SCC Elements Research Articles

Genomic portraits of methicillin-resistant staphylococci (MRS) from food fish unveiled the genes associated with staphylococcal food poisoning (SFP), virulence and antimicrobial resistance

BackgroundCharacteristics of non-clinical strains of methicillin-resistant Staphylococcus aureus (MRSA) especially from fishery environment are poorly understood. This research, in addition to comprehensive characterisation, sought to delineate the genetic relatedness between the MRSA strains originating from clinical as well as non-clinical settings. Out of 39 methicillin-resistant staphylococcal isolates from 197 fish samples, 6 (Three each of methicillin-resistant S. haemolyticus (MRSH) and MRSA) with distinct resistance profiles were selected for whole-genome sequencing. Using respective bioinformatics tools, MRSA genomes were comprehensively characterized for resistome, virulomes, molecular epidemiology and phylogenetic analysis. Simultaneously, MRSH genomes were specifically examined to characterize antimicrobial resistance genes (ARGs), owing to the fact that MRSH is often recognized as a reservoir for resistance determinants.ResultsThree MRSA clones identified in this study include ST672-IVd/t13599 (sequence type-SCCmec type/spa type), ST88-V/t2526, and ST672-IVa/t1309. Though, the isolates were phenotypically vancomycin-sensitive, five of the six genomes carried vancomycin resistance genes including the VanT (VanG cluster) or VanY (VanM cluster). Among the three MRSA, only one harbored the gene encoding Panton-Valentine Leukocidin (PVL) toxin, while staphylococcal enterotoxin (SEs) genes such as sea and seb, associated with staphylococcal food poisoning were identified in two other MRSA. Genomes of MRSH carried a composite of type V staphylococcal cassette chromosome mec (SCCmec) elements (5C2 & 5). This finding may be explained by the inversion and recombination events that may facilitate the integration of type V elements to the SCC elements of S. aureus with a methicillin-susceptible phenotype. Phylogenetically, MRSA from a non-clinical setting displayed a considerable relatedness to that from clinical settings.ConclusionThis study highlights the genetic diversity and resistance profiles of MRSA and MRSH, with non-clinical MRSA showing notable relatedness to clinical strains. Future research should explore resistance gene transfer mechanisms and environmental reservoirs to better manage MRSA spread.

Diagnostic validity of p16, E-cadherin, cyclin D1, p53, and HPV E6/E7 mRNA in CIN 3-like squamous cell carcinoma of the cervix.

Cervical intraepithelial neoplasia grade 3 (CIN 3)-like SCC is a recently identified deceptive growth pattern that closely mimics endocervical crypt involvement by CIN 3. As CIN 3-like SCC is indistinguishable from endocervical crypt involvement by CIN 3, it poses a significant challenge for pathologists. We examined 23 cases of CIN 3-like SCC, 6 of which also had concomitant conventional invasive SCC, and 9 cases of endocervical crypt involvement by CIN 3 as a control group. Immunohistochemistry was used to investigate the expression of p16, E-cadherin, cyclin D1, and p53, and the expression of human papillomavirus (HPV) E6/E7 mRNA, the key virus carcinogen of HPV, was detected. The clinicopathological, immunohistochemical, and molecular characteristics of endocervical crypt involvement by CIN 3, CIN 3-like SCC, and the concomitant conventional invasive SCC element were examined. CIN 3-like SCC exhibited a characteristic morphology similar to endocervical crypt involvement by CIN 3, with pushing borders invading into the wall of the cervix, often to a significant depth in most cases. Immunophenotypic features of E-cadherin, p16, cyclin D1, and p53 differed between CIN 3-like SCC and conventional invasive SCC, both in staining intensity and region. E6/E7 mRNA expression was higher in CIN 3-like SCC than in endocervical crypt involvement by CIN 3 (P < 0.05). CIN 3-like SCC is the type of cancer, presenting numerous challenges and potential for confusion as it mimics the phenotypes of endocervical crypt involvement by CIN 3.

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 Chimeric Staphylococcus aureus Strains from Waterfowl.

Staphylococcus aureus is a versatile pathogen that does not only occur in humans but also in various wild and domestic animals, including several avian species. When characterizing S. aureus isolates from waterfowl, isolates were identified as atypical CC133 by DNA microarray analysis. They differed from previously sequenced CC133 strains in the presence of the collagen adhesin gene cna; some also showed a different capsule type and a deviant spa type. Thus, they were subjected to whole-genome sequencing. This revealed multiple insertions of large regions of DNA from other S. aureus lineages into a CC133-derived backbone genome. Three distinct strains were identified based on the size and extent of these inserts. One strain comprised two small inserts of foreign DNA up- and downstream of oriC; one of about 7000 nt or 0.25% originated from CC692 and the other, at ca. 38,000 nt or 1.3% slightly larger one was of CC522 provenance. The second strain carried a larger CC692 insert (nearly 257,000 nt or 10% of the strain's genome), and its CC522-derived insert was also larger, at about 53,500 nt or 2% of the genome). The third strain carried an identical CC692-derived region (in which the same mutations were observed as in the second strain), but it had a considerably larger CC522-like insertion of about 167,000 nt or 5.9% of the genome. Both isolates of the first, and two out of four isolates of the second strain also harbored a hemolysin-beta-integrating prophage carrying "bird-specific" virulence factors, ornithine cyclodeaminase D0K6J8 and a putative protease D0K6J9. Furthermore, isolates had two different variants of SCC elements that lacked mecA/mecC genes. These findings highlight the role of horizontal gene transfer in the evolution of S. aureus facilitated by SCC elements, by phages, and by a yet undescribed mechanism for large-scale exchange of core genomic DNA.

Clonal Complexes Distribution of Staphylococcus aureus Isolates from Clinical Samples from the Caribbean Islands.

The aim of this study was to comprehensively characterise S. aureus from the Caribbean Islands of Trinidad and Tobago, and Jamaica. A total of 101 S. aureus/argenteus isolates were collected in 2020, mainly from patients with skin and soft tissue infections. They were characterised by DNA microarray allowing the detection of ca. 170 target genes and assignment to clonal complexes (CC)s and strains. In addition, the in vitro production of Panton-Valentine leukocidin (PVL) was examined by an experimental lateral flow assay. Two isolates were identified as S. argenteus, CC2596. The remaining S. aureus isolates were assigned to 21 CCs. The PVL rate among methicillin-susceptible S. aureus (MSSA) isolates was high (38/101), and 37 of the 38 genotypically positive isolates also yielded positive lateral flow results. The isolate that did not produce PVL was genome-sequenced, and it was shown to have a frameshift mutation in agrC. The high rate of PVL genes can be attributed to the presence of a known local CC8-MSSA clone in Trinidad and Tobago (n = 12) and to CC152-MSSA (n = 15). In contrast to earlier surveys, the USA300 clone was not found, although one MSSA isolate carried the ACME element, probably being a mecA-deficient derivative of this strain. Ten isolates, all from Trinidad and Tobago, were identified as MRSA. The pandemic ST239-MRSA-III strain was still common (n = 7), but five isolates showed a composite SCCmec element not observed elsewhere. Three isolates were sequenced. That showed a group of genes (among others, speG, crzC, and ccrA/B-4) to be linked to its SCC element, as previously found in some CC5- and CC8-MRSA, as well as in S. epidermidis. The other three MRSA belonged to CC22, CC72, and CC88, indicating epidemiological connections to Africa and the Middle East.

Characteristics of staphylococcal cassette chromosome mec and lugdunin operon genes in the complete genome of Staphylococcus lugdunensis.

Characteristics of staphylococcal cassette chromosome mec and lugdunin operon genes in the complete genome of Staphylococcus lugdunensis.

Characterizing a Novel Staphylococcal Cassette Chromosome mec with a Composite Structure from a Clinical Strain of Staphylococcus hominis, C34847.

Staphylococcal cassette chromosome mec (SCCmec) has predominantly been described in methicillin-resistant Staphylococcus aureus. However, studies have indicated that coagulase-negative staphylococci (CoNS) carry a larger diversity of SCC elements. We characterized a composite SCCmec element carrying an uncharacterized ccr1 and type A mec gene combination, in conjunction with a secondary element bearing ccr4, from a clinical strain of Staphylococcus hominis. The element's complex structure points to a high degree of recombination occurring in SCCmec in CoNS.

Characterisation of S. aureus/MRSA CC1153 and review of mobile genetic elements carrying the fusidic acid resistance gene fusC

While many data on molecular epidemiology of MRSA are available for North America, Western Europe and Australia, much less is known on the distribution of MRSA clones elsewhere. Here, we describe a poorly known lineage from the Middle East, CC1153, to which several strains from humans and livestock belong. Isolates were characterised using DNA microarrays and one isolate from the United Arab Emirates was sequenced using Nanopore technology. CC1153 carries agr II and capsule type 5 genes. Enterotoxin genes are rarely present, but PVL is common. Associated spa types include t504, t903 and t13507. PVL-positive CC1153-MSSA were found in Egyptian cattle suffering from mastitis. It was also identified among humans with skin and soft tissue infections in Saudi Arabia, France and Germany. CC1153-MRSA were mainly observed in Arabian Gulf countries. Some isolates presented with a previously unknown SCCmec/SCCfus chimeric element in which a mec B complex was found together with the fusidic acid resistance gene fusC and accompanying genes including ccrA/B-1 recombinase genes. Other isolates carried SCCmec V elements that usually also included fusC. Distribution and emergence of CC1153-MRSA show the necessity of molecular characterization of MRSA that are resistant to fusidic acid. These strains pose a public health threat as they combine resistance to beta-lactams used in hospitals as well as to fusidic acid used in the community. Because of the high prevalence of fusC-positive MRSA in the Middle East, sequences and descriptions of SCC elements harbouring fusC and/or mecA are reviewed. When comparing fusC and its surrounding regions from the CC1153 strain to available published sequences, it became obvious that there are four fusC alleles and five distinct types of fusC gene complexes reminiscent to the mec complexes in SCCmec elements. Likewise, they are associated with different sets of ccrA/B recombinase genes and additional payload that might include entire mec complexes or SCCmec elements.

Distribution of Staphylococcal cassette chromosome mecA (SCCmec) types among coagulase-negative Staphylococci isolates from healthcare workers in the North-West of Iran.

Objective(s):Methicillin-resistant coagulase-negative Staphylococci (MR-CoNS) are recognized as one of the major causes of healthcare-associated infections in hospitals. The present investigation aimed to study the prevalence of Staphylococcal cassette chromosome mec (SCCmec) types, along with aminoglycoside modifying enzymes (AMEs) genes in the nasal carriage of MR-CoNS in the north-west of Iran.Materials and Methods:To assess the potential of coagulase-negative Staphylococci as hidden reservoirs for antibiotic resistance, we analyzed the antimicrobial susceptibility of MR-CoNS using the disk diffusion method. In addition, PCR and multiplex PCR assays were performed to determine the prevalence of AME encoding genes and SCCmec types in methicillin-resistant coagulase-negative Staphylococci isolates.Results:A total of 51 MR-CoNS isolates were recovered from the anterior nares of healthcare workers. The observed resistance rates to tobramycin, gentamicin, cotrimoxazole, kanamycin, erythromycin, tetracycline, and ciprofloxacin were 74.5%, 68.5%, 57%, 53%, 51%, 49%, and 8%, respectively. Of the 51 tested MR-CoNS isolates, 2(4%) were harboring SCCmec type I, four (8%) were type II, six (12%) type III, eleven (21.6%) type IVa, two (4%) type IVb, two (4%) type IVc, six (12%) type IVd, and two (4%) type V. The rates of prevalence of the aminoglycoside modifying enzyme genes were as follows: aac (6′)/aph (2′′) (28 cases, 55 %), ant (4′)-Ia (20 cases, 39%), and the aph (3´)-IIIa gene (9 cases, 17.6 %).Conclusion:Subtypes IVa and IVd were the most prevalent SCC elements, and aac (6′)/aph (2′′) was the most common AME gene detected among the MR-CoNS isolates.

Staphylococcus epidermidis MSCRAMM SesJ Is Encoded in Composite Islands.

Staphylococcus epidermidis is a leading cause of nosocomial infections in patients with a compromised immune system and/or an implanted medical device. Seventy to 90% of S. epidermidis clinical isolates are methicillin resistant and carry the mecA gene, present in a mobile genetic element (MGE) called the staphylococcal cassette chromosome mec (SCCmec) element. Along with the presence of antibiotic and heavy metal resistance genes, MGEs can also contain genes encoding secreted or cell wall-anchored virulence factors. In our earlier studies of S. epidermidis clinical isolates, we discovered S. epidermidis surface protein J (SesJ), a prototype of a recently discovered subfamily of the microbial surface component recognizing adhesive matrix molecule (MSCRAMM) group. MSCRAMMs are major virulence factors of pathogenic Gram-positive bacteria. Here, we report that the sesJ gene is always accompanied by two glycosyltransferase genes, gtfA and gtfB, and is present in two MGEs, called the arginine catabolic mobile element (ACME) and the staphylococcal cassette chromosome (SCC) element. The presence of the sesJ gene was associated with the left-hand direct repeat DR_B or DR_E. When inserted via DR_E, the sesJ gene was encoded in the SCC element. When inserted via DR_B, the sesJ gene was accompanied by the genes for the type 1 restriction modification system and was encoded in the ACME. Additionally, the SCC element and ACME carry different isoforms of the SesJ protein. To date, the genes encoding MSCRAMMs have been seen to be located in the bacterial core genome. Here, we report the presence of an MSCRAMM in an MGE in S. epidermidis clinical isolates.IMPORTANCES. epidermidis is an opportunistic bacterium that has established itself as a successful nosocomial pathogen. The modern era of novel therapeutics and medical devices has extended the longevity of human life, but at the same time, we also witness the evolution of pathogens to adapt to newly available niches in the host. Increasing antibiotic resistance among pathogens provides an example of such pathogen adaptation. With limited opportunities to modify the core genome, most of the adaptation occurs by acquiring new genes, such as virulence factors and antibiotic resistance determinants present in MGEs. In this study, we describe that the sesJ gene, encoding a recently discovered cell wall-anchored protein in S. epidermidis, is present in both ACME and the SCC element. The presence of virulence factors in MGEs can influence the virulence potential of a specific strain. Therefore, it is critical to study the virulence factors found in MGEs in emerging pathogenic bacteria or strains to understand the mechanisms used by these bacteria to cause infections.

Characterisation of a novel SCCmec VI element harbouring fusC in an emerging Staphylococcus aureus strain from the Arabian Gulf region.

Fusidic acid is a steroid antibiotic known since the 1960s. It is frequently used in topical preparations, i.e., ointments, for the treatment of skin and soft tissue infections caused by Staphylococcus aureus. There is an increasing number of methicillin-resistant S. aureus (MRSA) strains that harbour plasmid-borne fusB/far1 or fusC that is localised on SCC elements. In this study we examined a series of related CC30-MRSA isolates from the Arabian Gulf countries that presented with SCCmec elements and fusC, including a variant that—to the best of our knowledge—has not yet formally been described. It consisted of a class B mec complex and ccrA/B-4 genes. The fusidic acid resistance gene fusC was present, but contrary to the previously sequenced element of HDE288, it was not accompanied by tirS. This element was identified in CC30 MRSA from Kuwait, Saudi Arabia and the United Arab Emirates that usually also harbour the Panton-Valentin leukocidin (PVL) genes. It was also identified in CC8 and ST834 isolates. In addition, further CC30 MRSA strains with other SCCmec VI elements harbouring fusC were found to circulate in the Arabian Gulf region. It can be assumed that MRSA strains with SCCmec elements that include fusC have a selective advantage in both hospital and community settings warranting a review of the use of topical antibiotics and indicating the necessity of reducing over-the-counter sale of antibiotics, including fusidic acid, without prescription.

The Enterococcus Cassette Chromosome, a Genomic Variation Enabler in Enterococci.

Enterococcus faecium has a highly variable genome prone to recombination and horizontal gene transfer. Here, we have identified a novel genetic island with an insertion locus and mobilization genes similar to those of staphylococcus cassette chromosome elements SCCmec This novel element termed the enterococcus cassette chromosome (ECC) element was located in the 3' region of rlmH and encoded large serine recombinases ccrAB similar to SCCmec Horizontal transfer of an ECC element termed ECC::cat containing a knock-in cat chloramphenicol resistance determinant occurred in the presence of a conjugative reppLG1 plasmid. We determined the ECC::cat insertion site in the 3' region of rlmH in the E. faecium recipient by long-read sequencing. ECC::cat also mobilized by homologous recombination through sequence identity between flanking insertion sequence (IS) elements in ECC::cat and the conjugative plasmid. The ccrABEnt genes were found in 69 of 516 E. faecium genomes in GenBank. Full-length ECC elements were retrieved from 32 of these genomes. ECCs were flanked by attR and attL sites of approximately 50 bp. The attECC sequences were found by PCR and sequencing of circularized ECCs in three strains. The genes in ECCs contained an amalgam of common and rare E. faecium genes. Taken together, our data imply that ECC elements act as hot spots for genetic exchange and contribute to the large variation of accessory genes found in E. faeciumIMPORTANCEEnterococcus faecium is a bacterium found in a great variety of environments, ranging from the clinic as a nosocomial pathogen to natural habitats such as mammalian intestines, water, and soil. They are known to exchange genetic material through horizontal gene transfer and recombination, leading to great variability of accessory genes and aiding environmental adaptation. Identifying mobile genetic elements causing sequence variation is important to understand how genetic content variation occurs. Here, a novel genetic island, the enterococcus cassette chromosome, is shown to contain a wealth of genes, which may aid E. faecium in adapting to new environments. The transmission mechanism involves the only two conserved genes within ECC, ccrABEnt, large serine recombinases that insert ECC into the host genome similarly to SCC elements found in staphylococci.

Staphylococcal Plasmids, Transposable and Integrative Elements.

Strains of Staphylococcus aureus, and to a lesser extent other staphylococcal species, are a significant cause of morbidity and mortality. An important factor in the notoriety of these organisms stems from their frequent resistance to many antimicrobial agents used for chemotherapy. This review catalogues the variety of mobile genetic elements that have been identified in staphylococci, with a primary focus on those associated with the recruitment and spread of antimicrobial resistance genes. These include plasmids, transposable elements such as insertion sequences and transposons, and integrative elements including ICE and SCC elements. In concert, these diverse entities facilitate the intra- and inter-cellular gene mobility that enables horizontal genetic exchange, and have also been found to play additional roles in modulating gene expression and genome rearrangement.

Crystal Structure of a New Single-Stranded DNA-Binding Protein Encoded by &lt;i&gt;Staphylococcal&lt;/i&gt; Cassette Chromosome Elements

Methicillin resistant Staphylococcus aureus (MRSA) is a global public-health threat. Methicillin resistance is carried on mobile genetic elements belonging to the Staphylococcal Cassette Chromosome or SCC family. The molecular mechanisms that SCC elements exploit for stable maintenance and for horizontal transfer between strains are poorly understand. Previously, we identified and characterized several conserved SCC genes with putative functions in DNA replication, including lp1413, which we found encodes a single-stranded (ss) DNA binding protein. We report here the 2.18 A crystal structure of LP1413, which shows that it adopts a winged helix-turn-helix fold rather than the OB-fold normally seen in replication-related ssDNA binding proteins. However, conserved residues form a hydrophobic pocket not normally found in winged helixturn-helix domains. LP1413 also has a conserved but disordered C-terminal tail. As we find that deletion of the tail does not affect cooperative binding to ssDNA, we propose that it mediates interactions with other proteins. We discuss roles that LP1413 could play in vivo.

Methicillin-resistant Staphylococcus pseudintermedius among dogs in the description of novel SCCmec variants

The presence and genetic characteristics of methicillin-resistant Staphylococcus pseudintermedius (MRSP) in Sri Lanka was investigated to add additional insight into global spread, emergence and evolution of MRSP. A total of 234 samples from dogs visiting veterinary clinics were cultured for staphylococci and the genomes of the MRSP isolates were sequenced, to identify resistance genes, the multilocus sequence types (MLST) and spa types. From a questionnaire the history of antimicrobial treatment and patient information was obtained.S. pseudintermedius was isolated from 116/229 samples, eight of these were MRSP. Six MRSP CC45 isolates contained a pseudo-SCC element ΨSCCmec57395. Two isolates belonging to ST429 (CC761) and ST121 (CC121) contained novel variants of the SCCmec Type V(T) element. The elements were designated SCCmecV(T)SL/066, that carried additional transposon-related genes, and SCCmecV(T)SL/154 that carried a type III restriction-modification system, a type 7 ccr gene complex, and a cadA coding sequence. Thirty-seven percent of the dogs received antimicrobial treatment at the time of sampling of which four dogs were MRSP-positive. The proportion of MRSP among S. pseudintermedius is low compared to other countries, despite the fact that in Sri Lanka antimicrobials for treatment of dogs are available over the counter. Important is the finding of novel type V(T) SCCmec elements, which further underlines the high recombination frequency of SCC elements. The ΨSCCmec57395 was found in isolates of CC45, which is the only sequence type of MRSP known to contain this pseudo-cassette.

Investigating a rare methicillin-resistant Staphylococcus aureus strain: first description of genome sequencing and molecular characterization of CC15-MRSA.

PurposeMethicillin resistant Staphylococcus aureus CC15 strains (CC15-MRSA) have only been sporadically described in literature. This study was carried out to describe the genetic make-up for this rare MRSA strain.MethodsFour CC15-MRSA isolates collected in Riyadh, Saudi Arabia, between 2013 and 2014 were studied. Two isolates were from clinical infection and 2 from retail meat products. Whole genome sequencing was carried out using Illumina HiSeq2500 genome analyzer.ResultsAll the CC15-MRSA isolates had the multilocus sequence typing profile ST1535, 13–13-1–1-81-11-13, which is a single locus variant of ST15. Of the 6 contigs related to the SCC element, one comprised a recombinase gene ccrAA, ccrC-PM1, fusC and a helicase, another one included mvaS, dru, mecA and 1 had yobV and Q4LAG7. The SCC element had 5 transposase genes, namely 3 identical paralogs of tnpIS431 and 2 identical paralogs of tnpIS256. Two identical copies of a tnpIS256-based insertion element flank the aacA-aphD gene. Two copies of this insertion element were present with 1 located in the SCC element and another inserted into the sasC gene. A short 3 kb region, which lacks any bacteriophage structural genes and site-specific DNA integrase, was inserted into the hlb gene. The hsdM and the 5’-part of the hsdS gene are replaced by a copy of the hsdM/hsdS paralogs from νSaβ giving rise to a new chimeric paralog of hsdS in νSaα.ConclusionCC15-MRSA shows a novel SCCmecV/SCCfus composite element. Its variant of hsdM/hsdS probably facilitated uptake of foreign mobile genetic elements that promoted emergence of CC15-MRSA. Close surveillance is needed to monitor spread and emergence of further CC15 MRSA strains.

Staphylococcus aureus CC395 harbours a novel composite staphylococcal cassette chromosome mec element.

Background: CoNS species are likely reservoirs of the staphylococcal cassette chromosome mec (SCCmec) in Staphylococcus aureus. S. aureus CC395 is unique as it is capable of exchanging DNA with CoNS via bacteriophages, which are also known to mediate transfer of SCCmec.Objectives: To analyse the structure and putative origin of the SCCmec element in S. aureus CC395.Methods: The only MRSA CC395 strain described in the literature, JS395, was subjected to WGS, and its SCCmec element was compared with those found in CoNS species and other S. aureus strains.Results: JS395 was found to carry an unusually large 88 kb composite SCCmec element. The 33 kb region downstream of orfX harboured a type V SCCmec element and a CRISPR locus, which was most similar to those found in the CoNS species Staphylococcus capitis and Staphylococcus schleiferi. A 55 kb SCC element was identified downstream of the type V SCCmec element and contained a mercury resistance region found in the composite SCC element of some Staphylococcus epidermidis and S. aureus strains, an integrated S. aureus plasmid containing genes for the detoxification of cadmium and arsenic, and a stretch of genes that was partially similar to the type IVg SCCmec element found in a bovine S. aureus strain.Conclusions: The size and complexity of the SCCmec element support the idea that CC395 is highly prone to DNA uptake from CoNS. Thus CC395 may serve as an entry point for SCCmec and SCC structures into S. aureus.

Diversity of SCCmec Elements in Staphylococcus aureus as Observed in South-Eastern Germany.

SCCmec elements are very important mobile genetic elements in Staphylococci that carry beta-lactam resistance genes mecA/mecC, recombinase genes and a variety of accessory genes. Twelve main types and a couple of variants have yet been described. In addition, there are also other SCC elements harbouring other markers. In order to subtype strains of methicillin-resistant S. aureus (MRSA) based on variations within their SCCmec elements, 86 markers were selected from published SCC sequences for an assay based on multiplexed primer extension reactions followed by hybridisation to the specific probes. These included mecA/mecC, fusC, regulatory genes, recombinase genes, genes from ACME and heavy metal resistance loci as well as several genes of unknown function. Hybridisation patterns for published genome or SCC sequences were theoretically predicted. For validation of the microarray based assay and for stringent hybridisation protocol optimization, real hybridization experiments with fully sequenced reference strains were performed modifying protocols until yielded the results were in concordance to the theoretical predictions. Subsequently, 226 clinical isolates from two hospitals in the city of Dresden, Germany, were characterised in detail. Beside previously described types and subtypes, a wide variety of additional SCC types or subtypes and pseudoSCC elements were observed as well as numerous composite elements. Within the study collection, 61 different such elements have been identified. Since hybridisation cannot recognise the localisation of target genes, gene duplications or inversions, this is a rather conservative estimate. Interestingly, some widespread epidemic strains engulf distinct variants with different SCCmec subtypes. Notable examples are ST239-MRSA-III, CC5-, CC22-, CC30-, and CC45-MRSA-IV or CC398-MRSA-V. Conversely, identical SCC elements were observed in different strains with SCCmec IVa being spread among the highest number of Clonal Complexes. The proposed microarray can help to distinguish isolates that appear similar or identical by other typing methods and it can be used as high-throughput screening tool for the detection of putative new SCC types or variants that warrant further investigation and sequencing. The high degree of diversity of SCC elements even within so-called strains could be helpful for epidemiological typing. It also raises the question on scale and speed of the evolution of SCC elements.

Multidrug-resistant Staphylococcus haemolyticus isolates from infected eyes and healthy conjunctivae in India.

This study aimed to determine the presence of antibiotic resistance genes (ARGs), SCCmec elements and genetic relatedness among Staphylococcus haemolyticus isolated from patients with a variety of eye infections (n=11) and from healthy conjunctiva (n=7). Minimum inhibitory concentrations were determined for 14 antimicrobials according to BSAC guidelines. PCR was used to identify the presence of mecA, mecC, SCCmec type and ARGs. Sequencing was used to determine mutations in gyrA, gyrB, topoisomerase IVA and IVB genes. Genetic relatedness was determined by PFGE. Of the 18 isolates, 17 showed resistance to at least one antibiotic, but none showed resistance to vancomycin or rifampicin. Ten isolates were oxacillin-resistant and carried the mecA gene, eight of which belonged to SCCmec type V. The presence of non-mec SCC elements in two meticillin-susceptible isolates and untypeable SCC elements in meticillin-resistant isolates suggests the involvement of S. haemolyticus in the diversification of SCC elements. Sequence analysis revealed point mutations in gyrA (Ser-84→Leu) and topoisomerase IVA genes (Ser-80→Leu) in 13 isolates, and additional variation in the QRDR (Asp-84→Asn) of two isolates, showing good correlation between mutations in gyrA and topoisomerase IV genes and the level of resistance to fluoroquinolones. PFGE analysis showed distinct pulsotypes forming two major clusters, indicating the existence of diversity among isolates, irrespective of the source of isolation. This study suggests that S. haemolyticus isolates from infected eyes and healthy conjunctivae invariably carried ARGs and SCCmec elements and showed diversity in their genomic content, irrespective of the source of isolation.

Novel Type XII Staphylococcal Cassette Chromosome mec Harboring a New Cassette Chromosome Recombinase, CcrC2.

Excision and integration of staphylococcal cassette chromosome mec (SCCmec) are mediated by cassette chromosome recombinases (Ccr), which play a crucial role in the worldwide spread of methicillin resistance in staphylococci. We report a novel ccr gene, ccrC2, in the SCCmec of a Staphylococcus aureus isolate, BA01611, which showed 62.6% to 69.4% sequence identities to all published ccrC1 sequences. A further survey found that the ccrC2 gene was mainly located among coagulase-negative staphylococci (CoNS) and could be found in staphylococcal isolates from China, the United States, France, and Germany. The ccr gene complex harboring the ccrC2 gene was designated a type 9 complex, and the SCCmec of BA01611 was considered a novel type and was designated type XII (9C2). This novel SCCmec element in BA01611 was flanked by a pseudo-SCC element (ΨSCCBA01611) carrying a truncated ccrA1 gene. Both individual SCC elements and a composite SCC were excised from the chromosome based on detection of extrachromosomal circular intermediates. We advocate inclusion of the ccrC2 gene and type 9 ccr gene complex during revision of the SCCmec typing method.