Epidemiology and molecular characterization of Staphylococcus aureus causing bovine mastitis in water buffaloes from the Hazara division of Khyber Pakhtunkhwa, Pakistan.

Buffalo represent a major source of milk in Pakistan. However, production is impacted by the disease bovine mastitis. Mastitis causes significant economic losses, with Staphylococcus aureus (S. aureus) being one of its major causative agents. While much work has been done understanding the epidemiology of bovine mastitis in Pakistan, detailed molecular characterization of the associated S. aureus is unavailable. In the current study both the epidemiological and molecular characterization of S. aureus from bovine mastitis in the Hazara division of Pakistan are examined. S. aureus was isolated from 18.41% of the animals, and left quarters more prone to infection (69.6%) than right quarters (30.4%). Sub-clinical mastitis (75.31%) was more prevalent than clinical mastitis (24.69%), with infections evenly distributed amongst the eight districts. Molecular characterization revealed that only 19.6% of the isolates were methicillin-resistant, and four strains types identified, including ST9-t7867-MSSA, ST9-MSSA, ST101-t2078-MSSA, and ST22-t8934-MRSA-IVa. Antiseptic resistance genes were not detected in the isolates, and low levels of antibiotic resistance were also noted, however the methicillin-resistant strains had higher overall antibiotic resistance. This study represents the most complete molecular typing data for S. aureus causing bovine mastitis in the Hazara district of Pakistan, and the country as a whole.

Staphylococcus aureus ST59: Concurrent but Separate Evolution of North American and East Asian Lineages.

Despite initially being described in North America, Staphylococcus aureus (SA) sequence type ST59 is the most commonly isolated sequence type in Eastern Asia. The origins and evolution of this strain type remains unclear and therefore we gathered a collection of ST59 isolates from Canada and mainland China for a detailed genetic analysis of the lineage. Bayesian inference phylogenomic analysis of our isolates, along with previously published ST59 sequences indicated that the lineage could be divided into 6 distinct subgroups (WGS-1 thorough 6), each having distinct molecular characteristics. Analysis also demonstrated the concurrent but separate evolution of North American and East Asian lineages, as well as the extensive diversification of the East Asian lineage. The presence of a mobile element structure (MES) was found to be the major difference between these two continental lineages, absent in all North American isolates, and present in all East Asian ones. Other mobile genetic elements, such as the Immune Evasion Complex (IEC), Panton Valentine Leukocidin (PVL), and Staphylococcal Cassette Chromosome mec (SCCmec), showed significant variability within each sub-group and likely represents local selective pressures rather than major characteristics defining the groups. Our analysis also demonstrated the existence of a more ancient ST59 sub-lineage from North America, which was MES negative and contained some of the earliest reported ST59 isolates. Combined with the existence of a MES negative isolate from Taiwan, predicted to have appeared prior to diversification of the East Asian lineages, these results hint at the possibility of a North American origin for the lineage, which gained hold in Eastern Asia following acquisition of MES, and subsequently diversified.

A Novel Assay for Detection of Methicillin-Resistant Staphylococcus aureus Directly From Clinical Samples.

The timely detection of Methicillin-resistant Staphylococcus aureus (MRSA) is crucial for antimicrobial therapy and a key factor to limit the hospital spread of MRSA. Currently available commercial MRSA detection assays target the 3' end of the orfX gene and the right extremity of Staphylococcal Cassette Chromosome mec (SCCmec). These assays suffer from both false positive due to SCC-like elements that lack mecA and false negative results due to the inability to detect new or variant SCCmec cassettes with the existing primers. We developed a novel MRSA detection scheme, designed to circumvent issues present in the existing commercial assays. Our assay demonstrated specificity and accuracy, capable of detecting prototypic strains of SCCmec types I-XIII [C(t) values ranged 8.58-26.29]. Previous false positive isolates (N = 19) by Xpert MRSA nasal assay were accurately classified with our assay. Further validation with 218 randomly selected clinical isolates (73 MRSA, 75 MSSA, 43 MR-CoNS, and 27 MS-CoNS) confirmed its feasibility and practicality. Testing assay performance with 88 direct clinical swabs from 33 patients showed that the assay was 96.6% in agreement with clinical culture results. Our novel MRSA detection assay targets both the S. aureus specific sequence and the mecA/mecC genes simultaneously to overcome the false positive and false negative deficits of currently available commercial assays. The results validate our assay and confirmed its feasibility and practicality. The assay is not affected by SCCmec types and only needs modification if new mec homologs emerge and establishes a new platform for other emerging SCCmec types.

Molecular Characterization and Pathogenicity of Staphylococcus aureus Isolated from Benin-City, Nigeria.

While numerous studies examine the epidemiology and molecular characterization of Staphylococcus aureus in most developed countries, the detailed molecular characterization and molecular epidemiology of S. aureus strains and clones in Africa is lacking. We determined the molecular epidemiology and virulence of 81 non-duplicate isolates of S. aureus from Benin-City, Nigeria, collected during January-July 2016, and compared with global strains. Forty-seven isolates (58.0%) were found to be methicillin-sensitive Staphylococcus aureus (MSSA), while 34 (42.0%) were methicillin-resistant Staphylococcus aureus (MRSA). ST152-MSSA (24.7%) and ST7-MRSA-V (19.8%) were the dominant groups identified, which were not genetically related to global predominant strains, but rather exhibited regional dominance. An interesting finding of the study was the presence of highly related strains in the region, which differed primarily in their methicillin resistance gene carriage, staphylococcal cassette chromosome mec (SCCmec), with 99.4-99.7% relatedness between the genomes of the strains within the MRSA-MSSA pairs. This suggest that the strains within a pair are experiencing gain or loss of SCCmec within local conditions, with evolution continuing to diversify the strains to a small degree. This study represents the most comprehensive genetic and virulence study of S. aureus in Nigeria.

The KLK5 protease suppresses breast cancer by repressing the mevalonate pathway.

Kallikrein-related peptidase 5 (KLK5) displays aberrant expression in cancer. However, any functional association is missing. Here, we show that reconstitution of KLK5 expression in non-expressing MDA-MB-231 breast cancer cells suppresses malignancy in vitro and in vivo dose-dependently. Reactivation of KLK5 suppressed key EMT genes. Unexpectedly, we identified altered expression of genes encoding enzymes of the mevalonate pathway typical of those observed upon cholesterol starvation. Consistently, we found that SREBF1, the master regulator of the mevalonate pathway was induced. KLK5 re-expression leads to reduced cellular cholesterol and fatty acid synthesis and enhanced uptake of LDL-cholesterol. Suppression of the mevalonate pathway in KLK5 transfectants was further shown by reduced synthesis of isoprenoids. Indeed, we found diminished levels of active RhoA, a signaling oncoprotein that requires prenylation for activation. We propose that reduced RhoA activation plays a dominant role in suppression of malignancy by KLK5, since geranylgeranyl pyrophosphate restored active RhoA in KLK5-reverted cells resulting in increased malignancy. For the first time, we suggest that a protease may suppress breast cancer by modulating the mevalonate pathway.

Phytochemical and comparative antibacterial studies on the crude ethanol and aqueous extracts of the leaves of Lecaniodiscus cupanoides Planch (Sapindaceae).

Phytochemical investigation was carried out on the leaves of Lecaniodiscus cupanoides Planch (Sapindaceae). The diameter of the zones of inhibition of the 90% ethanol and aqueous extracts of the leaves were compared in order to determine the relative activity of the extracts against the tested microorganisms and also to verify its claimed ethnomedicinal use in the treatment of microbial infections. Phytochemical tests were carried out employing standard procedures. The antimicrobial activity of the extract was tested against standard strains and clinical isolates of some aerobic bacteria using the agar well diffusion method. Commercial antibiotics were used as positive reference standards to determine the sensitivity of the strains. The minimum inhibitory concentration (MIC) values were also determined using the agar well diffusion method. Preliminary phytochemical studies revealed the presence of flavonoids, tannins, saponins and cardiac glycosides as the chemical classes of compounds present in the crude extract. The extracts showed inhibitory activity against clinical isolates of Bacillus subtilis. Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa Staphylococcus aureus and a standard strain of Staphylococcus aureus (NCTC 10788). The ethanol extract was more active than the aqueous extract against all the microorganisms tested, except against the clinical isolates of Staphylococcus aureus. The MIC values ranged from 2.5 to 6.25 mg/mL for all the organisms tested. The results showed that the ethanol extract was more potent than the aqueous extract. The broad spectrum of activity displayed by the extracts would appear to provide the scientific basis for the use of the leaves of Lecaniodicus cupanoides for dressing of boils, burns and cuts in ethnomedicine.

Antimicrobial evaluation of methanolic extract of Colliandra surinamensis on some pathogenic organisms.

The crude extract of Colliandra surinamensis is used in traditional medicine for the treatment of some diseases/infections. The crude extract obtained from the flower of Colliandra surinamensis was found to have antimicrobial activity against some Gram positive organisms such as Staphylococcus aureus, Bacillus subtilis and Steptococcus species. The performance of the extract against the bacteria isolates was favorably comparable with established commercial antibacterial agents.