Diversity of staphylococcal cassette chromosome in coagulase-negative staphylococci from animal sources

Aims:  To type the staphylococcal cassette chromosome (SCC) in coagulase-negative staphylococci (CoNS) from animal sources.
Methods and Results:  A total of 92 CoNS isolates recovered from farm animals was analysed. The top three staphylococcal species were Staphylococcus lentus (34), S. sciuri (31), and S. xylosus (13). The presence of the cassette chromosome recombinase (ccr) genes ccrA1 , ccrB1 , ccrA2 , ccrB2 , ccrA3 , ccrB3 and ccrC , the mec regulatory genes mecI and mecR1 , and Tn 554 was used to differentiate the SCC. A total of 60 of the 92 isolates were methicillin resistant. Among the 60 methicillin-resistant Staphylococcus spp. isolates, SCC mec ( mecA -carrying SCC) types I, III, IV and V were identified in 24 isolates based on the combinations of the ccr genes and the mec regulatory genes, with type III being predominant. The single S. epidermidis carried SCC mec type IV. SCC type III was also identified in two of 32 methicillin-susceptible isolates. Identical SCC mec types were present in different species of CoNS. Pulsed-field gel electrophoresis (PFGE) generated 64 patterns out of 81 PFGE typeable isolates. Indistinguishable clones were detected in animals from different farms.
Conclusions:  Heterogeneous SCC existed in CoNS of diverse genetic background. Both clonal transmission of methicillin-resistant CoNS and horizontal transfer of SCC mec occurred in the animal production environment.
Significance and Impact of the Study:  This study adds to our knowledge of SCC mec type and the diversity of SCC in CoNS.     

耐甲氧西林金黄色葡萄球菌染色体盒的研究进展

耐甲氧西林金黄色葡萄球菌（MRSA）的产生是由甲氧西林敏感的金黄色葡萄球菌（MSSA）获得外源性的SCCmec所致。MRSA菌株可以产生一种新的青霉素结合蛋白PBP2a,PBP2a降低了与β-内酰胺类抗生素的亲合力,从而对β-内酰胺类抗生素产生耐药性。PBP2a由mecA基因编码,mecA基因存在于葡萄球菌盒式染色体（Staphylococcal cassette chromosome mec,SCCmec）中,SCCmec是一种可移动的遗传元件,该元件还携带除mecA基因外的其他抗菌药物的耐药基因,造成多重耐药（Multidrug-resistance,MDR）。SCCmec目前主要分为8型,其中又分为若干亚型。SCCmec的基因型与MRSA的流行背景有关,不同地区的SCCmec基因分型分布可能不同。     

The emergence and evolution of methicillin-resistant Staphylococcus aureus

Significant advances have been made in recent years in our understanding of how methicillin resistance is acquired by Staphylococcus aureus. Integration of a staphylococcal cassette chromosome mec (SCCmec) element into the chromosome converts drug-sensitive S. aureus into the notorious hospital pathogen methicilin-resistant S. aureus (MRSA), which is resistant to practically all beta-lactam antibiotics. SCCmec is a novel class of mobile genetic element that is composed of the mec gene complex encoding methicillin resistance and the ccr gene complex that encodes recombinases responsible for its mobility. These elements also carry various resistance genes for non-beta-lactam antibiotics. After acquiring an SCCmec element, MRSA undergoes several mutational events and evolves into the most difficult-to-treat pathogen in hospitals, against which all extant antibiotics including vancomycin are ineffective. Recent epidemiological data imply that MRSA has embarked on another evolutionary path as a community pathogen, as at least one novel SCCmec element seems to have been successful in converting S. aureus strains from the normal human flora into MRSA.     

SCCmec in staphylococci: genes on the move

Staphylococcal cassette chromosome (SCC) elements are, so far, the only vectors described for the mecA gene encoding methicillin resistance in staphylococci. SCCmec elements are classified according to the type of recombinase they carry and their general genetic composition. SCCmec types I-V have been described, and SCC elements lacking mecA have also been reported. In this review, we summarize the current knowledge about SCC structure and distribution, including genetic variants and rudiments of the elements. Its origin is still unknown, but one assumes that staphylococcal cassette chromosome is transferred between staphylococci, and mecA-positive coagulase-negative staphylococci may be a potential reservoir for these elements. Staphylococcal genomes seem to change continuously as genetic elements move in and out, but no mechanism of transfer has been found responsible for moving SCC elements between different staphylococcal species. Observations suggesting de novo production of methicillin-resistant staphylococci and horizontal gene transfer of SCCmec will be discussed.     

Jumping the barrier to beta-lactam resistance in Staphylococcus aureus

Although the staphylococcal methicillin resistance determinant, mecA, resides on a mobile genetic element, staphylococcus cassette chromosome mec (SCCmec), its distribution in nature is limited to as few as five clusters of related methicillin-resistant Staphylococcus aureus (MRSA) clones. To investigate the potential role of the host chromosome in clonal restriction of the methicillin resistance determinant, we constructed plasmid pYK20, carrying intact mecA, and introduced it into several methicillin-susceptible Staphylococcus aureus strains, five of which were naive hosts (i.e., mecA not previously resident on the host chromosome) and five of which were experienced hosts (i.e., methicillin-susceptible variants of MRSA strains from which SCCmec was excised). We next assessed the effect of the recipient background on the methicillin resistance phenotype by population analysis, by assaying the mecA expression of PBP2a by Western blot analysis, and by screening for mutations affecting mecA. Each experienced host transformed with pYK20 had a resistance phenotype and expressed PBP2a similar to that of the parent with chromosomal SCCmec, but naive hosts transformed with pYK20 selected against its expression, indicative of a host barrier. Either inducible beta-lactamase regulatory genes blaR1-blaI or homologous regulatory genes mecR1-mecI, which control mecA expression, acted as compensatory elements, permitting the maintenance and expression of plasmid-carried mecA.     

SCCmec遗传元件及其在耐甲氧西林金黄色葡萄球菌分子分型中的应用

携带mec基因簇的葡萄球菌盒式染色体(Staphylococcal chromosome cassette mec, SCCmec)遗传元件的获得是耐甲氧西林金黄色葡萄球菌(Methicillin-resistant Staphylococcus aureus, MRSA)耐药的主要原因。SCCmec由一个mec基因簇、一个染色体重组酶(ccr)基因簇及3个J区组成。mec基因簇含有mecA及其调控基因,mecA基因编码的耐药决定簇使MRSA对β-内酰胺类抗生素耐药;ccr基因簇编码的重组酶负责SCCmec元件的整合与切离;J区差异大,导致不同来源MRSA菌株携带SCCmec的大小不一,在组成上也具有多样性。这些特征为利用SCCmec元件进行MRSA分型创造了条件。文章介绍了SCCmec元件的结构和功能,综述了基于SCCmec的MRSA分型研究。     

Distribution and regulation of the mobile genetic element-encoded phenol-soluble modulin PSM-mec in methicillin-resistant Staphylococcus aureus

The phenol-soluble modulin PSM-mec is the only known staphylococcal toxin that is encoded on a mobile antibiotic resistance determinant, namely the staphylococcal cassette chromosome (SCC) element mec encoding resistance to methicillin. Here we show that the psm-mec gene is found frequently among methicillin-resistant Staphylococcus aureus (MRSA) strains of SCCmec types II, III, and VIII, and is a conserved part of the class A mec gene complex. Controlled expression of AgrA versus RNAIII in agr mutants of all 3 psm-mec-positive SCCmec types demonstrated that expression of psm-mec, which is highly variable, is controlled by AgrA in an RNAIII-independent manner. Furthermore, psm-mec isogenic deletion mutants showed only minor changes in PSMα peptide production and unchanged (or, as previously described, diminished) virulence compared to the corresponding wild-type strains in a mouse model of skin infection. This indicates that the recently reported regulatory impact of the psm-mec locus on MRSA virulence, which is opposite to that of the PSM-mec peptide and likely mediated by a regulatory RNA, is minor when analyzed in the original strain background. Our study gives new insight in the distribution, regulation, and role in virulence of the PSM-mec peptide and the psm-mec gene locus.     

耐甲氧西林金黄色葡萄球菌的两种新SCCmec型别及其抗药性研究

为探明本地区耐甲氧西林金黄色葡萄球菌(Staphylococcusaureus)的耐药性、流行病学分布状况及携带的葡萄球菌染色体mec盒(SCCmec)型别,用K-B琼脂扩散法、E-test和多位点PCR,对临床分离的金黄色葡萄球菌菌株进行了SCCmec分型及耐药性测定。结果发现了两种新的SCCmec型别,新1型含Ⅱ型的mecA上游特异性位点B和位于mecA内的M位点以及Ⅲ型的下游位点F,缺乏Ⅱ型上游位点C和下游位点D、G;新2型含Ⅰ、Ⅱ型的上游特异性位点A、B和两个Ⅲ型的下游位点F、H,同样缺乏位点C、D、G,可能分别为原有Ⅱ型和Ⅰ、Ⅱ型与Ⅲ型的基因重组株;且携带有新SCCmec型别的MRSA菌株,其流行病学分布特点及抗药性也与国外已报导的菌株不同,多分自门诊病人,且耐药性高,抗药谱广,值得引起高度重视和关注。     

A novel type-III staphylococcal cassette chromosome mec (SCCmec) variant among Indian isolates of methicillin-resistant Staphylococcus aureus

We identified a novel type-III staphylococcal cassette chromosome mec (SCC mec ) element carried by eight methicillin-resistant Staphylococcus aureus (MRSA) strains from different wards and patients in an Indian hospital. Although the pulsed-field gel electrophoresis pattern and spa types of eight strains were identical and clonally related to other nosocomial Indian isolates that belonged to sequence type (ST) 239 and spa type t037, the minimum inhibitory concentration (MIC) of these eight variants was noticeably low compared with the typical type-III isolates from the same hospital, and we were unable to identify ccrC and hsdR by multiplex PCR, although mer operon and transposases A, B, and C of Tn 554 were amplified. By amplifying the entire SCC mec region by long-range PCR and determining parts of the nucleotide sequences of one isolate (V14), we found that the strain carried a novel SCC mec element containing a 422 bp sequence, which is highly homologous to that identified in strain CCR1-9583, mer operon and plasmid pT181 integrated in tandem via IS 431 in the J3 region. It also carried a cassette chromosome, previously reported to be an SCC-like element, downstream of type-III SCC mec . Because PCR amplification of representative genes showed that these eight strains carried the same genetic elements, they belong to a novel MRSA clone that differs from most nosocomial clones carrying type-III SCC mec and SCC mercury , despite belonging to the ST239 genotype.     

Community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) in Poland: further evidence for the changing epidemiology of MRSA

This study reports the isolation of CA-MRSA strain which was found to colonize the nasal mucosa of a patient undergoing haemodialysis treatment. The MRSA was subjected to molecular analysis by Pulsed Field Gel Electrophoresis (PFGE), multiplex PCR assay for staphylococcal cassette chromosome mec (SCCmec) typing, and PCR detection of the pvl gene encoding for Panton-Valentine leukocidin. The analyzed MRSA harbored the SCCmec type IV and the pvl gene-two unique genetic markers of CA-MRSA. The PFGE pattern of the strain corresponded to the common European CA-MRSA (MLST Type ST80). Moreover, the strain was only resistant to beta-lactam agents and tetracycline. This study adds further evidence for the changing epidemiology of MRSA and indicates the ability of CA-MRSA to affect persons with established risk factors in addition to previously healthy individuals.     

An unexpected location of the arginine catabolic mobile element (ACME) in a USA300-related MRSA strain

In methicillin resistant Staphylococcus aureus (MRSA), the arginine catabolic mobile element (ACME) was initially described in USA300 (t008-ST8) where it is located downstream of the staphylococcal cassette chromosome mec (SCCmec). A common health-care associated MRSA in Copenhagen, Denmark (t024-ST8) is clonally related to USA300 and is frequently PCR positive for the ACME specific arcA-gene. This study is the first to describe an ACME element upstream of the SCCmec in MRSA. By traditional SCCmec typing schemes, the SCCmec of t024-ST8 strain M1 carries SCCmec IVa, but full sequencing of the cassette revealed that the entire J3 region had no homology to published SCCmec IVa. Within the J3 region of M1 was a 1705 bp sequence only similar to a sequence in S. haemolyticus strain JCSC1435 and 2941 bps with no homology found in GenBank. In addition to the usual direct repeats (DR) at each extremity of SCCmec, M1 had two new DR between the orfX gene and the J3 region of the SCCmec. The region between the orfX DR (DR1) and DR2 contained the ccrAB4 genes. An ACME II-like element was located between DR2 and DR3. The entire 26,468 bp sequence between DR1 and DR3 was highly similar to parts of the ACME composite island of S. epidermidis strain ATCC12228. Sequencing of an ACME negative t024-ST8 strain (M299) showed that DR1 and the sequence between DR1 and DR3 was missing. The finding of a mobile ACME II-like element inserted downstream of orfX and upstream of SCCmec indicates a novel recombination between staphylococcal species.     

A study of the polymorphism of mec dna in methycillin-resistant strains of Staphylococcus aureus isolated at permanent stations in different regions of Russia

Polymorphism of the chromosome staphylococcus cassette mec (SCCmec), a mobile and heterological genetic element providing resistance to beta-lactam antibiotics was studied in methycillin-resistant strains of Staphylococcus aureus (MRSA) isolated at permanent stations situated in different regions of Russia. Type SCCmec was identified using the PCR method by determining allotypes of 3 different structural genetic complexes incorporated in the cassettes mec. It was found that the isolates studied in this work contained 3 different types of SCCmec: I, III, and IVb. Both isolates containing 2 different copies of SCCmec and isolates containing defective copies of SCCmec were identified. It was demonstrated that determination of the SCC-mec type provided an opportunity to differentiate the isolates studied in this work from one another. The isolates attributed to the same genotype variant (identified by polymorphism of coagulase gene) but isolated at different hospitals located in different regions of Russia were found to contain the same type of the chromosome staphylococcus cassette mec, whereas the isolates of different coagulase groups (i.e., different genotype variants) contained different types of SCCmec. It was found that at least 2 epidemic strains circulated in the permanent hospitals of Russia. The strains differ from one another by the polymorphism of the coagulase gene and the mec DNA polymorphism. According to results of studies of several molecular markers (including mec DNA), these strains proved to be identical to the international strains EMRSA-1 and EMRSA-2. Possible mechanisms of MRSA formation and circulation in Russia and CIS countries are discussed.     

Characterization of DNA sequences required for the CcrAB-mediated integration of staphylococcal cassette chromosome mec, a Staphylococcus aureus genomic island

The mobile element staphylococcal cassette chromosome mec (SCCmec), which carries mecA, the gene responsible for methicillin resistance in staphylococci, inserts into the chromosome at a specific site, attB, mediated by serine recombinases, CcrAB and CcrC, encoded on the element. This study sought to determine the sequence specificity for CcrB DNA binding in vitro and for CcrAB-mediated SCCmec insertion in vivo. CcrB DNA binding, as assessed in vitro by electrophoretic mobility shift assay (EMSA), revealed that a 14-bp sequence (CGTATCATAAGTAA; the terminal sequence of the orfX gene) was the minimal requirement for binding, containing an invariant sequence (TATCATAA) found in all chromosomal (attB) and SCCmec (attS) integration sites. The sequences flanking the minimal attB and attS binding sites required for insertion in vivo were next determined. A plasmid containing only 37 bp of attS and flanking sequences was required for integration into the attB site at 92% efficiency. In contrast, at least 200 bp of sequence within orfX, 5' to the attB core, and 120 bp of specific sequence 3' to the orfX stop site and attB core were required for the highest insertion frequency. Finally, an attS-containing plasmid was inserted into wild-type Staphylococcus aureus strains without integrated SCCmec (methicillin susceptible) at various frequencies which were determined both by sequences flanking the att site and by the presence of more than one att site on either the chromosome or the integration plasmid. This sequence specificity may play a role in the epidemiology of SCCmec acquisition.     

Methicillin-resistant coagulase-negative staphylococci on pig farms as a reservoir of heterogeneous staphylococcal cassette chromosome mec elements

Methicillin-resistant Staphylococcus aureus (MRSA) likely originated by acquisition of the staphylococcal cassette chromosome mec (SCCmec) from coagulase-negative staphylococci (CNS). However, it is unknown whether the same SCCmec types are present in MRSA and CNS that reside in the same niche. Here we describe a study to determine the presence of a potential mecA reservoir among CNS recovered from 10 pig farms. The 44 strains belonged to 10 different Staphylococcus species. All S. aureus strains belonged to sequence type 398 (ST398), with SCCmec types V and IVa. Type IVc, as well as types III and VI, novel subtypes of type IV, and not-typeable types, were found in CNS. S. aureus, S. epidermidis, and S. haemolyticus shared SCCmec type V. The presence of SCCmec type IVc in several staphylococcal species isolated from one pig farm is noteworthy, suggesting exchange of this SCCmec type in CNS, but the general distribution of this SCCmec type still has to be established. In conclusion, this study shows that SCCmec types among staphylococcal species on pig farms are heterogeneous. On two farms, more than one recovered staphylococcal species harbored the same SCCmec type. We conclude that staphylococci on pig farms act as a reservoir of heterogeneous SCCmec elements. These staphylococci may act as a source for transfer of SCCmec to S. aureus.     

Bridges from hospitals to the laboratory: genetic portraits of methicillin-resistant Staphylococcus aureus clones

Methicillin-resistant Staphylococcus aureus (MRSA) emerged in the early 1960's after the acquisition of the methicillin resistance gene mecA, which is carried by the staphylococcal cassette chromosome mec (SCCmec). MRSA seemed to have arisen by multiple introductions of SCCmec into successful methicillin-susceptible S. aureus (MSSA) lineages. MRSA is one of the most common agents of nosocomial infections worldwide increasing the cost and mortality compared to MSSA infections. Little by little, MRSA has acquired resistance to all antibiotics available in clinical practice, which complicates treatment. This situation was further aggravated by the recent reports of vanA-mediated vancomycin-resistant S. aureus. As a reaction to the emergence and spread of multidrug-resistant MRSA worldwide, international surveillance systems such as the CEM/NET initiative have been created. The characterization of over 3000 MRSA isolates from different regions of the world evidenced the existence of only a few epidemic clones spread worldwide, namely the Iberian, Brazilian, Hungarian, New York/Japan, Pediatric and EMRSA-16 clones. It was found that in surveillance or evolutionary studies strains should be characterized by a combination of different typing methods, namely pulsed-field gel electrophoresis, multi-locus sequence typing and SCCmec typing. In recent years, community-acquired MRSA (CA-MRSA) has become a growing public health concern. However, although many authors reported the emergence of CA-MRSA isolates, a standard definition has not been created and the prevalence of MRSA among persons without risk factors seems to remain very low. CA-MRSA has distinct properties compared to epidemic nosocomial clones and its origin is still unclear. Certain authors suggest there is MRSA transmission from the hospital setting to the community, namely transfer of nosocomial MRSA minor clones or sporadic isolates showing a high degree of similarity with CA-MRSA; others believe CA-MRSA strains represent new acquisitions of SCCmec DNA in susceptible backgrounds. Many questions concerning this extraordinarily versatile and threatening pathogen remain unanswered, needing future investigation     

Molecular characterization of methicillin-resistant Staphylococcus aureus in hospitals in Niigata, Japan: divergence and transmission

The major methicillin-resistant Staphylococcus aureus(MRSA) distributed among hospitals in Japan is New York/Japan clone [multilocus sequence type 5 (ST5), agr type 2 and methicillin resistance locus type (SCC mec) II] which possesses both the toxic shock syndrome toxin 1 gene (tst) and staphylococcal enterotoxin C gene (sec). In this study, we collected 245 MRSA strains from four hospitals during 2001 to 2005 in Niigata, Japan, and analyzed tst and sec genes and SCC mec type among them. A total of 13 strains were further examined for their genotypes, virulence gene patterns and drug resistance. Among the 245 strains four tst sec genes patterns were observed; tst(+) sec(+) strains represented a majority of 86.5% and 9.4% were tst(-) sec(-). SCCmec typing revealed that 91.4% had type II, 4.1% type IV and 4.1% type I. Multilocus sequence typing (MLST) revealed that 10 of the 13 typed strains belonged to clonal complex 5 (7 had ST5 while 3 were single locus variants of ST5) with similar characteristics to the New York/Japan clone and possessed multi-drug resistance with high virulence gene content. The remaining 3 strains were ST8 (n=2) and ST91 (n=1). The ST91 strain had SCC mec IV and seemed to originate in the community, while ST8 strains exhibited SCC mec type I, which is distinct from community type IV. The data suggest that MRSA in hospitals in Niigata now mainly includes the New York/Japan clone (undergoing genomic divergence and clonal expansion) and other minor types (e.g. ST8) as well as the community type.     

Characterisation of virulence genes in methicillin susceptible and resistant Staphylococcus aureus isolates from a paediatric population in a university hospital of Medellín, Colombia

Virulence and antibiotic resistance are significant determinants of the types of infections caused by Staphylococcus aureus and paediatric groups remain among the most commonly affected populations. The goal of this study was to characterise virulence genes of methicillin-susceptible S. aureus (MSSA) and methicillin-resistant S. aureus (MRSA) strains isolated from a paediatric population of a Colombian University Hospital during 2009. Sixty MSSA and MRSA isolates were obtained from paediatric patients between zero-14 years. We identified the genes encoding virulence factors, which included Panton-Valentine leucocidine (PVL), staphylococcal enterotoxins A-E, exfoliative toxins A and B and toxic shock syndrome toxin 1. Typing of the staphylococcal chromosome cassette mec (SCCmec) was performed in MRSA strains. The virulence genes were more diverse and frequent in MSSA than in MRSA isolates (83% vs. 73%). MRSA strains harboured SCCmec types IVc (60%), I (30%), IVa (7%) and V (3%). SCCmec type IVc isolates frequently carried the PVL encoding genes and harboured virulence determinants resembling susceptible strains while SCCmec type I isolates were often negative. PVL was not exclusive to skin and soft tissue infections. As previously suggested, these differences in the distribution of virulence factor genes may be due to the fitness cost associated with methicillin resistance.     

Comparative genomics and drug resistance of a geographic variant of ST239 methicillin-resistant Staphylococcus aureus emerged in Russia

Two distinct classes of methicillin-resistant Staphylococcus aureus (MRSA) are spreading in hospitals (as hospital-acquired MRSA, HA-MRSA) and in the community (as community-acquired MRSA, CA-MRSA). Multilocus sequence type (ST) 239 MRSA, one of the most worldwide-disseminated lineages, has been noted as a representative HA-MRSA. Here, we isolated ST239 MRSA (spa type 3 [t037] and staphylococcal cassette chromosome mec [SCCmec] type III.1.1.1) and its novel variant with ST239/spa351 (t030)/SCCmecIII.1.1.4 (SCCmecIII(R)) not only from hospitals but also from patients with urethritis in the community in Russia. The Russian variant (strain 16K) possessed a hybrid genome consisting of CC8 and CC30, similar to the ST239/spa3/SCCmecIII.1.1.1 HA-MRSA (TW20) genome, but with marked diversity. The 16K' CC30 section had SCCmecIII(R) carrying the dcs-carrying unit (which corresponded to the SCCmecIVc J3 joining region of ST30 CA-MRSA), lacked SCCmercury, and possessed a novel mobile element structure (MES16K) carrying the ccrC-carrying unit (with the recombinase gene ccrC1 allele 3) and drug resistance tranposons. The Russian variant included strains with a high ability to transfer its multiple drug resistance by conjugation; e.g., for strain 16K, the transfer frequency of a chloramphenicol resistance plasmid (p16K-1 with 2.9 kb in size) reached 1.4×10(-2), followed by Tn554 conjugative transfer at 3.6×l0(-4). The Russian variant, which has been increasing recently, included divergent strains with different plasmid patterns and pulsed field gel electrophoresis profiles. The data demonstrate the alternative nature of ST239 MRSA as CA-MRSA and also as a drug resistance disseminator, and its micro but dynamic evolution in Russia.