Objective: To understand the population structure of food-borne Staphylococcus (S.) aureus in China. Methods: Whole genome sequencing was used to analyze 763 food-borne S. aureus strains from 16 provinces in China from 2006 to 2020. Multilocus sequence typing (MLST), staphylococcal protein A gene (spa) typing, and staphylococcal chromosome cassettemec (SCCmec) typing were conducted, and minimum spanning tree based on ST types (STs) was constructed by BioNumerics 7.5 software. Thirty-one S. aureus strains isolated from imported food products were also included in constructing the genome phylogenetic tree. Results: A total of 90 STs (20 novel types) and 160 spa types were detected in the 763 S. aureus isolates. The 72 STs (72/90, 80.0%) were related to 22 clone complexes. The predominant clone complexes were CC7, CC1, CC5, CC398, CC188, CC59, CC6, CC88, CC15, and CC25, accounting for 82.44% (629/763) of the total. The STs and spa types in the predominant clone complexes changed over the years. The methicillin-resistant S. aureus (MRSA) detection rate was 7.60%, and 7 SCCmec types were identified. The ST59-t437-Ⅳa (17.24%, 10/58), ST239-t030-Ⅲ (12.07%, 7/58), ST59-t437-Ⅴb (8.62%, 5/58), ST338-t437-Ⅴb (6.90%, 4/58) and ST338-t441-Ⅴb (6.90%, 4/58) were the main types in MRSA strains. The genome phylogenetic tree had two clades, and the strains with the same CC, ST, and spa types clustered together. All CC7 methicillin sensitive S. aureus strains were included in Clade1, while 21 clone complexes and all MRSA strains were in Clade2. The MRSA strains clustered according to the SCCmec and STs. The strains from imported food products in CC398, CC7, CC30, CC12, and CC188 had far distances from Chinese strains in the tree. Conclusions: In this study, the predominant clone complexes of food-borne strains were CC7, CC1, CC5, CC398, CC188, CC59, CC6, CC88, CC15, and CC25, which overlapped with the previously reported clone complexes of hospital and community-associated strains in China, suggesting that close attention needs to be paid to food, a vehicle of pathogen transmission in community and food poisoning.
Humans ; Staphylococcus aureus/genetics* ; Methicillin-Resistant Staphylococcus aureus/genetics* ; Multilocus Sequence Typing ; Phylogeny ; Staphylococcal Infections/epidemiology* ; China/epidemiology*

Objective: To analyze the Staphylococcal enterotoxins, Staphylococcal enterotoxin genes, drug resistance and molecular typing of 41 Staphylococcus aureus isolates from 2 food-borne illness outbreaks on 21 August and 27 September 2020 in Guangzhou. Methods: A total of 41 Staphylococcus aureus isolates from 2 outbreaks were analyzed by multilocus sequence typing (MLST) and spa typing. The Staphylococcal enterotoxins typing and the Staphylococcal enterotoxin genes of the isolates were analyzed by ELISA and PCR, respectively. The antimicrobial susceptibility of the isolates was performed by disc diffusion. 21 Staphylococcus aureus isolates were characterized using whole genome sequencing (WGS). Based on the whole genome single nucleotide polymorphism (SNP), the phylogenetic tree was constructed by Snippy. Results: 41 Staphylococcus aureus isolates were divided into 2 types by MLST and spa typing: ST6-t701 and ST7-t091. 2 ST7-t091 isolates were identified as methicillin-resistant Staphylococcus aureus (MRSA). 25 ST7-t091 isolates and 14 ST6-t701 isolates were methicillin-sensitive Staphylococcus aureus (MSSA), and were resistant to 7 and 6 antibiotics, respectively. All isolates were positive for sea by PCR. WGS revealed all 21 isolates carried scn, sak, sea, hla, hld, hlgA, hlgB, hlgC, lukD virulence genes. The results showed the isolates contained an immune evasion cluster type D which located in bacteriophage ϕSa3. The SNP phylogenetic tree showed 2 MRSA ST7-t091 were constituted a separate clade from the 12 MSSA ST7-t091 isolates and 7 ST6-t701 isolates showed high similarity to each other. Conclusion: Base on the results of phylogenetic analysis, the 2 food-borne illness outbreaks occurred on 21 August and 27 September 2020 are caused by the combination of the MRSA ST7-t091 strain and the MSSA ST7-t091 strain, and the MSSA ST6-t701 strain, respectively. All isolates have high level of antibiotic resistance and carry high virulent genes.
Anti-Bacterial Agents/pharmacology* ; Disease Outbreaks ; Foodborne Diseases/epidemiology* ; Humans ; Methicillin-Resistant Staphylococcus aureus/genetics* ; Microbial Sensitivity Tests ; Multilocus Sequence Typing/methods* ; Phylogeny ; Staphylococcal Infections/epidemiology* ; Staphylococcus aureus/genetics*

Objective: To isolate and purify a bacteriophage against methicillin-resistant Staphylococcus aureus (MRSA), and to analyze its genomic information and biological characteristics. Methods: The experimental research methods were adopted. MRSA (hereinafter referred to as host bacteria) solution was collected from the wound of a 63-year-old female patient with the median sternum incision infection admitted to the Second Affiliated Hospital of Army Medical University (the Third Military Medical University). The bacteriophage, named bacteriophage SAP23 was isolated and purified from the sewage of the Hospital by sewage co-culture method and double-layer agar plate method, and the plaque morphology was observed. The morphology of bacteriophage SAP23 was observed by transmission electron microscope after phosphotungstic acid negative staining. The whole genome of bacteriophage SAP23 was sequenced with NovaSeq PE15 platform after its DNA was prepared by sodium dodecyl sulfonate/protease cleavage scheme, and genomic analysis including sequence assembly, annotation, and phylogenetic tree were completed. The bacteriophage SAP23 solution was co-incubated with the host bacterial solution for 4 h at the multiplicity of infection (MOI) of 10.000 0, 1.000 0, 0.100 0, 0.010 0, 0.001 0, and 0.000 1, respectively, and then the bacteriophage titer was measured by the drip plate method to select the optimal MOI, with here and the following sample numbers of 3. The bacteriophage SAP23 solution was co-incubated with the host bacterial solution at the optimal MOI for 5, 10, and 15 min, respectively, and the bacteriophage titer was measured by the same method as mentioned above to select the optimal adsorption time. After the bacteriophage SAP23 solution was co-incubated with the host bacterial solution at the optimal MOI for the optimal adsorption time, the bacteriophage titers were measured by the same method as mentioned above at 0 (immediately), 5, 10, 15, 20, 30, 40, 50, 60, 80, 100, and 120 min after culture, respectively, and a one-step growth curve was drawn. The bacteriophage SAP23 solution was incubated at 4, 37, 50, 60, 70, and 80 ℃ and pH 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12 for 1 h, respectively, to determine its stability. A total of 41 MRSA strains stored in the Department of Microbiology of Army Medical University (the Third Military Medical University) were used to determine the host spectrum of bacteriophage SAP23. Results: The bacteriophage SAP23 could form a transparent plaque on the host bacteria double-layer agar plate. The bacteriophage SAP23 has a polyhedral head with (88±4) nm in diameter and a tail with (279±21) nm in length and (22.6±2.6) nm in width. The bacteriophage SAP23 has a linear, double-stranded DNA with a full length of 151 618 bp and 11 681 bp long terminal repeats sequence in the sequence ends. There were 220 open reading frames predicted and the bacteriophage could encode 4 transfer RNAs, while no resistance genes or virulence factors were found. The annotation function of bacteriophage SAP23 genes could be divided into 5 groups. The GenBank accession number was MZ427930. According to the genomic collinearity analysis, there were 5 local collinear blocks in the whole genome between the bacteriophage SAP23 and the chosen 6 Staphylococcus bacteriophages, while within or outside the local collinear region, there were still some differences. The bacteriophage SAP23 belonged to the Herelleviridae family, Twortvirinae subfamily, and Kayvirus genus. The optimal MOI of bacteriophage SAP23 was 0.010 0, and the optimal adsorption time was 10 min. The bacteriophage SAP23 had a latent period of 20 min, and a growth phase of 80 min. The bacteriophage SAP23 was able to remain stable at the temperature between 4 and 37 ℃ and at the pH values between 4 and 9. The bacteriophage SAP23 could lyse 3 of the 41 tested MRSA strains. Conclusions: The bacteriophage SAP23 is a member of the Herelleviridae family, Twortvirinae subfamily, and Kayvirus genus. The bacteriophage SAP23 has a good tolerance for temperature and acid-base and a short latent period, and can lyse MRSA effectively. The bacteriophage SAP23 is a new type of potent narrow-spectrum bacteriophage without virulence factors and resistance genes.
Bacteriophages/genetics* ; Genomics ; Humans ; Methicillin-Resistant Staphylococcus aureus/genetics* ; Middle Aged ; Phylogeny ; Sternum

BACKGROUND: We evaluated the performance of the BD MAX StaphSR Assay (SR assay; BD, USA) for direct detection of Staphylococcus aureus and methicillin resistance not only in S. aureus but also in coagulase-negative Staphylococci (CNS) from positive blood cultures. METHODS: From 228 blood culture bottles, 103 S. aureus [45 methicillin-resistant S. aureus (MRSA), 55 methicillin-susceptible S. aureus (MSSA), 3 mixed infections (1 MRSA+Enterococcus faecalis, 1 MSSA+MRCNS, 1 MSSA+MSCNS)], and 125 CNS (102 MRCNS, 23 MSCNS) were identified by Vitek 2. For further analysis, we obtained the cycle threshold (Ct) values from the BD MAX system software to determine an appropriate cutoff value. For discrepancy analysis, conventional mecA/mecC PCR and oxacillin minimum inhibitory concentrations (MICs) were determined. RESULTS: Compared to Vitek 2, the SR assay identified all 103 S. aureus isolates correctly but failed to detect methicillin resistance in three MRSA isolates. All 55 MSSA isolates were correctly identified by the SR assay. In the concordant cases, the highest Ct values for nuc, mecA, and mec right-extremity junction (MREJ) were 25.6, 22, and 22.2, respectively. Therefore, we selected Ct values from 0-27 as a range of positivity, and applying this cutoff, the sensitivity/specificity of the SR assay were 100%/100% for detecting S. aureus, and 97.9%/98.1% and 99.0%/95.8% for detecting methicillin resistance in S. aureus and CNS, respectively. CONCLUSIONS: We propose a Ct cutoff value for nuc/mec assay without considering MREJ because mixed cultures of MSSA and MRCNS were very rare (0.4%) in the positive blood cultures.
Anti-Bacterial Agents/pharmacology ; Bacteremia/diagnosis/microbiology ; Coagulase/metabolism ; Humans ; Methicillin-Resistant Staphylococcus aureus/drug effects/genetics/*isolation & purification ; Microbial Sensitivity Tests ; Oxacillin/pharmacology ; Reagent Kits, Diagnostic ; Staphylococcus/drug effects/enzymology/genetics/isolation & purification ; Staphylococcus aureus/drug effects/genetics/*isolation & purification

OBJECTIVETo investigate the genotypic diversity of Methicillin-resistant Staphylococcus aureus (MRSA) isolated from pigs and retail foods from different geographical areas in China and further to study the routes and rates of transmission of this pathogen from animals to food.

METHODSSeventy-one MRSA isolates were obtained from pigs and retail foods and then characterized by multi-locus sequencing typing (MLST), spa typing, multiple-locus variable number of tandem repeat analysis (MLVA), pulsed-field gel electrophoresis (PFGE), and antimicrobial susceptibility testing.

RESULTSAll isolated MRSA exhibited multi-drug resistance (MDR). Greater diversity was found in food-associated MRSA (7 STs, 8 spa types, and 10 MLVA patterns) compared to pig-associated MRSA (3 STs, 1 spa type, and 6 MLVA patterns). PFGE patterns were more diverse for pig-associated MRSA than those of food-associated isolates (40 vs. 11 pulse types). Among the pig-associated isolates, CC9-ST9-t899-MC2236 was the most prevalent clone (96.4%), and CC9-ST9-t437-MC621 (20.0%) was the predominant clone among the food-associated isolates. The CC9-ST9 isolates showed significantly higher antimicrobial resistance than other clones. Interestingly, CC398-ST398-t034 clone was identified from both pig- and food-associated isolates. Of note, some community- and hospital-associated MRSA strains (t030, t172, t1244, and t4549) were also identified as food-associated isolates.

CONCLUSIONCC9-ST9-t899-MC2236-MDR was the most predominant clone in pigs, but significant genetic diversity was observed in food-associated MRSA. Our results demonstrate the great need for improved surveillance of MRSA in livestock and food and effective prevention strategies to limit MDR-MRSA infections in China.

Animals ; Anti-Bacterial Agents ; pharmacology ; China ; Food Microbiology ; Humans ; Methicillin ; pharmacology ; Methicillin Resistance ; Methicillin-Resistant Staphylococcus aureus ; genetics ; isolation & purification ; Nose ; microbiology ; Swine ; microbiology

Staphylococcus aureus, or methicillin-resistant S. aureus (MRSA), is a significant pathogen in both nosocomial and community infections. Community-associated MRSA (CA-MRSA) strains tend to be multi-drug resistant and to invade hospital settings. This study aimed to assess the antimicrobial resistance and molecular characteristicsof nasal S. aureus among newlyadmitted inpatients.In the present study, 66 S. aureus isolates, including 10 healthcare-associated MRSA (HA-MRSA), 8 CA-MRSA, and 48 methicillin-sensitive S. aureus (MSSA) strains, were found in the nasal cavities of 62 patients by screening 292 newlyadmitted patients. Antimicrobial resistance and molecular characteristics of these isolates, including spa-type, sequence type (ST) and SCCmec type, were investigated. All isolates were sensitive to linezolid, teicoplanin, and quinupristin/dalfopristin, but high levels of resistance to penicillin and erythromycin were detected. According to D-test and erm gene detection results, the cMLSB and iMLSB phenotypes were detected in 24 and 16 isolates, respectively. All 10 HA-MRSA strains displayed the cMLSB phenotypemediated by ermA or ermA/ermC, while the cMLSB CA-MRSA and MSSA strains carried the ermB gene. Molecular characterization revealedall 10 HA-MRSA strains were derived from the ST239-SCCmec III clone, and four out of eight CA-MRSA strains were t437-ST59-SCCmec V. The results suggest that patients play an indispensable role in transmitting epidemic CA-MRSA and HA-MRSA strains.
Anti-Bacterial Agents/*pharmacology ; Bacterial Proteins/genetics ; Drug Resistance, Multiple, Bacterial/genetics ; Humans ; Inpatients ; Methicillin-Resistant Staphylococcus aureus/*drug effects/genetics/isolation & purification ; Methyltransferases/genetics ; Microbial Sensitivity Tests ; Nasal Cavity/*microbiology ; Staphylococcal Infections/diagnosis/microbiology ; Staphylococcus aureus/*drug effects/genetics/isolation & purification

BACKGROUND: Methicillin-resistant Staphylococcus aureus (MRSA) are important pathogens causing nosocomial infections in Korean hospitals. This study aimed to investigate the epidemiological and genetic diversity of clinical S. aureus isolates in healthcare settings from 2001 to 2008. METHODS: Samples and data were obtained from 986 individuals as part of the National Antimicrobial Surveillance Project, involving 10 regions nationwide. Molecular typing studies, including multilocus sequence typing (MLST) and staphylococcal cassette chromosome mec (SCCmec) typing were performed, and a representative clone of Korean MRSA was classified by combinational grouping using a DiversiLab (DL; bioMérieux, France) repetitive element polymerase chain reaction (rep-PCR) system. RESULTS: Nine Korean MRSA clones (KMRSA-1 to -9) were identified by analysis of genetic backgrounds and molecular characteristics. KMRSA-1 to -3, expressing clonal complex (CC) 5 (carrying SCCmec II), CC8 (carrying SCCmec III), and CC72 (carrying SCCmec IV) were spread nationwide. In contrast, KMRSA-6 was highly prevalent in Gyeongsangnam-do, and KMRSA-4 was highly prevalent in Jeollanam-do and Jeollabuk-do. CONCLUSIONS: Epidemic KMRSA clones were genetically similar to major clones identified from the USA, with the exception of KMRSA-2, which had the SCCmec III type. Our results provide important insights into the distribution and molecular genetics of MRSA strains in Korea and may aid in the monitoring of MRSA spread throughout the country.
Bacterial Proteins/genetics ; DNA, Bacterial/genetics/metabolism ; Electrophoresis, Gel, Pulsed-Field ; Hospitals ; Humans ; Methicillin-Resistant Staphylococcus aureus/*genetics/isolation & purification ; Multilocus Sequence Typing ; Multiplex Polymerase Chain Reaction ; Prevalence ; Republic of Korea/epidemiology ; Staphylococcal Infections/diagnosis/*epidemiology/microbiology

OBJECTIVETo study the relationship between nasal carriage and Staphylococcus aureus (S. aureus) infection in hospitalized children.

METHODSFifty-six hospitalized children infected with S. aureus were recruited in this study. Nasal swabs were collected and cultured, and the nasal carriage rate of S. aureus was examined. PVL virulence gene and mecA resistance gene were both detected in clinical strains and nasal carriage strains by PCR.

RESULTSTwenty-two (39%) of the 56 children had nasal carriage of S. aureus, and most of them (18 cases) were younger than one year. Among these 22 children, 11 (50%) had previous hospitalization over the past year. In the infected strains, the rate of methicillin-resistant S. aureus (MRSA) was 29% (16/56), while it was 32% (7/22) in carriage strains. The mecA positive results in clinical strains were consistent with the results in nasal carriage strains. Among 5 PVL-positive nasal carriage strains, 4 (90%) could be matched with their clinical strains, all of which were MRSA.

CONCLUSIONSNasal carriage is a potential risk factor for S. aureus infection. Nosocomial transmission may lead to nasal carriage, which can cause S. aureus infection. The isolation rate of MRSA is high in hospitalized children infected with S. aureus, which implies that more attention is needed for this situation. The isolates from noses may be clonally identical to the isolates from clinical secretions, and the homology between them needs to be confirmed by multi-locus sequence typing.

Bacterial Proteins ; genetics ; Carrier State ; microbiology ; Child ; Child, Hospitalized ; Child, Preschool ; Female ; Humans ; Infant ; Infant, Newborn ; Male ; Methicillin-Resistant Staphylococcus aureus ; isolation & purification ; Nose ; microbiology ; Penicillin-Binding Proteins ; Staphylococcal Infections ; microbiology ; Staphylococcus aureus ; isolation & purification

This study was conducted to determine the prevalence of livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) in pigs, farm workers, and the environment in northern Thailand, and to assess LA-MRSA isolate phenotypic characteristics. One hundred and four pig farms were randomly selected from the 21,152 in Chiang Mai and Lamphun provinces in 2012. Nasal and skin swab samples were collected from pigs and farm workers. Environmental swabs (pig stable floor, faucet, and feeder) were also collected. MRSA was identified by conventional bacterial culture technique, with results confirmed by multiplex PCR and multi locus sequence typing (MLST). Herd prevalence of MRSA was 9.61% (10 of 104 farms). Among pigs, workers, and farm environments, prevalence was 0.68% (two of 292 samples), 2.53% (seven of 276 samples), and 1.28% (four of 312 samples), respectively. Thirteen MRSA isolates (seven from workers, four from environmental samples, and two from pigs) were identified as Staphylococcal chromosomal cassette mec IV sequences type 9. Antimicrobial sensitivity tests found 100% of the MRSA isolates resistant to clindamycin, oxytetracycline, and tetracycline, while 100% were susceptible to cloxacillin and vancomycin. All possessed a multidrug-resistant phenotype. This is the first evidence of an LA-MRSA interrelationship among pigs, workers, and the farm environment in Thailand.
*Animal Husbandry ; Animals ; Cross-Sectional Studies ; Genotype ; Humans ; Methicillin-Resistant Staphylococcus aureus/classification/*genetics/*isolation & purification ; Microbial Sensitivity Tests/veterinary ; Molecular Sequence Data ; Multilocus Sequence Typing/veterinary ; Multiplex Polymerase Chain Reaction/veterinary ; Occupational Diseases/*epidemiology/microbiology ; Phylogeny ; Prevalence ; Sequence Analysis, DNA/veterinary ; Staphylococcal Infections/*epidemiology/microbiology ; Swine ; Swine Diseases/*epidemiology/microbiology ; Thailand/epidemiology

Severe congenital neutropenia (SCN) is a heterogeneous group of disorders with a defect in granulopoiesis causing marked neutropenia and severe bacterial infections. A 17-month-old girl (patient 1) was admitted due to cervical lymphadenitis caused by methicillin-resistant Staphylococcus aureus, with neutropenia. She had Pseudomonas aeruginosa sepsis and peritonitis with perforated appendicitis at 8-month of age. Her sister, a 37-month-old girl (patient 2), had recurrent stomatitis with profound neutropenia, and her mother, a 32-yr-old woman (patient 3), had had recurrent stomatitis until her early 20s with neutropenia. We found an ELANE gene mutation (c.597+1G > A) from them in direct DNA sequencing analysis. Patients 1 and 2 did not respond to granulocyte colony stimulating factor and patient 1 was treated with prolonged antibiotics and excision. We demonstrated inherited SCN cases showing different severity even with the same mutation of the ELANE gene in a family.
Adult ; Child, Preschool ; DNA Mutational Analysis ; Female ; Granulocyte Colony-Stimulating Factor/therapeutic use ; Humans ; Infant ; Leukocyte Elastase/*genetics ; Methicillin-Resistant Staphylococcus aureus/isolation & purification ; Mutation/genetics ; Neutropenia/*congenital/diagnosis/drug therapy/genetics ; Pedigree ; *Phenotype ; Polymorphism, Single Nucleotide ; Recurrence ; Staphylococcal Infections/diagnosis/microbiology ; Stomatitis/diagnosis ; Tomography, X-Ray Computed