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Staphylococcus aureus (S. aureus) is a leading cause of bacteremia and blood stream infections. Methicillin-resistant S. aureus (MRSA) that first appeared in 1961 often caused hospital-acquired infections (HAIs) and community-acquired infections (CAIs) and was associated with high mortality rate. Accurate and rapid point-of-care testing (POCT) of MRSA is crucial for clinical management and treatment of MRSA infections, as well as the prevention and control of HAIs and CAIs. Here, we reported a novel extraction-free dual HiFi-LAMP assay for discriminative detection of methicillin-susceptible S. aureus and MRSA. The dual HiFi-LAMP assay can detect 30 copies/reaction of nuc and mecA genes with detection limits of 147 and 158 copies per 25 µL reaction, respectively. A retrospective clinical evaluation with 107 clinical S. aureus isolates showed both sensitivity and specificity of 100%. A prospective clinical evaluation with 35 clinical samples revealed a specificity of 100% and a sensitivity of 92.3%. The dual HiFi-LAMP assay can detect almost all S. aureus samples (141/142; 99.3%) within 20 min, implying that the entire HiFi-LAMP assay (including sample process) can be completed within 40 min, extremely significantly shorter than 3-5 days by the traditional clinical microbial culture and antibiotic susceptibility testing. The novel extraction-free dual HiFi-LAMP assay can be used as a robust POCT tool to promote precise diagnosis and treatment of MRSA infections in hospitals and to facilitate surveillance of MRSA at hospital and community settings.IMPORTANCEMethicillin-resistant Staphylococcus aureus (MRSA) was associated with high mortality rate and listed as a "priority pathogen" by the World Health Organization. Accurate and rapid point-of-care testing (POCT) of MRSA is critically required for clinical management and treatment of MRSA infections. Some previous LAMP-based POCT assays for MRSA might be questionable due to their low specificity and the lack of appropriate evaluation directly using clinical samples. Furthermore, they are relatively tedious and time-consuming because they require DNA extraction and lack multiplex detection capacity. Here, we reported a novel extraction-free dual HiFi-LAMP assay for discriminative detection of MRSA and methicillin-susceptible S. aureus. The assay has high specificity and sensitivity and can be completed within 40 min. Clinical evaluation with real clinical samples and clinical isolates showed excellent performance with 100% specificity and 92.3%-100% sensitivity. The novel extraction-free assay may be a robust POCT tool to promote precise diagnosis of MRSA infections and facilitate surveillance of MRSA at hospital and community settings.

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The frequency of Staphylococcus aureus strains associated with oral cavity microbiota has prodigious consideration. Although S. aureus has been reflected as an ephemeral member of the human oral cavity microbiota, the isolation, identification, and characterization of S. aureus is important. The present study aimed to characterize S. aureus strains from the oral cavity microflora, isolation of S. aureus from the human oral cavity microbiota, and demographic information of the participants to evaluate exogenous factors associated with the presence of S. aureus and their genetic analysis linkage with different factors. The method used in this study is the isolation of oral cavity microbiomes using sheep blood agar and Mannitol salt agar. We performed antibiotic profiling with various antibiotics and genetic analysis utilizing gene-specific primers for specific genes, including nuc, mecA, pvl, agr, and coa. A significant number of S. aureus isolates were found in the oral cavity of humans 18/84 (21.42%), and all 18 strains tested positive for the confirmatory nuc gene. Antibiotic resistance-conferring gene mecA was positive in 10 (55.6%) isolates. It was found that the occurrence of pvl, agr, and coagulase (coa) genes was 9 (50%), 6 (33.33%), and 10 (55.6%), respectively. The genetic analysis reported that significant associations were present between male and mecA gene (P = 0.03) and coa (P = 0.03), smokers with the occurrence of mecA (P = 0.02), agr (P = 0.048) and coa (P = 0.02) genes. Likewise, the association of antibiotic usage was significantly found with mecA (P = 0.02), coa (P = 0.02); however, the individuals who have taken orthodontic treatment recently have a significant association with agr (P = 0.017). The use of mouth rinse was significantly associated with the prevalence of the pvl gene (P = 0.01), and tooth brushing frequency and inflammation of the buccal cavity were also statistically significant in relation to pvl gene prevalence (P = 0.02, 0.00, respectively). Moreover, calories and weight-controlled diet were significantly associated with mecA, agr, and highly significant with coa (P = 0.02, 0.048, 0.000), so all P < 0.05, and no significant association was found between the socioeconomic status of individuals with aforementioned analyzed genes.

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Background and Aim: Staphylococcus aureus is a bacterium that causes several infectious diseases, including mastitis, endocarditis, and osteomyelitis, and poses a threat to human and animal health. This study aims to phenotypically and genetically identify S. aureus from the isolates collected from humans, animals, environment, and Dangke products in the dairy farms of South Sulawesi Province, Indonesia, as well as to establish a genetic relationship among the isolated S. aureus strains. Materials and Methods: The total number of samples was 142, comprising 30 humans (skin swab), 58 animals (raw milk), 14 dairy products (Dangke), and 40 environmental samples (water). S. aureus was phenotypically identified using the culture method, followed by Gram staining, catalase test, and coagulase test. Simultaneously, genotypic identification of S. aureus was performed using the conventional polymerase chain reaction and sequencing methods. Sequencing data were analyzed using the MEGA X software by comparing BLAST National Center for Biotechnology Information databases. Results: The phenotypic methods revealed that 56/142 (39.4%) animal, human, and Dangke samples grew on culture, and 56/56 (100%) were Gram stain positive, 56/56 (100%) catalase-positive, and 23/56 (41.1%) coagulase positive. The genotypic method revealed that 32/56 (57.1%) samples amplified the nuc gene. The phylogenetic analysis of 12 isolates revealed that they are all closely related and do not belong to distinct clades. Conclusion: It indicates that S. aureus isolates from animals (S30) are probably the same strain as human isolates (H2, H3, H4, and H5). The findings of this study can be used as information regarding the importance of preventing and controlling diseases caused by S. aureus using a health approach involving the human, animal, and environmental sectors. This study was limited to the sequencing analysis of the nuc gene.

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OBJECTIVES: This study aims to investigate the prevalence and antibiogram of Staphylococcus aureus and methicillin-resistance S. aureus (MRSA) in rabbits, rabbit handlers, and rabbitry environments in Terengganu. MATERIALS AND METHODS: Swab samples from 183 rabbits (183 oral and 183 ear swabs), 45 rabbit handlers (45 oral and 45 nasal), and environmental (n = 180) samples from rabbitries were collected from 10 rabbit farms in Terengganu. The associated S. aureus isolates from the swabs were isolated using phenotypic microbiology tests. The bacteria were confirmed by polymerase chain reaction targeting nuc (S. aureus) and mecA (MRSA) genes. The antibiogram of all S. aureus isolates was determined using the Kirby-Bauer test. RESULTS: Staphylococcus aureus was detected in 19% of rabbits, 26.7% of rabbit handlers, and 8.8% of swabs from the rabbitry environment. However, MRSA (0%) could not be detected. Antibiotic susceptibility test revealed that S. aureus from rabbits showed low resistance (<20%) against 15 different antibiotics while fully susceptible to 4 antibiotics. Meanwhile, S. aureus from rabbit handlers showed high resistance against penicillin (86%), oxacillin (64%), and amoxicillin (50%). CONCLUSIONS: This study suggests the emergence of antibiotic-resistant S. aureus in rabbit farms settings. Therefore, careful selection of antimicrobial agents will be essential to preserve the effectiveness of treatments toward S. aureus infections.

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Pneumonia is the acute inflammation of lung tissue and is multi-factorial in etiology. Staphylococcus aureus (S. aureus) is a harmful pathogen present as a normal flora of skin and nares of dairy cattle. In bovine pneumonia, S. aureus triggers to activates Toll-Like Receptors (TLRs), that further elicits the activation of the inflammation via NF-κB pathway, oxidative stress and apoptotic pathways. In the current study, pathogen-associated gene expression of the pro-inflammatory cytokines, oxidative stress and apoptotic markers in the lung tissue of cattle was explored in bovine pneumonia. Fifty lung samples collected from abattoir located in Wuhan city, Hubei, China. Histopathologically, thickening of alveolar wall, accumulation of inflammatory cells and neutrophils in perivascular space, hyperemia, hemorrhages and edema were observed in infected lungs as compared to non-infected lung samples. Furthermore, molecular identification and characterization were carried by amplification of S. aureus-specific nuc gene (270 base pairs) from the infected and non-infected lung samples to identify the S. aureus. Moreover, qPCR results displayed that relative mRNA levels of TLR2, TLR4, pro-inflammatory gene (IL-1ß, IL-6 and TNF-α) and apoptosis-associated genes (Bax, caspase-3 and caspase-9) were up-regulated except Bcl-2, which is antiapoptotic in nature, and oxidative stress related genes (Nrf2, NQO1, HO-1 and GCLC) which was down-regulated in infected pulmonary group. The relative protein expression of NF-κB, mitochondria-mediated apoptosis gene was up-regulated while Bcl-2 and Nrf2 pathway genes were downregulated in infected cattle lungs. Our findings revealed that genes expression levels of inflammatory mediators, oxidative stress and apoptosis were associated with host immunogenic regulatory mechanisms in the lung tissue during infection. Conclusively, the present study provides insights of active immune response via TLRs-mediated inflammatory, oxidative damage, and apoptotic paradox.

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Staphylococcus aureus (S. aureus) is one of the most common pathogens for nosocomial and community infections, which is closely related to the occurrence of pyogenic and toxic diseases in human beings. In the current study, a lab-built microchip capillary electrophoresis (microchip CE) system was employed for the rapid determination of S. aureus, while a simple-to-use space domain internal standard (SDIS) method was carried out for the reliable quantitative analysis. The precision, accuracy, and reliability of SDIS were investigated in detail. Noted that these properties could be elevated in SDIS compared with traditional IS method. Remarkably, the PCR products of S. aureusnuc gene could be identified and quantitated within 80 s. The theoretical detection limit could achieve a value of 0.066 ng/µL, determined by the using SDIS method. The current work may provide a promising detection strategy for the high-speed and highly efficient analysis of pathogens in the fields of food safety and clinical diagnosis.

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Rapid detection of Methicillin Resistant Staphylococcus aureus (MRSA) is an important concern for both treatment and implementation of infection control policies. The present study provides an 'in house' real-time PCR assay to detect directly nuc, pvl, and mecA genes. The assay is able to perform identification of MRSA, Methicillin-Sensitive S. aureus, Methicillin-Resistant Coagulase Negative Staphylococci and the Panton-Valentine leukocidin virulence gene from rectal and pharyngeal swab samples in a screening context. We found an analytical sensitivity of this current Triplex PCR assay of 514 CFU/mL. Analytical specificity was tested with different Gram-positive and Gram-negative species and yielded no false-positive PCR signal. The sensitivity and specificity of the Triplex Real Time PCR were both 100% for these targets when compared with the culture and conventional methods. This assay is readily adaptable for routine use in a microbiology laboratory, as it will enable the implementation of timely and properly guided therapy and infection control strategies.

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A single-tube multiplex real-time PCR targeting the nuclease (nuc) gene and subsequent high-resolution melting analysis (HRMA) were used to identify 13 different Staphylococcus species. The nuc gene was targeted due to its low intraspecies variation and the greater interspecies variation than the 16S rRNA gene in Staphylococcus. We used HRMA software that can store and compare HRMA profiles from different runs as long as the runs contain the same reference reaction. Thus, we reduced the 14 PCRs to 2 different PCRs, one targeting the unknown sequence and the other targeting the reference sequences to screen 13 different Staphylococcus species. The specificity of the developed method was tested on 16 different Staphylococcus reference strains and 115 different field strains that were isolated from the milk of cattle with subclinical mastitis. We conclude that the method can be used to quickly and cost-effectively differentiate Staphylococcus aureus (S. aureus) from other Staphylococcus species (S. epidermidis, S. lugdunensis, S. schleiferi, S. hyicus, S. chromogenes, S. lentus, S. haemolyticus, S. xylosus, S. saprophyticus, S. warneri, S. simulans and S. hominis).

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Methicillin-resistant Staphylococcus aureus (MRSA) has long been a common pathogen in healthcare facilities, but now, it has emerged as a problematic pathogen in the community setting as well. This study reported source, diagnosis and treatment of HA-MRSA and CA-MRSA. A total of sixty-five clinical samples (urine, pus, wound swab) were collected from clinical origin of Dhaka city, Bangladesh. All the isolates were tested phenotypically by conventional methods and genotypically by PCR targeting nuc, pvl and mecA genes. Finally sequencing was carried out for pvl gene to know the mutagenic variation or any amino acid changes in pvl gene. Chi square test was employed for statistical analysis. Patients of age group 51-60 years are more susceptible (46.15%) to MRSA, CA-MRSA or HA-MRSA infection. Female are (32.30%) more susceptible to MRSA infection. Among 65 isolates 53 isolates identified phenotypically as S. aureus. These were positive for amplification of nuc (270 bp) gene of S. aureus. Moreover, among 53 isolates 33 phenotypically considered as MRSA and 38 (72%) showed positive amplification for mecA (162 bp) gene. Among 38 MRSA isolates 22 (57.89%) confirmed as CA-MRSA and 16 (42.10%) as HA-MRSA. Finally, sequence analysis for lukS/F-PV genes from 4 representative isolates detected a new single nucleotide polymorphism in comparison with the control sequence. However, no amino acid changes were found. Statistical analysis showed HA-MRSA isolates were more commonly found in urine sample and CA-MRSA in pus and wound swab. CA-MRSA isolates were more resistant to tested antibiotics than HA-MRSA.

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The development of molecular techniques of research in the end of XX century permitted to broaden nomenclature of species forming genus Staphylococcus that nowadays numbers 51 species and 27 sub-species. The pathogenic species of genus have a capacity to coagulate blood plasma of mammals forming group of coagulase-positive staphylococci including 7 species: S. aureus, S. delphini, S. intermedius, S. pseudintermedius, S. lutrae, S. schleiferi ssp. сoagulans, S hyicus. In clinical practice, S.aureus is considered as the most virulent among staphylococci. The cumulated data testifies increasing etiologic significance of other representatives of group of coagulase-positive staphylococci in human and animal infection pathology. The keen attention is needed to be paid to Staphylococcus intermedius of group (SIG), uniting three close kindred species: S. pseudintermedius, S. intermedius, S. delphini. Among them the most broadly prevailed are methicillin-resistant clones of S. pseudintermedius, capable to bring on in patient various pyoinflammatory diseases. The laboratory methods based on phenotype tests, provide no opportunity to differentiate coagulase-positive staphylococci because of significant similarity of phenotype characteristics in certain representatives of this group. Te comparative analysis was implemented concerning efficiency of various methods of species identification of coagulase-positive staphylococci: biochemical, molecular genetic (multi-primer polymerase chain reaction for identifying differences in gene structure of thermonuclease, analysis of polymorphism of lengths of restricting fragments of catalase gene and their sequencing), matrix-activated laser desorptional/ionizing time-of-flight mass-spectrometry (MALDI-ToF MS) with various modes of probe preparation. The analysis was applied to 117 isolates of representatives of SIG, separated from ill and healthy individuals of small domestic animals, clinical isolates form patients of hospitals. The multi-primer polymerase chain reaction permitted to identify 97% of isolates, analysis of polymorphism of lengths of restricting fragments of catalase gene - 100% of isolates that confirms efficiency of molecular genetic methods of analysis. The MALDI-ToF MS requires replenishment data base of mass-spectrometer and application of the mode of preliminary protein extraction of samples fo increasing efficiency of species identification of coagulase-positive staphylococci.

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ABSTRACT: This research aimed to detect coagulase-positive Staphylococcus (CPS) directly in samples of artificially contaminated milk, using multiplex PCR (mPCR). Standard and isolated bacterial strains of S. aureus, S. epidermidis, S. hyicus, S. intermedius, Listeria monocytogenes and Escherichia coli species were used, evaluating the specificity and detection limit of mPCR, for artificially contaminated UHT milk. Primers specific for the nuc gene (NUC1-NUC2 were used for S. aureus, NUC3-NUC4 for S. hyicus and NUC5-NUC6 for S. intermedius). It was possible to detect the three target species by mPCR, directly from bovine whole milk, with adequate specificity and acceptable detention limit for identification of coagulase-positive Staphylococcus (CPS) in foods. The specificity was determined by the amplification of species-specific fragments, and the detection limit was assessed by the detection thresholds obtained for the three species (103 CFU mL-1). From these results, the mPCR described, with the proposed set of primers, has the potential for use in precise identification and differentiation between CPSs in milk samples.

RESUMO: Esta pesquisa teve como objetivo detectar diretamente em amostras de leite contaminado artificialmente Staphylococcus coagulase positiva (ECP) por multiplex PCR (mPCR). Cepas padrão e isolados de S. aureus, S. epidermidis, S. hyicus, S. intermedius, Listeria monocytogenes e Escherichia coli foram utilizados no estudo. Foram utilizados primers específicos para o gene nuc (NUC1-NUC2 para o S. aureus, NUC3-NUC4 para o S. hyicus e NUC5-NUC6 para o S. intermedius ). Foi possível detectar as três espécies-alvo por mPCR, formar diretamente nas amostras de leite integral bovino, com especificidade adequada e limite de detecção aceitável para identificação de espécies de Staphylococcus coagulase positiva (ECP) em alimentos. A especificidade foi determinada por meio da amplificação de fragmentos específicos das espécies e o limite de detecção foi avaliado pelos limiares de detecção obtidos para as três espécies (103 UFC mL-1 para as espécies presentes nas amostras de leite contaminadas artificialmente).

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Objective To understand the colonization ,distribution and prevention of methicillin‐resistant Staphylococcus aureus (MRSA) in ICU .Methods The nasal vestibule swabs were immediately taken from new patients in ICU and the sputum suction catheter specimens were collected from the patients conforming to respiratory tract infection at 72 h after entering ICU .The fluores‐cence PCR was adopted to rapidly detect mecA ,nuc gene .The related environment specimens such as nasal cavity and hands in med‐ical staffs were performed the regular tracking monitoring ,prevention and control .Results The MRSA‐detection rates were 9 .9%for nasal vestibule swabs ,2 .7% for sputum suction catheter ;7 .1% for nasal vestibule swabs of medical staffs ,4 .0% for hands and 4 .4% for cuffs;the medical staffs and patient′s nasal cavity with MRSA‐positive adopted mupirocin for scrubing ,MRSA detection rate in hands after reinforce disinfection was 0 .0% ,but nasal cavity MRSA in medical staff was still detected out every quarter . Conclusion The PCR method has significantly higher positive detection rate compared with the conventional culture method .Nasal vestibule is a major colonization site of M RSA and the main infection source of infection ,the hands are the important route of trans‐mission ,monitoring nasal vestibule in ICU patients and medical staffs and hands in medical staffs is important to control MRSA nosocomial infection .

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Sixty-five strains of coagulase positive staphylococci (Staphylococcus aureus, S. intermedius and S. hyicus) were identified at species level by PCR amplification of the coa gene, specific for S. aureus, and of the nuc gene, specific for S. intermedius and for S. hyicus.

Sessenta e cinco cepas de estafilococos coagulase positiva foram identificadas em nível de espécie, através da amplificação, por PCR, de seqüências do gene coa, específicas para S. aureus, e do gene nuc, específicas para S. intermedius e para S. hyicus.

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Sixty-five strains of coagulase positive staphylococci (Staphylococcus aureus, S. intermedius and S. hyicus) were identified at species level by PCR amplification of the coa gene, specific for S. aureus, and of the nuc gene, specific for S. intermedius and for S. hyicus.

Sessenta e cinco cepas de estafilococos coagulase positiva foram identificadas em nível de espécie, através da amplificação, por PCR, de seqüências do gene coa, específicas para S. aureus, e do gene nuc, específicas para S. intermedius e para S. hyicus.