Phylogenomic and molecular markers based studies on Staphylococcaceae and Gemella species. Proposals for an emended family Staphylococcaceae and three new families (Abyssicoccaceae fam. nov., Salinicoccaceae fam. nov. and Gemellaceae fam. nov.) harboring four new genera, Lacicoccus gen. nov., Macrococcoides gen. nov., Gemelliphila gen. nov., and Phocicoccus gen. nov.

The family Staphylococcacae and genus Gemella contain several organisms of clinical or biotechnological importance. We report here comprehensive phylogenomic and comparative analyses on 112 available genomes from species in these taxa to clarify their evolutionary relationships and classification. In a phylogenomic tree based on 678 core proteins, Gemella species were separated from Staphylococcacae by a long branch indicating that they constitute a distinct family (Gemellaceae fam. nov.). In this tree, Staphylococcacae species formed two main clades, one encompassing the genera Aliicoccus, Jeotgalicoccus, Nosocomiicoccus and Salinicoccus (Family "Salinicoccaceae"), while the other clade consisted of the genera Macrococcus, Mammaliicoccus and Staphylococcus (Family Staphylococcaceae emend.). In this tree, species from the genera Gemella, Jeotgalicoccus, Macrococcus and Salinicoccus each formed two distinct clades. Two species clades for these genera are also observed in 16S rRNA gene trees and supported by average amino acid identity analysis. We also report here detailed analyses on protein sequences from Staphylococcaceae and Gemella genomes to identify conserved signature indels (CSIs) which are specific for different genus and family-level clades. These analyses have identified 120 novel CSIs robustly demarcating different proposed families and genera. The identified CSIs provide independent evidence that the genera Gemella, Jeotgalicoccus, Macrococcus and Salinicoccus consist of two distinct clades, which can be reliably distinguished based on multiple exclusively shared CSIs. We are proposing transfers of the species from the novel clades of the above four genera into the genera Gemelliphila gen. nov., Phocicoccus gen. nov., Macrococcoides gen. nov. and Lacicoccus gen. nov., respectively. The identified CSIs also provide strong evidence for division of Staphylococcaceae into an emended family Staphylococcaceae and two new families, Abyssicoccaceae fam. nov. and Salinicoccaceae fam. nov. All of these families can be reliably demarcated based on several exclusively shared CSIs.

The emergence of novel macrolide resistance island in Macrococcus caseolyticus and Staphylococcus aureus of food origin.

Food-derived Staphylococcaceae species with severe antimicrobial resistance, especially Staphylococcus aureus, is a major threat to public health. Macrococcus caseolyticus (M. caseolyticus) is a member of the Staphylococcaceae family which plays a vital role in fermented products and disease causation in animals. In our previous study, several Staphylococcus aureus antibiotic-resistant island msr (SaRImsr) were found in multidrug-resistant S. aureus. In this study, novel SaRImsr, SaRImsr-III emerged from S. aureus. Another novel SaRImsr-like further emerged in M. caseolyticus from food. These isolates' prevalence and genetic environment were investigated and characterized to understand the distribution and transmission of these novel SaRImsr strains. All SaRImsr-positive S. aureus isolates exhibited a multidrug resistance (MDR) phenotype, within which a series of antimicrobial resistance genes (ARGs) and virulence factor genes (VFs) were identified. In addition, three SaRImsr types, SaRImsr-I (15.1 kb), SaRImsr-II (16-17 kb), and SaRImsr-III (18 kb) carrying mef(D)-msr(F), were identified in these isolates' chromosomes. SaRImsr-(I-III) contains a site-specific integrase gene int and operon mef(D)-msr(F). SaRImsr-III has an additional orf3-orf4-IS30 arrangement downstream of mef(D) and msr(F). Moreover, the SaRImsr-like and macrolide-resistant transposon Tn6776 forming a novel mosaic structure coexisted in one M. caseolyticus isolate. Within this mosaic structure, the macrolide-resistant genes mef(D)-msr(F) were absent in SaRImsr-like, whereas an operon, mef(F)-msr(G), was identified in Tn6776. The SaRImsr-(I-III) and SaRImsr-like structure were inserted into the rpsI gene encoding the 30S ribosomal protein S9 in the chromosome. Excision and cyclisation of SaRImsr-III, SaRImsr-like, operon mef(D)-msr(F), and orf3-orf4-IS30 arrangements were confirmed using two-step PCR. This study is the first to report MDR S. aureus harbouring novel SaRImsr-III and M. caseolyticus containing novel mosaic structures isolated from retail foods. Similar SaRImsr-type resistant islands' occurrence and propagation in Staphylococcaceae species require continuous monitoring and investigation.

A prevalence and molecular characterization of novel pathogenic strains of Macrococcus caseolyticus isolated from external wounds of donkeys in Khartoum State -Sudan.

A pathogenic strain of Macrococcus caseolyticus (M. caseolyticus) was isolated from wounds infection during an investigation on donkeys in Khartoum State. (122) samples were collected from external wounds (head, abdomen, back and leg) during different seasons. One isolate (124B) was identified using whole-genome sequence analysis. RAST software identified 31 virulent genes of disease and defense, including methicillin-resistant genes, TatR family and ANT(4')-Ib. Plasmid rep22 was identified by PlasmidFindet-2.0 Server and a CRISPR. MILST-2.0 predicted many novel alleles. NCBI notated the genome as a novel M. caseolyticus strain (DaniaSudan). The MLST-tree-V1 revealed that DaniaSudan and KM0211a strains were interrelated. Strain DaniaSudan was resistant to ciprofloxacin, ceftazidime, erythromycin, oxacillin, clindamycin and kanamycin. Mice modeling showed bacteremia and many clinical signs (swelling, allergy, wounds, and hair loss). Enlargement, hyperemia, adhesions and abscesses were observed in many organs.Constructive conclusionThe prevalence of the strain was 4.73%, with significant differences between collection seasons and locations of wounds. A highly significant association between doses (105 CFU/ml, 102 CFU/ml, Intra-peritoneum and sub-cutaneous) and swelling, developing of allergy and loss of hair (p = 0.001, p = 0.000 and p = 0.005) respectively were seen.This result represents the first report of pathogenic strains of M. caseolyticus worldwide.

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.

Identification and characterization of a novel GNAT superfamily Nα -acetyltransferase from Salinicoccus halodurans H3B36.

Nα -acetyl-α-lysine was found as a new type of compatible solutes that acted as an organic cytoprotectant in the strain of Salinicoccus halodurans H3B36. A novel lysine Nα -acetyltransferase gene (shkat), encoding an enzyme that catalysed the acetylation of lysine exclusively at α position, was identified from this moderate halophilic strain and expressed in Escherichia coli. Sequence analysis indicated ShKAT contained a highly conserved pyrophosphate-binding loop (Arg-Gly-Asn-Gly-Asn-Gly), which was a signature of the GNAT superfamily. ShKAT exclusively recognized free amino acids as substrate, including lysine and other basic amino acids. The enzyme showed a wide range of optimal pH value and was tolerant to high-alkali and high-salinity conditions. As a new member of the GNAT superfamily, the ShKAT was the first enzyme recognized free lysine as substrate. We believe this work gives an expanded perspective of the GNAT superfamily, and reveals great potential of the shkat gene to be applied in genetic engineering for resisting extreme conditions.

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.

Microbiome signatures in neonatal central line associated bloodstream infections.

Neonates are at high risk for central line associated bloodstream infections (CLABSI). Biofilm formation is universal on indwelling catheters but why some biofilms seed the bloodstream to cause CLABSI is not clearly understood. With the objective to test the hypothesis that catheter biofilm microbiome in neonates with CLABSI differs than those without infection, we prospectively enrolled neonates (n = 30) with infected and uninfected indwelling central catheters. Catheters were collected at the time of removal, along with blood samples and skin swabs at the catheter insertion sites. Microbiomes of catheter biofilms, skin swabs and blood were evaluated by profiling the V4 region of the bacterial 16S rRNA gene using Illumina MiSeq sequencing platform. The microbial DNA load was higher from catheter biofilms of CLABSI patients without differences in alpha diversity when compared to that of the non-CLABSI neonates. Proteus and unclassified Staphylococcaceae were more abundant in infected catheter biofilms while Bradyrhizobium, Cloacibacterium, and Sphingomonas were more abundant in the uninfected catheters. A blood microbiome was detected in uninfected samples. The blood microbiome in CLABSI neonates clustered separately from the uninfected blood samples in beta diversity plots. We found that the microbiome signature in catheter biofilm and blood of neonates with CLABSI is different than the microbiomes of non-CLABSI neonates.

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.

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.

Investigation of the viral and bacterial microbiota in intestinal samples from mink (Neovison vison) with pre-weaning diarrhea syndrome using next generation sequencing.

Pre-weaning diarrhea (PWD) in mink kits is a common multifactorial syndrome on commercial mink farms. Several potential pathogens such as astroviruses, caliciviruses, Escherichia coli and Staphylococcus delphini have been studied, but the etiology of the syndrome seems complex. In pooled samples from 38 diarrheic and 42 non-diarrheic litters, each comprising of intestinal contents from 2-3 mink kits from the same litter, the bacterial populations were studied using Illumina Next Generation Sequencing technology and targeted 16S amplicon sequencing. In addition, we used deep sequencing to determine and compare the viral intestinal content in 31 healthy non-diarrheic and 30 diarrheic pooled samples (2-3 mink kits from the same litter per pool). The results showed high variations in composition of the bacterial species between the pools. Enterococci, staphylococci and streptococci dominated in both diarrheic and non-diarrheic pools. However, enterococci accounted for 70% of the reads in the diarrheic group compared to 50% in the non-diarrheic group and this increase was at the expense of staphylococci and streptococci which together accounted for 45% and 17% of the reads in the non-diarrheic and diarrheic group, respectively. Moreover, in the diarrheic pools there were more reads assigned to Clostridia, Escherichia-Shigella and Enterobacter compared to the non-diarrheic pools. The taxonomically categorized sequences from the virome showed that the most prevalent viruses in all pools were caliciviruses and mamastroviruses (almost exclusively type 10). However, the numbers of reads assigned to caliciviruses were almost 3 times higher in the diarrheic pools compared the non-diarrheic pools and Sapporo-like caliciviruses were more abundant than the Norwalk-like caliciviruses. The results from this study have contributed to the insight into the changes in the intestinal microbiota associated with the PWD syndrome of mink.

The Genus Macrococcus: An Insight Into Its Biology, Evolution, and Relationship With Staphylococcus.

The Gram-positive genus Macrococcus is composed of eight species that are evolutionarily closely related to species of the Staphylococcus genus. In contrast to Staphylococcus species, species of Macrococcus are generally regarded to be avirulent in their animal hosts. Recent reports on Macrococcus have focused on the presence of novel methicillin resistance genes in Macrococcus caseolyticus and Macrococcus canis, with the discovery of the first plasmid-encoded methicillin resistance gene in clinical Staphylococcus aureus of probable macrococcal origin generating further interest in these organisms. Furthermore, M. caseolyticus has been associated with flavor development in certain fermented foods and its potential as a food bio-preservative has been documented. The potential application of these organisms in food seems at odds with the emerging information regarding antibiotic resistance and is prompting further examination of the potential safety issues associated with such strains, given the European Food Safety Authority framework for the safety evaluation of microorganisms in the food chain. A comprehensive understanding of the genus would also contribute to understanding the evolution of staphylococci in terms of its acquisition of antibiotic resistance and pathogenic potential. In this review, we discuss the current knowledge on Macrococcus with regard to their phenotypic capabilities, genetic diversity, and evolutionary history with Staphylococcus. Comparative genomics of the sequenced Macrococcus species will be discussed, providing insight into their unique metabolic features and the genetic structures carrying methicillin resistance. An in-depth understanding of these antibiotic resistance determinants can open the possibilities for devising better preventative strategies for an unpredictable future.

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.

Culturable symbionts associated with the reproductive and digestive tissues of the Neotropical brown stinkbug Euschistus heros.

Symbionts are widely distributed in eukaryotes, and potentially affect the physiology, ecology and evolution of their host. Most insects harbour free-living bacteria in their haemocoel and gut lumen, intracellular-living bacteria in a range of tissues or bacteria in host-derived specialized cells. Stinkbugs, as do many arthropods, harbour extracellular bacteria in the gut that may affect the fitness of their host. This study identified the culturable symbionts associated with the ovaries, spermatheca, seminal vesicle and posterior midgut region (V4) of males and females of Euschistus heros (F.) (Hemiptera: Pentatomidae). Several culture media were used to isolate the bacteria associated with these structures. The selected colonies (morphotypes) were cultured in liquid medium, subjected to genomic DNA extraction, 16S rRNA gene amplification, and restriction fragment length polymorphism (RFLP) analyses. Morphotypes with distinct RFLP patterns were purified and sequenced, and the sequences obtained were used for putative identification and phylogenetic analysis. Comparison of the sequences with those available in the EzTaxon-e database and the use of a matrix of paired distances grouped the isolates in phylotypes belonging to the Phylum Proteobacteria. Proteobacteria was represented by γ-Proteobacteria phylotypes belonging to Enterobacteriaceae, while Firmicutes had Bacilli phylotypes distributed in Enterococcaceae and Staphylococcaceae. Some of the phylotypes identified were associated exclusively with single structures, such as ovaries, spermatheca and the V4 midgut region of males and females. All culturable bacteria associated with the seminal vesicle were also associated with other tissues.

A rapid PCR-based method to discriminate Macrococcus caseolyticus and Macrococcus canis from closely-related Staphylococcus species based on the ctaC gene sequence.

Our method exploits the amplification of the cytochrome c oxidase subunit II (ctaC) gene for the screening of Macrococcus caseolyticus and Macrococcus canis in complex microbial communities, and discriminating these species from strains of their sister genus Staphylococcus. Thirteen novel strains of these species were isolated using this approach.

Genome analysis of methicillin resistance in Macrococcus caseolyticus from dairy cattle in England and Wales.

Species of the genus Macrococcus are widespread commensals of animals but are becoming increasingly recognised as veterinary pathogens. They can encode methicillin resistance and are implicated in its spread to the closely-related, but more pathogenic, staphylococci. In this study we have identified 33 isolates of methicillin-resistant Macrococcus caseolyticus from bovine bulk tank milk from England and Wales. These isolates were characterised to provide insight into the molecular epidemiology of M. caseolyticus and to discern the genetic basis for their methicillin resistance. Antimicrobial susceptibility testing was performed by Vitek2 and disc diffusion. Isolates were whole-genome sequenced to evaluate phylogenetic relationships and the presence of methicillin resistance determinants, mecA-D. All 33 isolates were phenotypically methicillin-resistant according to cefoxitin disc diffusion, cefoxitin Etest and oxacillin resistance assessed by Vitek2. In contrast only a single isolate was resistant in the Vitek2 cefoxitin screen. Twenty-seven isolates were positive for mecD and six were positive for mecB. mecA and mecC were not detected. The results of phylogenetic analysis indicated that these methicillin-resistant isolates represented a heterogeneous population with both mecB and mecD found in diverse isolates. Isolates had a widespread distribution across the sampled region. Taken together with the role of M. caseolyticus in veterinary infections, including bovine mastitis, and in the potential spread of methicillin resistance to more pathogenic staphylococci, this work highlights the need to better understand their epidemiology and for increased awareness among veterinary microbiology laboratories.

Emergence of pathogenic and multiple-antibiotic-resistant Macrococcus caseolyticus in commercial broiler chickens.

Macrococcus caseolyticus is generally considered to be a non-pathogenic bacterium that does not cause human or animal diseases. However, recently, a strain of M. caseolyticus (SDLY strain) that causes high mortality rates was isolated from commercial broiler chickens in China. The main pathological changes caused by SDLY included caseous exudation in cranial cavities, inflammatory infiltration, haemorrhages and multifocal necrosis in various organs. The whole genome of the SDLY strain was sequenced and was compared with that of the non-pathogenic JCSC5402 strain of M. caseolyticus. The results showed that the SDLY strain harboured a large quantity of mutations, antibiotic resistance genes and numerous insertions and deletions of virulence genes. In particular, among the inserted genes, there is a cluster of eight connected genes associated with the synthesis of capsular polysaccharide. This cluster encodes a transferase and capsular polysaccharide synthase, promotes the formation of capsules and causes changes in pathogenicity. Electron microscopy revealed a distinct capsule surrounding the SDLY strain. The pathogenicity test showed that the SDLY strain could cause significant clinical symptoms and pathological changes in both SPF chickens and mice. In addition, these clinical symptoms and pathological changes were the same as those observed in field cases. Furthermore, the anti-microbial susceptibility test demonstrated that the SDLY strain exhibits multiple-antibiotic resistance. The emergence of pathogenic M. caseolyticus indicates that more attention should be paid to the effects of this micro-organism on both poultry and public health.

Development of the first gene expression system for Salinicoccus strains with potential application in bioremediation of hypersaline wastewaters.

Salinicoccus salsiraiae IM408 (=CGMCC13032) is a novel halophilic bacterium that we isolated from the saline soil of Da Gang Oilfield. It tolerates 60 g/l sodium chloride and up to 123 g/l (1.5 M) sodium acetate and has shown a potential application in bioremediation of wastewater with high salt and high chemical oxygen demand (COD). Two plasmids, pS408-1 and pS408-2, were identified in S. salsiraiae IM408, and the sequences and copy numbers of the plasmids were determined. Based on these plasmids, two shuttle vectors containing a replicon for Escherichia coli, ampicillin, and chloramphenicol resistance genes, as well as the replicon from pS408-1 or pS408-2, were constructed and named as pTCS101 and pTCS201, respectively. A suitable host strain, named S. salsiraiae PE01, was also developed from the wild-type by plasmid elimination. Using the plasmid pTCS101 as an expression vector, L-lactate dehydrogenase from Staphylococcus aureus was expressed successfully in S. salsiraiae PE01. This is the first gene expression system for the Salinicoccus genus. It has provided the potential for expression of desired proteins or for establishment of desired pathways in Salinicoccus strains, which would make these halophiles more advantageous in future biotechnological applications.

Novel methicillin resistance gene mecD in clinical Macrococcus caseolyticus strains from bovine and canine sources.

Methicillin-resistant Macrococcus caseolyticus strains from bovine and canine origins were found to carry a novel mecD gene conferring resistance to all classes of ß-lactams including anti-MRSA cephalosporins. Association of ß-lactam resistance with mecD was demonstrated by gene expression in S. aureus and deletion of the mecD-containing island in M. caseolyticus. The mecD gene was located either on an 18,134-bp M. caseolyticus resistance island (McRImecD-1) or a 16,188-bp McRImecD-2. Both islands were integrated at the 3' end of the rpsI gene, carried the mecD operon (mecD-mecR1m-mecIm), and genes for an integrase of the tyrosine recombinase family and a putative virulence-associated protein (virE). Apart from the mecD operon, that shared 66% overall nucleotide identity with the mecB operon, McRImecD islands were unrelated to any mecB-carrying elements or staphylococcal cassette chromosome mec. Only McRImecD-1 that is delimitated at both ends by direct repeats was capable of circular excision. The recombined excision pattern suggests site-specific activity of the integrase and allowed identification of a putative core attachment site. Detection of rpsI-associated integrases in Bacillus and S. aureus reveals a potential for broad-host range dissemination of the novel methicillin resistance gene mecD.

Mutagenesis and redox partners analysis of the P450 fatty acid decarboxylase OleTJE.

The cytochrome P450 enzyme OleTJE from Jeotgalicoccus sp. ATCC 8456 is capable of converting free long-chain fatty acids into α-alkenes via one-step oxidative decarboxylation in presence of H2O2 as cofactor or using redox partner systems. This enzyme has attracted much attention due to its intriguing but unclear catalytic mechanism and potential application in biofuel production. Here, we investigated the functionality of a select group of residues (Arg245, Cys365, His85, and Ile170) in the active site of OleTJE through extensive mutagenesis analysis. The key roles of these residues for catalytic activity and reaction type selectivity were identified. In addition, a range of heterologous redox partners were found to be able to efficiently support the decarboxylation activity of OleTJE. The best combination turned out to be SeFdx-6 (ferredoxin) from Synechococcus elongatus PCC 7942 and CgFdR-2 (ferredoxin reductase) from Corynebacterium glutamicum ATCC 13032, which gave the highest myristic acid conversion rate of 94.4%. Moreover, Michaelis-Menton kinetic parameters of OleTJE towards myristic acid were determined.