Staphylococcal Genes Research Articles

Antibacterial Activity and Phytochemical Analysis of Moringa oleifera Extract against Staphylococcus aureus Identified by Routine and Molecular Methods

Abstract Background: Many diseases caused by bacterial and viral infections may now be treated using herbal extracts instead of chemical drugs. Objectives: This study aimed to investigate the antibacterial effect of Moringa oleifera leaf extracts against the bacterium employed in the experiment, as a commensal. Staphylococcus aureus can be found on the skin and in the nasal flora. It can also cause invasive, localized illnesses. Many virulence factors are present in S. aureus. It is recognized by routine and molecular methods depending on Vick Staphylococcal gene. Material and Methods: Phytochemical analysis of high polarity solvent leaves was done by using ethanol. Phytochemical study revealed the presence of tannins, alkaloids, flavonoids, steroids, saponins, and other compounds in the extract. Using the well-diffusion method, the antibacterial effect of the extracts on microorganisms was investigated. Results: The ethanol extract proved potent against pathogenic microorganisms, with S. aureus showing the highest activity (10–100 mg/mL). In comparison to the concentrations of alcoholic extracts, the zones’ inhibition of bacterial growth in diameters increased. However, dosages of 80–100 mg/mL were highly effective and significant against S. aureus growth, whereas concentrations of 10–20 mg/mL had low post-detected efficiency and concentrations of 40–60 mg/mL had medium post-detected efficiency. Conclusion: These studies demonstrate the validity of the plant’s traditional medicinal properties.

Repurposing of non-steroidal anti-inflammatory drugs for combination therapies to combat multidrug-resistant S. aureus of bovine reproductive tract origin.

Multidrug-resistant bacteria have become the predominant etiology in bovine female reproductive tract infections and thus require effective treatment approaches. The main goal of this study was the molecular detection of mecA, blaZ, tetK, and aacA-aphD genes in Staphylococcus aureus (S. aureus) responsible for methicillin, beta-lactam, tetracycline, and aminoglycoside resistance respectively. Phylogenetic analysis was conducted to check the homology of staphylococcal genes with NCBI sequences. The in-vitro efficacy of non-steroidal anti-inflammatory drugs (NSAIDs) in combination therapies against MDR S. aureus was evaluated using well diffusion assay and checkerboard method. Vaginal swab samples (n = 384) collected from bovines suffering from endometritis, pyometra, and retained placenta were tested for S. aureus. Results showed a 17.96% overall prevalence. Both phenotypic and genotypic resistance was observed among S. aureus isolates with 50.72% and 37.68% isolates being confirmed as methicillin-resistant (MRSA), 36.23% and 18.84% isolates exhibiting beta-lactam, 40.58%, and 27.54% isolates showing tetracycline, and 33.33% and 36.23% isolates showing aminoglycosides resistance based on disc diffusion and gene confirmation, respectively. Phylogenetic analysis indicated homology with previously reported Pakistani isolates suggesting the possibility of MDR S. aureus transmission within and between animals. Synergy testing indicated that combinations of ceftriaxone-ketoprofen (153.77%), ceftriaxone-meloxicam (149.55%), amoxiclav-flunixin meglumine (106.06%), and oxytetracycline-flunixin meglumine (104.47%) showed synergy on well diffusion assay. Based on the fractional inhibitory concentration index by checkerboard method, oxytetracycline-meloxicam and gentamicin-ketoprofen combinations exhibited synergistic interaction. In conclusion, MDR S. aureus resistance was mitigated in-vitro through the combination of antibiotics (oxytetracycline, gentamicin) with NSAIDs (meloxicam, ketoprofen) that could be used to create therapeutic strategies for bovine reproductive issues.

The role of GpsB in Staphylococcus aureus cell morphogenesis.

Staphylococcus aureus is a Gram-positive clinical pathogen, which is currently the second cause of death by antibiotic-resistant infections worldwide. For decades, S. aureus cells were thought to be spherical and lack the ability to undergo elongation. However, super-resolution microscopy techniques allowed us to observe the minor morphological changes that occur during the cell cycle of this pathogen, including cell elongation. S. aureus elongation is not required for normal growth in laboratory conditions. However, it seems to be essential in the context of some infections, such as osteomyelitis, during which S. aureus cells apparently elongate to invade small channels in the bones. In this work, we uncovered new determinants required for S. aureus cell elongation. In particular, we show that GpsB has an important role in the spatio-temporal regulation of PBP2 and PBP4, two proteins involved in peptidoglycan synthesis, contributing to the maintenance of the correct cell morphology in S. aureus.

Biofilms: A developmental niche for vancomycin-intermediate Staphylococcus aureus

Staphylococcus aureus are gram-positive bacteria responsible for a wide array of diseases, ranging from skin and soft tissue infections to more chronic illnesses such as toxic shock syndrome, osteomyelitis, and endocarditis. Vancomycin is currently one of the most effective antibiotics available in treating patients infected with methicillin-resistant S. aureus (MRSA), however the emergence of vancomycin-resistant S. aureus (VRSA), and more commonly vancomycin-intermediate S. aureus (VISA), threaten the future efficacy of vancomycin. Intermediate resistance to vancomycin occurs due to mutations within the loci of Staphylococcal genes involved in cell wall formation such as rpoB, graS, and yycG. We hypothesized the VISA phenotype may also arise as a result of the natural stress occurring within S. aureus biofilms, and that this phenomenon is mediated by the RecA/SOS response. Wildtype and recA null mutant/lexAG94E strains of S. aureus biofilms were established in biofilm microtiter assays or planktonic cultures with or without the addition of sub-inhibitory concentrations of vancomycin (0.063 mg/l - 0.25 mg/L ciprofloxacin, 0.5 mg/l vancomycin). Efficiency of plating techniques were used to quantify the subpopulation of biofilm-derived S. aureus cells that developed vancomycin-intermediate resistance. The results indicated that a greater subpopulation of cells from wildtype biofilms (4.16 × 102 CFUs) emerged from intermediate-resistant concentrations of vancomycin (4 μg/ml) compared with the planktonic counterpart (1.53 × 101 CFUs). Wildtype biofilms (4.16 × 102 CFUs) also exhibited greater resistance to intermediate-resistant concentrations of vancomycin compared with strains deficient in the recA null mutant (8.15 × 101 CFUs) and lexA genes (8.00 × 101 CFUs). While the VISA phenotype would be an unintended consequence of genetic diversity and potentially gene transfer in the biofilm setting, it demonstrates that mutations occurring within biofilms allow for S. aureus to adapt to new environments, including the presence of widely used antibiotics.

Integrated genomic and functional analyses of human skin-associated Staphylococcus reveal extensive inter- and intra-species diversity.

Human skin is stably colonized by a distinct microbiota that functions together with epidermal cells to maintain a protective physical barrier. Staphylococcus, a prominent genus of the skin microbiota, participates in colonization resistance, tissue repair, and host immune regulation in strain-specific manners. To unlock the potential of engineering skin microbial communities, we aim to characterize the diversity of this genus within the context of the skin environment. We reanalyzed an extant 16S rRNA amplicon dataset obtained from distinct body sites of healthy volunteers, providing a detailed biogeographic depiction of staphylococcal species that colonize our skin. S. epidermidis, S. capitis, and S. hominis were the most abundant staphylococcal species present in all volunteers and were detected at all body sites. Pan-genome analysis of isolates from these three species revealed that the genus-core was dominated by central metabolism genes. Species-restricted-core genes encoded known host colonization functions. The majority (~68%) of genes were detected only in a fraction of isolate genomes, underscoring the immense strain-specific gene diversity. Conspecific genomes grouped into phylogenetic clades, exhibiting body site preference. Each clade was enriched for distinct gene sets that are potentially involved in site tropism. Finally, we conducted gene expression studies of select isolates showing variable growth phenotypes in skin-like medium. In vitro expression revealed extensive intra- and inter-species gene expression variation, substantially expanding the functional diversification within each species. Our study provides an important resource for future ecological and translational studies to examine the role of shared and strain-specific staphylococcal genes within the skin environment.

Co-expression network analysis for the identification of potential prostate cancer genes and in vitro confirmation of their expression in cell model in the presence of Staphylococcal tsst-1 gene.

Prostate cancer has arisen as an important life-threatening malignancy in males worldwide. Therefore, it is important to study underlying molecular pathways to be able to proposed appropriate a novel pathway of apoptosis in prostate cancer. This study aimed to explore the molecular effects of Staphylococcal tsst-1 gene on PC3 cell line apoptosis by in silico and in vitro studies. In this work, the differential expression of genes in prostate cancer was predicted by analyzing the public dataset GSE132063. Then, the pcDNA3.1 (+) vector was used to transfer tsst-1 gene to the PC3 cells and its effects was investigated using flow cytometry and qPCR. Co-expression network analysis indicated that lncRNAs had strong relationship with apoptosis genes in prostate cancer. Results of protein-protein docking indicated that BCL2L11, GRAMD3 and EGR1 interacted with tsst-1. Finally, the flow cytometry results showed that transfection by pcDNA3.1 (+)- tsst-1 could increase cellular death rates (48.15%) compared with the pcDNA3.1 (+) groups (6.35%); and the expression levels of GRAMD3, EGR1, BCL2L11 and PLAC4 were dysregulated in tsst-1 -transfected PC3 compared with empty-transfected PC3 (p < .05). In conclusion, our data will provide a novel landscape to understanding the mechanism of Staphylococcal tsst-1 gene on the PC3 cells apoptosis pathways.

Detection of New Staphylococcal Enterotoxin Encoding Genes in Staphylococcus aureus Isolated from Animals and Humans in Yogyakarta

Staphylococcal enterotoxins (SE) are essential in human and animal infection and food poisoning. This study analyzed ten genes (sek, sel, sem, sen, seo, sep, seq, ser, ses, and set) encoding new staphylococcal enterotoxin isolated from animals and humans by multiplex polymerase chain reaction (PCR). In Yogyakarta, Indonesia, samples from human infection cases (174 isolates), samples from cattle (5 isolates), and samples from goats (5 isolates) resulted in a total of 183 Staphylococcus aureus isolates.. All isolates were confirmed to be Staphylococcus aureus based on bacterial culture and biochemistry as well as identification of 23S rRNA. The sel gene was most often observed in Staphylococcus aureus isolates from goats (4 isolates, 80%), followed by the 20% sek gene. Isolates Staphylococcus aureus from cattle, the sel gene was most often found 44% (4 isolates), followed by sek gene 22%, sep gene 22% and ser gene 11%. Staphylococcus aureus isolates from humans were found with the most sem genes 22% (38 isolates), followed by 21% sel genes, 14% sek genes, 13% sep genes, 8% sen genes, and 2% ser genes. Staphylococcus aureus isolates from goats and cattle most frequently included the sel gene, whereas those from humans contained the sem gene. Detecting a new type of SE among animals and humans indicates a public health threat due to SE infection. The occurrence of this new type of SE might be used as an approach for controlling infections and food poisoning diseases.

Pathogenic role of the staphylococcal accessory gene regulator quorum sensing system in atopic dermatitis.

The skin is home to various bacteria, archaea, fungi, and viruses, collectively referred to as the skin microbiota. Patients with certain skin diseases reportedly have unique skin "dysbiosis," a condition involving imbalanced microbiota, suggesting that dysbiosis in the skin may be either causal or a consequence of specific skin diseases. Atopic dermatitis (AD) is the most common allergic skin disease that affects 15-20% of children and 2-10% of adults worldwide. Both intrinsic genetic factors, such as susceptibility to type 2 inflammation or skin barrier dysfunction, and extrinsic environmental factors, such as air pollen and skin microbiota, contribute to AD. Staphylococcus aureus, which does not often colonize the skin of healthy individuals, is commonly identified in the lesional skin of patients with AD and is correlated with the disease flare. However, the role of S. aureus in the pathogenesis of AD has not been elucidated. Here, we discuss the pathological behavior of S. aureus, focusing on accessory gene regulator (Agr) quorum sensing, which is a fundamental bacterial cell-to-cell interaction mechanism that affects the behavior of S. aureus and other members of the microbial community. Importantly, beyond bacteria-bacteria interactions, the Agr quorum sensing system also regulates various virulence factors, which induce type 2 and IL-17-dependent skin inflammation in the host. Furthermore, the colonization of Agr-positive S. aureus in early life accelerates the development of pediatric AD. Finally, we aim to highlight the current efforts to establish novel therapeutic methods to ameliorate or prevent AD through Agr-targeted intervention.

In Vivo Gene Expression Profiling of Staphylococcus aureus during Infection Informs Design of Stemless Leukocidins LukE and -D as Detoxified Vaccine Candidates.

Staphylococcus aureus is a clinically important bacterial pathogen that has become resistant to treatment with most routinely used antibiotics. Alternative strategies, such as vaccination and phage therapy, are therefore actively being investigated to prevent or combat staphylococcal infections. Vaccination requires that vaccine targets are expressed at sufficient quantities during infection so that they can be targeted by the host's immune system. While our knowledge of in vitro expression levels of putative vaccine candidates is comprehensive, crucial in vivo expression data are scarce and promising vaccine candidates during in vitro assessment often prove ineffective in preventing S. aureus infection. Here, we show how a newly developed high-throughput quantitative reverse transcription-PCR (qRT-PCR) assay monitoring the expression of 84 staphylococcal genes encoding mostly virulence factors can inform the selection and design of effective vaccine candidates against staphylococcal infections. We show that this assay can accurately quantify mRNA expression levels of these genes in several host organs relying only on very limited amounts of bacterial mRNA in each sample. We selected two highly expressed genes, lukE and lukD, encoding pore-forming leukotoxins, to inform the design of detoxified recombinant proteins and showed that immunization with recombinant genetically detoxified LukED antigens conferred protection against staphylococcal skin infection in mice. Consequently, knowledge of in vivo-expressed virulence determinants can be successfully deployed to identify and select promising candidates for optimized design of effective vaccine antigens against S. aureus. Notably, this approach should be broadly applicable to numerous other pathogens. IMPORTANCE Vaccination is an attractive strategy for preventing bacterial infections in an age of increased antimicrobial resistance. However, vaccine development frequently suffers significant setbacks when candidate antigens that show promising results in in vitro experimentation fail to protect from disease. An alluring strategy is to focus resources on developing bacterial virulence factors that are expressed during disease establishment or maintenance and are critical for bacterial in-host survival as vaccine targets. While expression profiles of many virulence factors have been characterized in detail in vitro, our knowledge of their in vivo expression profiles is still scarce. Here, using a high-throughput qRT-PCR approach, we identified two highly expressed leukotoxins in a murine infection model and showed that genetically detoxified derivatives of these elicited a protective immune response in a murine skin infection model. Therefore, in vivo gene expression can inform the selection of promising candidates for the design of effective vaccine antigens.

Prevalence, antimicrobial resistance, and staphylococcal toxin genes of blaTEM‐1a‐producing Staphylococcus aureus isolated from animals in Chongqing, China

Staphylococcus aureus infection of livestock animals and humans is a major public health issue. There are reports of antimicrobial resistance and multiple staphylococcal superantigen genes in many countries and several provinces of China, but the status in Chongqing, China is uncertain. The aim of this study was to determine the prevalence, antimicrobial susceptibility, and other molecular characteristics of S. aureus isolates from livestock animals in Chongqing. Staphylococcus aureus was isolated and identified by selective enrichment and amplification of the nuc gene from 1371 samples collected at farms in Chongqing. The agar dilution method was used to determine the resistant phenotype, and extended spectrum β-lactamase genes were amplified by PCR. Methicillin-resistant S. aureus was verified by the presence of the mecA gene, and the presence or absence of SE, SEl, and TSST-1 genes was detected in the isolates. We cultured 89 S. aureus isolates from 1371 samples between March 2014 and December 2017. These isolates were from pigs, cattle, goats, rabbits, and chickens. There were four methicillin-resistant S. aureus strains (three from pigs and one from a chicken). The 89 isolates had high resistance to penicillin (93.3%) and ampicillin (92.1%), but most were susceptible to amikacin and ofloxacin, with resistance rates below 10%. A total of 62.9% of the isolates had varying degrees of multidrug resistance. Almost all strains, except for three isolates from chickens, were positive for blaTEM-1a . There were 19 of 20 tested staphylococcal SE/SEl/TSST-1 genes present (all except for seq), and the predominant genes were sei (58.4%), tst-1 (56.2%), and seg (51.7%). The high antimicrobial resistance and prevalence of blaTEM-1a reinforce the need to reduce the usage of antimicrobials in livestock. The universal existence of staphylococcal toxin genes implies a potential threat to public health by animal-to-human transmission via the food chain.

Low-level colonization of methicillin-resistant Staphylococcus aureus in pigs is maintained by slowly evolving, closely related strains in Finnish pig farms

BackgroundOver the past two decades, livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) has become widely prevalent in pig production in Europe. The carriage status of LA-MRSA is known to vary among individual pigs, but bacterial load in pigs has rarely been studied. We assessed the quantity of LA-MRSA in nasal and skin samples of pigs and investigated the genetic diversity of the strains together with sequenced strains from national surveillance and pathology samples from the Finnish Food Authority. On two farms with assumed MRSA-positive status, farm 1 and farm 2, 10 healthy pigs were sampled three times during 2 weeks from the nares and skin (study A). On farm 1, 54 additional pigs were sampled and from confirmed MRSA-positive animals, 10 were randomly selected and transported to a clean, controlled environment for further sampling (study B). From the samples taken on farms 1 and 2 and in the controlled environment, MRSA was isolated both by direct plating and enrichment on selective media. spa types, multilocus sequence types, staphylococcal cassette chromosome mec types, resistance and virulence genes were determined. Core genome multilocus sequence typing (cgMLST) analysis was performed, including the sequences deriving from the surveillance/pathology samples from the Finnish Food Authority.ResultsAll pigs on farm 1 carried LA-MRSA in the nares at all three time points and five pigs on farm 2 at one time point. Nasal quantity varied between 10 and 103 CFU/swab and quantity on the skin between 10 and 102 CFU/swab. In the controlled environment, MRSA was detected in at least one of the nasal samples from each animal. spa type t034 was predominant. cgMLST showed one cluster with minimum allele differences between 0 and 11.ConclusionsThe study shows predominantly low-level carriage (< 103 CFU/swab) of LA-MRSA on farms. In the controlled environment we observed a decline in nasal carriage but constant skin carriage. cgMLST showed that strains of spa type t034 are closely related at the national level.

Characterization of coagulase-positive Staphylococcus spp., antimicrobial resistance profile, and presence of enterotoxin-producing genes in goat milk in Paraná State

Goat milk presents interesting characteristics to consumers, but the production of this food faces several challenges that influence its quality. Process failures from milking to processing and commercialization can expose milk to contamination by pathogenic microorganisms, including the coagulase-positive Staphylococcus group. Although Staphylococcus aureus is the most relevant species in mastitis and food poisoning, other species in this group are also important, especially those related to food poisoning. This study aimed to identify the coagulase-positive Staphylococcus species in goat milk using biochemical tests, determine the prevalence of antimicrobial resistance using the disc diffusion test, and investigate enterotoxin-producing genes, sea, seb, sec, sed, see, seg, seh, and sei by multiplex PCR. A total of 384 coagulase-positive Staphylococcus strains obtained from raw goat milk collected from nine farms in Paraná during four seasons of the year were studied. Biochemical tests showed that 85.69% of the 384 strains were S. aureus, followed by 9.38% of S. intermedius, 4.17% of S. hyicus and 0.78% of S. delphini. For the antimicrobial resistance test, up to three strains of each species identified as coagulase-positive Staphylococci were chosen from each farm, with a total of 74 strains. Of these, 27% (20/74) showed resistance to at least one antibiotic, and among all penicillin-resistant strains, 45% (9/20) also presented resistance to tetracycline. In the search for staphylococcal enterotoxin-producing genes, 49 S. aureus strains were studied; among them, 40.81% (20/49) presented enterotoxin-producing genes. The highest prevalence was detected for the sec gene, which was present in 22.44% (11/49) of the strains, followed by the seh gene in 18.36% (9/49), and the see gene was detected in 4.08% (2/49). It was concluded that S. aureus was the predominant species in raw goat milk, with a high prevalence of penicillin- and tetracycline-resistant Staphylococci and a significant number of strains with staphylococcal enterotoxin-producing genes. The strains studied carried enterotoxin-producing genes involved in food poisoning in humans, indicating that goat milk is a possible source of contamination and, therefore, a potential danger to public health.

Transcriptional adaptation of staphylococci during colonization of the authentic human environment: An overview of transcriptomic changes and their relationship to physiological conditions.

Staphylococci are commensals of human skin and mucous membranes, but some species can also cause serious infections. Host niches during both colonization and infection differ greatly and are characterized by specific environmental conditions (pH, temperature, oxygen, nutrient availability, and microbiota) that can affect gene expression and virulence of microbes. To successfully occupy extremely different habitats at different anatomical sites, Staphylococci are equipped with a variety of regulatory elements that allow specific adaptation to the changing environments. Not surprisingly, gene expression in vivo can be significantly different from the expression pattern observed in vitro. Niche specific stimuli that influence the bacterial ability to either cause infection or maintain colonization are only partially understood. Here, we describe habitat specific conditions and discuss the available literature analyzing staphylococcal gene expression, focusing on Staphylococcus aureus and S. epidermidis during colonization of the nose and skin.

Lactic acid bacteria as biocontrol agents to reduce Staphylococcus aureus growth, enterotoxin production and virulence gene expression

The capacity of six lactic acid bacteria (LAB) strains to decrease both the growth of Staphylococcus aureus UCLM-1 and the production of staphylococcal enterotoxins growing in co-culture in sheep's milk, was evaluated. During incubation of the cultures, samples were taken to monitor LABs and Staphylococcus growth, pH, production of organic acids, presence of the staphylococcal enterotoxins A, B, C, D, and E and the expression of 11 staphylococcal genes involved in virulence and enterotoxin production. Strains Lactiplantibacillus plantarum UCLM-101 and UCLM-56 yielded the highest decreases of both Staphylococcus aureus viable cell counts and pH, followed by the strain Levilactobacillus brevis UCLM-99. Analysis of organic acids and enterotoxins production by these three strains displayed that the main acid produced was lactic acid, while enterotoxins were not detected in the co-cultures with any of them. In addition, a decrease in the expression of some staphylococcal genes was observed, which varied depending on the LAB strain and the incubation time. Results allow us to propose the use of any of these three strains as biocontrol agents in the manufacture of “free of preservatives” dairy products, to avoid the risk of staphylococcal foodborne infections.

Evaluation of aminoglycoside modifying enzymes, SCCmec, coagulase gene and PCR-RFLP coagulase gene typing of Staphylococcus aureus isolates from hospitals in Shiraz, southwest of Iran

Staphylococcus aureus is an important human pathogen that causes various infections. Aminoglycosides are broad-spectrum antibiotics used to treat methicillinresistant S. aureus (MRSA) infections. Typing of S. aureus isolates by coagulase gene typing and PCR-RFLP coa gene is a fast and suitable method for epidemiological studies. Aim of the present study was to evaluate the resistance to aminoglycosides, staphylococcal chromosomal cassette mec (SCCmec) types, coagulation typing and PCR-RFLP coa gene in clinical isolates of S. aureus. 192 S. aureus isolates were collected from Namazi and Shahid Faghihi hospitals. Antibiotic resistance was measured by disk diffusion method and MIC was determined for gentamicin. The presence of genes encoding aminoglycoside modifying enzymes (AME) and mecA gene were assessed by PCR. Also the coagulase typing, PCR-RFLP coa gene, and SCCmec typing were performed. Out of 192 isolated S. aureus isolates, 83 (43.2%) MRSA isolates were identified. In this study, a high resistance to streptomycin and gentamicin (98.7%) were observed. Among the AME genes, the aac (6′)-Ie-aph (2″) gene was the most common. Based on the SCCmec typing, it was determined that the prevalence of SCCmec type III (45.8%) was highest. From the amplification of the coa gene, 5 different types were obtained. Also, in digestion of coa gene products by HaeIII enzyme, 10 different RFLP patterns were observed. According to this study, aminoglycoside resistance is increasing among MRSA isolates. As a result, monitoring and control of aminoglycoside resistance can be effective in the treatment of MRSA isolates. Also, typing of S. aureus isolates based on coagulase gene polymorphism is a suitable method for epidemiological studies.

Novel macrolide-lincosamide-streptogramin B resistance gene erm(54) in MRSA ST398 from Germany.

Macrolides are important broad-spectrum antimicrobial agents in human and veterinary medicine. Macrolide resistance genes are often located on mobile genetic elements (MGEs) in staphylococci, which can be horizontally transferred across strain, species or genus boundaries.1 The erm genes encode 23S rRNA methylases, which confer combined resistance to macrolide, lincosamide and streptogramin B (MLS) compounds by adding one or two methyl groups to the adenine A2058 in the 23S rRNA.1,2 Livestock-associated MRSA (LA-MRSA) are often multiresistant and mainly associated with ST398 in many parts of the world.3 They have their main reservoir in food-producing animals and, therefore, represent a considerable health risk for humans, especially for those having frequent contact with livestock.3 Macrolides are commonly used to treat bacterial infections in livestock and, consequently, different erm genes, including erm(A), erm(B), erm(C) and erm(T), have so far been detected on MGEs among LA-MRSA ST398.1,4,5 LA-MRSA ST398 isolate 3860 was obtained from the nasal swab of a pig suffering from respiratory disease in the German National Resistance Monitoring programme GERM-Vet in 2007. WGS on Illumina MiSeq and PacBio Sequel II platforms, followed by hybrid assembly and consecutive sequence analysis, revealed the presence of a novel erm gene, designated erm(54) by the Nomenclature Center for MLS Resistance Genes (https://faculty.washington.edu/marilynr), on the non-conjugative 36 929 bp plasmid pHKS3860 (GenBank accession number OM362497) (Figure 1a). Upstream of the erm(54) gene, a regulatory region, known as translational attenuator, was identified. This regulatory region was more complex compared with the corresponding regions of other known staphylococcal erm genes.1,6,7 Besides an open reading frame for a 30 amino acid (aa) regulatory peptide, seven pairs of inverted repeats (IRs), IR1a,b to IR7a,b, were detected. A putative stable mRNA secondary structure formed by the IR sequences was identified, in which the IR7b sequence, which covered in part the erm(54)-associated ribosome binding site and the start of the erm(54) gene, was freely accessible to ribosomes and thereby allowed the translation of the erm(54) transcript.6 Screening for inducible macrolide/lincosamide resistance by agar disc diffusion8 using erythromycin (15 μg), clindamycin (2 μg) and pirlimycin (2 μg) discs confirmed the constitutive expression of erm(54).

Livestock-associated methicillin-resistant Staphylococcus aureus in Czech retailed ready-to-eat meat products

This study was aimed on the detection of methicillin resistant Staphylococcus aureus (MRSA) in different categories of retailed ready-to-eat (RTE) meat products from the Czech producers and determination of their genetic properties, antimicrobial resistance and virulence. In RTE meat products, 2% (4/181) of examined samples were MRSA positive. MRSA strains were detected only in durable fermented meat products made exclusively from pork meat. Detection of livestock-associated MRSA (LA-MRSA) clonal lineages (ST398 and ST4999), SCCmec cassette type V and tetracycline resistance indicate a source of contamination from raw pork. The study confirms the ability of these strains to survive the technological process rather than contamination of meat products from the food processing environment. MRSA strains did not carry any of the tested genes encoding staphylococcal enterotoxins or virulence genes (for Panton-Valentine leukocidin, exfoliative toxins A, B and toxic shock syndrome). Our results point out the spread of LA-MRSA through the meat processing chain.

Expression of Staphylococcal Virulence Genes In Situ in Human Skin and Soft Tissue Infections.

Background: Staphylococcus aureus, the most common pathogen in skin and soft tissue infections (SSTI), harbors many well-characterized virulence genes. However, the expression of many of them in SSTIs is unknown. In this study, S. aureus virulence genes expressed in SSTI were investigated. Methods: Fifty-three subjects presenting to the outpatient’s care and emergency departments with a purulent SSTI at two medical centers in Wisconsin, USA, were enrolled in the study. Total mRNA was extracted from the purulent or swab materials, made into cDNA and sequenced on MiSeq platform. The relative cDNA counts to gmk and identifications of the transcripts were carried out with respect to USA300 reference genome and using SAMTOOLS v.1.3 and BWA, respectively. Result: A significantly higher cDNA count was observed for many of the virulence and regulatory gene transcripts in the pus samples compared to the swab samples relative to the cDNA counts for gmk, a housekeeping gene. They were for lukS-PV (18.6 vs. 14.2), isaA (13.4 vs. 8.5), ssaA (4.8 vs. 3.1), hlgC (1.4 vs. 1.33), atl (17.7 vs. 8.33), clfA (3.9 vs. 0.83), eno (6.04 vs. 3.16), fnbA (5.93 vs. 0.33), saeS (6.3 vs. 1.33), saeR (5.4 vs. 3.33) and agrC (5.6 vs. 1.5). Conclusions: A relative increase in the transcripts of several toxins, adhesion and regulatory genes with respect to a gmk in purulent materials suggests their role in situ during SSTIs, perhaps in an orchestrated manner.

Genomic Analysis of the Unusual Staphylococcus aureus ST630 Isolates Harboring WTA Glycosyltransferase Genes tarM and tagN.

ABSTRACTStaphylococcus aureus (S. aureus) can cause a broad spectrum of diseases ranging from skin infections to life-threatening diseases in both community and hospital settings. The surface-exposed wall teichoic acid (WTA) has a strong impact on host interaction, pathogenicity, horizontal gene transfer, and biofilm formation in S. aureus. The unusual S. aureus ST630 strains containing both ribitol-phosphate (RboP) WTA glycosyltransferase gene tarM and glycerol-phosphate (GroP) WTA glycosyltransferase gene tagN have been found recently. Native PAGE analysis showed that the WTA of tagN, tarM-encoding ST630 strains migrated slower than that of non-tagN-encoding ST630 strains, indicating the differences in WTA structure. Some mobile genetic elements (MGEs) such as the unique GroP-WTA biosynthetic gene cluster (SaGroWI), SCCmec element, and prophages that probably originated from the CoNS were identified in tagN, tarM-encoding ST630 strains. The SaGroWI element was first defined in S. aureus ST395 strain, which was refractory to exchange MGEs with typical RboP-WTA expressing S. aureus but could undergo horizontal gene transfer events with other species and genera via the specific bacteriophage Φ187. Overall, our data indicated that this rare ST630 was prone to acquire DNA from CoNS and might serve as a novel hub for the exchange of MGEs between CoNS and S. aureus.IMPORTANCE The structure of wall-anchored glycopolymers wall teichoic acid (WTA) produced by most Gram-positive bacteria is highly variable. While most dominant Staphylococcus aureus lineages produce poly-ribitol-phosphate (RboP) WTA, the tagN, tarM-encoding ST630 lineage probably has a poly-glycerol-phosphate (GroP) WTA backbone like coagulase-negative staphylococci (CoNS). There is growing evidence that staphylococcal horizontal gene transfer depends largely on transducing helper phages via WTA as the receptor. The structural difference of WTA greatly affects the transfer of mobile genetic elements among various bacteria. With the growing advances in sequencing and analysis technologies, genetic analysis has revolutionized research activities in the field of the important pathogen S. aureus. Here, we analyzed the molecular characteristics of ST630 and found an evolutionary link between ST630 and CoNS. Elucidating the genetic information of ST630 lineage will contribute to understanding the emergence and diversification of new pathogenic strains in S. aureus.

Characteristics of Virulence Genes of Clinically Isolated Staphylococci in Jingzhou Area.

Purpose The aim of this study was to further understand the distribution characteristics of staphylococcal virulence genes in the Jingzhou area, in order to provide a basis for clinically effective treatments and prevention and control measures. Methods A total of 181 strains of staphylococci were collected from Jingzhou Hospital Affiliated to Yangtze University from April 2013 to April 2021, which were divided into the methicillin-resistant Staphylococcus aureus (MRSA) strains and the methicillin-sensitive Staphylococcus aureus (MSSA) strains and coagulase-negative staphylococci (CoNS) by an antimicrobial susceptibility test and PCR method. The 73 MRSA strains were classified by staphylococcal cassette chromosome mec (SCCmec) and Staphylococcus protein A (Spa). Sea, sec, seh, sek, seb, seq, sep, Tsst-1, clfA, clfB, fnbA, hla, hld, hlg, lukE, bbp, cna, eap, ebpS, sdrC, sdrD, sdrE, and Pvl genes were also detected in all strains. The χ2 test was used for statistical analysis for comparison between groups. Results The 181 strains of staphylococci were divided into 97 strains of MRSA, 54 strains of MSSA, and 30 strains of CoNS. 73 MRSA strains were derived from clinical specimens such as lower respiratory tract, secretions, sepsis secreted by tissue infection, urine, and hydrothorax. There were 70 strains that can be identified including SCCmec types and 15 Spa types of all strains, while the most popular types were SCCmecIII-t030 and SCCmecIV-t437 from lower respiratory tract specimens. There were four virulence genes that were detected including seb, seq, clfB, and hld in CoNS strains, while the detection rates of these four virulence genes in Staphylococcus aureus were higher than that of CoNS, and the differences were statistically significant, P < 0.05(P=0.004, P=0.001, P=0.001, P=0.001). 23 virulence genes were detected in 151 strains of Staphylococcus aureus, among which the detection rate of the Tsst-1 gene was the lowest and that of the clfB gene was the highest, and the other genes were 4.6%∼98.0%. The detection rates of sea, sek, seb, seq, sep, cna, eap, ebpS, sdrC, and sdrE virulence genes in MRSA were higher than that of MSSA, and the differences were statistically significant, P < 0.05 (P=0.001, P=0.001, , P=0.001, P=0.001, P=0.009, , P=0.019, P=0.001, , P=0.001, P=0.001, , P=0.003). Conclusion The mainly prevalent type of MRSA strains in Jingzhou is SCCmecIII-t030 in lower respiratory tract specimens. Virulence genes of Staphylococcus aureus and antibiotic drug resistance rates are also different from other regions. In this experiment, virulence genes were also detected in CoNS, suggesting that more attention should be paid to the prevention and treatment of these strains clinically.