Agr Genes Research Articles

Antibiotic resistance and pathogenicity factors in Staphylococcus aureus isolated from mastitic Sahiwal cattle

Methicillin-resistant Staphylococcus aureus (MRSA) poses a serious problem in dairy animals suffering from mastitis. In the present study, the distribution of mastitic MRSA and antibiotic resistance was studied in 107 strains of S. aureus isolated from milk samples from 195 infected udders. The characterizations pathogenic factors (adhesin and toxin genes) and antibiotic susceptibility of isolates were carried out using gene amplification and disc diffusion assays, respectively. A high prevalence of MRSA was observed in the tested isolates (13.1%). The isolates were also highly resistant to antibiotics, i.e. 36.4% were resistant to streptomycin, 33.6% to oxytetracycline, 29.9% to gentamicin and 26.2% each to chloramphenicol, pristinomycin and ciprofloxacin. A significant variation in the expression of pathogenic factors (Ig, coa and clf) was observed in these isolates. The overall distribution of adhesin genes ebp, fib, bbp, fnbB, cap5, cap8, map and cna in the isolates was found to be 69.1, 67.2, 6.5, 20.5, 60.7, 26.1, 81.3 and 8.4%, respectively. The presence of fib, fnbB, bbp and map genes was considerably greater in MRSA than in methicillin-susceptible S. aureus (MSSA) isolates. The proportions of toxin genes, namely, hlb, seb, sec, sed, seg and sei, in the isolates were found to be 94.3, 0.9, 8.4, 0.9, 10.2 and 49.5%, respectively. The proportions of agr genes I, II, III and IV were found to be 39.2, 27.1, 21.5 and 12.1%, respectively. A few isolates showed similar antibiotic-resistance patterns, which could be due to identical strains or the dissemination of the same strains among animals. These findings can be utilized in mastitis treatment programmes and antimicrobials strategies in organized herd.

Molecular mapping of genes for resistance to the bean pod weevil (Apion godmani Wagner) in common bean

The bean pod weevil (Apion godmani Wagner) is a serious insect pest of common beans (Phaseolus vulgaris L.) grown in Mexico and Central America that is best controlled by host-plant resistance available in Durango or Jalisco genotypes such as J-117. Given unreliable infestation by the insect, the use of marker-assisted selection is desirable. In the present study, we developed a set of nine molecular markers for Apion resistance and mapped them to loci on chromosomes 2, 3, 4 and 6 (linkage groups b01, b08, b07 and b11, respectively) based on genetic analysis of an F (5:10) susceptible x resistant recombinant inbred line population (Jamapa x J-117) and two reference mapping populations (DOR364 x G19833 and BAT93 x JaloEEP558) for which chromosome and linkage group designations are known. All the markers were derived from randomly amplified polymorphic DNA (RAPD) bands that were identified through bulked segregant analysis and cloned for conversion to sequence tagged site (STS) markers. One of the markers was dominant while four detected polymorphism upon digestion with restriction enzymes. The other markers were mapped as RAPD fragments. Phenotypic data for the population was based on the evaluation of percentage seed damage in replicated trials conducted over four seasons in Mexico. In single point regression analysis, individual markers explained from 3.5 to 22.5% of the variance for the resistance trait with the most significant markers overall being F10-500S, U1-1400R, R20-1200S, W9-1300S and Z4-800S, all markers that mapped to chromosome 2 (b01). Two additional significant markers, B1-1400R and W6-800R, were mapped to chromosome 6 (b11) and explained from 4.3 to 10.2% of variance depending on the season. The latter of these markers was a dominant STS marker that may find immediate utility in marker-assisted selection. The association of these two loci with the Agr and Agm genes is discussed as well as the possibility of additional resistance genes on chromosome 4 (b07) and chromosome 3 (b08). These are among the first specific markers developed for tagging insect resistance in common bean and are expected to be useful for evaluating the mechanism of resistance to A. godmani.

Pheno and genotyping ofStaphylococcus aureus, isolated from bovine milk samples from São Paulo State, Brazil

In the present study, 87 Staphylococcus aureus isolates obtained from milk samples of 87 cows with mastitis in 6 different municipal districts of 2 regions of Sao Paulo State, Brazil, were compared pheno- and genotypically. Pulsed-field gel electrophoresis (PFGE) analysis of the strains was performed, and PCR was carried out to detect genes for a number of staphylococcal cell surface proteins, exoproteins, and 3 classes of agr genes. Nine distinct S. aureus lineages (LA-LI) were identified by PFGE. The lineages LA and LE, which accounted together for 63 strains (72.2%), were prevalent and had been collected from all of the 6 municipal districts, indicating a broad geographic distribution of these lineages; LB, LC, LD, LF, LG, LH, and LI, however, were isolated sporadically and accounted for 24 strains (27.8%). Some characteristics, like penicillin resistance and the presence of cap8 and agr class II genes, were associated with the prevalent lineages (LA and LE), and penicillin susceptibility and the presence of cna and cap5 genes were associated with sporadic lineages. According to the present results, some S. aureus lineages possess a combination of genes that confer the propensity to cause and disseminate infection, and only a limited number of clones are responsible for the cases of bovine mastitis on the various farms.

Host-parasite interactions in Staphylococcus aureus keratitis.

Ulcerative keratitis is among the leading ocular bacterial infections, and Streptococcus aureus accounts for approximately 25% of cases in some surveys. Although S. aureus expresses numerous virulence factors, many of which are under the control of staphylococcal global regulatory genes, their pathophysiologic roles in keratitis are largely unknown. Similarly, the nature of the host response during S. aureus keratitis is unclear. Following a review of previously published research on the pathophysiology of S. aureus ocular infection, we present the results of a study designed to assess the host-parasite relationship between S. aureus and human corneal epithelial cells (HCECs) in vitro. In this model system, a wild-type S. aureus strain and its isogenic mutants harboring mutations in agr and sar global regulatory genes or fibronectin-binding proteins A and B (fnbAB) were tested for their ability to bind and invade confluent HCEC monolayers. The contribution of host cell factors was assessed by preincubating HCECs with various inhibitory agents. These studies demonstrated that S. aureus not only adhered to the surface of HCECs but was also internalized, as has been previously observed in other nonocular cell lines. Adherence and invasion of HCECs was saturable at 1 h of incubation in the presence of approximately 10(7) CFU per HCEC monolayer (multiplicity of infection approximately 10). A mutant defective in both agr and sar global regulators was not significantly different in invasive capacity compared to its isogenic wild-type parent strain. In contrast, mutations in fibronectin-binding proteins A and B (fnbAB) reduced the invasiveness of S. aureus by 99% compared to the wild-type strain. Pretreatment of HCECs with colchicine had little effect on S. aureus invasion. In sharp contrast, cytochalasin D and genistein were each capable of inhibiting invasion by >99%. In summary, the results of this study point to fibronectin-binding protein as a key S. aureus surface adhesin facilitating invasion of HCECs in vitro. Furthermore, these results suggest an active mechanism for S. aureus internalization by HCECs, likely involving actin polymerization and tyrosine kinase activity. Additional studies are warranted to determine the applicability of these findings in vivo, and to facilitate the rational design of therapeutic agents aimed at blocking the establishment and progression of S. aureus keratitis.