Comparative genotypic characterization related to antibiotic resistance phenotypes of clinical carbapenem-resistant Acinetobacter baumannii MTC1106 (ST2) and MTC0619 (ST25).

BACKGROUND: The prevalence of Acinetobacter baumannii in nosocomial infections and its remarkable ability to develop antimicrobial resistance have been a critical issue in hospital settings. Here, we examined the genomic features related to resistance phenotype displayed by carbapenem-resistant A. baumannii (CRAB) MTC1106 (ST2) and MTC0619 (ST25). RESULTS: Resistome analysis of both strains revealed that MTC1106 possessed higher numbers of antimicrobial resistance genes compared to MTC0619. Some of those genetic determinants were present in accordance with the susceptibility profile of the isolates. The predicted ISAba1 region upstream of blaOXA-23 gene was related to carbapenem resistance since this IS element was well-characterized to mediate overexpression of carbapenemase genes and eventually provided capability to confer resistance. Unlike MTC0619 strain, which only carried class B and D ß-lactamase genes, MTC1106 strain also possessed blaTEM-1D, a class A ß-lactamase. Regarding to aminoglycosides resistance, MTC0619 contained 5 related genes in which all of them belonged to three groups of aminoglycosides modifying enzyme (AME), namely, N-acetyltransferase (AAC), O-nucleotidyltransferase (ANT), and O-phosphotransferase (APH). On the other hand, MTC1106 lacked only the AAC of which found in MTC0619, yet it also carried an armA gene encoding for 16S rRNA methyltransferase. Two macrolides resistance genes, mph(E) and msr(E), were identified next to the armA gene of MTC1106 isolate in which they encoded for macrolide 2'-phosphotransferase and ABC-type efflux pump, respectively. Besides acquired resistance genes, some chromosomal genes and SNPs associated with resistance to fluoroquinolones (i.e. gyrA and parC) and colistin (i.e. pmrCAB, eptA, and emrAB) were observed. However, gene expression analysis suggested that the genetic determinants significantly contributing to low-level colistin resistance remained unclear. In addition, similar number of efflux pumps genes were identified in both lineages with only the absence of adeC, a part of adeABC RND-type multidrug efflux pump in MTC0619 strain. CONCLUSIONS: We found that MTC1106 strain harbored more antimicrobial resistance genes and showed higher resistance to antibiotics than MTC0619 strain. Regarding genomic characterization, this study was likely the first genome comparative analysis of CARB that specifically included isolates belonging to ST2 and ST25 which were widely spread in Thailand. Taken altogether, this study suggests the importance to monitor the resistance status of circulating A. baumannii clones and identify genes that may contribute to shifting the resistance trend among isolates.

Cocultures of Enterococcus faecium and Aeromonas veronii Induce the Secretion of Bacteriocin-like Substances against Aeromonas.

Lactic acid bacteria (LAB) were screened from Lutjanus russellii (red sea bass), and their antimicrobial activities were evaluated against two Aeromonas species isolated from the Nile tilapia, namely, Aeromonas veronii (AV) and Aeromonas jandaei (AJ). Three LAB isolates, Enterococcus faecium MU8 (EF_8), Enterococcus faecalis MU2 (EFL_2), and E. faecalis MU9 (EFL_9), were found to inhibit both AV and AJ; however, their cell-free supernatant (CFS) did not do so. Interestingly, bacteriocin-like substances (BLS) induced by cocultures of EF_8 with AV exhibited the highest antimicrobial activity against both Aeromonas sp. The size of BLS was less than 1.0 kDa; the purified BLS were susceptible to proteinase K digestion, indicating that they are peptides. BLS contained 13 identified peptides derived from E. faecium, as determined by liquid chromatography-tandem mass spectrometry. Cocultures of Gram-positive-producing and -inducing LAB strains have been used to increase bacteriocin yields. To our knowledge, this is the first report describing inducible BLS produced by cocultures of Gram-positive-producing and Gram-negative-inducing strains.

CRISPR-Based Gene Editing in Acinetobacter baumannii to Combat Antimicrobial Resistance.

Antimicrobial resistance (AMR) poses a significant threat to the health, social, environment, and economic sectors on a global scale and requires serious attention to addressing this issue. Acinetobacter baumannii was given top priority among infectious bacteria because of its extensive resistance to nearly all antibiotic classes and treatment options. Carbapenem-resistant A. baumannii is classified as one of the critical-priority pathogens on the World Health Organization (WHO) priority list of antibiotic-resistant bacteria for effective drug development. Although available genetic manipulation approaches are successful in A. baumannii laboratory strains, they are limited when employed on newly acquired clinical strains since such strains have higher levels of AMR than those used to select them for genetic manipulation. Recently, the CRISPR-Cas (Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein) system has emerged as one of the most effective, efficient, and precise methods of genome editing and offers target-specific gene editing of AMR genes in a specific bacterial strain. CRISPR-based genome editing has been successfully applied in various bacterial strains to combat AMR; however, this strategy has not yet been extensively explored in A. baumannii. This review provides detailed insight into the progress, current scenario, and future potential of CRISPR-Cas usage for AMR-related gene manipulation in A. baumannii.

Classification of Southeast Asian mints (Mentha spp.) based on simple sequence repeat markers.

Mentha is a complex genus encompassing many species as a consequence of their interspecific hybridization and polyploidy. Southeast Asian mints have been poorly distinguished though they are widely used for culinary and medical purposes. In this study, we have analyzed Southeast Asian mints and known varieties as well as a related Lamiaceae species (Nepeta sp.) using simple sequence repeat (SSR) markers and leaf morphology. Two types of mints were clearly distinguished based on their venation pattern and leaf shape index. We developed 12 SSR markers that allowed good amplification in the Mentha and another Lamiaceae species. In the SSR-based phylogram, the Mentha lines could be delimited into groups I-VI. The Southeast Asian mints divided into groups I and II, and the phylogram separated most of the available species, with groups I and II containing the known species M. × cordifolia and M. arvensis, respectively. The separation of the two groups was supported by a population structure analysis. The SSR markers developed in this study enabled the simultaneous classification of mints and will help improve our understanding of the genetic composition of known mint varieties and as yet unclassified Southeast Asian mints.

Toxin-Antitoxin Systems: A Key Role on Persister Formation in Salmonella enterica Serovar Typhimurium.

The toxin and antitoxin modules in bacteria consist of a toxin molecule that has activity to inhibit various cellular processes and its cognate antitoxin that neutralizes the toxin. This system is considered taking part in the formation of persister cells, which are a subpopulation of recalcitrant cells able to survive antimicrobial treatment without any resistance mechanisms. Importantly, persisters have been associated with long-term infections and treatment failures in healthcare settings. It is a public health concern since persisters can be involved in the evolution and dissemination of antimicrobial resistance amidst the aggravating spread of multidrug-resistant bacteria and insufficient novel antimicrobial therapy to tackle this issue. Salmonella enterica serovar Typhimurium is one of the most prevalent Salmonella serotypes in the world and is a leading cause of food-borne salmonellosis. S. Typhimurium has been known to cause persistent infection and a wealth of investigations on Salmonella persisters indicates that toxin and antitoxin modules play a role in mediating the phenotypic switch of persisters, rendering its survival ability in the presence of antimicrobial agents. In this review, we discuss findings regarding mechanisms that underly persistence in S. Typhimurium, especially the involvement of toxin and antitoxin modules.

An Update of Mobile Colistin Resistance in Non-Fermentative Gram-Negative Bacilli.

Colistin, the last resort for multidrug and extensively drug-resistant bacterial infection treatment, was reintroduced after being avoided in clinical settings from the 1970s to the 1990s because of its high toxicity. Colistin is considered a crucial treatment option for Acinetobacter baumannii and Pseudomonas aeruginosa, which are listed as critical priority pathogens for new antibiotics by the World Health Organization. The resistance mechanisms of colistin are considered to be chromosomally encoded, and no horizontal transfer has been reported. Nevertheless, in November 2015, a transmissible resistance mechanism of colistin, called mobile colistin resistance (MCR), was discovered. Up to ten families with MCR and more than 100 variants of Gram-negative bacteria have been reported worldwide. Even though few have been reported from Acinetobacter spp. and Pseudomonas spp., it is important to closely monitor the epidemiology of mcr genes in these pathogens. Therefore, this review focuses on the most recent update on colistin resistance and the epidemiology of mcr genes among non-fermentative Gram-negative bacilli, especially Acinetobacter spp. and P. aeruginosa.

Comparative study of phenotypic-based detection assays for carbapenemase-producing Acinetobacter baumannii with a proposed algorithm in resource-limited settings.

The increasing incidence of carbapenem resistance in Acinetobacter baumannii is a critical concern worldwide owing to the limitations of therapeutic alternatives. The most important carbapenem resistance mechanism for A. baumannii is the enzymatic hydrolysis mediated by carbapenemases, mostly OXA-type carbapenemases (class D) and, to a lesser extent, metallo-ß-lactamases (class B). Therefore, early and accurate detection of carbapenemase-producing A. baumannii is required to achieve the therapeutic efficacy of such infections. Many methods for carbapenemase detection have been proposed as effective tests for A. baumannii; however, none of them are officially recommended. In this study, three carbapenemase detection methods, namely, CarbaAcineto NP test, modified carbapenem inactivation method (mCIM), and simplified carbapenem inactivation method (sCIM) were evaluated for phenotypic detection of clinically isolated A. baumannii. The MICs of imipenem, meropenem, and doripenem were determined for 123 clinically isolated A. baumannii strains before performing three phenotypic detections. The overall sensitivity and specificity values were 89.09%/100% for the carbAcineto NP test, 71.82%/100% for sCIM, and 32.73%/33.13% for mCIM. CarbAcineto NP test and sCIM performed excellently (100% sensitivity) when both Class B and Class D carbapenemases were present in the same isolate. Based on the results, the combined detection method of sCIM and CarbAcineto NP test was proposed to detect carbapenemase-producing A. baumannii rather than a single assay, significantly increasing the sensitivity of detection to 98.18%. The proposed algorithm was more reliable and cost-effective than the CarbAcineto NP test alone. It can be easily applied in routine microbiology laboratories for developing countries with limited resources.

Predominance of international clone 2 multidrug-resistant Acinetobacter baumannii clinical isolates in Thailand: a nationwide study.

BACKGROUND: Acinetobacter baumannii has emerged as one of the common multidrug resistance pathogens causing hospital-acquired infections. This study was conducted to elucidate the distribution of antimicrobial resistance genes in the bacterial population in Thailand. Multidrug-resistant A. baumannii (MDR A. baumannii) isolates were characterized phenotypically, and the molecular epidemiology of clinical isolates in 11 tertiary hospitals was investigated at a country-wide level. METHODS: A total of 135 nonrepetitive MDR A. baumannii isolates collected from tertiary care hospitals across 5 regions of Thailand were examined for antibiotic susceptibility, resistance genes, and sequence types. Multilocus sequence typing (MLST) was performed to characterize the spread of regional lineages. RESULTS: ST2 belonging to IC2 was the most dominant sequence type in Thailand (65.19%), and to a lesser extent, there was also evidence of the spread of ST164 (10.37%), ST129 (3.70%), ST16 (2.96%), ST98 (2.96%), ST25 (2.96%), ST215 (2.22%), ST338 (1.48%), and ST745 (1.48%). The novel sequence types ST1551, ST1552, ST1553, and ST1557 were also identified in this study. Among these, the blaoxa-23 gene was by far the most widespread in MDR A. baumannii, while the blaoxa-24/40 and blaoxa-58 genes appeared to be less dominant in this region. The results demonstrated that the predominant class D carbapenemase was blaOXA-23, followed by the class B carbapenemase blaNDM-like, while the mcr-1 gene was not observed in any isolate. Most of the MDR A. baumannii isolates were resistant to ceftazidime (99.23%), gentamicin (91.85%), amikacin (82.96%), and ciprofloxacin (97.78%), while all of them were resistant to carbapenems. The results suggested that colistin could still be effective against MDR A. baumannii in this region. CONCLUSION: This is the first molecular epidemiological analysis of MDR A. baumannii clinical isolates at the national level in Thailand to date. Studies on the clonal relatedness of MDR A. baumannii isolates could generate useful data to understand the local epidemiology and international comparisons of nosocomial outbreaks.

Prevalence of OXA-Type ß-Lactamase Genes among Carbapenem-Resistant Acinetobacter baumannii Clinical Isolates in Thailand.

Carbapenem-resistant Acinetobacter baumannii (CRAB) is a critical health concern for the treatment of infectious diseases. The aim of this study was to investigate the molecular epidemiology of CRAB emphasizing the presence of oxacillinase (OXA)-type ß-lactamase-encoding genes, one of the most important carbapenem resistance mechanisms. In this study, a total of 183 non-repetitive CRAB isolates collected from 11 tertiary care hospitals across Thailand were investigated. As a result, the blaoxa-51-like gene, an intrinsic enzyme marker, was detected in all clinical isolates. The blaoxa-23-like gene was presented in the majority of isolates (68.31%). In contrast, the prevalence rates of blaoxa-40/24-like and blaoxa-58-like gene occurrences in CRAB isolates were only 4.92% and 1.09%, respectively. All isolates were resistant to carbapenems, with 100% resistance to imipenem, followed by meropenem (98.91%) and doripenem (94.54%). Most isolates showed high resistance rates to ciprofloxacin (97.81%), ceftazidime (96.72%), gentamicin (91.26%), and amikacin (80.87%). Interestingly, colistin was found to be a potential drug of choice due to the high susceptibility of the tested isolates to this antimicrobial (87.98%). Most CRAB isolates in Thailand were of ST2 lineage, but some belonged to ST25, ST98, ST129, ST164, ST215, ST338, and ST745. Further studies to monitor the spread of carbapenem-resistant OXA-type ß-lactamase genes from A. baumannii in hospital settings are warranted.

Investigation on the Epoxidation of Piperitenone, and Structure-activity Relationships of Piperitenone Oxide for Differentiation-inducing Activity.

Piperitenone oxide, a major chemical constituent of the essential oil of spearmint, Mentha spicata, induces differentiation in human colon cancer RCM-1 cells. In this study, piperitenone oxide and trans-piperitenone dioxide were prepared as racemic forms by epoxidation of piperitenone. The relative configuration between two epoxides in piperitenone dioxide was determined to be trans by 1H NMR analysis and nuclear Overhauser effect spectroscopy (NOESY) in conjunction with density functional theory (DFT) calculations. Optical resolution of (±)-piperitenone oxide by high-performance liquid chromatography (HPLC) using a chiral stationary phase (CSP) afforded both enantiomers with over 98% enantiomeric excess (ee). Evaluation of the differentiation-inducing activity of the synthetic compounds revealed that the epoxide at C-1 and C-6 in piperitenone oxide is important for the activity, and (+)-piperitenone oxide has stronger activity than (-)-piperitenone oxide. The results obtained in this study provide new information on the application of piperitenone oxide and spearmint for differentiation-inducing therapy. Furthermore, natural piperitenone oxide was isolated from M. spicata. The enantiomeric excess of the isolated natural piperitenone oxide was 66% ee. Epoxidation of piperitenone with hydrogen peroxide proceeded in a phosphate buffer under weak basic conditions to give (±)-piperitenone oxide. These results suggest that the nonenzymatic epoxidation of piperitenone, which causes a decrease in the enantiomeric excess of natural piperitenone oxide, is accompanied by an enzymatic epoxidation in the biosynthesis of piperitenone oxide.

Effect of Sucrier Banana Peel Extracts on Inhibition of Melanogenesis through the ERK Signaling Pathway.

Hyperpigmentation is a type of pigmentary disorder induced by overexpression of melanin content activated severe esthetic problems as melasma, freckle, ephelides, lentigo and other forms on human skin. Several whitening agents have restricted use because of their side effects or stability such as kojic acid, ascorbic acid and hydroquinone can act as cytotoxic substance which associated to dermatitis and skin cancer. To find for the safe substance, this study aimed to find for the ability of several components in Sucrier banana peel (SBP) extracts to inhibit melanogenesis process through p38 signaling pathway in B16F10 mouse melanoma cells. Tyrosinase activity and the cellular melanin content were dose dependent manner decreasing after SBP treatment. Furthermore, SBP decreased the expression of melanogenesis relate protein as microphthalmia-associated transcription factor (MITF) and tyrosinase protein after 24 hours incubation with α-melanocyte stimulating hormones (MSH) stimulating. The findings demonstrated that SBP contained an effective agent for hyperpigmentation inhibitor through p38 signaling pathways without any effect to ERK pathway, and subsequent down-regulate MITF expression and tyrosinase enzyme family production.

Cladogynos orientalis Zipp. extracts inhibit cell culture-derived hepatitis C virus genotype 2a replication in Huh-7 cells through NS5B inhibition

Objective: To evaluate the potential anti-hepatitis C virus (HCV) activities of Cladogynos orientalis Zipp. ex Span and to investigate the molecular mode of action. Methods: Ethanolic and water extracts from various parts of Cladogynos orientalis were examined for cytotoxicity by MTT assay. Sub-cytotoxic concentrations of the extracts were used for further determining anti-HCV activity using cell culture-derived HCV genotype 2a propagated in HepaRG cell line. Immunofluorescence assay was performed to observe the effect on viruses at the pre-entry step. Mode of action at the post-entry step was investigated for the viral RNA and protein expressions by real time RT-PCR and Western blotting assays, respectively. Results: Although Cladogynos orientalis water extracts exhibited lower cytotoxicity than ethanolic extracts, all ethanolic extracts from roots, stems, and leaves of Cladogynos orientalis exhibited higher anti-HCV activities than water extracts. The highest anti-HCV activity was observed in infected cells treated with the extracts 5 h after absorption. No extracts showed pre-viral entry effect. At the post-viral entry step, only leaf ethanolic extracts inhibited NS5B expression, while all extracts did not inhibit HCV NS3 expression. Conclusions: Cladogynos orientalis ethanolic extracts could be further studied and the major active compound needs to be identified as a promising source for anti-HCV agents.

Phenotypic and genotypic characterizations of extended-spectrum beta-lactamase-producing Escherichia coli in Thailand.

PURPOSE: Extended-spectrum ß-lactamases (ESBLs) have become an issue in community worldwide due to an increase in antibiotic resistance over the past decade. This study was aimed to investigate the phenotypic and genotypic characteristics of ESBL-producing Escherichia coli in Thailand. MATERIALS AND METHODS: In this study, all clinical isolates collected from tertiary hospitals in Thailand were identified as E. coli by biochemical tests and MALDI-TOF mass spectrometry. ESBL-producing E. coli was preliminary screened with disk diffusion method by cephalosporin disks and confirmed by the method of combination disk diffusion. Antimicrobial susceptibility test was used to determine MIC values of all ESBL-producing E. coli. For genotypic detection, a variety of ESBL genes were determined by PCR. Moreover, multilocus sequence typing (MLST) analysis was performed on internal portions of seven housekeeping genes for the diversity and phylogenetic relatedness of E. coli clonal group. RESULTS: Of the 285 ESBL-producing E. coli, most were susceptible to carbapenems. These strains showed a high resistance rate to ciprofloxacin (85.26%). The most frequently detected gene was bla CTX-M1 group at about 71.23% followed by bla CTX-M9 group (38.95%). The bla TEM, bla PER, bla GES, bla VEB, and bla SHV genes were identified in 31.93%, 5.96%, 4.56%, 3.51%, and 0.70% of ESBL-producing isolates, respectively. The bla OXA-10 gene was detected in only one strain. ESBL-producing E. coli isolates with high antimicrobial resistance were further investigated. Among those, E. coli sequence type ST38 was mostly found, followed by ST405, ST410, and ST131. It is noteworthy that the bla CTX-M gene was mainly detected in all four ST-type E. coli clones (ST38, ST405, ST410, and ST131). CONCLUSION: This study provided a recent evidence of the genetic diversity of ESBL-producing E. coli in Thailand. In addition, the profile related to antimicrobial resistance pattern in this region was also demonstrated.

In vitro assessment of Enterococcus faecalis MTC 1032 as the potential probiotic in food supplements.

Probiotics become important bacteria in our daily life due to their benefit on human health. In this study, a subset of bacterial strains from children was isolated and evaluated for beneficial probiotic traits such as antimicrobial activity, bile and acid tolerance, and pathogenic cell adherence inhibition. The strain with the best antimicrobial activity was selected for further characterization on the basis of morphological, biochemical characteristics and gene sequence. This strain was Gram-positive, oxidase and catalase-negative, and it produced acids by fermenting sugar and starch as carbon sources. Additionally, it could only hydrolyze bile-esculin, but not red blood cells. The 16S rDNA gene sequence revealed that this strain was Enterococcus faecalis. Interestingly, this strain effectively inhibited a variety of pathogens by acid and bacteriocin production and was bile-tolerant, able to survive under acidic condition. In the safety assessments, E. faecalis MTC 1032 could adhere to host epithelial cells and evidently inhibited pathogenic cell adhesion as demonstrated by cell reduction over time of E. coli ATCC 25922 and S. typhimurium ATCC 13311 on Caco-2 cell line. In summary, it was clearly represented that E. faecalis MTC 1032 provided suitable properties and could be a candidate as a probiotic strain in food supplements.

Chemopreventive and biological activities of Helicteres isora L. fruit extracts.

Helicteres isora L. (H. isora) has been used in traditional medicine in Asia. This study was aimed to determine biological activities of H. isora fruit extracts. Chemopreventive effect was examined by cell proliferation assay and differentiation-inducing effect. Anti-inflammatory activity of extracts was studied on the levels of nitric oxide (NO), tumor necrosis factor alpha (TNF-α), production of prostaglandin E2 (PGE-2), and cyclooxygenas-2 (COX-2). Cell proliferation assay revealed that H. isora extracts and its major compound, rosmarinic acid, showed no cytotoxicity in THP-1 and RCM-1 cells. Methylthio acetic acid from Cucumis melo var.conomon used as a positive control and 80% ethanol extracts demonstrated significant cell differentiation induction. Hexane extract of H. isora could lower the levels of TNF-α, PGE-2, and NO in THP-1 cells with 51.61 ± 0.79%, 69.68 ± 0.017%, and 69.93 ± 9.41% inhibition, respectively. The highest inhibitory effect on COX-2 was obtained from dichloromethane extract. Dexamethasone inhibited the secretion of TNF-α with 95.82 ± 0.50% while celecoxib showed the inhibitory effect on COX-2 and PGE-2 with 100% and 99.86%, respectively. The ethanol extract showed the best antioxidant activity by DPPH and FRAP assays at IC50 of 5.43 ± 1.01 µg/mL and 22.83 ± 0.13 mmol FeSO4/g sample, respectively, while the positive control, trolox, showed the antioxidant activity with IC50 and FRAP values at 4.08 ± 0.85 µg/mL and 10.84 ± 0.04 mmol FeSO4/g sample, respectively. Taken together, H. isora possess chemopreventive and antioxidant activity. Further studies on in vivo activities of this plant are suggested.

Genotypic detection of the blaCTX-M-1 gene among extended-spectrum ß-lactamase-producing Enterobacteriaceae.

OBJECTIVES: Extended-spectrum ß-lactamases (ESBLs), a group of ß-lactamase enzymes produced by bacteria in the family Enterobacteriaceae, are becoming a major problem in the healthcare community worldwide. Although many attempts have been made in the detection of ESBL-producing bacteria, the cost and speed of detection remains an important challenge. Therefore, this study aimed to develop a rapid, effective and affordable method for detection of the blaCTX-M-1 ESBL gene by a loop-mediated isothermal amplification (LAMP) technique. METHODS: Clinical ESBL-producing Enterobacteriaceae, including Escherichia coli and Klebsiella pneumoniae, were isolated and were used as representative strains. The double-disk synergy method was performed to detect ESBL-producing Enterobacteriaceae. Performance of the LAMP method in the detection of blaCTX-M-1 was compared with conventional PCR in terms of sensitivity and specificity. RESULTS: The developed LAMP method efficiently identified the presence of the blaCTX-M-1 gene in ESBL-producing Enterobacteriaceae. It provided similar results to conventional PCR, but the LAMP technique required only 20min of testing time. The accuracy of the LAMP method was confirmed by restriction digestion, which showed the predicted size of the blaCTX-M-1 gene. In addition, the developed method was comparable with PCR that amplified only the target blaCTX-M-1 gene in terms of specificity, but LAMP was ca. 1000-fold more sensitive than PCR. CONCLUSIONS: A rapid assay to detect ESBL-producing Enterobacteriaceae by a LAMP technique was developed in this study. The developed method is sensitive and suitable for rapid screening of blaCTX-M-1 in routine laboratories with limited resources.

Loop-mediated isothermal amplification assay targeting the blaCTX-M9 gene for detection of extended spectrum ß-lactamase-producing Escherichia coli and Klebsiella pneumoniae.

Extended-spectrum ß-lactamases (ESBLs) produced by Enterobacteriaceae are one of the resistance mechanisms to most ß-lactam antibiotics. ESBLs are currently a major problem in both hospitals and community settings worldwide. Rapid and reliable means of detecting ESBL-producing bacteria is necessary for identification, prevention and treatment. Loop-mediated isothermal amplification (LAMP) is a technique that rapidly amplifies DNA with high specificity and sensitivity under isothermal conditions. This study was aimed to develop a convenient, accurate and inexpensive method for detecting ESBL-producing bacteria by a LAMP technique. ESBLs-producing Escherichia coli and Klebsiella pneumoniae were isolated from a tertiary hospital in Bangkok, Thailand and reconfirmed by double-disk synergy test. A set of four specific oligonucleotide primers of LAMP for detection of bla(CTX-M9) gene was designed based on bla(CTX-M9) from E. coli (GenBank Accession No. AJ416345). The LAMP reaction was amplified under isothermal temperature at 63°C for 60 min. Ladder-like patterns of band sizes from 226 bp of the bla(CTX-M9) DNA target was observed. The LAMP product was further analyzed by restriction digestion with MboI and TaqI endonucleases. The fragments generated were approximately 168, 177 and 250 bp in size for MboI digestion and 165, 193, 229, 281 and 314 bp for TaqI digestion, which is in agreement with the predicted sizes. The sensitivity of the LAMP technique to bla(CTX-M9) was greater than that of the PCR method by at least 10,000-fold. These results showed that the LAMP primers specifically amplified only the bla(CTX-M9) gene. Moreover, the presence of LAMP amplicon was simply determined by adding SYBR Green I in the reaction. In conclusion, this technique for detection of ESBLs is convenient, reliable and easy to perform routinely in hospitals or laboratory units in developing countries.

Purification and characterization of anti-HIV-1 protein from Canna indica L. leaves.

A novel 10 kDa protein with anti-HIV-1 reverse transcriptase (RT) inhibitory activity was isolated from leaves of Canna indica L. using a combination of native-PAGE and ammonium sulfate precipitation. HIV-1 and RT inhibitory activity was measured using a syncytium forming (deltaTat/Rev) MC99 virus in Tat/Rev transfected 1A2 cell line and ELISA technique, respectively. Edman N-terminal and internal amino acid sequence (using LC-MS-MS) determination revealed the 10 kDa Canna indica L. leaf protein as a putative plastocyanin. This is the first report of a plant plastocyanin with HIV-1 RT inhibitory property.

Further studies on the soluble form (gs) of rabies virus glycoprotein (g): molecular structure of gs protein and possible mechanism of the shedding.

In this study, we investigated the antigenic structures and maturation of some C-terminal-deficient derivatives of rabies virus glycoprotein (G). The Gs protein, a soluble form of G protein shed from infected cells, displayed antigenicity to most of our conformational epitope-specific anti-G mAbs, but took the 1-30-44 epitope-deficient conformation (termed G(C) form). (The 1-30-44 epitope was acid-sensitive and dependent on two separate regions, the Lys-202-containing and Asn-336-containing regions; Kankanamge et al., Microbiol. Immunol., 47: 507-519). Intact G proteins took the 1-30-44 epitope-positive form (referred to as G(B) form) on the cell surface, but not inside the cell. A deletion mutant G(1-429) (termed GDeltaTC), lacking the transmembrane (TM) and cytoplasmic domains, was shown to be accumulated in the rough endoplasmic reticulum (rER) with BiP and did not seem to be shed. Another C-terminal-deficient mutant G(1-462) (termed CT1) was deprived of the whole cytoplasmic domain except for a basic amino acid left at the C-terminus, but was transported to the cell surface, where it showed pH-dependent cell fusion activity and almost full antigenicity to most of the anti-G mAbs with the exception of very weak antigenicity to mAb #1-30-44. No Gs protein could be detected in the CT1-producing cultures. Based on these results, we think that the cytoplasmic domain was not necessary for the G protein to be transported to the cell surface, but was necessary to keep its 1-30-44 epitope-positive G(B) conformation. Gs proteins might have lost the C-terminal regions during the maturation process after being exported from the rER.