Unearthing New ccr Genes and Staphylococcal Cassette Chromosome Elements in Staphylococci Through Genome Mining.

Staphylococcal cassette chromosome mec (SCCmec) typing is crucial for investigating methicillin-resistant Staphylococcus aureus (MRSA), relying primarily on the combination of ccr and mec gene complexes. To date, 19 ccr genes and 10 ccr gene complexes have been identified, forming 15 SCCmec types. With the vast release of bacterial genome sequences, mining the database for novel ccr gene complexes and SCC/SCCmec elements could enhance MRSA epidemiological studies. In this study, we identified 12 novel ccr genes (6 ccrA, 3 ccrB, and 3 ccrC) through mining of the National Center for Biotechnology Information (NCBI) database, forming 12 novel ccr gene complexes and 10 novel SCC elements. Overexpression of 5 groups of novel Ccr recombinases (CcrA9B3, CcrA10B1, CcrC3, CcrC4, and CcrC5) in a mutant MRSA strain lacking the ccr gene and extrachromosomal circular intermediate (ciSCC) production significantly promoted ciSCC production, demonstrating their biological activity. This discovery provides an opportunity to advance MRSA epidemiological research and develop database-based bacterial typing methods.

Genetic mutations in adaptive evolution of growth-independent vancomycin-tolerant Staphylococcus aureus.

BACKGROUND: Antibiotic tolerance allows bacteria to overcome antibiotic treatment transiently and potentially accelerates the emergence of resistance. However, our understanding of antibiotic tolerance at the genetic level during adaptive evolution of Staphylococcus aureus remains incomplete. We sought to identify the mutated genes and verify the role of these genes in the formation of vancomycin tolerance in S. aureus. METHODS: Vancomycin-susceptible S. aureus strain Newman was used to induce vancomycin-tolerant isolates in vitro by cyclic exposure under a high concentration of vancomycin (20× MIC). WGS and Sanger sequencing were performed to identify the genetic mutations. The function of mutated genes in vancomycin-tolerant isolates were verified by gene complementation. Other phenotypes of vancomycin-tolerant isolates were also determined, including mutation frequency, autolysis, lysostaphin susceptibility, cell wall thickness and cross-tolerance. RESULTS: A series of vancomycin-tolerant S. aureus (VTSA) strains were isolated and 18 mutated genes were identified by WGS. Among these genes, pbp4, htrA, stp1, pth and NWMN_1068 were confirmed to play roles in VTSA formation. Mutation of mutL promoted the emergence of VTSA. All VTSA showed no changes in growth phenotype. Instead, they exhibited reduced autolysis, decreased lysostaphin susceptibility and thickened cell walls. In addition, all VTSA strains were cross-tolerant to antibiotics targeting cell wall synthesis but not to quinolones and lipopeptides. CONCLUSIONS: Our results demonstrate that genetic mutations are responsible for emergence of phenotypic tolerance and formation of vancomycin tolerance may lie in cell wall changes in S. aureus.

Antibiotics trigger initiation of SCCmec transfer by inducing SOS responses.

The rise of antimicrobial resistance limits therapeutic options for infections by methicillin-resistant staphylococci. The staphylococcal cassette chromosome mec (SCCmec) is a mobile genetic element as the only carrier of the methicillin-resistance determinants, the mecA or mecC gene. The use of antibiotics increases the spread of antibiotic resistance, but the mechanism by which antibiotics promote horizontal dissemination of SCCmec is largely unknown. In this study, we demonstrate that many antibiotics, including ß-lactams, can induce the expression of ccrC1 and SCCmec excision from the bacterial chromosome. In particular, three widely used antibiotics targeting DNA replication and repair (sulfamethoxazole, ciprofloxacin and trimethoprim) induced higher levels of ccrC1 expression and higher rates of SCCmec excision even at low concentrations (1/8 × minimum inhibitory concentration). LexA was identified as a repressor of ccrC1 and ccrAB by binding to the promoter regions of ccrC1 and ccrAB. The activation of RecA after antibiotic induction alleviated the repression by LexA and increased the expression of ccrC1 or ccrAB, consequently increasing the excision frequency of the SCCmec for SCCmec transfer. These findings lead us to propose a mechanism by which antimicrobial agents can promote horizontal gene transfer of the mecA gene and facilitate the spread of methicillin resistance.

Effect of bla regulators on the susceptible phenotype and phenotypic conversion for oxacillin-susceptible mecA-positive staphylococcal isolates.

OBJECTIVES: The bla regulatory system is critical in the regulation of mecA expression particularly when staphylococci lack the mec regulator. We sought to evaluate the effect of bla regulators on the cryptic methicillin-resistant phenotype and resistance conversion under ß-lactam exposure in oxacillin-susceptible or oxacillin-resistant mecA-positive staphylococcal isolates. METHODS: Methicillin-resistant staphylococci isolates, NW19 and DY39, and their mutants, were used in this study. Both NW19 and DY39 carried intact mecA, a truncated mecR1 gene and a single copy of the bla regulatory system. Oxacillin MICs were determined using the agar dilution method. Increased and reduced expression of bla regulators was achieved by overexpression and antisense RNA, respectively. Expression of mecA, blaR1 and blaI was quantified in the presence or absence of oxacillin. RESULTS: NW19 had high expression of blaR1-blaI, low expression of mecA and was oxacillin susceptible, while DY39 expressed a low level of blaR1-blaI, expressed a high level of mecA and was oxacillin resistant. Increased expression of blaR1-blaI in DY39-RI led to an oxacillin-susceptible phenotype. Overexpressing blaR1 in DY39-R did not result in any phenotypic change. Under serial exposure of oxacillin, NW19, DY39-RI and DY39-R, with higher expression of blaR1, converted to be highly resistant at a faster speed compared with DY39 and NW19-KD, which had lower expression of blaR1. CONCLUSIONS: The expression level of BlaI was mainly responsible for the oxacillin-resistant phenotype in oxacillin-susceptible mecA-positive Staphylococcus without mec regulators. The initial amount of BlaR1 was determinative for the phenotypic conversion speed under ß-lactam exposure.

Screening of immunomodulatory and adhesive Lactobacillus with antagonistic activities against Salmonella from fermented vegetables.

The purpose of this study was to select strains of lactic acid bacteria (LAB) by their in vitro adhesive and immunomodulatory properties for potential use as probiotics. In this study, 16 randomly selected LAB strains from fermented vegetables (sauerkraut, bean and cabbage) were first screened for their tolerance to acid, bile salts, pepsin and pancreatin, bacterial inhibitory activities and abilities to adherence to Caco-2 cells. Then, 4 strains with the highest adhesion abilities were selected for further studies of their immunomodulatory properties and inhibitory effects against Salmonella adhesion and invasion to Caco-2 cells in vitro. The results showed that these 16 LAB strains effectively survived in simulated gastrointestinal condition and inhibited growth of six tested pathogens. Lactobacillus rhamnosus P1, Lactobacillus plantarum P2, Lactobacillus rhamnosus P3 and Lactobacillus casei P4 had the highest abilities to adhere to Caco-2 cells. Furthermore, L. plantarum P2 strain showed higher abilities to induce expression of tumor necrosis factor-α and interleukin-12 by splenic monocytes and strongly inhibited the adhesion and invasion of S. enteritidis ATCC13076 to Caco-2 cells. These results suggest that Lactobacillus strains P2 could be used as a probiotic candidate in food against Salmonella infection.

The use of fine nets to prevent the breeding of mosquitoes on dry farmland in southern Taiwan.

OBJECTIVES: The stagnate water stored in buckets traditionally used by farmers in southern Taiwan to irrigate their dry farmland, serves as favorable breeding ground for Aedes aegypti, the vector of the dengue virus. The public health bureau there distributed fine nets to the farmers to cover their buckets to reduce vector breeding sites. The goal of this study was to compare the container index (CI) in Alian and Tianliao Townships, Kaohsiung County in southern Taiwan over a 2-year period, for 1 year before the fine nets were distributed and 1 year after. METHODS: In March 2005, we selected eight villages in Alian Township and 10 in Tianliao Township. Specialists monitored the local Stegomyia indices every month. We compared the 2005 CI to the 2004 CI, representing the years before and after the nets were used. The results of the comparisons were analyzed by paired t-test and Wilcoxon rank test. RESULTS: In Alian Township, mean CI had significantly reduced to 1.63 levels (May 2005) from a mean 5.88 level in May 2004 (paired t-test, p=0.001 and Wilcoxon rank signed test, p=0.0012). In Tianliao Township, CI was reduced to 1.2 from 2.3 in May 2004 (p<0.0001 and p=0.007, paired t-test and Wilcoxon rank signed test, respectively). CONCLUSIONS: The nets effectively reduced the CIs in these townships, and might be considered for wide scale use. The mosquito nets were generally welcomed by the farmers who found them to be convenient, inexpensive and practical.