[Emergence of novel variants of gyrA, parC, qnrS genes in multi-drug resistant Klebsiella caused pneumonia].

OBJECTIVE: To investigate the resistant mechanism of quinolones on multi-drug resistant Klebsiella caused pneumonia (MDR-KPN). METHODS: From August 2008 to May 2010, 47 strains of MDR-KPN were collected from 6 hospitals in Hangzhou and Huzhou in Zhejiang province in China. Drug target genes to quinolones (gyrA, parC) and quinolone-resistance genes mediated by mobile genetic elements [qnrA, qnrB, qnrS, aac (6')-Ib-cr, qepA] were analyzed by PCR and verified by DNA sequencing. RESULTS: Positive results were found in 47 strains of MDR-KPN, 43 strains (91.5%) of gyrA mutation, 40 strains (85.1%) of parC mutation, 3 strains (6.4%) of qnrB2, 1 strain (2.1%) of qnrB 4, 8 strains (17.0%) of qnrS 1, 5 strains (10.6%) of qnrS 4, 2 strains (4.3%) of aac (6')-Ib-cr respectively. Moreover, 5 novel variants of gyrA (GenBank accession number: JN811952, JN811953, JN811954, JN811955, JN811956), 5 novel variants of parC (GenBank accession number: JN817432, JN817433, JN817434, JN817435, JN817436) were also identified. In addition, qnrS4 (GenBank accession number: JN836269) appeared to be the novel variants of qnrS. CONCLUSION: Quinolone-resistance-determining region played a key role on the resistance to quinolones in this group of MDR-KPN, and quinolone-resistance genes mediated by mobile genetic elements [qnrB2, qnrB4, qnrS1, qnrS4, aac (6')-Ib-cr] showed positive in some parts of the strains. This was the first report on emergence of qnrS4 in the world.

Salvianolate inhibits cytokine gene expression in small intestine of cirrhotic rats.

AIM: To study the effect of salvianolate on expression of tumor necrosis factor (TNF)-α and interleukin (IL)-6 mRNA in small intestine of cirrhotic rats. METHODS: Cirrhosis in rats was induced using CCl4 (0.3 mL/kg). Rats were randomly divided into non-treatment group, low-dose salvianolate (12 mg/kg) treatment group, medium-dose salvianolate (24 mg/kg) treatment group, and high-dose salvianolate (48 mg/kg) treatment group, and treated for 2 wk. Another 10 healthy rats served as a normal control group. Mortality of cirrhotic rats in each group was evaluated after treatment with salvianolate. Serum samples were taken from portal vein for the detection of endotoxin. Morphological changes in tissue samples from the ileocecum were observed under a light microscope. Expression of TNF-α and IL-6 mRNA in the small intestine of rats was analyzed by real-time reverse-transcriptase polymerase chain reaction. RESULTS: The mortality of cirrhotic rats in the non-treatment group was 37.5%. No cirrhotic rat died in the high-dose salvianolate treatment group. The serum endotoxin level was significantly higher in the non-treatment group than in the salvianolate treatment and normal control groups. The intestinal mucosal and villous atrophy, necrosis and shedding of the intestinal mucosal epithelium, observed in the non-treatment group, were reversed in different salvianolate treatment groups. The TNF-α and IL-6 mRNA expression levels in small intestine were significantly lower in different salvianolate treatment groups than in the non-treatment group. CONCLUSION: Salvianolate can reduce the endotoxin level, ameliorate the injury of intestinal mucosa, and inhibit the expression of TNF-α and IL-6 mRNA in small intestine of cirrhotic rats.

In vitro susceptibility testing of Aspergillus spp. against voriconazole, itraconazole, posaconazole, amphotericin B and caspofungin.

BACKGROUND: During recent years, the incidence of serious infections caused by opportunistic fungi has increased dramatically due to alterations of the immune status of patients with hematological diseases, malignant tumors, transplantations and so forth. Unfortunately, the wide use of triazole antifungal agents to treat these infections has lead to the emergence of Aspergillus spp. resistant to triazoles. The present study was to assess the in vitro activities of five antifungal agents (voriconazole, itraconazole, posaconazole, amphotericin B and caspofungin) against different kinds of Aspergillus spp. that are commonly encountered in the clinical setting. METHODS: The agar-based Etest MIC method was employed. One hundred and seven strains of Aspergillus spp. (5 species) were collected and prepared according to Etest Technique Manuel. Etest MICs were determined with RPMI agar containing 2% glucose and were read after incubation for 48 hours at 35°C. MIC(50), MIC(90) and MIC range were acquired by Whonet 5.4 software. RESULTS: The MIC(90) of caspofungin against A. fumigatus, A. flavus and A. nidulans was 0.094 µg/ml whereas the MIC(90) against A. niger was 0.19 µg/ml. For these four species, the MIC(90) of caspofungin was the lowest among the five antifungal agents. For A. terrus, the MIC(90) of posaconazole was the lowest. For A. fumigatus and A. flavus, the MIC(90) in order of increasing was caspofungin, posaconazole, voriconazole, itraconazole, and amphotericin B. The MIC of amphotericin B against A. terrus was higher than 32 µg/ml in all 7 strains tested. CONCLUSIONS: The in vitro antifungal susceptibility test shows the new drug caspofungin, which is a kind of echinocandins, has good activity against the five species of Aspergillus spp. and all the triazoles tested have better in vitro activity than traditional amphotericin B.

[Analysis of detection and antimicrobial resistance of pathogens in prostatic secretion from 1186 infertile men with chronic prostatitis].

OBJECTIVE: To investigate the distribution and the antimicrobial resistance of the bacteria, mycoplasma and Chlamydia trachomatis isolated from the prostatic secretion of infertile men with chronic prostatitis, and to provide clinicians with grounds for choosing antibiotic agents. METHODS: The bacteria obtained were isolated and identified, the Chlamydia trachomatis was detected by FLO-PCR, and the results were analysed statistically. RESULTS: In 1 186 specimens of EPS, the total positive rate of isolates was 51.7%. Among them, there were 364 strains of gram-positive coccus, 20 gram-negative bacillus, 5 other strains and 157 mycoplasma, including 116 Ureaplasma urealyticum and 41 Mycoplasma hominis, and 67 Chlamydia trachomatis DNA. As for the isolated staphylococci, their antimicrobial resistance was the lowest against vancomycin (0.0%), but the highest against penicillins (76.9%-100%); for the Streptococcus agalactiae, it was the highest against erythromycin and clindamycin (100%), and the lowest against beta-lactams, aminoglycosides, trimethoprim + sulfamethoxazole, rifampin and vancomycin (0.0%); for the Ureaplasma urealyticum, it was the highest against ciprofloxacin (59.5%), and the lowest against josamycin, tetracycline and fosfomycin (1.7%); for the Mycoplasma hominis, it was the highest against erythromycin (100%), and the lowest against doxycycline and fosfomycin (0.0%). CONCLUSION: Bacteria, mycoplasma and Chlamydia trachomatis are the possible etiological factors of male infertility. Isolated bacterial strains differ greatly in their resistance against different antibiotics.

[Effects of sodium nitroprusside on P38MAPK/STAT3 activation and telomerase reverse transcriptase mRNA expression in inducing apoptosis of K562 cell line].

This study was aimed to investigate the activation of P38MAPK/STAT3 and expression of telomerase reverse transcriptase during sodium nitroprusside (SNP) inducing apoptosis of human leukemia cell line K562 and to explore the molecular mechanisms of SNP-inducing apoptosis in K562 cells. The K562 cell were treated with different concentrations of SNP and were cultured for different time. Cell apoptosis was analysed by cell morphology, DNA agarose gel electrophoresis, DNA content, and Annexin-V/PI labeling method. The TdT-mediated dUTP nick end labeling (TUNEL) assay was used to quantitate the in situ cell apoptosis. The expressions of phosphorylated p38MAPK or STAT3 were analysed by flow cytometry, while the expression of hTERT mRNA in transcriptional level was measured by fluorescence quantitative RT-PCR. The results showed that SNP inhibited K562 cell growth. The K562 cell apoptosis was confirmed by typical cell morphology and DNA fragment, peak of sub-G1 phase, TUNEL and Annexin-V/PI labeling. A majority of K562 cells were arrested in G0/G1 phase. After treatment with SNP at 0.5-3.0 mmol/L, the expression of phosphorylated-P38MAPK and phosphorylated-STAT3 increased first and decreased afterwards. Incubation of K562 cell with SNP (2 mmol/L) could increase the expression of phosphorylated-P38MAPK and phosphorylated-STAT3 at 12 hours and 24 hours respectively, and down-regulated at 72 hours and 48 hours. SNP could decrease the expression of hTERT-mRNA in time-and dose-dependent manner. It is concluded that SNP can significantly induce K562 cells apoptosis, its mechanism may be related to the activation of P38MAPK and suppression of phosphorylated-STAT3 and hTRET-mRNA.

[Distribution of MIF -173 single nucleotide polymorphism checked by tetra-primer amplification refractory mutation system].

OBJECTIVE: To study the distribution of macrophagemigration inhibitory factor gene (MIF) -173 single nucleotide polymorphism (SNP) of Chinese Han population in Zhejiang province. METHODS: The DNA samples were extracted from EDTA blood of 142 unrelated healthy individuals. Alleles of MIF -173 SNP were genotyped by using the techniques of tetra-primer amplification refractory mutation system (ARMS) and restriction fragment length polymorphisms (RFLP)-PCR Meantime the PCR products were cloned and sequenced. RESULTS: The authors detected three kinds of genotypes at the MIF -173 locus, and no deviation was observed from Hardy-Weinberg equilibrium. The final results were the same completely, whatever either tetra-primer ARMS or RFLP-PCR was used to check the MIF -173 single nucleotide polymorphism. Statistical analysis showed that the distributions of MIF -173 SNP alleles and genotype frequencies were significantly different between Chinese Han population and European Caucasian(P< 0.01), but no significant difference demonstrated to happen between Chinese and Japanese(P> 0.05). CONCLUSION: Tetra-primer ARMS is an accurate, rapid and economical method for SNP genotyping.There exists ethnic difference in the distribution of MIF -173 SNP alleles.