Increasing trend of multiple resistance and genomic mobility of Neisseria gonorrhoeae to penicillin and quinolone

A significant decline of gonorrhea incidence has been observed during the years 1990-99. However, a slight increase in the number of cases has been reported in 2000. In addition, an increase in resistant strains has been found in Thailand. In this study, 207 isolates of N. gonorrhoeae from patients attending Bangrak hospital (National Centre of Sexually Transmitted Infections), 67 isolates obtained during January-March 2000, 74 isolates obtained during January-March 2002, and 66 isolates obtained during October-December 2002, were tested. All isolates were susceptible to ceftriaxone while 71.5% and 74.4% were resistant to penicillin and quinolone, respectively. The high level of ciprofloxacin resistance (MIC =4 mg/L) also increased from 13.4% during January-March 2000 to 25.8% during October-December 2002. Multiple resistance determinants commonly coexisted in a single isolate so that the level of resistance was increased. The incidence of double resistance determinants, penicillin and quinolone resistance, were significantly increased from 34.3% among isolates during January to March 2000 up to 77.3% among isolates during October to December 2002 (P 1.024 g/L). Several plasmid patterns have been identified and various patterns of the plasmid can be artificially transferred and maintained their expression in Escherichia coli transformants. Such evidences infer the high mobility of resistant genome among microorganisms in the region. Moreover, the significant increase in penicillin and quinolone resistance herein, indicates the selective pressure and the diversity of genomic distribution among N. gonorrhoeae in Thailand. Primers JDA (5-TAC TCA ATC GGT AAT TGG CTT C-3) and JDB (5-CCA TAT CAC CGT CGG TAC TG-3) have been designed from sequences of the Asia, the Africa, and the Toronto ß-lactamase plasmids. By using the JDA and the JDB as PCR primers, our data reveal the highest prevalence and a significantly increasing trend of the epidemic Africa type of genomic ß-lactamase

Activities of thiotetrahydropyridines as antioxidant and antimicrobial agents

Tetrahydropyridines have been reported previously as important medicinal agents. The present study, thiotetrahydropyridines were prepared and tested for antioxidants (DPPH and SOD assays) and antimicrobials (agar dilution method). The results show that 1-acetyl-1,2,3,4- and 1,2,3,6-thiotetrahydropyridines 15a-b, 16, 17 and 18a are new antioxidants that scavenge superoxide and free radicals. Whereas the analogs 15a and 16 are novel antimicrobials. Significantly, 1-acetyl-2-(1-adamantylthio)-3,4-diacetoxy-1,2,3,4-tetrahydropyridine (15a) is the most potent compound that inhibits the growth of Streptococcus pyogenes and Moraxella catarrhalis with MIC of 32 µg/mL, of Corynebacterium diphtheriae NCTC 10356 and of Vibrio cholerae (MIC of 64 µg/mL). Remarkably, the analog 15a is the most potent antioxidant and antimicrobial agent. This finding reveals a new and unique group of 1-acetyl-1,2,3,4-thiotetrahydropyridines as interesting lead compound with potential to be further developed for medicinal applications

Retinol-binding protein 4 and its potential roles in hypercholesterolemia revealed by proteomics

Effects of hypercholesterolemia on alterations of serum proteins have not been fully elucidated. Herein, using two-dimensional gel electrophoresis (2-DE) in conjunction with LC-MS searching has successfully been carried out to investigate the change of protein expression profiles as consequences of raised blood cholesterol at different levels (normal group: total cholesterol < 200 mg/dL; borderline high group: total cholesterol 200-239 mg/dL; and high group: total cholesterol ≥ 240 mg/dL) (n = 45). Results revealed that down-regulation of retinol-binding protein 4 (RBP4) (-2.26 fold), transthyretin (-1.25 fold) and gelsolin (-1.47 fold) was observed in the high group. Meanwhile, the other proteins such as haptoglobin, complement factor B and CD5 antigen-like protein were upregulated upto +3.24, +1.96 and +2.04 fold, respectively. Confirmation by Western blotting revealed a significant reduction of RBP4 (approximately 50 %) in individual samples derived from the high group. Presumptive conclusion can be drawn that down-regulation of RBP4 might be attributable to the inflammation of adipocytes caused by the release of proinflammatory cytokines (e.g. tumor necrosis factor α and interleukin-1β) from adipose tissues. Moreover, the decrease of transthyretin might also be taken into accounts since it is known that the transthyretin usually forms complex with RBP4 to prevent glomerular filtration and excretion through the kidney. The suppressing effect on RBP4 should be potentiated by the increase of complement factor B and CD5 antigen-like protein, which rendered the adipose tissues to overwhelm the liberation of RBP4 to blood circulation by metabolic and inflammatory processes. Such inflammation could further modulate the induction of cytokine release (e.g. IL-6 and IL-1β), resulting in the synthesis of acute phase protein, in particular, haptoglobin and Creactive proteins from hepatocytes. However, the mechanism of gelsolin reduction remains unclear. Among these differentially expressed proteins, the RBP4 has been proposed as a major linkage between hypercholesterolemia, adipose tissues, liver and kidney, which is believed to be a potential biomarker for metabolic and cardiovascular disorders associated with dyslipidemia in the future

Roles of kininogen-1, basement membrane specific heparan sulfate proteoglycan core protein, and roundabout homolog 4 as potential urinary protein biomarkers in diabetic nephropathy

Diabetic nephropathy, a major complication of diabetes mellitus (DM), is increasing worldwide and the large majority of patients have type 2 DM. Microalbuminuria has been used as a diagnostic marker of diabetic nephro­pathy. But owing to its insufficient sensitivity and specificity, other biomarkers are being sought. In addition, the pathophysiology of diabetic nephropathy is not fully understood and declines in renal function occur even without microalbuminuria. In this study, we investigated urinary proteins from three study groups (controls, and type 2 diabetic subjects with or without microalbuminuria). Non-targeted label-free Nano-LC QTOF analysis was conducted to discover underlying mechanisms and protein networks, and targeted label-free Nano-LC QTOF with SWATH was performed to qualify discovered protein candidates. Twenty-eight proteins were identified as candidates and functionally analyzed via String DB, gene ontology and pathway analysis. Four predictive mechanisms were analyzed: i) response to stimulus, ii) platelet activation, signaling and aggregation, iii) ECM-receptor interaction, and iv) angiogenesis. These mechanisms can provoke kidney dysfunction in type 2 diabetic patients via endothelial cell damage and glomerulus structural alteration. Based on these analyses, three proteins (kininogen-1, basement membrane-specific heparan sulfate proteoglycan core protein, and roundabout homolog 4) were proposed for further study as potential biomarkers. Our findings provide insights that may improve methods for both prevention and diagnosis of diabetic nephropathy

Beta-Lactamase in Haemophilus ducreyi: Purification, Characterization, and Mutagenesis

Chancroid caused by Haemophilus ducreyi, has been described as a significantly predisposing factor of HIV heterosexual transmission in an endemic region of both diseases. Antibiotic resistances are extensively found in H. ducreyi and many of the resistances are due to the presence of resistance plasmids. A high tendency of drug resistances has commonly been found among isolates derived in Thailand. In the research described in this thesis, plasmids of sixty-three H. ducreyi isolates from Thailand were purified and analyzed. Plasmid DNA was further cloned into Escherichia coli and ampicillin resistant transformants were selected and characterized by plasmid profiles and susceptibility patterns to various b-lactams. A 3.6-kb ampicillin resistance plasmid (pCb) was subsequently purified and characterized. The pCb b-lactamase could be isolated from a culture filtrate and further purified by ammonium sulfate and Chelating Sepharose Fast Flow loaded with Zn2+. The enzyme belonged to a class A b-lactamase which had 99% identity to the ampicillin resistance transposon Tn3 of pBR322 (TEM-1). Two nucleotides were different between the E. coli (Tn3) and H. ducreyi (pCb) genes that affected the amino acid sequence. Beta-lactamase TEM-1 (pCb) derivatives; E104K, R164D, D179R, E104K+R164D, E104K+D179R, R164D+D179R, and E104K+R164D+D179R were constructed by site-directed mutagenesis. E104K and D179R showed less of a catalytic effect than R164D. The mutant clones containing the R164D substitution showed a reduction in the minimal inhibitory concentration (MIC) of ampicillin. However, the E104K+R164D and E104K+R164D+D179R mutant clones elevated the MIC of ceftazidime and cefotaxime. Furthermore, the dissociation constant for the inhibitor binding value (Ki) of clavulanic acid for the mutant b-lactamases carrying the R164D substitution was much higher than the parental enzyme. In addition, two b-lactamases, penicillinase type I from Bacillus cereus and TEM-1 (pCb) b-lactamase, were immobilized on a Chelating Sepharose Fast Flow column loaded with Ni2+ in an active form. Flow-injection analysis of b-lactams was performed by using an enzyme column reactor fitted into the enzyme thermistor. With both enzymes it was possible to monitor both penicillins and cephalosporins

Bioactive Metabolites from Spilanthes acmella Murr.

Spilanthes acmella Murr. (Compositae) has been used as a traditional medicine for toothache, rheumatism and fever. Its extracts had been shown to exhibit vasorelaxant and antioxidant activities. Herein, its antimicrobial, antioxidant and cytotoxic activities were evaluated. Agar dilution method assays against 27 strains of microorganisms were performed. Results showed that fractions from the chloroform and methanol extracts inhibited the growth of many tested organisms, e.g. Corynebacterium diphtheriae NCTC 10356 with minimum inhibitory concentration (MIC) of 64-256 mg/mL and Bacillus subtilis ATCC 6633 with MIC of 128-256 mg/mL. The tested fractions all exhibited antioxidant properties in both DPPH and SOD assays. Potent radical scavenging activity was observed in the DPPH assay. No cytotoxic effects of the extracts against KB and HuCCA-1 cell lines were evident. Bioassay-guided isolation resulted in a diverse group of bioactive compounds such as phenolics [vanillic acid (2), trans-ferulic acid (5) and trans-isoferulic acid (6)], coumarin (scopoletin, 4) and triterpenoids like 3-acetylaleuritolic acid (1), b-sitostenone (3), stigmasterol and stigmasteryl-3-O-b-D-glucopyranosides, in addition to a mixture of stigmasteryl-and b-sitosteryl-3-O-b-D-glucopyranosides. The compounds 1–6 represent bioactive metabolites of S. acmella Murr. that were never previously reported. Our findings demonstrate for the first time the potential benefits of this medicinal plant as a rich source of high therapeutic value compounds for medicines, cosmetics, supplements and as a health food

Antimicrobial resistance markers as a monitoring index of gonorrhoea in Thailand

Multiplex PCR was applied to explore the antimicrobial-resistance profiles of 145 gonococci isolated from Bangrak Hospital, Thailand in 2007. All isolates were clearly identified for the plasmid-mediated resistant types of penicillin (Asia, Africa and Toronto) and tetracycline (American and Dutch). This method can also predict the decreased susceptibility to ciprofloxacin by detection of Ser-91 mutation. Prevalence rates of penicillinase-producing Neisseria gonorrhoeae (PPNG) and high-level tetracycline-resistance N. gonorrhoeae (TRNG) were shown to be high as 82.1% and 84.1%, respectively. Most PPNG carried the Africa-type (78.2%) while the American-type (61.8%) was harboured in most TRNG. Mono- and triple-resistance patterns were presented in 2.6% and 79.5% of male, 20.7% and 62.1% of men who have sex with men (MSM), 0% and 75.0% of female, and 10% and 70% of female sex workers (FSW). Additionally, the rate of the Dutch type was high in patients among the age of 35–44 years (57.1%) and female patients (43.8%). The changing types of plasmids have been noticed during the time period of study. The multi-resistance patterns of the gonococcal isolates can be used as an epidemiological index of gonorrhoea and human sexual behaviours. This information will support the management of individual patients as well as the public health surveillance

Bioactive Metabolites from Spilanthes acmella Murr

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Antibiograms and Randomly Amplified Polymorphic DNA-Polymerase Chain Reactions (RAPD-PCR) as Epidemiological Markers of Gonorrhea

The development of antimicrobial resistance of Neisseria gonorrhoeae arising from wide dissemination of resistant clones is a major global health problem. In this study, a total of 235 isolates of N. gonorrhoeae isolated from patients of Bangrak Hospital were tested for their antibiotic susceptibilities to penicillin, norfloxacin, ofloxacin, ciprofloxacin, spectinomycin, and ceftriaxone. Mutation (Ser-91) in the quinolone resistance determining regions of gyrA and random amplification of the polymorphic DNA polymerase chain reaction (RAPD-PCR) were examined from 145 isolates. Among these, 55 isolates were obtained during January-March 2000, 46 isolates during January-March 2002, and 44 isolates during October-December 2002. The occurrence of combination resistance between penicillin and quinolone was 20% in January-March 2000, which was increased to 57.8% during the period of October-December 2002 (P<0.0001). Mutation of Ser-91 in gyrA could be directly linked with the resistance or declining of susceptibility to ciprofloxacin. Using RAPD-PCR, we could classify the 145 isolates into 4 and 5 groups by primers D11344 (5'-AGTGAATTCGCGGTGAGATGCCA-3') and D8635 (5'-GAGCGGCCAAAGGGAG-CA GAC-3'), respectively. Combination of the data obtained from these two primers produced 11 fingerprint groups. Our findings conclude that monitoring of the Ser-91 mutation of gyrA and RAPD-PCR methods are most useful for epidemiological screening. (C) 2010 Wiley-Liss, Inc

Nitroxoline

Antimicrobial resistance has become a prime global concern. An ability of the microbes to produce enzymes to destroy antimicrobial drugs is one of the well-known mechanisms underlying the resistance. 8-Hydroxyquinoline (8HQ) and derivatives were reported to exert diverse biological effects such as antimicrobial, antioxidant and an- tineurodegenerative activities. Herein, 8HQ (1), nitroxoline (NQ, 2) and 7-Br-8HQ (3) were investigated for antimicrobial activity against Enterobacteriaceae including extended spectrum β-lactamase (ESBL)-producing and carbapenemase-producing strains as well as the effect of metal ions .These compounds (1-3) displayed the great antimicrobial activity against fifty-eight bacterial isolates of Escherichia coli, Providencia rettgeri and Klebsiella pneumoniae, in which NQ (2) exerted the highest antimicrobial activity with a MIC50 of 42.04 ❍M (8 µg/mL) and MBC50 of 168.28 ❍M (32 µg/mL). The MIC values of NQ (2) and 7-Br-8HQ (3) were significantly increased in the presence of Cu2+ and Fe3+. This finding reveals that NQ could be an effective compound to be further developed as an antimicrobial agent for combating Enterobacteriaceae infections