonotic risk evaluation. Our data indicate that MRSA has likely decreased in prevalence since earlier survey work in England and Wales during 2011-12 and highlights the diversity of methicillin resistance and other resistance determinants among bovine-associated staphylococci with implications for veterinary and human medicine.

Unconventional SCCmec types and low prevalence of the Panton-Valentine Leukocidin exotoxin in South African blood culture Staphylococcus aureus surveillance isolates, 2013-2016.

Staphylococcus aureus is a healthcare-associated pathogen that can harbour multiple antimicrobial resistance determinants and express multiple virulence factors e.g. Panton-Valentine Leukocidin (PVL). Unknown staphylococcal cassette chromosome mec (SCCmec) typing patterns were previously observed among 11% (n = 52) of methicillin-resistant S. aureus (MRSA) isolates; we further investigated these as well as the proportion of PVL, encoded by lukS/F-PV, in 761 S. aureus isolates from patients with a diagnosis of pneumonia/lower respiratory tract, skin/soft tissue, bone and joint infection. S. aureus isolates from blood culture were identified and antimicrobial susceptibility testing was performed using automated systems. Conventional PCR assays were used to identify the ccr and mec gene complexes in mecA-positive isolates with an unknown SCCmec type and screen for lukS/F-PV. Epidemiological data was used to classify isolates as healthcare- or community-associated infections. Antimicrobial susceptibility profiles according to SCCmec type and PVL were reported. Of the unknown SCCmec types, isolates were interpreted as type I-like (86%, 38/44), type II-like (9%, 4/44) and type III-like (5%, 2/44). Eight isolates did not produce definitive results. Of all MRSA isolates, majority were multidrug-resistant as indicated by their non-susceptibility to most antimicrobial agents; 92% were healthcare-associated. PVL was seen in 14% of the isolates (MRSA: 25%, MSSA: 75%); 56% were classified as healthcare-associated infection. The SCCmec typing method did not definitively classify all unknown isolates into clearly defined types. It showed that majority of these isolates were not the conventional types; untypeable elements appeared to be composite SCCmec elements, consisting of multiple ccr gene complexes. Majority of the MRSA isolates were non-susceptible to most antibiotics indicating that multiple resistance genes are present in our population. Furthermore, the proportion of PVL was low and more prevalent in MSSA.

Effects of gamma-irradiation on antibiotic resistance and diagnostic molecular markers of methicillin-resistant Staphylococcus aureus in Egyptian cancer patients

:Purpose: This in-vitro study aimed to assess in 120 [40 community-acquired (CA-MRSA) & 80 hospital-acquired (HA-MRSA)] isolates from cancer patients whether the transmissible staphylococcal cassette chromosome mec (SCCmec) typing, and the Panton–Valentine leukocidin (PVL) virulence genes detection could be employed as tools for molecular diagnostic purposes to distinguish both methicillin-resistant Staphylococcus aureus (MRSA) categories in radiotherapy treated cancer patients.Materials and methods: SCCmec typing was determined by the combination of the type of the cassette chromosome recombinase genes (ccr) gene complex and the class of the methicillin resistance (mec) gene complex. Besides, a rapid slide latex agglutination test (LAT) and antibiotic resistance spectrum determination before and after irradiation were performed.Results: In the strict sense, with the effect of irradiation; the presence of SCCmec subtypes IVa (22.5% vs. 10.0%), b (47.5% vs. 25.0%), & d (7.5 vs. 2.5%) or type V (15.0% vs. 7.5%) genetic elements and PVL genes (p < .001) were not proved as a signature for CA-MRSA. While, the larger SCCmec types II, and III elements were not detected in 14, and 19 from the 38, and 36 typed HA-MRSA isolates (p < .001), respectively. Remarkable effects on class A & class B mec gene complex and type2, type 3 & type 5 ccr gene complex and an increase in agglutination reaction strength in response to gamma irradiation external stimulus were observed.Conclusions: Different heterogeneous genetic composition with upregulation mecA gene expression was detected after irradiation in the HA- MRSA studied population. CA-MRSA showed remarkable ability to acquire multi-antibiotic resistance after irradiation and propose a novel paradigm for future chemotherapy against the multi-resistant pathogens whose proliferation especially among immunocompromised cancer patients is on the increase.

Staphylococcal Cassette Chromosome mec (SCCmec) Analysis of MRSA.

Methicillin-susceptible S. aureus changes to methicillin-resistant S. aureus (MRSA) upon the acquisition of staphylococcal cassette chromosome mec (SCCmec), a genomic island that encodes methicillin resistance. SCCmec elements in S. aureus are classified into different types based on the combination of mec gene complexes and ccr gene complexes, which share variations, five classes in mec and eight in ccr. To date, at least 13 types of SCCmec elements have been identified and each SCCmec type has individual characteristics. It is known that hospital-associated MRSA strains carry SCCmec elements of types, I, II, and III, and the majority of community-acquired MRSA strains carry characteristic SCCmec elements, type IV SCCmec or type V SCCmec. We herein describe multiplex PCR methods to type SCCmec elements by identifying the mec gene complex class and ccr gene complex type.

Characterization of mecC gene-carrying coagulase-negative Staphylococcus spp. isolated from various animals

The presence of the methicillin resistance gene mecC in coagulase-negative Staphylococcus spp. (CoNS) is scarce. The aim of this study was to characterize mecC-positive CoNS isolated from various wild and domestic animals. The presence of the mecC gene was screened in 4299 samples from wild animals and domestic animals. Fifteen coagulase-negative staphylococci, that displayed a cefoxitin-resistant phenotype, were tested mecC-positive by PCR. Antimicrobial susceptibility testing was performed for all isolates. The 15 isolates were genotyped by sequencing of the entire class E mec gene complex (blaZ-mecC-mecR1-mecI), the ccrA and ccrB recombinase genes and other determinants within the type XI SCCmec element. DNA microarray analysis was performed and five selected isolates were additionally whole genome sequenced and analyzed. S. stepanovicii (n = 3), S. caprae (n = 1), S. warneri (n = 1), S. xylosus (n = 1) and S. sciuri (n = 9) were detected. All but the S. sciuri isolates were found to be susceptible to all non-beta lactams. The entire class E mec gene complex was detected in all isolates but ccrA and ccrB genes were not identified in S. stepanovicii and S. xylosus. The genes erm(B) and fexA (n = 4, each) were the most predominant non-beta lactam resistance genes detected in the S. sciuri isolates. Even though the presence of the mecC gene among CoNS is a rare observation, this study further expands our knowledge by showing that the mecC gene, including its allotypes, are present in more staphylococcal species from different animal species than has been previously described.

Real-time PCR followed by high-resolution melting analysis - a new robust approach to evaluate SCCmec typing of methicillin-resistant Staphylococcus aureus.

We designed a novel approach based on real-time PCR followed by high-resolution melting (HRM) analysis to determine the Staphylococcal cassette chromosome mec (SCCmec) types of methicillin-resistant Staphylococcus aureusstrains, which we compared against the results of conventional multiplex PCR SCCmec typing. Multiplex PCR (for ccr and mec gene complexes) was carried out as conventional method. The HRM analysis was then designed using standard strains of each SCCmec type. The M-PCR results included types III (33.33%), IV (43.33%) and V (23.33%). HRM analysis was able to distinguish all five types, which were used to set up the protocol with a sensitivity and specificity of 100% compared with the conventional method. This novel method for SCCmec typing has high specificity and sensitivity and can be conducted in a shorter period of time at lower costs.

Description and Comparative Genomics of Macrococcus caseolyticus subsp. hominis subsp. nov., Macrococcus goetzii sp. nov., Macrococcus epidermidis sp. nov., and Macrococcus bohemicus sp. nov., Novel Macrococci From Human Clinical Material With Virulence Potential and Suspected Uptake of Foreign DNA by Natural Transformation.

The genus Macrococcus is a close relative of the genus Staphylococcus. Whilst staphylococci are widespread as human pathogens, macrococci have not yet been reported from human clinical specimens. Here we investigated Gram-positive and catalase-positive cocci recovered from human clinical material and identified as Macrococcus sp. by a polyphasic taxonomic approach and by comparative genomics. Relevant phenotypic, genotypic and chemotaxonomic methods divided the analyzed strains into two separate clusters within the genus Macrococcus. Comparative genomics of four representative strains revealed enormous genome structural plasticity among the studied isolates. We hypothesize that high genomic variability is due to the presence of a com operon, which plays a key role in the natural transformation of bacilli and streptococci. The possible uptake of exogenous DNA by macrococci can contribute to a different mechanism of evolution from staphylococci, where phage-mediated horizontal gene transfer predominates. The described macrococcal genomes harbor novel plasmids, genomic islands and islets, as well as prophages. Capsule gene clusters, intracellular protease, and a fibronectin-binding protein enabling opportunistic pathogenesis were found in all four strains. Furthermore, the presence of a CRISPR-Cas system with 90 spacers in one of the sequenced genomes corresponds with the need to limit the burden of foreign DNA. The highly dynamic genomes could serve as a platform for the exchange of virulence and resistance factors, as was described for the methicillin resistance gene, which was found on the novel composite SCCmec-like element containing a unique mec gene complex that is considered to be one of the missing links in SCC evolution. The phenotypic, genotypic, chemotaxonomic and genomic results demonstrated that the analyzed strains represent one novel subspecies and three novel species of the genus Macrococcus, for which the names Macrococcus caseolyticus subsp. hominis subsp. nov. (type strain CCM 7927T = DSM 103682T), Macrococcus goetzii sp. nov. (type strain CCM 4927T = DSM 103683T), Macrococcus epidermidis sp. nov. (type strain CCM 7099T = DSM 103681T), and Macrococcus bohemicus sp. nov. (type strain CCM 7100T = DSM 103680T) are proposed. Moreover, a formal description of Macrococcus caseolyticus subsp. caseolyticus subsp. nov. and an emended description of the genus Macrococcus are provided.

Genetic features of livestock-associated Staphylococcus aureus ST9 isolates from Chinese pigs that carry the lsa(E) gene for quinupristin/dalfopristin resistance

Whole-genome sequencing (WGS) was used to investigate the genetic features of the recently identified lsa(E) gene in porcine S. aureus ST9 isolates. Three quinupristin/dalfopristin-resistant isolates harboring the lsa(E) gene (two MRSA and one MSSA) were sequenced. Phylogenetic analysis of 184S. aureus genomes showed that ST9 porcine isolates belong to a distinct sequence cluster. Further analysis showed that all isolates were deficient in the recently described type IV restriction-modification system and SCCmec type XII was identified in the two MRSA isolates, which included a rare class C2 mec gene complex. A 24kb ΨSCC fragment was found in the MRSA and MSSA isolates sharing 99% nucleotide sequence homology with the ΨSCCJCSC6690 (O-2) element of a ST9 MRSA isolate from Thailand (accession number AB705453). Comparison of these ST9 isolates with 181 publically available S. aureus genomes identified 24 genes present in all (100%) ST9 isolates, that were absent from the most closely related human isolate. Our analysis suggests that the sequenced quinupristin/dalfopristin-resistant ST9 lineage represent a reservoir of mobile genetic elements associated with resistance and virulence features.

The presence of a single MALDI-TOF mass spectral peak predicts methicillin resistance in staphylococci

The class A mec gene complex also carries the psm-mec gene. The psm-mec gene product can be detected as a peak near 2415m/z using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). The correlation of a 2415m/z peak with methicillin resistance (mecA carriage) in consecutive staphylococcal blood culture isolates was evaluated. A 2415±2.00m/z peak was observed in 37% (51/137) of methicillin-resistant Staphylococcus aureus, in none (0/146) of the methicillin-susceptible S. aureus, in 6% (10/180) of methicillin-resistant S. epidermidis, and in 2% (1/63) of methicillin-susceptible S. epidermidis isolates. Although the sensitivity of the 2415m/z peak for mecA carriage in S. aureus and S. epidermidis was low (37% and 6%, respectively), the specificity was high (≥98%). This peak can be identified during routine MALDI-TOF MS clinical testing, and the analysis adds no reagent cost and requires minimal time expenditure.