ObjectiveTo explore the microbiological characteristics of Staphylococcus epidermidis with hemolytic phenotype （SEHP）. MethodsHemolytic phenotype was detected using the three-point inoculation method， involving a total of 5 strains of SEHP and 5 strains of Staphylococcus epidermidis with non-hemolytic phenotype （SENHP） . Bacterial species were identified using the Microflex LT MALDI-TOF mass spectrometer， and a phylogenetic tree was constructed through 16S rRNA sequence alignment. Growth curves were monitored through the microcultivation assay. Biofilm formation ability was assessed by microplate crystal violet staining. Red blood cell toxicity was detected using the microplate method. Antimicrobial susceptibility testing of SEHP and SENHP against commonly used antibiotics was performed using a VITEK 2 GP639 test kit. Antagonistic effects of SEHP and SENHP against Staphylococcus aureus and Corynebacterium striatum were evaluated by the Oxford cup inhibition assay. ResultsCompared with SENHP， SEHP exhibited a marked decrease in growth rate during the late logarithmic phase， accompanied by significant hemolytic toxicity. Additionally， it showed lower resistance rates to levofloxacin and moxifloxacin， and could antagonize Staphylococcus aureus and Corynebacterium striatum. ConclusionThe microbiological characteristics of SEHP differ from those of SENHP in that SEHP demonstrates antagonistic effects against S. aureus and C. striatum.

Objective: To explore identification and resistance characteristics of CAMP-negativeStreptococcus agalactiae. Methods : Using matrix-assisted laser desorption ionization time-of-flight mass spectrometry(MALDI-TOF MS) and the CAMP assay, 33 presumptive strains ofStreptococcus agalactiaewere identified. The CAMP-negative strains were further validated through 16S rDNA, while the CAMP factor encoding gene(cfb) was detected using real-time fluorescence quantitative polymerase chain reaction(qPCR). Antimicrobial susceptibility testing was conducted using the microbroth dilution method, and the resistance rates of CAMP-negative and CAMP-positive strains were compared. Results : Based on MALDI-TOF MS identification, all 33 strains were classified asStreptococcus agalactiae. Among them, 7 strains tested negative for CAMP were subsequently confirmed asStreptococcus agalactiaethrough 16S rDNA. The qPCR results indicated that, only 1 strain showedcfbpresence. The CAMP-negative and CAMP-positive strains were sensitive to penicillin G, cefepime, cefotaxime, vancomycin, and linezolid. The resistance rates of the former to chloramphenicol and tetracycline(28.57%, 85.71%) were slightly higher than the latter(15.38%, 57.69%), while the resistance rates to moxifloxacin, levofloxacin, and erythromycin(14.29%, 14.29%, 42.86%) were slightly lower than the latter(34.62%, 34.62%, 57.69%), but was not significant. Conclusion Drug risistance of CAMP-negativeStreptococcus agalactiaeis the same as CAMP-positive strains, but traditional CAMP assay andcfb-targeted qPCR can result in missed detections. MALDI-TOF MS offers a quick, simple, and accurate identification method that merits wider adoption.

Objective : To investigate the antagonistic activity of Lactococcus garvieae SHAMU-LG6 against Staphy- lococcus . Methods : VITEK 2 GP identification card , Microflex LT MALDI-TOF mass spectrometer and 16S rDNA amplification sequencing were used to identify the strain species . The antagonistic activity of L. garvieae SHAMU- LG6 against different Staphylococcus was detected by Oxford cup method for bacterial inhibition ; the antimicrobial active components were preliminarily isolated and purified by adsorption on XAD16 nonionic macroporous resin , gradient ethanol elution and rotary evaporation drying. Results : L. garvieae SHAMU-LG6 exhibited potent antago- nistic effect against methicillin-resistant Staphylococcus aureus , methicillin-susceptible S. aureus , S. epidermidis , S. saprophyticus , S. lugdunensis , S. hominis , S. capitis and S. warneri , with inhibitory indices of 3 . 3 , 3 . 0 , 4. 3 , 2. 0 , 4. 0 , 3 . 5 , 3 . 8 , and 3 . 5 , respectively. The antimicrobial active components produced by L. garvieae SHAMU-LG6 were mainly present in 70% and 80% ethanol eluates . Conclusion L. garvieae SHAMU-LG6 ex- hibits a potent antagonistic effect on Staphylococcus , and the antimicrobial active components produced by it are ex- pected to be a lead compound for the development of novel antimicrobial agents .

Objective : To explore the molecular characteristics of four CAMP negativeStreptococcus agalactiae(S.agalactiae) in whole genome sequencing. Methods : The identification of suspicious bacterial strains was conducted using matrix-assisted laser desorption ionization time-of-flight mass spectrometry(MALDI-TOF MS). For the strains confirmed asS.agalactiaethrough identification, further CAMP experiments were conducted. For CAMP negative strains, whole genome sequencing was performed using MGI DNBSEQ-T7 and MinION Flow Cell sequencing platforms. Subsequently, multi-locus sequence typing(MLST), virulence genes and resistance genes of the strains were compared and analyzed using various databases. Phoenix M50 fully automatic drug sensitivity analyzer was employed to determine the sensitivity of the bacterial strains to commonly used antibiotics. Results: Four CAMP-negativeS.agalactiaestrains were included. Whole-genome sequencing analysis revealed that all four CAMP-negativeS.agalactiaestrains belonged to the ST862 type. These strains harbored 22 virulence genes associated with capsular polysaccharides, β-hemolysin, and hyaluronidase, as well as seven resistance genes linked to macrolides, lincosamides, polypeptides, and aminoglycosides. Antimicrobial susceptibility testing revealed that CAMP-negativeS.agalactiaewas susceptible to penicillin G, cefepime, cefotaxime, and vancomycin. However, three strains exhibited resistance to erythromycin, and one strain demonstrated resistance to clindamycin. Conclusion Four CAMP negativeS.agalactiaeof the ST862 type possess multiple virulence and drug resistance genes, showing high resistance to erythromycin, warranting clinical attention.

Objective : To investigate the causes of false negative results in the detection of Streptococcus agalactiae using fluorescent quantitative PCR(qPCR) targeting the CAMP factor gene(cfb),and to perform a comprehensive analysis of the associated molecular mechanisms. Methods: A total of 76 vaginal secretion samples were evaluated using both qPCR based on cfb gene and bacterial culture methods. Four suspicious strains exhibiting negative qPCR results but positive culture findings were identified using matrix-assisted laser desorption ionization time-of-flight mass spectrometry(MALDI-TOF MS),latex agglutination antigen detection,and the CAMP test. Whole genome analysis was conducted utilizing the MGI DNBSEQ-T7 and Nanopore-PromethION 48 sequencing platforms. Phylogenetic and circular evolutionary trees were constructed using the 16S rRNA gene for strain verification. Multilocus sequence typing(MLST) was conducted,and cfb sequences were aligned and analyzed based on spliced sequences and original data. Specific primers targeting the cfb gene were designed for full-length amplification,followed by verification through agarose gel electrophoresis. Results: The four strains identified as suspicious were classified as S. agalactiae through MALDI-TOF MS,antigen detection,and 16S rRNA gene analysis,with MLST typing indicating ST-862. Phenotypic analysis revealed a negative CAMP test. Whole genome sequence alignment failed to detect the cfb gene or any homologous sequences,and molecular testing confirmed the absence of cfb gene PCR amplification products,thereby confirming its complete deletion. Conclusion This deletion is identified as the molecular mechanism responsible for the false negative qPCR detection of S. agalactiae when targeting this specific gene.It is recommended that the qPCR detection targeting a single cfb gene has limitation,and this may impact clinical diagnosis and treatment decisions. This limitation warrants carefulconsideration.

Objective: To investigate the hemolytic phenotypes of Staphylococcus aureus clinical isolates． Methods: The hemolytic phenotypes of 105 Staphylococcus aureus isolates were analyzed and summarized using the three-point inoculation method．Real-time fluorescence quantitative PCR was used to measure the mRNA expression levels of four hemolysin genes (hla，hlb，hlc，and hld) ; The VITEK 2 GP639 antimicrobial susceptibility card was used to detect resistance to commonly used antibiotics ; DNA gel electrophoresis was performed to determine the prevalence of the mecA，sea，tst，and pvl genes ; The microtiter plate crystal violet staining method was used to assess biofilm formation ability ; The CCK-8 assay was used to evaluate cytotoxicity against macrophages． Results: Seven hemo- lytic phenotypes were identified among the Staphylococcus aureus clinical isolates． Differences were found among Staphylococcus aureus clinical isolates with different hemolytic phenotypes in terms of mRNA expression levels of he- molysin genes，antibiotic resistance，virulence gene prevalence，biofilm formation ability，and cytotoxicity to mouse macrophages (P ＜0. 05 ) ． Conclusion Staphylococcus aureus clinical isolates exhibit diverse hemolytic pheno- types，which should be a focus across multiple dimensions，including microbiological testing，clinical treatment， and nosocomial infection prevention and control．

Objective To explore the microbiological characteristics of Staphylococcus aureus(S.aureus)with no-vel incomplete hemolytic phenotype(SIHP).Methods Hemolytic phenotypes were detected and categorized by u-sing the three-point inoculation method.A total of 11 novel SIHP and 33 randomly matched S.aureus with com-plete hemolytic phenotype(SCHP)were included.Antibiotic susceptibility test was performed using broth microdi-lution method.Coagulase test was performed with freeze-dried rabbit plasma.Catalase activity was detected by slide catalase test.Expression of hemolysin genes was detected by qRT-PCR.Toxicity to human red blood cells was as-sessed by microplate method.Microplate biofilm formation was measured using crystal violet staining method.Growth kinetic determination was performed through microcultivation assay.Results Compared with SCHP,the expression profiles of the four hemolysin genes(hla,hlb,hlc,and hld)in the new SIHP were different.The new SIHP had higher resistance rates to penicillin,oxacillin,gentamicin,quinolones,clindamycin,and trimethoprim-sulfamethoxazole.Furthermore,the new SIHP had stronger hemolytic toxicity,plasma coagulase activity,and bio-film formation ability.Additionally,the new SIHP grown faster in the logarithmic phase.Conclusion Taken to-gether,the microbiological characteristics of the new SIHP are different from those of SCHP,including stronger an-tibiotic resistance and pathogenicity,which should be paid more attention by clinicians.