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.

Detection and characterization of potential virulence determinants in Staphylococcus pseudintermedius and S. schleiferi strains isolated from canine otitis externa in Korea.

BACKGROUND: A recent increase in the occurrence of canine skin and soft tissue infections, including otitis externa and pyoderma, caused by antimicrobial-resistant Staphylococcus pseudintermedius and S. schleiferi has become a significant public and veterinary health issues. OBJECTIVE: We investigated the virulence potentials associated with the occurrence of canine otitis externa in S. pseudintermedius and S. schleiferi. METHODS: In this study, the prevalence of genes encoding leukocidins, exfoliative toxins, and staphylococcal enterotoxins (SEs) was investigated using previously characterized S. pseudintermedius (n = 26) and S. schleiferi (n = 19) isolates derived from canine otitis externa. Susceptibility to cathelicidins (K9CATH and PMAP-36) and hydrogen peroxide (H2O2) was also examined in both staphylococcal species. RESULTS: A high prevalence of genes encoding leukocidins (lukS/F-I, lukS1/F1-S, and lukS2/F2-S), exfoliative toxins (siet, expB, and sset), and SEs was identified in both S. pseudintermedius and S. schleiferi isolates. Notably, S. pseudintermedius isolates possessed higher number of SE genes, especially newer SE genes, than S. schleiferi isolates harboring egc clusters. Although no significant differences in susceptibility to K9CATH and H2O2 were observed between the two isolate groups, S. pseudintermedius isolates exhibited enhanced resistance to PMAP-36 compared to S. schleiferi isolates. CONCLUSIONS: These findings suggest that high a prevalence of various toxin genes together with enhanced resistance to cathelicidins may contribute to the pathogenicity of S. pseudintermedius and S. schleiferi in canine cutaneous infections.

Species Profiles and Antimicrobial Resistance of Non-aureus Staphylococci Isolated from Healthy Broilers, Farm Environments, and Farm Workers.

Non-aureus staphylococci (NAS), particularly antimicrobial-resistant NAS, have a substantial impact on human and animal health. In the current study, we investigated (1) the species profiles of NAS isolates collected from healthy broilers, farm environments, and farm workers in Korea, (2) the occurrence of antimicrobial-resistant NAS isolates, especially methicillin resistance, and (3) the genetic factors involved in the methicillin and fluoroquinolone resistance. In total, 216 NAS isolates of 16 different species were collected from healthy broilers (n=178), broiler farm environments (n=18), and farm workers (n=20) of 20 different broiler farms. The two most dominant broiler-associated NAS species were Staphylococcus agnetis (23.6%) and Staphylococcus xylosus (22.9%). Six NAS isolates were mecA-positive carrying staphylococcal cassette chromosome mec (SCCmec) II (n=1), SCCmec IV (n=1), SCCmec V (n=2), or non-typeable SCCmec element (n=2). While two mecA-positive Staphylococcus epidermidis isolates from farm workers had SCCmec II and IV, a mecA-positive S. epidermidis isolate from broiler and a Staphylococcus haemolyticus isolate farm environment carried SCCmec V. The occurrence of multidrug resistance was observed in 48.1% (104/216 isolates) of NAS isolates with high resistance rates to ß-lactams (>40%) and fusidic acid (59.7%). Fluoroquinolone resistance was confirmed in 59 NAS isolates (27.3%), and diverse mutations in the quinolone resistance determining regions of gyrA, gyrB, parC, and parE were identified. These findings suggest that NAS in broiler farms may have a potential role in the acquisition, amplification, and transmission of antimicrobial resistance.

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.

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.

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.

Clonal distribution and antimicrobial resistance of methicillin-susceptible and -resistant Staphylococcus aureus strains isolated from broiler farms, slaughterhouses, and retail chicken meat.

Colonization of food-producing animals by antimicrobial-resistant Staphylococcus aureus, especially methicillin-resistant S. aureus (MRSA), has become a serious public health problem worldwide. In the current study, clonal diversities of livestock-associated S. aureus isolates collected from broiler farms, slaughterhouses, and retail chicken meat were examined. Two-hundred S. aureus isolates (43 MRSA and 157 methicillin-susceptible S. aureus [MSSA] isolates) were analyzed to determine 1) the genotypes of the isolates (multilocus sequence, agr, and spa types), 2) the methicillin resistance phenotype and staphylococcal cassette chromosome mec (SCCmec) types, 3) the antimicrobial resistance profiles, and 4) the mutational changes in gyrA, gyrB, parC, and parE in fluoroquinolone-resistant isolates. Fifteen different sequence types (STs) of MSSA strains displaying a relatively high degree of genetic diversity were detected in broiler farms, slaughterhouses, and retail chicken meat. In contrast to MSSA, 2 dominant genetic lineages of MRSA (ST692-SCCmecV with t2249 spa type, and ST188-SCCmecIVa with spa type t189) were found in healthy broilers. The high prevalence of ST692 and ST188 in healthy broilers is associated with high levels of multiple antimicrobial-resistance phenotypes, particularly fluoroquinolone resistance. All fluoroquinolone-resistant isolates carried double point mutations in gyrA (S84L) and parC (S80F), regardless of STs or methicillin resistance. Notably, only the ST188 lineage carried an additional third mutation in gyrB (D494N), correlating with enhanced ciprofloxacin minimum inhibitory concentration values versus the strains with double mutations. These results provide important insights into the genetic diversity of antimicrobial-resistant S. aureus strains associated with the chicken meat production chain, including healthy broilers, in Korea.

Multilocus sequence type-dependent activity of human and animal cathelicidins against community-, hospital-, and livestock-associated methicillin-resistant Staphylococcus aureus isolates.

Sequence type (ST) 5 methicillin-resistant Staphylococcus aureus (MRSA) with staphylococcal cassette chromosome mec (SCCmec) type II (ST5-MRSA-II) and ST72-MRSA-IV represent the most significant genotypes for healthcare- (HA) and community-associated (CA) MRSA in Korea, respectively. In addition to the human-type MRSA strains, the prevalence of livestock-associated (LA) MRSA clonal lineages, such as ST541 and ST398 LA-MRSA-V in pigs and ST692 LA-MRSA-V and ST188 LA-MRSA-IV in chickens, has recently been found. In this study, clonotype-specific resistance profiles to cathelicidins derived from humans (LL-37), pigs (PMAP-36), and chickens (CATH-2) were examined using six different ST groups of MRSA strains: ST5 HA-MRSA-II, ST72 CA-MRSA-IV, ST398 LA-MRSA-V, ST541 LA-MRSA-V, ST188 LA-MRSA-IV, and ST692 LA-MRSA-V. Phenotypic characteristics often involved in cathelicidin resistance, such as net surface positive charge, carotenoid production, and hydrogen peroxide susceptibility were also determined in the MRSA strains. Human- and animal-type MRSA strains exhibited clonotype-specific resistance profiles to LL-37, PMAP-36, or CATH-2, indicating the potential role of cathelicidin resistance in the adaptation and colonization of human and animal hosts. The ST5 HA-MRSA isolates showed enhanced resistance to all three cathelicidins and hydrogen peroxide than ST72 CA-MRSA isolates by implementing increased surface positive charge and carotenoid production. In contrast, LA-MRSA strains employed mechanisms independent of surface charge regulation and carotenoid production for cathelicidin resistance. These results suggest that human- and livestock-derived MRSA strains use different strategies to counteract the bactericidal action of cathelicidins during the colonization of their respective host species.

Profiles of Non-aureus Staphylococci in Retail Pork and Slaughterhouse Carcasses: Prevalence, Antimicrobial Resistance, and Genetic Determinant of Fusidic Acid Resistance.

As commensal colonizers in livestock, there has been little attention on staphylococci, especially non-aureus staphylococci (NAS), contaminating meat production chain. To assess prevalence of staphylococci in retail pork and slaughterhouse carcass samples in Korea, we collected 578 samples from Korean slaughterhouses (n=311) and retail markets (n=267) for isolation of staphylococci and determined antimicrobial resistance phenotypes in all the isolates. The presence of and prevalence of fusB-family genes (fusB, fusC, fusD, and fusF) and mutations in fusA genes were examined in fusidic acid resistant isolates. A total of 47 staphylococcal isolates of 4 different species (Staphylococcus aureus, n=4; S. hyicus, n=1; S. epidermidis, n=10; Mammaliicoccus sciuri, n=32) were isolated. Fusidic acid resistance were confirmed in 9/10 S. epidermidis and all of the 32 M. sciuri (previously S. sciuri) isolates. Acquired fusidic acid resistance genes were detected in all the resistant strains; fusB and fusC in S. epidermidis and fusB/C in M. sciuri. Multi-locus sequence type analysis revealed that ST63 (n=10, 31%) and ST30 (n=8, 25%) genotypes were most prevalent among fusidic acid resistant M. sciuri isolates. In conclusion, the high prevalence of fusB-family genes in S. epidermidis and M. sciuri strains isolated from pork indicated that NAS might act as a reservoir for fusidic acid resistance gene transmissions in pork production chains.

Co-occurrence of cfr-mediated linezolid-resistance in ST398 LA-MRSA and non-aureus staphylococci isolated from a pig farm.

Linezolid resistance, mediated by the cfr gene, which confers resistant phenotypes to phenicols, lincosamides, oxazolidinones, pleuromutilins, and streptogramin A antimicrobials, has emerged in S. aureus and non-aureus staphylococci (NAS). Moreover, due to the transferable potential via plasmids, the spread of cfr among staphylococci is of great concern. In the present study, we investigated the prevalence of cfr-mediated linezolid resistance in ST398 methicillin-resistant S. aureus (MRSA) and NAS strains isolated from a pig farm. Among the 26 staphylococci isolates collected from a pig farm, 14 cfr-harboring ST398 MRSA and NAS (S. epidermidis, S. pasteuri, S. cohnii, and S. rostri) strains were resistant to linezolid and also carried the fexA gene. Comparative genome analysis of cfr-carrying linezolid-resistant ST398 MRSA and NAS (S. pasteuri, S. cohnii, and S. epidermidis) strains revealed that the segments harboring cfr in different staphylococcal strains showed ≥ 99 % sequence identity and the corresponding region containing the cfr, fexA, and Tn558 elements were located in a 38-kb plasmid, designated pSA12 of ST398 MRSA. These observations indicate that the cfr-carrying plasmids and/or fragments may be disseminated among staphylococci in a pig farm and possibly transmitted to staphylococci of human origin, subsequently posing a threat to public health. This is the first report of the co-existence of cfr in linezolid-resistant ST398 MRSA and NAS isolated from a pig farm in South 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.

Profiles of coagulase-positive and -negative staphylococci in retail pork: prevalence, antimicrobial resistance, enterotoxigenicity, and virulence factors.

OBJECTIVE: The present study aimed to investigate the occurrence and species of coagulasepositive staphylococci (CoPS) and coagulase-negative staphylococci (CoNS) in retail pork meat samples collected during nationwide monitoring. The staphylococcal isolates were characterized for antimicrobial and zinc chloride resistance and enterotoxigenic potential. METHODS: A total of 260 pre-packaged pork meat samples were collected from 35 retail markets in 8 provinces in Korea for isolation of staphylococci. Antimicrobial and zinc chloride resistance phenotypes, and genes associated with the resistance phenotypes were determined on the isolates. Furthermore, the presence and distribution of 19 staphylococcal enterotoxin (SE) genes and enterotoxin-like genes among the pork-associated staphylococci were determined by multiplex polymerase chain reaction-based assays using the specific primer sets. RESULTS: A total of 29 staphylococcal strains (29/260, 11.1%) were isolated from samples of retail pork meat, 24 (83%) of which were CoNS. The four CoNS species identified were S. saprophyticus (n = 16, 55%), S. sciuri (n = 3, 10%), S. warneri (n = 3, 10%), and S. epidermidis (n = 2, 7%). Among the 29 isolates, four methicillin-resistant CoNS (MR-CoNS; three S. sciuri and one S. epidermidis) and one methicillin-resistant CoPS (MR-CoPS; one S. aureus) were identified. In addition, a relatively high level of tetracycline (TET) resistance (52%) was confirmed in CoNS, along with a predominant distribution of tet(K). The most prevalent SEs were sep (45%), and sen (28%), which were carried by 81% of S. saprophyticus. CONCLUSION: These findings suggest that CoNS, especially S. saprophyticus strains, in raw pork meat could be a potential risk factor for staphylococcal food poisoning (SFP), and therefore, requires further investigation to elucidate the role of SEls in SFP and virulence of the pathogen. Our results also suggest that CoNS from raw pork meat may act as a source for transmission of antimicrobial resistance genes such as staphylococcal cassette chromosome mec and tet(K).

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.

Genetic Factors Associated with Increased Host Defense Antimicrobial Peptide Resistance in Sequence Type 5 Healthcare-Associated MRSA Clinical Isolates.

Sequence type (ST) 72 methicillin-resistant Staphylococcus aureus with staphylococcal cassette chromosome mec (SCCmec) type IV (ST72-MRSA-IV) and ST5-MRSA-II are the most significant lineages found in community-associated (CA) and healthcare-associated (HA) environments in Korea, respectively. ST5 HA-MRSA-II tend to display enhanced resistance to host defense-cationic antimicrobial peptides (HD-CAPs) compared to ST72 CA-MRSA-IV and ST72 livestock-associated (LA)-MRSA-IV due to mechanisms involving a higher surface positive charge. Thus, the present study explored the genetic factors contributing to the enhanced HD-CAP resistance phenotype in ST5 MRSA strains. The ST5 HA-MRSA-II strains displayed higher levels of mprF and dltABCD expression compared to the ST72 CA-/LA-MRSA-IV strains. The increase in expression of mprF and dltABCD in ST5 HA-MRSA-II strains was correlated with dysregulation of the upstream transcriptional regulator, graRS. However, single nucleotide polymorphisms (SNPs) within mprF and graRS ORFs were not involved in the enhanced surface positive charge or the altered expression of mprF/dltABCD.

Chryseobacterium vaccae sp. nov., isolated from raw cow's milk.

Strain CA7T, a Gram-stain-negative, non-motile, non-spore-forming, aerobic and rod-shaped bacterial strain, was isolated from raw cow's milk collected from a farm affiliated with Chung-Ang University, Anseong, Korea, and characterized by a polyphasic approach. Optimal growth of strain CA7T was observed on tryptic soy agar at 30 °C and pH 7.0 with 0 % NaCl. Phylogenetic analysis based on the 16S rRNA gene sequence revealed that strain CA7T belonged to the genus Chryseobacterium. The most closely related strains (16S rRNA gene sequence similarity indicated in parentheses), based on the phylogenetic analysis, were Chryseobacterium rhizosphaerae KCTC 22548T (98.08 %), Chryseobacterium nakagawai CCUG 60563T (98.61 %), Chryseobacterium jejuense KACC 12501T (97.85 %) and Chryseobacterium aurantiacum KCTC 62135T (97.78 %). Whole genome sequencing indicated that the genome size was 5 125 723 bp and had a DNA G+C content of 37.4 mol%. Average nucleotide identity values for strain CA7T with C. rhizosphaerae, C. nakagawai, C. jejuense, C. aurantiacum, and the type species of the genus Chryseobacterium, C. gleum, were 80.2, 79.8, 79.8, 79.6 and 80.4 %, respectively. The digital DNA-DNA hybridization values of CA7T compared to C. rhizosphaerae, C. nakagawai, C. jejuense, C. aurantiacum and C. gleum were 24.1, 23.9, 23.9, 23.7 and 24.3 %, respectively. The major fatty acids were iso-C15 : 0, summed feature 9 (iso-C17 : 1 ω9c and/or C16 : 0 10-methyl), iso-C17 : 0 3-OH and summed feature 3 (iso-C15 : 0 2-OH and/or C16 : 1 ω7c). Menaquinone-6 was the only respiratory quinone. The major polar lipid was phosphatidylethanolamine. Based on this polyphasic taxonomic study, strain CA7T represents a novel species of the genus Chryseobacterium for which the name Chryseobacterium vaccae sp. nov. is proposed. The type strain is CA7T (=KACC 21402T=JCM 33749T).

Occurrence and Characteristics of Methicillin-Resistant and -Susceptible Staphylococcus aureus Isolated from the Beef Production Chain in Korea.

The emergence and persistence of methicillin-susceptible Staphylococcus aureus (MSSA) and methicillin-resistant S. aureus (MRSA) in livestock animals have been reported as a potential risk factor for transmission to humans. In this study, we investigated the nationwide prevalence and characteristics of MRSA and MSSA in the Korean beef production system, including retail markets, slaughterhouses, and cattle farms. From a total of 1,285 samples, only 5 MRSA strains were isolated: from a farmer (1 ST72 MRSA), a carcass sample from a slaughterhouse (1 ST72 MRSA), and beef cattle (3 ST5 MRSA). In addition, 11 MSSA strains were isolated from beef cattle (n=3), humans (1 farmer, 1 slaughterhouse worker, and 4 retail market workers), and carcass samples (n=1) and slaughterhouse environment (n=1). Although the prevalence of MRSA and MSSA in beef cattle was much lower than that reported in pigs, 5/5 MRSA and 2/11 MSSA strains displayed multiple drug resistance (MDR) phenotypes. Unlike the swine-associated MRSA, no correlation was found between tetracycline/zinc resistance and MDR phenotype. However, MRSA strains had an identical set of staphylococcal enterotoxins and exhibited enhanced levels of resistance to antimicrobial peptides (PMAP-36 and LL-37) compared to the MSSA strains. In conclusion, continued and systemic surveillance of livestock, meat products, and humans in close contact with livestock/meat products is necessary to prevent the transmission of MRSA and MSSA to humans.

Acinetobacter pullorum sp. nov., Isolated from Chicken Meat.

A bacterial strain, designated B301T and isolated from raw chicken meat obtained from a local market in Korea, was characterized and identified using a polyphasic taxonomic approach. Cells were gram-negative, non-motile, obligate-aerobic coccobacilli that were catalase-positive and oxidase-negative. The optimum growth conditions were 30°C, pH 7.0, and 0% NaCl in tryptic soy broth. Colonies were round, convex, smooth, and cream-colored on tryptic soy agar. Strain B301T has a genome size of 3,102,684 bp, with 2,840 protein-coding genes and 102 RNA genes. The 16S rRNA gene analysis revealed that strain B301T belongs to the genus Acinetobacter and shares highest sequence similarity (97.12%) with A. celticus ANC 4603T and A. sichuanensis WCHAc060041T. The average nucleotide identity and digital DNA-DNA hybridization values for closely related species were below the cutoff values for species delineation (95-96% and 70%, respectively). The DNA G+C content of strain B301T was 37.0%. The major respiratory quinone was Q-9, and the cellular fatty acids were primarily summed feature 3 (C16:1 ω6c/C16:1 ω7c), C16:0, and C18:1 ω9c. The major polar lipids were phosphatidylethanolamine, diphosphatidyl-glycerol, phosphatidylglycerol, and phosphatidyl-serine. The antimicrobial resistance profile of strain B301T revealed the absence of antibiotic-resistance genes. Susceptibility to a wide range of antimicrobials, including imipenem, minocycline, ampicillin, and tetracycline, was also observed. The results of the phenotypic, chemotaxonomic, and phylogenetic analyses indicate that strain B301T represents a novel species of the genus Acinetobacter, for which the name Acinetobacter pullorum sp. nov. is proposed. The type strain is B301T (=KACC 21653T = JCM 33942T).

Livestock-associated methicillin-resistant Staphylococcus aureus in Korea: antimicrobial resistance and molecular characteristics of LA-MRSA strains isolated from pigs, pig farmers, and farm environment.

The emergence of livestock-associated (LA)-methicillin-resistant Staphylococcus aureus (MRSA) in livestock animal has become a significant zoonotic concern. In the present study, we investigated nationwide prevalence of LA-MRSA across pork production chain including pig farms, slaughterhouses, and retail markets. A total of 40 MRSA strains were isolated during the investigation and the overall prevalence of MRSA was 3.4% (n = 37), 0.6% (n = 2), and 0.4% (n = 1) in pig farms, slaughterhouses, and retail markets, respectively. Multilocus sequence typing analyses revealed that the 2 most significant clonal lineages in pork production chain in Korea were ST398 (n = 25) and ST541 (n = 6). All of the 40 MRSA isolates were further characterized to investigate key genotypic and phenotypic correlates associated with the emergence and spread of clonal complex 398 (CC398; ST398, and ST541) LA-MRSA. Although the prevalence of swine-associated MRSA was still relatively low and mostly restricted to pig farms, multidrug-resistant CC398 LA-MRSA isolates with new spa types (t18102 and t18103) were identified as a major clonal lineage. The CC398 LA-MRSA strains tended to exhibit increased levels of multiple drug resistance (MDR) phenotype compared with non-CC398 MRSA strains. Of note, in comparison with non-CC398 MRSA isolates, CC398 LA-MRSA isolates exhibited significantly enhanced tetracycline (TET) and zinc resistance. These findings suggested that co-selection pressure associated with MDR phenotype, especially TET resistance, and zinc resistance may have played a significant role in the emergence and persistence of CC398 LA-MRSA in pig farms in Korea.

Complete genome sequence of a methicillin-resistant Staphylococcus schleiferi strain from canine otitis externa in Korea.

The increase in canine skin and soft tissue infections, such as pyoderma and otitis, caused by Staphylococcus schleiferi strains, is of significant zoonotic concern. In this study, we report the first complete genome sequence for a methicillin-resistant clinical isolate of S. schleiferi (MRSS) designated as SS4, obtained from a dog with otitis externa, in Korea. The genome of SS4 strain was of 2,539,409 bp and presented high G+C content ratio (35.90%) with no plasmid. Comparative analysis of SS4 genome revealed that it is closely related to 2142-05 and 5909-02 strains isolated from the canine skin infections in the USA.

Comparative assessment of genotypic and phenotypic correlates of Staphylococcus pseudintermedius strains isolated from dogs with otitis externa and healthy dogs.

Staphylococcus pseudintermedius is considered a primary pathogen of canine skin and soft tissue infections, and the rapid emergence of methicillin-resistant S. pseudintermedius worldwide is a major issue. In the current study, genotypic and phenotypic correlates associated with S. pseudintermedius causing canine otitis externa were evaluated using 41 S. pseudintermedius strains isolated from dogs with otitis externa (n = 26) and healthy dogs (n = 15). The S. pseudintermedius strains were subjected to a comparative analysis of (i) genotypes (multilocus sequence typing, agr, and spa types), (ii) methicillin resistance and SCCmec types, (iii) multidrug resistance (MDR), (iv) biofilm formation, and (v) susceptibility to canine cathelicidin (K9CATH). A high degree of genetic diversity was observed in both groups of S. pseudintermedius strains, regardless of methicillin resistance. Almost all methicillin-resistant strains (>95%) harbored SCCmec V and displayed MDR. Although there was no difference in biofilm formation, S. pseudintermedius strains derived from otitis externa exhibited enhanced resistance to cationic antimicrobial peptide (K9CATH) compared with strains from healthy dogs. The high degree of heterogeneity in MLST, agr, and spa types prevented the identification of correlations between any specific genotype and virulence phenotype in otitis externa caused by S. pseudintermedius, These findings provide an important basis for monitoring and treating canine skin and soft tissue infections in Korea.