The bla and mec families of ß-lactam resistance genes in the genera Macrococcus, Mammaliicoccus and Staphylococcus: an in-depth analysis with emphasis on Macrococcus.

ß-Lactamases (Bla) and low-affinity penicillin-binding proteins (PBP2A) are responsible for ß-lactam resistance in the genera Macrococcus, Mammaliicoccus and Staphylococcus. These resistance mechanisms are in most species acquired through mobile genetic elements that carry a blaZ-like ß-lactamase gene for penicillin resistance and/or a mec gene (mecA, mecB, mecC,mecD) encoding a PBP2A for resistance to virtually all classes of ß-lactams. The mecA and mecC genes can be acquired through staphylococcal cassette chromosome mec (SCCmec) elements in Staphylococcus and Mammaliicoccus. The mecB and mecD genes are found in Macrococcus on SCCmec elements, as well as on unrelated mecD-carrying Macrococcus resistance islands (McRImecD) and large mecB-carrying plasmids. This review provides a phylogenetic overview of Macrococcus, Mammaliicoccus and Staphylococcus species and an in-depth analysis of the genetic structures carrying bla and mec genes in these genera. Native bla genes were detected in species belonging to the novobiocin-resistant Staphylococcus saprophyticus group and Mammaliicoccus. The evolutionary relatedness between Macrococcus and Mammaliicoccus is illustrated on the basis of a similar set of intrinsic PBPs, especially, the presence of a second class A PBP. The review further focuses on macrococcal elements carrying mecB and mecD, and compares them with structures present in Staphylococcus and Mammaliicoccus. It also discusses the different recombinases (ccr of SCCmec) and integrases (int of McRI) that contribute to the mobility of methicillin resistance genes, revealing Macrococcus as an important source for mobilization of antibiotic resistance genes within the family of Staphylococcaceae.

Macrococcus armenti sp. nov., a novel bacterium isolated from the skin and nasal cavities of healthy pigs and calves.

Gram-positive coccoid bacteria were isolated from the nasal cavities of pigs and calves as well as from axillar and inguinal skin regions of pigs. Phylogenetic analysis of seven strains based on complete genome, 16S rRNA, hsp60, dnaJ, rpoB and sodA gene sequences and MALDI-TOF MS profiles revealed that they belonged to the genus Macrococcus with the closest relatedness to Macrococcus canis, Macrococcus caseolyticus subsp. caseolyticus and Macrococcus caseolyticus subsp. hominis. DNA relatedness of the type strain JEK37T with the type strains of M. canis, M. caseolyticus subsp. caseolyticus and M. caseolyticus subsp. hominis was 23.4, 23.1 and 23.0 % by digital DNA-DNA hybridization and 80.39, 80.45 and 80.87 % by average nucleotide identity (ANI) calculations, confirming that they do not belong to the same species. The DNA G+C content of JEK37T was 35.65 mol%. The novel strains can be differentiated from M. canis KM 45013T by the ability to fermentate d-ribose and by the absence of DNAase production and haemolysis, from M. caseolyticus subsp. caseolyticus CCUG 15606T by the ability to fermentate sucrose and from both species by the inability to grow in 9 and 12% NaCl. They differ from M. caseolyiticus subsp. hominis by the presence of α-glucosidase. The most common fatty acids of JEK37T were C14 : 0, C18 : 1 ω9c and C18 : 0. Known polar lipids consisted of diphosphatidylglycerol, phosphatidylglycerol, aminolipid, aminoglycolipid, aminophospholipid, glycolipid and phospholipid. Cell-wall peptidoglycan of JEK37T was of type A3α l-Lys-Gly2-L-Ser-Gly (similar to A11.3) and the respiratory quinolone was menaquinone 6. Based on their genotypic and chemotaxonomic characteristics, these strains represent a novel species of the genus Macrococcus, for which we propose the name Macrococcus armenti sp. nov. The type strain is JEK37T (=DSM 112712T=CCOS 1982T).

The novel macrolide resistance genes mef(F) and msr(G) are located on a plasmid in Macrococcus canis and a transposon in Macrococcus caseolyticus.

OBJECTIVES: To analyse macrolide resistance in a Macrococcus canis strain isolated from a dog with an ear infection, and determine whether the resistance mechanism is also present in other bacteria, and associated with mobile genetic elements. METHODS: The whole genome of M. canis Epi0082 was sequenced using PacBio and Illumina technologies. Novel macrolide resistance determinants were identified through bioinformatic analysis, and functionality was demonstrated by expression in Staphylococcus aureus. Mobile genetic elements containing the novel genes were analysed in silico for strain Epi0082 as well as in other bacterial strains deposited in GenBank. RESULTS: M. canis Epi0082 contained a 3212 bp operon with the novel macrolide resistance genes mef(F) and msr(G) encoding a efflux protein and an ABC-F ribosomal protection protein, respectively. Cloning in S. aureus confirmed that both genes individually confer resistance to the 14- and 15-membered ring macrolides erythromycin and azithromycin, but not the 16-membered ring macrolide tylosin. A reduced susceptibility to the streptogramin B pristinamycin IA was additionally observed when msr(G) was expressed in S. aureus under erythromycin induction. Epi0082 carried the mef(F)-msr(G) operon together with the chloramphenicol resistance gene fexB in a novel 39 302 bp plasmid pMiCAN82a. The mef(F)-msr(G) operon was also found in macrolide-resistant Macrococcus caseolyticus strains in the GenBank database, but was situated in the chromosome as part of a novel 13 820 bp or 13 894 bp transposon Tn6776. CONCLUSIONS: The identification of mef(F) and msr(G) on different mobile genetic elements in Macrococcus species indicates that these genes hold potential for further dissemination of resistance to the clinically important macrolides in the bacterial population.

The Novel Macrolide Resistance Genes mef(D), msr(F), and msr(H) Are Present on Resistance Islands in Macrococcus canis, Macrococcus caseolyticus, and Staphylococcus aureus.

Chromosomal resistance islands containing the methicillin resistance gene mecD (McRI mecD ) have been reported in Macrococcus caseolyticus Here, we identified novel macrolide resistance genes in Macrococcus canis on similar elements, called McRI msr These elements were also integrated into the 3' end of the 30S ribosomal protein S9 gene (rpsI), delimited by characteristic attachment (att) sites, and carried a related site-specific integrase gene (int) at the 5' end. They carried novel macrolide resistance genes belonging to the msr family of ABC subfamily F (ABC-F)-type ribosomal protection protein [msr(F) and msr(H)] and the macrolide efflux mef family [mef(D)]. Highly related mef(D)-msr(F) fragments were found on diverse McRI msr elements in M. canis, M. caseolyticus, and Staphylococcus aureus Another McRI msr -like element identified in an M. canis strain lacked the classical att site at the 3' end and carried the msr(H) gene but no neighboring mef gene. The expression of the novel resistance genes in S. aureus resulted in a low-to-moderate increase in the MIC of erythromycin but not streptogramin B. In the mef(D)-msr(F) operon, the msr(F) gene was shown to be the crucial determinant for macrolide resistance. The detection of circular forms of McRI msr and the mef(D)-msr(F) fragment suggested mobility of both the island and the resistance gene subunit. The discovery of McRI msr in different Macrococcus species and S. aureus indicates that these islands have a potential for dissemination of antibiotic resistance within the Staphylococcaceae family.

The integrase of the Macrococcus caseolyticus resistance island mecD (McRImecD ) inserts DNA site-specifically into Staphylococcus and Bacillus chromosomes.

The methicillin resistance gene mecD has been recently identified on chromosomal islands in Macrococcus caseolyticus (McRImecD ). The 5' end of McRImecD carries an integrase (int) of the tyrosine recombinase family and two genes (intR and xis) encoding putative DNA-binding proteins. The islands are integrated site-specifically at the 3' end of the rpsI gene, a highly conserved locus in Gram-positive bacteria. Moreover, the rpsI gene of some Staphylococcus and Bacillus strains was found to be followed by a related integrase, raising the question of whether McRImecD could be transferred to these species. We used circular model elements carrying 5' end fragments of McRImecD -1 to demonstrate that the int enzyme and the attachment (att) site were sufficient to mediate site-specific DNA integration into the rpsI locus of Staphylococcus aureus, Staphylococcus pseudintermedius and Bacillus thuringiensis in vivo. Including xis in the model element stimulated both integrative and excisive recombination reactions and influenced the Int enzyme in att site selection. The intR gene functions as a negative regulator of int and xis. The int-xis genes of McRImecD -1 encode a site-specific recombination function that enables the acquisition of McRImecD in new hosts and the potential dissemination of broad-spectrum ß-lactam resistance across genus barriers.

The tva(A) Gene from Brachyspira hyodysenteriae Confers Decreased Susceptibility to Pleuromutilins and Streptogramin A in Escherichia coli.

The tva(A) gene suspected to confer resistance to pleuromutilins in Brachyspira hyodysenteriae was tested for functionality in Escherichia coli AG100A and Staphylococcus aureus RN4220. Expression of the cloned tva(A) gene conferred decreased susceptibility to pleuromutilin (P) and streptogramin A (SA) antibiotics in E. coli and had a minor effect in S. aureus The finding provides evidence of the direct association of tva(A) with the PSA resistance phenotype.

Macrococcus canis contains recombinogenic methicillin resistance elements and the mecB plasmid found in Staphylococcus aureus.

OBJECTIVES: To analyse the genetic context of mecB in two Macrococcus canis strains from dogs, compare the mecB-containing elements with those found in other Macrococcus and Staphylococcus species, and identify possible mobilizable mecB subunits. METHODS: Whole genomes of the M. canis strains Epi0076A and KM0218 were sequenced using next-generation sequencing technologies. Multiple PCRs and restriction analysis confirmed structures of mecB-containing elements, circularization and recombination of mecB subunits. RESULTS: Both M. canis strains contained novel composite pseudo (Ψ) staphylococcal cassette chromosome mec (SCCmec) elements. Integration site sequences for SCC flanked and subdivided composite ΨSCCmecEpi0076A (69569 bp) into ΨSCC1Epi0076A-ΨSCCmecEpi0076A-ΨSCC2Epi0076A and composite ΨSCCmecKM0218 (24554 bp) into ΨSCCKM0218-ΨSCCmecKM0218. Putative γ-haemolysin genes (hlgB and hlgC) were found at the 3' end of both composite elements. ΨSCCmecKM0218 contained a complete mecB gene complex (mecIm-mecR1m-mecB-blaZm) downstream of a new IS21-family member (ISMaca1). ΨSCCmecEpi0076A carried a blaZm-deleted mecB gene complex similar to that reported in 'Macrococcus goetzii' CCM4927T. A second mecB gene was found on the 81325 bp MDR plasmid pKM0218 in KM0218. This plasmid contained a complete Tn6045-associated mecB gene complex distinct from that of ΨSCCmecKM0218. pKM0218 was almost identical to the mecB-containing plasmid recently reported in Staphylococcus aureus (overall 99.96% nucleotide identity). Mobilization of mecB within an unconventional circularizable structure was observed in Epi0076A as well as chromosomal plasmid insertion via recombination of mecB operons in KM0218. CONCLUSIONS: Our findings provide evidence of both the continuing evolution of mecB-containing elements in macrococci and M. canis as a potential source of the mecB-containing plasmid found in staphylococci.

Typing of mecD Islands in Genetically Diverse Methicillin-Resistant Macrococcus caseolyticus Strains from Cattle.

Macrococcus caseolyticus belongs to the normal bacterial flora of dairy cows and does not usually cause disease. However, methicillin-resistant M. caseolyticus strains were isolated from bovine mastitis milk. These bacteria had acquired a chromosomal island (McRI mecD -1 or McRI mecD -2) carrying the methicillin resistance gene mecD To gain insight into the distribution of McRI mecD types in M. caseolyticus from cattle, 33 mecD-containing strains from Switzerland were characterized using molecular techniques, including multilocus sequence typing, antibiotic resistance gene identification, and PCR-based McRI mecD typing. In addition, the same genetic features were analyzed in 27 mecD-containing M. caseolyticus strains isolated from bovine bulk milk in England/Wales using publicly available whole-genome sequences. The 60 strains belonged to 24 different sequence types (STs), with strains belonging to ST5, ST6, ST21, and ST26 observed in both Switzerland and England/Wales. McRI mecD -1 was found in different STs from Switzerland (n = 19) and England/Wales (n = 4). McRI mecD -2 was only found in 7 strains from Switzerland, all of which belonged to ST6. A novel island, McRI mecD -3, which contains a complete mecD operon (mecD-mecR1m-mecIm [where the subscript m indicates Macrococcus]) combined with the left part of McRI mecD -2 and the right part of McRI mecD -1, was found in heterogeneous STs from both collections (Switzerland, n = 7; England/Wales, n = 21). Two strains from England/Wales carried a truncated McRI mecD -3. Phylogenetic analyses revealed no clustering of strains according to geographical origin or carriage of McRI mecD -1 and McRI mecD -3. Circular excisions were also detected for McRI mecD -1 and McRI mecD -3 by PCR. The analyses indicate that these islands are mobile and may spread by horizontal gene transfer between genetically diverse M. caseolyticus strains.IMPORTANCE Since its first description in 2017, the methicillin resistance gene mecD has been detected in M. caseolyticus strains from different cattle sources and countries. Our study provides new insights into the molecular diversity of mecD-carrying M. caseolyticus strains by using two approaches to characterize mecD elements: (i) multiplex PCR for molecular typing of McRI mecD and (ii) read mapping against reference sequences to identify McRI mecD types in silico In combination with multilocus sequence typing, this approach can be used for molecular characterization and surveillance of M. caseolyticus carrying mecD.

New Macrolide-Lincosamide-Streptogramin B Resistance Gene erm(48) on the Novel Plasmid pJW2311 in Staphylococcus xylosus.

Whole-genome sequencing of Staphylococcus xylosus strain JW2311 from bovine mastitis milk identified the novel 49.3-kb macrolide-lincosamide-streptogramin B (MLSB) resistance plasmid pJW2311. It contained the macrolide resistance gene mph(C), the macrolide-streptogramin B resistance gene msr(A), and the new MLSB resistance gene erm(48) and could be transformed into Staphylococcus aureus by electroporation. Functionality of erm(48) was demonstrated by cloning and expression in S. aureus.

Characterization of a Novel Composite Staphylococcal Cassette Chromosome mec in Methicillin-Resistant Staphylococcus pseudintermedius from Thailand.

A novel staphylococcal cassette chromosome mec (SCCmec) composite island (SCCmecAI16-SCCczrAI16-CI) was identified in Staphylococcus pseudintermedius. Four integration site sequences for SCC subdivided the 60,734-bp island into 41,232-bp SCCmecAI16, 19,400-bp SCCczrAI16, and 102-bp SCC-likeAI16 elements. SCCmecAI16 represents a new combination of ccrA1B3 genes with a class A mec complex. SCCczrAI16 contains ccrA1B6 and genes related to restriction modification and heavy metal resistance. SCCmecAI16-SCCczrAI16-CI was found in methicillin-resistant S. pseudintermedius sequence type 112 (ST112) and ST111 isolated from dogs and veterinarians in Thailand.

First Staphylococcal Cassette Chromosome mec Containing a mecB-Carrying Gene Complex Independent of Transposon Tn6045 in a Macrococcus canis Isolate from a Canine Infection.

A methicillin-resistant mecB-positive Macrococcus canis (strain KM45013) was isolated from the nares of a dog with rhinitis. It contained a novel 39-kb transposon-defective complete mecB-carrying staphylococcal cassette chromosome mec element (SCCmecKM45013). SCCmecKM45013 contained 49 coding sequences (CDSs), was integrated at the 3' end of the chromosomal orfX gene, and was delimited at both ends by imperfect direct repeats functioning as integration site sequences (ISSs). SCCmecKM45013 presented two discontinuous regions of homology (SCCmec coverage of 35%) to the chromosomal and transposon Tn6045-associated SCCmec-like element of M. caseolyticus JCSC7096: (i) the mec gene complex (98.8% identity) and (ii) the ccr-carrying segment (91.8% identity). The mec gene complex, located at the right junction of the cassette, also carried the ß-lactamase gene blaZm (mecRm-mecIm-mecB-blaZm). SCCmecKM45013 contained two cassette chromosome recombinase genes, ccrAm2 and ccrBm2, which shared 94.3% and 96.6% DNA identity with those of the SCCmec-like element of JCSC7096 but shared less than 52% DNA identity with the staphylococcal ccrAB and ccrC genes. Three distinct extrachromosomal circularized elements (the entire SCCmecKM45013, ΨSCCmecKM45013 lacking the ccr genes, and SCCKM45013 lacking mecB) flanked by one ISS copy, as well as the chromosomal regions remaining after excision, were detected. An unconventional circularized structure carrying the mecB gene complex was associated with two extensive direct repeat regions, which enclosed two open reading frames (ORFs) (ORF46 and ORF51) flanking the chromosomal mecB-carrying gene complex. This study revealed M. canis as a potential disease-associated bacterium in dogs and also unveiled an SCCmec element carrying mecB not associated with Tn6045 in the genus Macrococcus.

The new macrolide-lincosamide-streptogramin B resistance gene erm(45) is located within a genomic island in Staphylococcus fleurettii.

Genome alignment of a macrolide, lincosamide, and streptogramin B (MLSB)-resistant Staphylococcus fleurettii strain with an MLSB-susceptible S. fleurettii strain revealed a novel 11,513-bp genomic island carrying the new erythromycin resistance methylase gene erm(45). This gene was shown to confer inducible MLSB resistance when cloned into Staphylococcus aureus. The erm(45)-containing island was integrated into the housekeeping gene guaA in S. fleurettii and was able to form a circular intermediate but was not transmissible to S. aureus.

New shuttle vector-based expression system to generate polyhistidine-tagged fusion proteins in Staphylococcus aureus and Escherichia coli.

Four Staphylococcus aureus-Escherichia coli shuttle vectors were constructed for gene expression and production of tagged fusion proteins. Vectors pBUS1-HC and pTSSCm have no promoter upstream of the multiple cloning site (MCS), and this allows study of genes under the control of their native promoters, and pBUS1-Pcap-HC and pTSSCm-Pcap contain the strong constitutive promoter of S. aureus type 1 capsule gene 1A (Pcap) upstream of a novel MCS harboring codons for the peptide tag Arg-Gly-Ser-hexa-His (rgs-his6). All plasmids contained the backbone derived from pBUS1, including the E. coli origin ColE1, five copies of terminator rrnB T1, and tetracycline resistance marker tet(L) for S. aureus and E. coli. The minimum pAMα1 replicon from pBUS1 was improved through either complementation with the single-strand origin oriL from pUB110 (pBUS1-HC and pBUS1-Pcap-HC) or substitution with a pT181-family replicon (pTSSCm and pTSSCm-Pcap). The new constructs displayed increased plasmid yield and segregational stability in S. aureus. Furthermore, pBUS1-Pcap-HC and pTSSCm-Pcap offer the potential to generate C-terminal RGS-His6 translational fusions of cloned genes using simple molecular manipulation. BcgI-induced DNA excision followed by religation converts the TGA stop codon of the MCS into a TGC codon and links the rgs-his6 codons to the 3' end of the target gene. The generation of the rgs-his6 codon-fusion, gene expression, and protein purification were demonstrated in both S. aureus and E. coli using the macrolide-lincosamide-streptogramin B resistance gene erm(44) inserted downstream of Pcap. The new His tag expression system represents a helpful tool for the direct analysis of target gene function in staphylococcal cells.

The novel macrolide-Lincosamide-Streptogramin B resistance gene erm(44) is associated with a prophage in Staphylococcus xylosus.

A novel erythromycin ribosome methylase gene, erm(44), that confers resistance to macrolide, lincosamide, and streptogramin B (MLSB) antibiotics was identified by whole-genome sequencing of the chromosome of Staphylococcus xylosus isolated from bovine mastitis milk. The erm(44) gene is preceded by a regulatory sequence that encodes two leader peptides responsible for the inducible expression of the methylase gene, as demonstrated by cloning in Staphylococcus aureus. The erm(44) gene is located on a 53-kb putative prophage designated ΦJW4341-pro. The 56 predicted open reading frames of ΦJW4341-pro are structurally organized into the five functional modules found in members of the family Siphoviridae. ΦJW4341-pro is site-specifically integrated into the S. xylosus chromosome, where it is flanked by two perfect 19-bp direct repeats, and exhibits the ability to circularize. The presence of erm(44) in three additional S. xylosus strains suggests that this putative prophage has the potential to disseminate MLSB resistance.

Initial bacterial adherence and biofilm formation on novel restorative materials used in paediatric dentistry.

OBJECTIVE: To evaluate the initial bacterial adherence and biofilm formation on novel restorative materials in paediatric dentistry and compare the results to stainless steel crown and primary enamel. MATERIALS AND METHODS: Twenty-five samples (Diameter = 4 mm) from five restorative materials (Tetric Power Fill light cured for 3 s or 10 s, Fuji II LC, Equia Forte HT Fil, Cention Forte, Stainless-steel crown) and primary enamel were prepared. Four samples served for recording of surface roughness (Ra) using a contact profilometer, 21 samples were incubated in stimulated human saliva for 2 h (initial bacterial adherence) and 72 h (biofilm formation) and served to determine ion releasing and bacterial growth. After 2 and 72 h, the number of colony-forming units (CFU) per ml was counted and expressed in Log10 CFU/ml. Data were analysed with two-way ANOVA and Tuckey's multiple comparisons test (p < 0.05). RESULTS: All tested materials showed similar initial bacterial adherence (p > 0.1). Stainless steel crown showed statistically significantly less biofilm formation than all other tested materials (p ≤ 0.02), except for Fuji II LC (p = 0.06). In terms of biofilm formation, the differences between all tested materials were not statistically significant (p ≥ 0.9). SIGNIFICANCE: Novel restorative materials in paediatric dentistry show similar initial bacterial adherence and biofilm formation. However, compared to other restorative materials, stainless steel crowns demonstrate the lowest level of biofilm formation. Ion-releasing materials may not necessarily show better antimicrobial properties than conventional materials.

Novel pseudo-staphylococcal cassette chromosome mec element (ψSCCmec57395) in methicillin-resistant Staphylococcus pseudintermedius CC45.

Genetic characterization of methicillin-resistant Staphylococcus pseudintermedius (MRSP) from Thailand and Israel revealed the presence of a predominant atypical clonal lineage which was not typeable by SmaI-PFGE and SCCmec typing. All the atypical isolates (n = 34) belonged to CC45 (30 ST45 and 2 ST179 isolates, 1 ST57 isolate, and 1 ST85 isolate). The isolates originated from healthy and diseased dogs and cats, as well as from the environment of one clinic. Cfr9I-pulsed-field gel electrophoresis (Cfr9I-PFGE) and dru typing permitted the further distinction of CC45 isolates from the two different countries. Microarray analysis identified genes that confer resistance to ß-lactams (mecA; blaZ), aminoglycosides [aac(6')-Ie-aph(2')-Ia; aph(3')-III; ant(6)-Ia], macrolides and lincosamides [erm(B)], tetracyclines [tet(M)], trimethoprim [dfr(G)], streptothricin (sat4), and chloramphenicol (catpC221). Fluoroquinolone resistance was attributed to specific amino acid substitutions, i.e., Ser84Leu in GyrA and Ser80Ile and Asp84Asn in GrlA. A novel pseudo-staphylococcal cassette chromosome (ΨSCCmec57395) element was identified in MRSP strain 57395 (sequence type ST45) by whole-genome sequencing. The 12,282-bp ΨSCCmec57395 element contained a class C1 mec gene complex but no ccr genes. In addition to the methicillin resistance gene mecA, ΨSCCmec57395 also carried determinants of resistance to heavy metals, such as arsenic, cadmium, and copper. Bsu36I restriction analysis of the ΨSCCmec57395 element amplified by long-range PCR revealed the presence of ΨSCCmec57395 in the 33 additional isolates of MRSP CC45. The ΨSCCmec57395 element represents a new class of SCCmec and has been identified in MRSP of CC45, which is a predominant clonal lineage in Israel and Thailand.

Genetic characterization of antimicrobial resistance in coagulase-negative staphylococci from bovine mastitis milk.

Coagulase-negative staphylococci (CNS; n=417) were isolated from bovine milk and identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Nineteen different species were identified, and Staphylococcus xylosus, Staphylococcus chromogenes, Staphylococcus haemolyticus, and Staphylococcus sciuri were the most prevalent species. Resistance to oxacillin (47.0% of the isolates), fusidic acid (33.8%), tiamulin (31.9%), penicillin (23.3%), tetracycline (15.8%), streptomycin (9.6%), erythromycin (7.0%), sulfonamides (5%), trimethoprim (4.3%), clindamycin (3.4%), kanamycin (2.4%), and gentamicin (2.4%) was detected. Resistance to oxacillin was attributed to the mecA gene in 9.7% of the oxacillin-resistant isolates. The remaining oxacillin-resistant CNS did not contain the mecC gene or mecA1 promoter mutations. The mecA gene was detected in Staphylococcus fleurettii, Staphylococcus epidermidis, Staph. haemolyticus, and Staph. xylosus. Resistance to tetracycline was attributed to the presence of tet(K) and tet(L), penicillin resistance to blaZ, streptomycin resistance to str and ant(6)-Ia, and erythromycin resistance to erm(C), erm(B), and msr. Resistance to tiamulin and fusidic acid could not be attributed to an acquired resistance gene. In total, 15.1% of the CNS isolates were multidrug resistant (i.e., resistant to 2 or more antimicrobials). The remaining CNS isolates were susceptible to antimicrobials commonly used in mastitis treatment. Methicillin-resistant CNS isolates were diverse, as determined by mecA gene sequence analysis, staphylococcal cassette chromosome mec typing, and pulsed-field gel electrophoresis. Arginine catabolic mobile element types 1 and 3 were detected in both methicillin-resistant and methicillin-susceptible Staph. epidermidis and were associated with sequence types ST59 and ST111. Because this study revealed the presence of multidrug-resistant CNS in a heterogeneous CNS population, we recommend antibiogram analysis of CNS in persistent infections before treatment with antimicrobials.

New MLSB resistance gene erm(43) in Staphylococcus lentus.

The search for a specific rRNA methylase motif led to the identification of the new macrolide, lincosamide, and streptogramin B resistance gene erm(43) in Staphylococcus lentus. An inducible resistance phenotype was demonstrated by cloning and expressing erm(43) and its regulatory region in Staphylococcus aureus. The erm(43) gene was detected in two different DNA fragments, of 6,230 bp and 1,559 bp, that were each integrated at the same location in the chromosome in several S. lentus isolates of human, dog, and chicken origin.

Human RECQL5beta stimulates flap endonuclease 1.

Human RECQL5 is a member of the RecQ helicase family which is implicated in genome maintenance. Five human members of the family have been identified; three of them, BLM, WRN and RECQL4 are associated with elevated cancer risk. RECQL1 and RECQL5 have not been linked to any human disorder yet; cells devoid of RECQL1 and RECQL5 display increased chromosomal instability. Here, we report the physical and functional interaction of the large isomer of RECQL5, RECQL5beta, with the human flap endonuclease 1, FEN1, which plays a critical role in DNA replication, recombination and repair. RECQL5beta dramatically stimulates the rate of FEN1 cleavage of flap DNA substrates. Moreover, we show that RECQL5beta and FEN1 interact physically and co-localize in the nucleus in response to DNA damage. Our findings, together with the previous literature on WRN, BLM and RECQL4's stimulation of FEN1, suggests that the ability of RecQ helicases to stimulate FEN1 may be a general feature of this class of enzymes. This could indicate a common role for the RecQ helicases in the processing of oxidative DNA damage.