High Prevalence of Clonal Complex 398 Methicillin-Susceptible and -Resistant Staphylococcus aureus in Pig Farms: Clonal Lineages, Multiple Drug Resistance, and Occurrence of the Staphylococcal Cassette Chromosome mec IX.

High prevalence of livestock-associated methicillin-susceptible and -resistant Staphylococcus aureus (LA-MSSA and LA-MRSA, respectively) in livestock farms, particularly pig farms, is an increasingly serious threat to food safety and public health. In this study, 173 S. aureus (84 MRSA and 89 MSSA) isolates from healthy pigs, farm environments, and farm workers in Korea were examined to determine the (1) genetic diversity of S. aureus isolates (sequence type [ST], spa, and agr types), (2) staphylococcal cassette chromosome mec (SCCmec) types of MRSA isolates, and (3) multidrug resistance (MDR) phenotypes of MRSA and MSSA isolates. Clonal complex 398 (CC398) genotypes of MRSA and MSSA isolates, particularly CC398-spa type t571-agr I lineages, displaying MDR phenotypes were highly prevalent in pig farms. High prevalence of CC398-t571 MRSA and MSSA was more frequently associated with weaning piglets and growing pigs. Moreover, the same clonal lineages of S. aureus isolates colonized both pigs and farm workers, suggesting the transmission of antimicrobial-resistant CC398 MRSA and MSSA between pigs and humans in the pig farms. Furthermore, two dominant SCCmec types, SCCmec V and SCCmec IX, were identified in CC398 MRSA isolates colonizing healthy pigs. To the best of our knowledge, this is the first report of a CC398 LA-MRSA isolate carrying SCCmec IX in Korea. Collectively, these results suggest widespread distribution of the CC398 lineage among MRSA and MSSA isolates in pigs, farm environments, and farm workers in Korea.

Role of the Staphylococcus aureus Extracellular Loop of GraS in Resistance to Distinct Human Defense Peptides in PMN and Invasive Cardiovascular infections.

GraS is a membrane sensor in Staphylococcus aureus that induces mprF and dltABCD expression to alter the surface positive charge upon exposure to cationic human defense peptides (HDPs). The sensing domain of GraS likely resides in the 9-residue extracellular loop (EL). In this study, we assessed a hospital-acquired methicillin-resistant S. aureus (HA-MRSA) strain (COL) for the specific role of two distinct EL mutations: F38G (bulk) and D/35/37/41K (charged inversion). Activation of mprF by polymyxin B (PMB) was reduced in the D35/37/41K mutant versus the D35/37/41G mutant, correlating with reduced surface positive charge; in contrast, these effects were less prominent in the F38G mutant but still lower than those in the parent. These data indicated that both electrostatic charge and steric bulk of the EL of GraS influence induction of genes impacting HDP resistance. Using mprF expression as a readout, we confirmed GraS signaling was pH dependent, increasing as pH was lowered (from pH 7.5 down to pH 5.5). In contrast to PMB activation, reduction of mprF was comparable at pH 5.5 between the P38G and D35/37/41K point mutants, indicating a mechanistic divergence between GraS activation by acidic pH versus cationic peptides. Survival assays in human blood and purified polymorphonuclear leukocytes (PMNs) revealed lower survival of the D35/37/41K mutant versus the F38G mutant, with both being lower than that of the parent. Virulence studies in the rabbit endocarditis model mirrored whole blood and PMN killing assay data described above. Collectively, these data confirmed the importance of specific residues within the EL of GraS in conferring essential bacterial responses for MRSA survival in infections.

Genomic Information on Linezolid-Resistant Sequence-Type 398 Livestock-Associated Methicillin-Resistant Staphylococcus aureus Isolated from a Pig.

The frequent occurrence of sequence-type 398 (ST398) livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) in pigs has become a major public health concern owing to the increased zoonotic potential of the pathogen. Recently, a novel oxazolidinone resistance gene, chloramphenicol-florfenicol resistant (cfr), conferring multiresistance phenotypes to phenicols, lincosamides, oxazolidinones, pleuromutilins, and streptogramin A (PhLOPSA), has been found among ST398 LA-MRSA strains isolated from pigs. In this study, we report the first in silico genome analysis of a linezolid-resistant ST398 LA-MRSA strain, designated PJFA-521M, recovered from a pig in Korea. Genomic analyses revealed that the presence of the cfr gene was responsible for the observed linezolid resistance in the PJFA-521M strain. Moreover, newer antimicrobial resistance genes, such as the dfrG, aadE, spw, lsa(E), lnu(B), and fexA genes, were found in the PJFA-521M strain. In addition to the genetic elements for antimicrobial resistance, the carriage of various virulence genes for adherence, invasion, and immunomodulation was identified in the genome, especially within several mobile genetic elements (MGEs). The presence of multiple antimicrobial resistance genes and virulence genes on MGEs in the genome of a linezolid-resistant ST398 LA-MRSA should raise awareness regarding the use of other antimicrobial agents in pig farms and may also provide selective pressure for the prevalence of the cfr gene and the associated multidrug-resistant phenotype.

Detection of antibodies against hepatitis E virus in pet veterinarians and pet dogs in South Korea.

Hepatitis E virus (HEV) is a zoonotic pathogen commonly considered an important foodborne virus. Pet dogs are important reservoirs of zoonotic agents. In the present study, the seroprevalence of HEV in pet dogs and pet veterinarians were found to be 28.2 and 5.0%, respectively. It remains unclear whether pet veterinarians are at higher risk of HEV transmission. However, pet animals and individuals who have contact with infected animals must be continually monitored for public health concerns.

Genetic alterations responsible for reduced susceptibility to vancomycin in community-associated MRSA strains of ST72.

Objectives: We previously reported the first case of vancomycin treatment failure due to development of vancomycin-intermediate resistance in a patient with an MRSA of ST72, a community genotype in Korea. We investigated two isogenic MRSA strains from this patient, who experienced treatment failure with vancomycin and rifampicin. Methods: We tracked the genetic alterations that confer reduced susceptibility to vancomycin on those two isogenic MRSA strains by WGS. Results: Five non-synonymous mutations were identified, including rpoB (H481Y), dprA (G196C), femA (F92C), vraR (E127K) and agrC (E391stop). We further studied the role of a mutation of vraR in reduced susceptibility to vancomycin. Introduction of the mutated vraR (E127K) into a vancomycin-susceptible Staphylococcus aureus strain resulted in an increase in vraSR mRNA expression and vancomycin MIC and development of the hetero-VISA phenotype, which was confirmed by the population analysis profile (PAP)/AUC. Electron microscopy showed increased cell wall thickness in the strains with mutated vraR. Conclusions: Based on the genomic data, molecular experiments and PAP and cell wall analyses, we propose that a single mutation of vraR is associated with the reduced susceptibility to vancomycin in MRSA and further treatment failure.

TLR2 contributes to trigger immune response of pleural mesothelial cells against Mycobacterium bovis BCG and M. tuberculosis infection.

Mycobacterium tuberculosis is a causative agent leading to pleural effusion, characterized by the accumulation of fluid and immune cells in the pleural cavity. Although this phenomenon has been described before, detailed processes or mechanisms associated with the pleural effusion are still not well understood. Pleural mesothelial cells (PMCs) are specialized epithelial cells that cover the body wall and internal organs in pleural cavity playing a central role in pleural inflammation. Toll-like receptors are expressed in various cell types including mesothelial cells and initiate the recognition and defense against mycobacterial infection. In the present study, we investigated direct immune responses of PMCs against two mycobacterial strains, M. bovis vaccine strain Bacille Calmette-Guérin (BCG) and M. tuberculosis virulent strain H37Rv, and the role of TLR2 in such responses. Infection with BCG and H37Rv increased the production of IL-6, CXCL1, and CCL2 in WT PMCs, which was partially impaired in TLR2-deficient cells. In addition, the activation of NF-κB and MAPKs induced by BCG and H37Rv was suppressed in TLR2-deficient PMCs, as compared with the WT cells. TLR2 deficiency led to the decrease of nitric oxide (NO) production through the delayed gene expression of iNOS in PMCs. TLR2 was also shown to be essential for optimal expression of cellular adhesion molecules such as ICAM-1 and VCAM-1 in PMCs in response to BCG and H37Rv. These findings strongly suggest that TLR2 participates in mycobacteria-induced innate immune responses in PMCs and may play a role in pathogenesis of tuberculosis pleural effusion.

The GraS Sensor in Staphylococcus aureus Mediates Resistance to Host Defense Peptides Differing in Mechanisms of Action.

Staphylococcus aureus uses the two-component regulatory system GraRS to sense and respond to host defense peptides (HDPs). However, the mechanistic impact of GraS or its extracellular sensing loop (EL) on HDP resistance is essentially unexplored. Strains with null mutations in the GraS holoprotein (ΔgraS) or its EL (ΔEL) were compared for mechanisms of resistance to HDPs of relevant immune sources: neutrophil α-defensin (human neutrophil peptide 1 [hNP-1]), cutaneous ß-defensin (human ß-defensin 2 [hBD-2]), or the platelet kinocidin congener RP-1. Actions studied by flow cytometry included energetics (ENR); membrane permeabilization (PRM); annexin V binding (ANX), and cell death protease activation (CDP). Assay conditions simulated bloodstream (pH 7.5) or phagolysosomal (pH 5.5) pH contexts. S. aureus strains were more susceptible to HDPs at pH 7.5 than at pH 5.5, and each HDP exerted a distinct effect signature. The impacts of ΔgraS and ΔΕL on HDP resistance were peptide and pH dependent. Both mutants exhibited defects in ANX response to hNP-1 or hBD-2 at pH 7.5, but only hNP-1 did so at pH 5.5. Both mutants exhibited hyper-PRM, -ANX, and -CDP responses to RP-1 at both pHs and hypo-ENR at pH 5.5. The actions correlated with ΔgraS or ΔΕL hypersusceptibility to hNP-1 or RP-1 (but not hBD-2) at pH 7.5 and to all study HDPs at pH 5.5. An exogenous EL mimic protected mutant strains from hNP-1 and hBD-2 but not RP-1, indicating that GraS and its EL play nonredundant roles in S. aureus survival responses to specific HDPs. These findings suggest that GraS mediates specific resistance countermeasures to HDPs in immune contexts that are highly relevant to S. aureus pathogenesis in humans.

Dysregulation of mprF and dltABCD expression among daptomycin-non-susceptible MRSA clinical isolates.

BACKGROUND: In small series or individual reports, SNPs within the mprF ORF and dysregulation of its expression in Staphylococcus aureus have been linked to daptomycin resistance (DAP-R) via a proposed gain-in-function mechanism. Similarly, dysregulation of dltABCD has also been associated with DAP-R. METHODS: Using 22 well-characterized, isogenic daptomycin-susceptible (DAP-S)/DAP-R clinical MRSA strain pairs, we assessed potential relationships of the DAP-R phenotype with: (i) regulation of mprF transcription; (ii) regulation of dltABCD transcription; (iii) expression of the two-component regulatory system, graRS (upstream regulator for both mprF and dltABCD transcription); (iv) SNPs within the graRS promoter or its ORF; and (v) altered mprF transcription and lysyl-phosphatidylglycerol (L-PG) synthesis. RESULTS: Enhanced expression of mprF occurred with SNPs in highly distinct and well-chronicled MprF domain 'hot spots' and rarely occurred without such mutations. Increased expression and/or dysregulation of mprF and dltABCD were not uncommon in DAP-R strains, occurring in 27% of strains for each gene. In these latter strains, neither graRS expression profiles nor polymorphic sequences within the graRS promoter or ORF could be significantly linked to altered transcription of mprF or dlt. CONCLUSIONS: Although graRS can co-regulate mprF and dltABCD expression, loci outside of this regulon appear to be involved in dysregulation of these latter two genes and the DAP-R phenotype. Finally, DAP-R strains exhibiting significantly altered mprF transcription profiles produced significantly increased levels of L-PG.

Toll/IL-1 domain-containing adaptor inducing IFN-ß (TRIF) mediates innate immune responses in murine peritoneal mesothelial cells through TLR3 and TLR4 stimulation.

Mesothelial cells are composed of monolayer of the entire surface of serosal cavities including pleural, pericardial, and peritoneal cavity. Although mesothelial cells are known to express multiple Toll-like receptors (TLRs) which contribute to trigger innate immune responses against infections, the precise molecular mechanism remains still unclear. In the present study, we investigated the role of Toll/IL-1 domain-containing adaptor inducing IFN-ß (TRIF), one of the two major TLRs-adaptor molecules, on innate immune response induced by TLR3 and TLR4 stimulation in murine peritoneal mesothelial cells (PMCs). TRIF was strongly expressed in PMCs and its deficiency led to impaired production of cytokines and chemokines by poly I:C and LPS in the cells. Activation of NF-κB or MAPKs through poly I:C and LPS stimulation was reduced in TRIF-deficient PMCs as compared to the WT cells. TRIF was also necessary for optimal nitric oxide synthesis and gene expression of inducible nitric oxide synthase (iNOS) and IFN-ß in PMCs in response to poly I:C and LPS. Furthermore, both Escherichia coli and Pseudomonas aeruginosa induced high level of IL-6, CXCL1, and CCL2 production in PMCs, which was significantly impaired by TRIF deficiency. These results demonstrated that TRIF is required for optimal activation of innate immune responses in mesothelial cells against microbial infections.

Characterization of a novel two-component regulatory system, HptRS, the regulator for the hexose phosphate transport system in Staphylococcus aureus.

Hexose phosphate is an important carbon source within the cytoplasm of host cells. Bacterial pathogens that invade, survive, and multiply within various host epithelial cells exploit hexose phosphates from the host cytoplasm through the hexose phosphate transport (HPT) system to gain energy and synthesize cellular components. In Escherichia coli, the HPT system consists of a two-component regulatory system (UhpAB) and a phosphate sensor protein (UhpC) that tightly regulate expression of a hexose phosphate transporter (UhpT). Although growing evidence suggests that Staphylococcus aureus also can invade, survive, and multiply within various host epithelial cells, the genetic elements involved in the HPT system in S. aureus have not been characterized yet. In this study, we identified and characterized the HPT system in S. aureus that includes the hptRS (a novel two-component regulatory system), the hptA (a putative phosphate sensor), and the uhpT (a hexose phosphate transporter) genes. The hptA, hptRS, and uhpT markerless deletion mutants were generated by an allelic replacement method using a modified pMAD-CM-GFPuv vector system. We demonstrated that both hptA and hptRS are required to positively regulate transcription of uhpT in response to extracellular phosphates, such as glycerol-3-phosphate (G3P), glucose-6-phosphate (G6P), and fosfomycin. Mutational studies revealed that disruption of the hptA, hptRS, or uhpT gene impaired the growth of bacteria when the available carbon source was limited to G6P, impaired survival/multiplication within various types of host cells, and increased resistance to fosfomycin. The results of this study suggest that the HPT system plays an important role in adaptation of S. aureus within the host cells and could be an important target for developing novel antistaphylococcal therapies.

Frequency and Distribution of Single-Nucleotide Polymorphisms within mprF in Methicillin-Resistant Staphylococcus aureus Clinical Isolates and Their Role in Cross-Resistance to Daptomycin and Host Defense Antimicrobial Peptides.

MprF is responsible for the lysinylation of phosphatidylglycerol (PG) to synthesize the positively charged phospholipid (PL) species, lysyl-PG (L-PG). It has been proposed that the single-nucleotide polymorphisms (SNPs) within the mprF open reading frame (ORF) are associated with a gain-in-function phenotype in terms of daptomycin resistance in Staphylococcus aureus. (Note that although the official term is daptomycin nonsusceptibility, we use the term daptomycin resistance in this paper for ease of presentation.) Using 22 daptomycin-susceptible (DAP(s))/daptomycin-resistant (DAP(r)) clinical methicillin-resistant S. aureus (MRSA) strain pairs, we assessed (i) the frequencies and distribution of putative mprF gain-in-function SNPs, (ii) the relationships of the SNPs to both daptomycin resistance and cross-resistance to the prototypical endovascular host defense peptide (HDP) thrombin-induced platelet microbicidal protein (tPMP), and (iii) the impact of mprF SNPs on positive surface charge phenotype and modifications of membrane PL profiles. Most of the mprF SNPs identified in our DAP(r) strains were clustered within the two MprF loci, (i) the central bifunctional domain and (ii) the C-terminal synthase domain. Moreover, we were able to correlate the presence and location of mprF SNPs in DAP(r) strains with HDP cross-resistance, positive surface charge, and L-PG profiles. Although DAP(r) strains with mprF SNPs in the bifunctional domain showed higher resistance to tPMPs than DAP(r) strains with SNPs in the synthase domain, this relationship was not observed in positive surface charge assays. These results demonstrated that both charge-mediated and -unrelated mechanisms are involved in DAP resistance and HDP cross-resistance in S. aureus.

Site-specific mutation of the sensor kinase GraS in Staphylococcus aureus alters the adaptive response to distinct cationic antimicrobial peptides.

The Staphylococcus aureus two-component regulatory system, GraRS, is involved in resistance to killing by distinct host defense cationic antimicrobial peptides (HD-CAPs). It is believed to regulate downstream target genes such as mprF and dltABCD to modify the S. aureus surface charge. However, the detailed mechanism(s) by which the histidine kinase, GraS, senses specific HD-CAPs is not well defined. Here, we studied a well-characterized clinical methicillin-resistant S. aureus (MRSA) strain (MW2), its isogenic graS deletion mutant (ΔgraS strain), a nonameric extracellular loop mutant (ΔEL strain), and four residue-specific ΔEL mutants (D37A, P39A, P39S, and D35G D37G D41G strains). The ΔgraS and ΔEL strains were unable to induce mprF and dltA expression and, in turn, demonstrated significantly increased susceptibilities to daptomycin, polymyxin B, and two prototypical HD-CAPs (hNP-1 and RP-1). Further, P39A, P39S, and D35G-D37G-D41G ΔEL mutations correlated with moderate increases in HD-CAP susceptibility. Reductions of mprF and dltA induction by PMB were also found in the ΔEL mutants, suggesting these residues are pivotal to appropriate activation of the GraS sensor kinase. Importantly, a synthetic exogenous soluble EL mimic of GraS protected the parental MW2 strain against hNP-1- and RP-1-mediated killing, suggesting a direct interaction of the EL with HD-CAPs in GraS activation. In vivo, the ΔgraS and ΔEL strains displayed dramatic reductions in achieved target tissue MRSA counts in an endocarditis model. Taken together, our results provide new insights into potential roles of GraS in S. aureus sensing of HD-CAPs to induce adaptive survival responses to these molecules.

Heterogeneity of mprF sequences in methicillin-resistant Staphylococcus aureus clinical isolates: role in cross-resistance between daptomycin and host defense antimicrobial peptides.

Over the past several years, single-nucleotide polymorphisms (SNPs) within the mprF open reading frame (ORF) have been proposed to be associated with a gain-of-function phenotype in terms of daptomycin (DAP) nonsusceptibility (referred to as daptomycin resistance [DAP-R] herein for ease of presentation) in Staphylococcus aureus. We investigated the frequencies of SNPs within the mprF ORF and the relationships of such SNPs to cross-resistance between DAP and cationic host defense peptides (HDPs). Thirty-five well-characterized, unique DAP-susceptible (DAP-S) and DAP-R methicillin-resistant S. aureus (MRSA) isolates of the clonal complex 5 genotype were used. In addition to mprF SNPs and DAP-HDP cross-resistance, several other key genotypic and phenotypic metrics often associated with DAP-R were delineated, as follows: (i) mprF expression, (ii) membrane phospholipid content, (iii) positive surface charge, (iv) DAP binding, and (v) cell wall thickness profiles. A number of DAP-S strains (MICs of ≤ 1 µg/ml) exhibited mprF SNPs, occasionally with high-level mprF sequence variation from the genotype reference strain. However, none of these SNPs were localized to well-chronicled mprF hot spot locations associated with DAP-R in S. aureus. In contrast, all 8 DAP-R isolates demonstrated SNPs within such known mprF hot spots. Moreover, only the DAP-R strains showed MprF gain-of-function phenotypes, enhanced mprF expression, higher survival against two prototypical HDPs, and reduced DAP binding. Although a heterogenous array of mprF SNPs were often found in DAP-S strains, only selected hot spot SNPs, combined with concurrent mprF dysregulation, were associated with the DAP-R phenotype.

Virulence of endemic nonpigmented northern Australian Staphylococcus aureus clone (clonal complex 75, S. argenteus) is not augmented by staphyloxanthin.

Staphylococcus aureus clonal complex 75 (herein referred to as S. argenteus) lacks the carotenoid pigment operon, crtOPQMN, responsible for production of the putative virulence factor, staphyloxanthin. Although a common cause of community-onset skin infections among Indigenous populations in northern Australia, this clone is infrequently isolated from hospital-based patients with either bacteremic or nonbacteremic infections. We hypothesized that S. argenteus would have attenuated virulence compared to other S. aureus strains due to its staphyloxanthin "deficiency." Compared to prototypical S. aureus strains, S. argenteus was more susceptible to oxidative stress and neutrophil killing in vitro and had reduced virulence in murine sepsis and skin infection models. Transformation with pTX-crtOPQMN resulted in staphyloxanthin expression and increased resistance to oxidative stress in vitro. However, neither resistance to neutrophil killing nor in vivo virulence was increased. Thus, reduced virulence of S. argenteus in these models is due to mechanisms unrelated to lack of staphyloxanthin production.

Oral bacteria may affect conjunctival microorganisms in brachycephalic dogs: a preliminary study.

OBJECTIVE: To evaluate the prevalence of oral bacteria in the conjunctiva of brachycephalic and nonbrachycephalic dogs. ANIMALS: 12 brachycephalic (9.58 ± 3.55 years) and 12 nonbrachycephalic (8.33 ± 4.92 years) dogs without systemic disease, regardless of breed and sex, were included in the study, and half of the dogs in each group had periodontitis. METHODS: This prospective study investigated clinical data including craniofacial ratio, ophthalmic examination results, and periodontal status of the included dogs. Bacterial samples were collected by swabbing the oral mucosa and conjunctival surfaces. The presence and quantity of bacteria were analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, 16S rRNA sequencing analysis, and the 10-fold dilution method. Statistical analyses were performed to assess correlations and factors influencing the presence of oral bacteria in the conjunctiva. RESULTS: The most common bacteria in the conjunctival flora in both groups were Micrococcus luteus, Corynebacterium spp, and Staphylococcus spp. The prevalence of oral bacteria on the conjunctival surface was 33%, with a significantly higher incidence in brachycephalic dogs (P = .027). Oral bacteria detected in the conjunctiva were predominantly Frederiksenia canicola, Neisseria spp, and Moraxella spp. Multiple regression analysis identified age, craniofacial ratio, and gingival index as factors influencing the presence of oral bacteria in the conjunctival flora. CLINICAL RELEVANCE: Oral resident bacteria have often been isolated from severe infectious corneal ulcers. This study provided evidence that brachycephalic dogs may require dental prophylaxis to reduce their oral bacterial load and that the association of oral bacteria in ocular diseases should be considered.

Causal role of single nucleotide polymorphisms within the mprF gene of Staphylococcus aureus in daptomycin resistance.

Single nucleotide polymorphisms (SNPs) within the mprF open reading frame (ORF) have been commonly observed in daptomycin-resistant (DAP(r)) Staphylococcus aureus strains. Such SNPs are usually associated with a gain-in-function phenotype, in terms of either increased synthesis or enhanced translocation (flipping) of lysyl-phosphatidylglycerol (L-PG). However, it is unclear if such mprF SNPs are causal in DAP(r) strains or are merely a biomarker for this phenotype. In this study, we used an isogenic set of S. aureus strains: (i) Newman, (ii) its isogenic ΔmprF mutant, and (iii) several in trans plasmid complementation constructs, expressing either a wild-type or point-mutated form of the mprF ORF cloned from two isogenic DAP-susceptible (DAP(s))-DAP(r) strain pairs (616-701 and MRSA11/11-REF2145). Complementation of the ΔmprF strain with singly point-mutated mprF genes (mprFS295L or mprFT345A) revealed that (i) individual and distinct point mutations within the mprF ORF can recapitulate phenotypes observed in donor strains (i.e., changes in DAP MICs, positive surface charge, and cell membrane phospholipid profiles) and (ii) these gain-in-function SNPs (i.e., enhanced L-PG synthesis) likely promote reduced DAP binding to S. aureus by a charge repulsion mechanism. Thus, for these two DAP(r) strains, the defined mprF SNPs appear to be causally related to this phenotype.

Role of the LytSR two-component regulatory system in adaptation to cationic antimicrobial peptides in Staphylococcus aureus.

Many host defense cationic antimicrobial peptides (HDPs) perturb the staphylococcal cell membrane (CM) and alter transmembrane potential (ΔΨ) as key parts of their lethal mechanism. Thus, a sense-response system for detecting and mediating adaptive responses to such stresses could impact organism survival; the Staphylococcus aureus LytSR two-component regulatory system (TCRS) may serve as such a ΔΨ sensor. One well-known target of this system is the lrgAB operon, which, along with the related cidABC operon, has been shown to be a regulator in the control of programmed cell death and lysis. We used an isogenic set of S. aureus strains: (i) UAMS-1, (ii) its isogenic ΔlytS and ΔlrgAB mutants, and (iii) plasmid-complemented ΔlytSR and ΔlrgAB mutants. The ΔlytS strain displayed significantly increased in vitro susceptibilities to all HDPs tested (neutrophil-derived human neutrophil peptide 1 [hNP-1], platelet-derived thrombin-induced platelet microbicidal proteins [tPMPs], and the tPMP-mimetic peptide RP-1), as well as to calcium-daptomycin (DAP), a cationic antimicrobial peptide (CAP). In contrast, the ΔlrgAB strain exhibited no significant changes in susceptibilities to these cationic peptides, indicating that although lytSR positively regulates transcription of lrgAB, increased HDP/CAP susceptibilities in the ΔlytS mutant were lrgAB independent. Further, parental UAMS-1 (but not the ΔlytS mutant) became more resistant to hNP-1 and DAP following pretreatment with carbonyl cyanide m-chlorophenylhydrazone (CCCP) (a CM-depolarizing agent). Of note, lytSR-dependent survival against CAP/HDP killing was not associated with changes in either surface positive charge, expression of mprF and dlt, or CM fluidity. The ΔlytS strain (but not the ΔlrgAB mutant) displayed a significant reduction in target tissue survival in an endocarditis model during DAP treatment. Collectively, these results suggest that the lytSR TCRS plays an important role in adaptive responses of S. aureus to CM-perturbing HDPs/CAPs, likely by functioning as a sense-response system for detecting subtle changes in ΔΨ.

Alleviation of weight-gain in mice by an ethanolic extract from Rubus coreanus under conditions of a high-fat diet and exercise.

The administration of an ethanolic extract (RCE) from Rubus coreanus significantly reduced the body weight and epididymal fat tissue of mice under conditions of a high-fat diet (HFD) and exercise. The mice also displayed enhanced muscular carnitine palmitoyltransferase 1 (CPT1) expression and increased superoxide dismutase and glutathione levels. These results suggest that RCE exerted an anti-obesity effect by up-regulating CPT1 and elevating the level of antioxidants.

Occurrence of cfr-Positive Linezolid-Susceptible Staphylococcus aureus and Non-aureus Staphylococcal Isolates from Pig Farms.

The emergence and spread of cfr-mediated resistance to linezolid in staphylococci have become a serious global concern. The acquisition of cfr confers multidrug resistance to phenicols, lincosamides, oxazolidinones, pleuromutilins, and streptogramin A (PhLOPSA phenotype). However, occurrence of cfr-positive and linezolid-susceptible staphylococci has been identified. To investigate the mechanism underlying linezolid susceptibility in cfr-positive Staphylococcus aureus and non-aureus staphylococci (NAS) isolates from pig farms in Korea. Eleven cfr-positive and linezolid-susceptible staphylococci were analyzed for mutations in domain V of 23S rRNA, ribosomal proteins (L3, L4, and L22), cfr open reading frames (ORFs), and cfr promoter regions. The effect of the cfr mutation (Q148K) on the PhLOPSA phenotype was determined using plasmid constructs expressing either the mutated (cfrQ148K) or nonmutated cfr genes. All 11 (six S. aureus and five NAS) cfr-positive and linezolid-susceptible isolates had a point mutation at position 442 in cfr ORFs (C to A) that resulted in the Q148K mutation. No mutations were detected in 23S rRNA, L3, L4, or L22. The Q148K mutation in Cfr is responsible for phenotypes susceptible to PhLOPSA antimicrobial agents. To our knowledge, this is the first study to report the causal role of a single nucleotide mutation (Q148K) in cfr of S. aureus and NAS isolates in PhLOPSA resistance. Continued nationwide surveillance is necessary to monitor the occurrence and dissemination of mutations in cfr that affect resistance phenotypes in staphylococci of human and animal origin.

Antimicrobial resistance profiles and clonal diversity of Staphylococcus epidermidis isolates from pig farms, slaughterhouses, and retail pork.

Livestock-associated coagulase-negative staphylococci (CoNS), such as Staphylococcus (S.) epidermidis, have emerged as a significant reservoir of antimicrobial resistance (AMR). In the current study, the AMR profiles and genetic diversity of S. epidermidis isolates obtained from pig farms, slaughterhouses, and retail pork were analyzed. A total of 89 S. epidermidis isolates, comprising 22 methicillin-resistant (MRSE) and 67 methicillin-susceptible S. epidermidis (MSSE) isolates, were assessed to determine (i) the clonal lineages of the isolates [multilocus sequence (MLST), agr, and staphylococcal cassette chromosome mec (SCCmec) types], (ii) the profiles of AMR phenotypes, and (iii) the carriage of genetic factors associated with major AMR phenotypes and zinc chloride resistance. Two dominant clonal lineages of S. epidermidis, ST100 and ST570, were observed on pig farms, especially in healthy pigs. In addition, potential transmission of pig-associated ST100 MRSE-SCCmec V and ST100 MSSE to farm workers was identified. The high prevalence of ST100 and ST570 isolates in pig farms was associated with high levels of AMR and zinc chloride resistance. In relation to resistance phenotypes, higher carriage rates of resistance genes, such as ß-lactams (mecA), phenicols (fexA), and tetracyclines [tet(K), tet(L), tet(S), tet(M), and tet(O)], were identified in pig farm-associated isolates. Furthermore, cfr-mediated linezolid resistance was detected in 14 MSSE isolates from pig farms. This study is the first to provide important insights into the clonal structures and AMR profiles of S. epidermidis isolates collected from healthy pigs, carcass/pork samples, and human workers in Korea.