Complete reference genomes of two ceftriaxone-resistant Neisseria gonorrhoeae strains identified in routine surveillance in Bangkok, Thailand, using Nanopore Q20+ chemistry, VolTRAX V2b, and Illumina sequencing.

Ceftriaxone-resistant Neisseria gonorrhoeae strains, mostly associated with Asia, threaten gonorrhea treatment. We report the reference genomes of two ceftriaxone-resistant isolates found in routine surveillance in Bangkok, Thailand. The genomes belonged to the more antimicrobial-susceptible genomic lineage B, illustrating that both ceftriaxone-resistant strains and the mosaic penA-60.001 ceftriaxone-resistance determinant are spreading.

Occurrence of mcr-mediated colistin resistance in Salmonella clinical isolates in Thailand.

Nontyphoidal Salmonella, an important zoonotic pathogen and a major cause of foodborne illnesses, could be a potential reservoir of plasmids harbouring mobile colistin resistance gene (mcr). This study reported, for the first time, a high rate of mcr-carrying Salmonella clinical isolates (3.3%, 24/724) in Thailand, associated with mcr-3 gene (3.0%, 22/724) in S. 4,[5],12:i:-(15.4%, 4/26), S. Typhimurium (8.8%, 5/57), and S. Choleraesuis (5.6%, 13/231). Remarkably, the increasing trends of colistin and extended-spectrum cephalosporin resistances have displayed a high agreement over the years, with a dramatic rise in the mcr-carrying Salmonella from 1.1% (6/563) during 2005-2007 to 11.2% (18/161) during 2014-2018 when CTX-M-55 became abundant. Clonal and plasmid analysis revealed that the self-transferable IncA/C and a novel hybrid IncA/C-FIIs MDR plasmids were the major vehicles to disseminate both mcr-3 and blaCTX-M55 genes among diverse Salmonella strains, from as early as 2007. To our knowledge the occurrence of mcr-3 and the co-existence of it with blaCTX-M-55 in S. Choleraesuis are reported here for the first time, leading to clinical concern over the treatment of the invasive salmonellosis. This study provides evidence of the potential reservoirs and vectors in the dissemination of the mcr and highlights the co-selection by colistin and/or cephalosporins.

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.

Discovery of novel halogenated 8-hydroxyquinoline-based anti-MRSA agents: In vitro and QSAR studies.

Methicillin-resistant Staphylococcus aureus (MRSA) infection has been considered to be one of global health problems due to limited classes of effective antimicrobial drugs. Herein, 8-hydroxyquinoline (8HQ) and its derivatives (1-7) were investigated for their anti-MRSA and antioxidant activities. Cloxyquin (2), a halogenated 8HQ, exerted the highest antimicrobial activity (MIC50 ≤ 5.57 µM) with high safety index, whereas an amino-derivative 7 showed the strongest antioxidant activity. Additionally, quantitative structure-activity relationship (QSAR) study demonstrated that mass, polarizability, topological charge, and van der Waals volume are essential properties governing the anti-MRSA activity. Taken together, cloxyquin was highlighted as a promising compound for further development as a novel anti-MRSA agent. QSAR findings would also benefit for further rational design of novel 8HQ-based compounds to combat the MRSA resistance.

Nitroxoline: a potent antimicrobial agent against multidrug resistant Enterobacteriaceae.

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 antineurodegenerative activities. Herein, 8HQ (1), nitroxoline (NQ, 2) and 7-Br-8HQ (3) were investigated for antimicrobial activity against Enterobacteriaceae including extended spectrum ß-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 µM (8 µg/mL) and MBC50 of 168.28 µM (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.

Repositioning of 8-hydroxyquinoline derivatives as a new promising candidate for combating multidrug resistant Neisseria gonorrhoeae.

The multidrug resistance of Neisseria gonorrhoeae becomes a public health problem worldwide, especially the strain H041 that showed the decrease susceptibility to ceftriaxone which is the last resort for gonorrhea treatment. Therefore, the simultaneous discovery and development of a new compound to fight this pathogen is urgently required. In this study, 8-hydroxyquinoline (8HQ) and derivatives were evaluated for their antimicrobial activities against the gonococcal pathogen using spectinomycin as the reference drug. The results showed that 8HQ derivatives gave an excellent antimicrobial potency. Particularly, the dihalogenated 8HQ (iodoquinol, clioquinol and 5,7-diCl-8HQ) exerted the high activity with MIC range of 0.08-0.15 µM, 0.10-0.20 µM and 0.28-0.56 µM, respectively, compared with the reference drug (MIC = 16 µg/mL or 48.14 µM). Moreover, these compounds were also shown to be non-cytotoxic/very high safety index. The findings reveal that these three compounds could be further developed as a new antimicrobial agent for fighting the gonorrheal disease.

Multiplex PCR scheme for variant plasmid mediated class C ß-lactamase typing.

BACKGROUND: An increasing of prevalence and diversification of plasmid-mediated AmpC (pAmpC) has been emerged worldwide. The incidence of pAmpC resulted in increasing ß-lactamase production and conferred resistance to almost all ß-lactam antibiotics excluding carbapenems. The lack of standard method for pAmpC identification and classification exert a challenge in epidemiological surveillance and infection control practices. METHODS: A robust, single tube multiplex PCR has been developed to classify six different pAmpC groups including CIT (CMY-2 like, LAT and CFE), ECB (ACT, MIR), MOX & CMY-1 like, DHA, ACC, and FOX. The developed method was optimized and validated by testing of sensitivity and specificity. RESULTS: Developed method can detect crude extracted DNA template at nano-scale (2.5 ηg) and has high discriminatory power as compared to phenotypic and commercial genotypic method. CONCLUSION: The developed method can be utilized for tracking the changes of clinically important resistance patterns and further investigation of occurrence and distribution of plasmid-mediated AmpC types.

Comparative proteomics analysis of Neisseria gonorrhoeae strains in response to extended-spectrum cephalosporins.

Neisseria gonorrhoeae strains displaying reduced susceptibility and resistance to extended-spectrum cephalosporins (ESCs) are major public health concerns. Although resistance mechanisms of ESCs have extensively been studied, the proteome-wide investigation on the biological response to the antibiotic stress is still limited. Herein, a proteomics approach based on two-dimensional gel electrophoresis and MALDI-TOF/TOF-MS analysis was applied to investigate the global protein expression under ESC stresses of ESC-susceptible and ESC-reduced susceptible N. gonorrhoeae strains. Upon exposure to ceftriaxone, 14 and 21 proteins of ESC-susceptible and ESC-reduced susceptible strains, respectively, were shown to be differentially expressed. In the meanwhile, differential expressions of 13 and 17 proteins were detected under cefixime stress for ESC-susceptible and ESC-reduced susceptible strains, respectively. ESC antibiotics have been proven to trigger the expression of several proteins implicated in a variety of biological functions including transport system, energy metabolism, stress response and pathogenic virulence factors. Interestingly, macrophage infectivity potentiators (Ng-MIP) showed increased expression for ESC-reduced susceptible strain under ESC stress. The altered expression of Ng-MIP was found to be a unique response to ESC stresses. Our finding proposes a broad view on proteomic changes in N. gonorrhoeae in response to ESC antibiotics that provides further insights into the gonococcal antimicrobial resistance and physiological adaptation mechanism.

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 up-regulated 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-1ß) 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-1ß), resulting in the synthesis of acute phase protein, in particular, haptoglobin and C-reactive 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.

Proteochemometric model for predicting the inhibition of penicillin-binding proteins.

Neisseria gonorrhoeae infection threatens to become an untreatable sexually transmitted disease in the near future owing to the increasing emergence of N. gonorrhoeae strains with reduced susceptibility and resistance to the extended-spectrum cephalosporins (ESCs), i.e. ceftriaxone and cefixime, which are the last remaining option for first-line treatment of gonorrhea. Alteration of the penA gene, encoding penicillin-binding protein 2 (PBP2), is the main mechanism conferring penicillin resistance including reduced susceptibility and resistance to ESCs. To predict and investigate putative amino acid mutations causing ß-lactam resistance particularly for ESCs, we applied proteochemometric modeling to generalize N. gonorrhoeae susceptibility data for predicting the interaction of PBP2 with therapeutic ß-lactam antibiotics. This was afforded by correlating publicly available data on antimicrobial susceptibility of wild-type and mutant N. gonorrhoeae strains for penicillin-G, cefixime and ceftriaxone with 50 PBP2 protein sequence data using partial least-squares projections to latent structures. The generated model revealed excellent predictability (R2=0.91, Q2=0.77, QExt2=0.78). Moreover, our model identified amino acid mutations in PBP2 with the highest impact on antimicrobial susceptibility and provided information on physicochemical properties of amino acid mutations affecting antimicrobial susceptibility. Our model thus provided insight into the physicochemical basis for resistance development in PBP2 suggesting its use for predicting and monitoring novel PBP2 mutations that may emerge in the future.

Reference map and comparative proteomic analysis of Neisseria gonorrhoeae displaying high resistance against spectinomycin.

A proteome reference map of Neisseria gonorrhoeae was successfully established using two-dimensional gel electrophoresis in conjunction with matrix-assisted laser desorption ionization-time of flight mass spectrometry. This map was further applied to compare protein expression profiles of high-level spectinomycin-resistant (clinical isolate) and -susceptible (reference strain) N. gonorrhoeae following treatment with subminimal inhibitory concentrations (subMICs) of spectinomycin. Approximately 200 protein spots were visualized by Coomassie brilliant blue G-250 staining and 66 spots representing 58 unique proteins were subsequently identified. Most of the identified proteins were analysed as cytoplasmic proteins and belonged to the class of energy metabolism. Comparative proteomic analysis of whole protein expression of susceptible and resistant gonococci showed up to 96% similarity while eight proteins were found to be differentially expressed in the resistant strain. In the presence of subMICs of spectinomycin, it was found that 50S ribosomal protein L7/L12, an essential component for ribosomal translocation, was upregulated in both strains, ranging from 1.5- to 3.5-fold, suggesting compensatory mechanisms of N. gonorrhoeae in response to antibiotic that inhibits protein synthesis. Moreover, the differential expression of proteins involved in energy metabolism, amino acid biosynthesis, and the cell envelope was noticeably detected, indicating significant cellular responses and adaptation against antibiotic stress. Such knowledge provides valuable data, not only fundamental proteomic data, but also knowledge of the mode of action of antibiotic and secondary target proteins implicated in adaptation and compensatory mechanisms.

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.

Protocol for a facile multiplex PCR for multi-antimicrobial resistance and Gonococcus detection.

Gonorrhea is a continuing problem worldwide particularly in terms of the spread of multiple drug resistance. We have successfully developed an efficient PCR method for the simultaneous identification of gonococci and detection of the antimicrobial-resistant profile. By this method, penicillinase-producing Neisseria gonorrhoeae (PPNG), high-level tetracycline-resistant N. gonorrhoeae (TRNG), and ciprofloxacin-resistant N. gonorrhoeae (CRNG) can be clearly identified. Moreover, the plasmid-types of penicillin and tetracycline resistance are also characterized. The method has 100 % sensitivity and specificity. It is also time- and labor-saving compared to the conventional method. Thus, the procedure is suitable for epidemiological surveillance.

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'-GAGCGGCCAAAGGGAGCA 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.

One-step PCR for the identification of multiple antimicrobial resistance in Neisseria gonorrhoeae.

One-step multiplex PCR was developed for the identification of gonococci and antimicrobial-resistant profiles. From forty Neisseria gonorrhoeae isolates, the penicillinase-producing N. gonorrhoeae (PPNG), the high-level tetracycline-resistant N. gonorrhoeae (TRNG), and the ciprofloxacin-resistant N. gonorrhoeae (CRNG) were successfully classified. Our method provides expediency and benefit to epidemiology and antimicrobial-resistance mobility with 100% sensitivity and specificity for gonococcal-detection. The detection limit was 500 CFU/reaction.

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.