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.

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.

Dissemination of Carbapenem-Resistance among Multidrug Resistant Pseudomonas aeruginosa carrying Metallo-Beta-Lactamase Genes, including the Novel blaIMP-65 Gene in Thailand.

BACKGROUND: Pseudomonas aeruginosa is considered as one of the most emerging threats in this century. Serious infections caused by this pathogen are often treated by carbapenems which are the last resource of antibiotics. Metallo-beta-lactamases (MBLs) production is one of the most important carbepenem resistance mechanisms and is usually related with nosocomial infections caused by P. aeruginosa. This study was aimed to determine the prevalence of MBL genes and distribution pattern of MBLs producing P. aeruginosa strains in Thailand. MATERIALS AND METHODS: Specific primers were designed to detect MBL genes including IMP-, VIM-, and NDM-type MBL genes. Multilocus sequence typing method was used to determine the dissemination pattern of carbapenem-resistance among multidrug-resistant (CR-MDR) P. aeruginosa. RESULTS: A total of 153 P. aeruginosa clinical isolates were characterized as CR-MDR. Among those, 31 P. aeruginosa clinical isolates (20.3%) presented metallo-beta-lactamase genes which could be divided into VIM-type (14 strains) and IMP-type (17 strains). blaIMP-1, blaIMP-13, blaIMP-14a, and blaVIM-2 genes were detected. Moreover, a novel IMP-type MBL, blaIMP-65 was discovered and it was demonstrated to be the unique group of MBLs in Thailand. It was of interest that ST235 was the major ST type in Thailand followed by ST964 and ST111 and ST235 was detected in both MBL harboring and non-MBL harboring strains. CONCLUSION: This study reported the dissemination of MBL gene including novel MBL, blaIMP-65. This study was also demonstrated major ST of P. aeruginosa which was ST235, followed by ST964 and ST111. Moreover, it is also the first report on many P. aeruginosa STs in Thailand: ST273, ST292, ST621, ST1584, and ST1816 which emphasized the dissemination trait difference of MBLs harboring P. aeruginosa in Thailand.

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.

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.

Development of Acyclovir-Loaded Albumin Nanoparticles and Improvement of Acyclovir Permeation Across Human Corneal Epithelial T Cells.

PURPOSE: The aim of the present study was to develop acyclovir (ACV) ocular drug delivery systems of bovine serum albumin (BSA) nanoparticles as well as to assess their in vitro transcorneal permeation across human corneal epithelial (HCE-T) cell multilayers. METHODS: The ACV-loaded BSA nanoparticles were prepared by desolvation method along with physicochemical characterization, cytotoxicity, as well as in vitro transcorneal permeation studies across HCE-T cell multilayers. RESULTS: The nanoparticles appeared to be spherical in shape and nearly uniform in size of about 200 nm. The size of nanoparticles became smaller with decreasing BSA concentration, while the ratios of water to ethanol seemed not to affect the size. Increasing the amount of ethanol in desolvation process led to significant reduction of drug entrapment of nanoparticles with smaller size and more uniformity. The ACV-loaded BSA nanoparticles prepared were shown to have no cytotoxic effect on HCE-T cells used in permeation studies. The in vitro transcorneal permeation results revealed that ACV could permeate through the HCE-T cell multilayers significantly higher from BSA nanoparticles than from aqueous ACV solutions. CONCLUSION: The ACV-loaded BSA nanoparticles could be prepared by desolvation method without glutaraldehyde in the formulation. ACV could increasingly permeate through the multilayers of HCE-T cells from the ACV-loaded BSA nanoparticles. Therefore, the ACV-loaded BSA nanoparticles could be a highly potential ocular drug delivery system.

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.

Synergism and the mechanism of action of the combination of α-mangostin isolated from Garcinia mangostana L. and oxacillin against an oxacillin-resistant Staphylococcus saprophyticus.

BACKGROUND: Globally, staphylococci have developed resistance to many antibiotics. New approaches to chemotherapy are needed and one such approach could be to use plant derived actives with conventional antibiotics in a synergestic way. The purpose of this study was to isolate α-mangostin from the mangosteen (Garcinia mangostana L.; GML) and investigate antibacterial activity and mechanisms of action when used singly and when combined with oxacillin against oxacillin-resistant Staphylococcus saprophyticus (ORSS) strains. The isolated α-mangostin was confirmed by HPLC chromatogram and NMR spectroscopy. The minimum inhibitory concentration (MIC), checkerboard and killing curve were determined. The modes of action of these compounds were also investigated by enzyme assay, transmission electron microscopy (TEM), confocal microscopic images, and cytoplasmic membrane (CM) permeabilization studies. RESULTS: The MICs of isolated α-mangostin and oxacillin against these strains were 8 and 128 µg/ml, respectively. Checkerboard assays showed the synergistic activity of isolated α-mangostin (2 µg/ml) plus oxacillin (16 µg/ml) at a fractional inhibitory concentration index (FICI) of 0.37. The kill curve assay confirmed that the viability of oxacillin-resistant Staphylococcus saprophyticus DMST 27055 (ORSS-27055) was dramatically reduced after exposure to isolated α-mangostin (2 µg/ml) plus oxacillin (16 µg/ml). Enzyme assays demonstrated that isolated α-mangostin had an inhibitory activity against ß-lactamase in a dose-dependent manner. TEM results clearly showed that these ORSS-27055 cells treated with this combination caused peptidoglycan and cytoplasmic membrane damage, irregular cell shapes and average cell areas were significantly larger than the control. Clearly, confocal microscopic images confirmed that this combination caused considerable peptidoglycan damage and DNA leakage. In addition, the CM permeability of ORSS-27055 was also increased by this combination of actives. CONCLUSIONS: These findings provide evidence that isolated α-mangostin alone has not only some activity but also shows the synergistic activity with oxacillin against ORSS-27055. The chromone and isoprenyl structures could play a significant role in its action. This synergistic activity may involve three mechanisms of action. Firstly, potential effects of cytoplasmic membrane disruption and increases permeability. Secondly, inhibit ß-lactamase activity. Finally, also damage to the peptidoglycan structure. We proposes the potential to develop a novel adjunct phytopharmaceutical to oxacillin for the treatment of ORSS. Future studies require clinical trials to establish if the synergy reported can be translated to animals and humans.

Efficacy of cinnamon bark oil and cinnamaldehyde on anti-multidrug resistant Pseudomonas aeruginosa and the synergistic effects in combination with other antimicrobial agents.

BACKGROUND: The emergence of drug resistant pathogens becomes a crucial problem for infectious diseases worldwide. Among these bacteria, Pseudomonas aeruginosa is one of which highly resists to many currently used drugs and becomes a major concern in public health. Up till now, the search for potential antimicrobial agents has been still a challenge for researchers. METHODS: Broth microdilution assay was used to determine minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) of the essential oils and antibiotics against P. aeruginosa. Inhibition activity of the essential oils under vapor condition was examined to obtain the minimum inhibitory dose (MID). Time-kill assay included in this study was performed according to CLSI guideline. Bioautographic assay was used to detect active components of the essential oil. Synergistic effect with currently used antibiotics was further examined by checkerboard assay. RESULTS AND DISCUSSION: In this study, a variety of essential oils were examined for anti-multidrug resistant P. aeruginosa (MDR-PA) activity, of which cinnamon bark oil showed the strongest antimicrobial activity against all clinical-isolated MDR-PA strains with MIC of 0.0562-0.225 % v/v and MBC of 0.1125-1.8 % v/v. Bioautographic results demonstrated that the active compounds of cinnamon bark oil were cinnamaldehyde and eugenol which showed strong inhibitory effect against P. aeruginosa. Interestingly, cinnamaldehyde, a major constituent of cinnamon bark oil, possessed stronger antimicrobial effect to P. aeruginosa than eugenol. Under gaseous condition, cinnamon bark oil and cinnamaldehyde showed antibacterial activity against MDR-PA strains with MID of 0.5-1 mg/L. Moreover, combination of cinnamon bark oil or cinnamaldehyde with currently used antibiotics was further studied by checkerboard assay to examine synergistic interactions on clinically isolated MDR-PA strains. Cinnamon bark oil and cinnamaldehyde combined with colistin demonstrated synergistic rates at 16.7 and 10 %, respectively. CONCLUSION: These results indicated that cinnamon bark oil and cinnamaldehyde might be active natural compounds which could be further examined as alternative treatment for multidrug-resistant P. aeruginosa infection.

Development of molecular approach based on PCR assay for detection of histamine producing bacteria.

Histamine fish poisoning becomes highly concern not only in public health but also economic aspect. Histamine is produced from histidine in fish muscles by bacterial decarboxylase enzyme. Several techniques have been developed to determine the level of histamine in fish and their products but the effective method for detecting histamine producing bacteria is still required. This study was attempted to detect histamine producing bacteria by newly developed PCR condition. Histamine producing bacteria were isolated from scombroid fish and determined the ability to produce histamine of isolated bacteria by biochemical and TLC assays. PCR method was developed to target the histidine decarboxylase gene (hdc). The result showed that fifteen histamine producing bacterial isolates and three standard strains produced an amplicon at the expected size of 571 bp after amplified by PCR using Hdc_2F/2R primers. Fifteen isolates of histamine producing bacteria were classified as M. morganii, E. aerogenes, and A. baumannii. The lowest detection levels of M. morganii and E. aerogenes were 10(2) and 10(5) Cfu/mL in culture media and 10(3) and 10(6) Cfu/mL in fish homogenates, respectively. The limit of detection by this method was clearly shown to be sensitive because the primers could detect the presence of M. morganii and E. aerogenes before the histamine level reached the regulation level at 50 ppm. Therefore, this PCR method exhibited the potential efficiency for detecting the hdc gene from histamine producing bacteria and could be used to prevent the proliferation of histamine producing bacteria in fish and fish products.