Abundance of Colistin-Resistance Genes in Retail Meats in Vietnam.

The degree of contamination of retail meat with colistin-resistant bacteria and its potential contribution to dissemination within communities remains to be determined. Thus, we aimed to elucidate the contamination status of colistin-resistance genes, indicative of colistin-resistant bacteria, in retail meats in Vietnam. In total, 46 chicken and 49 pork meats from stores in Vietnam and Japan were examined. Multiplex real-time polymerase chain reaction with TaqMan probes was performed for detecting mcr-1, mcr-3, and Escherichia coli 16S rRNA. Colistin-resistant bacteria in meats were isolated using selective media. The minimum inhibitory concentrations of colistin were determined using the broth microdilution method. The results showed that 70.7% of chicken meats in Vietnam were contaminated with both mcr-1 and mcr-3. Meanwhile, mcr-1 and mcr-3 were detected in 15.9% and 40.9% of pork meat, respectively. Only mcr-3 was detected in 40% of chicken in Japan. In addition, mcr-1-harboring E. coli and mcr-3-harboring Aeromonas were isolated from chicken meats in Vietnam. Some of these isolates showed colistin resistance. These results showed that most retail meats were highly contaminated with colistin-resistance genes. Notably, our results suggest that mcr-3 is more prevalent in the contaminated samples compared with mcr-1.

Effects of Subchronic Administrations of Vortioxetine, Lurasidone, and Escitalopram on Thalamocortical Glutamatergic Transmission Associated with Serotonin 5-HT7 Receptor.

The functional suppression of serotonin (5-HT) type 7 receptor (5-HT7R) is forming a basis for scientific discussion in psychopharmacology due to its rapid-acting antidepressant-like action. A novel mood-stabilizing atypical antipsychotic agent, lurasidone, exhibits a unique receptor-binding profile, including a high affinity for 5-HT7R antagonism. A member of a novel class of antidepressants, vortioxetine, which is a serotonin partial agonist reuptake inhibitor (SPARI), also exhibits a higher affinity for serotonin transporter, serotonin receptors type 1A (5-HT1AR) and type 3 (5-HT3R), and 5-HT7R. However, the effects of chronic administration of lurasidone, vortioxetine, and the selective serotonin reuptake inhibitor (SSRI), escitalopram, on 5-HT7R function remained to be clarified. Thus, to explore the mechanisms underlying the clinical effects of vortioxetine, escitalopram, and lurasidone, the present study determined the effects of these agents on thalamocortical glutamatergic transmission, which contributes to emotional/mood perception, using multiprobe microdialysis and 5-HT7R expression using capillary immunoblotting. Acute local administration of a 5-HT7R agonist and antagonist into the mediodorsal thalamic nucleus (MDTN) enhanced and reduced thalamocortical glutamatergic transmission, induced by N-methyl-D-aspartate (NMDA)/glutamate receptor inhibition in the reticular thalamic nucleus (RTN). Acute local administration of a relevant therapeutic concentration of vortioxetine and lurasidone into the MDTN suppressed the thalamocortical glutamatergic transmission via 5-HT7R inhibition, whereas that of escitalopram activated 5-HT7R. Subchronic administration of effective doses of vortioxetine and lurasidone (for 7 days) reduced the thalamocortical glutamatergic transmission, but escitalopram did not affect it, whereas subchronic administration of these three agents attenuated the stimulatory effects of the 5-HT7R agonist on thalamocortical glutamatergic transmission. Subchronic administration of effective doses of vortioxetine, lurasidone, and escitalopram downregulated the 5-HT7R expression of the plasma membrane in the MDTN; the 5-HT7R downregulation induced by vortioxetine and lurasidone was observed at 3 days, but that induced by escitalopram required a longer duration of 7 days. These results indicate that chronic administration of vortioxetine, escitalopram, and lurasidone generate downregulation of 5-HT7R in the thalamus; however, the direct inhibition of 5-HT7R associated with vortioxetine and lurasidone generates more rapid downregulation than the indirect elevation of the extracellular serotonin level via serotonin transporter inhibition by escitalopram.

Entropic and Energetic Elasticities of Natural Rubber with a Nanomatrix Structure.

The entropic and energetic elasticities of natural rubber with a nanomatrix structure are investigated by measuring the viscoelastic properties of deproteinized natural rubber (DPNR)-graft-polystyrene (PS). A nanomatrix structure is formed by graft copolymerization of styrene onto the surface of natural rubber particles, followed by coagulation. The morphology of the nanomatrix structure is observed with transmission electron microscopy. Natural rubber particles with about 1 µm diameter are well dispersed in a nanomatrix of PS. The horizontal shift factor (aT) and vertical shift factor (bT) are determined by superposition to create a master curve according to the time-temperature superposition principle. The positive slope of DPNR appears in the plot of bT versus temperature, suggesting that entropic elasticity occurs. In contrast, the slopes of DPNR-graft-PS are negative at lower temperatures but positive at higher temperatures. The negative slope, which suggests entropic elasticity, may be attributed to the formation of a nanomatrix structure. Natural rubber with a nanomatrix structure realizes both energetic elasticity and entropic elasticity.

Detection of Genetic Elements Carrying vanA in Vancomycin-Resistant Enterococcus saigonensis VE80T Isolated from Retail Chicken Meat.

In this study, we aimed to detect genetic elements carrying vanA in Enterococcus saigonensis VE80T isolated from retail chicken in Vietnam. The structures of vancomycin-resistance determinants and the location of vancomycin-resistance genes were detected by sequencing the vanA gene cluster, Southern hybridization analyses, and whole-genome sequence analyses. The Tn1546-related elements harboring vanA clusters, which exhibited a characteristic structure with five point mutations compared with the prototype Tn1546, were located on the 76-kb plasmid pVE80-1 of VE80T. The vanS sequence of VE80T harboring three point mutations was 100% identical to those of vancomycin-resistant enterococci isolated from poultry in Taiwan and Japan, indicating that the element may be prevalent in poultry production farms in Asia.

Consumption of edible ice contaminated with Acinetobacter, Pseudomonas, and Stenotrophomonas is a risk factor for fecal colonization with extended-spectrum ß-lactamase-producing Escherichia coli in Vietnam.

Although Vietnamese residents frequently harbor extended-spectrum ß-lactamase-producing Escherichia coli (ESBL-E), it is unclear which foods/beverages are risk factors for acquiring these bacteria. The aim of this study was to evaluate the frequency with which edible ice served in restaurants is contaminated with antibiotic-resistant bacteria, and thereby clarify whether this product poses a risk for ESBL-E carriage in humans. Ice from restaurants in Vietnam and Japan was screened for bacteria capable of growing on agar containing cefotaxime (BG-CTX). Of the 119 BG-CTX strains isolated in Vietnam, 40%, 39%, and 12% were identified as Pseudomonas spp., Acinetobacter spp., and Stenotrophomonas maltophilia, respectively. Meanwhile, of the six such strains isolated in Japan, five were identified as Acinetobacter spp. and one as Pseudomonas spp. More than 10% of the Acinetobacter isolates exhibited cefotaxime, ceftazidime, and sulfa/trimethoprim resistance, while 21% of Pseudomonas and 14% of S. maltophilia isolates exhibited meropenem and sulfa/trimethoprim resistance, respectively. Subsequent multiplex polymerase chain reaction (PCR) analyses detected ESBL-encoding genes in 10% of the BG-CTX. Notably, feces harvested from mice administered water contaminated with BG-CTX contained E. coli harboring the blaCTX-M-9 gene. In conclusion, our findings indicate that consumption of contaminated edible ice is a risk factor for human ESBL-E carriage.

Detection of antibiotics in chicken eggs obtained from supermarkets in Ho Chi Minh City, Vietnam.

The residual levels of antibiotics in Vietnamese eggs were monitored from 2014 to 2015. A total of 111 egg packages, distributed by 11 different companies, were collected from supermarkets in Ho Chi Minh City and the levels of 28 antibiotics were analyzed using liquid chromatography-tandem mass spectrometry (LC-MS/MS) screening method. Sixteen samples tested positive for antibiotics; a total of eight compounds (enrofloxacin, ciprofloxacin, norfloxacin, sulfadimethoxine, sulfamethazine, sulfamonomethoxine, tilmicosin and trimethoprim) were detected. Enrofloxacin was detected in eight samples, with two samples exhibiting concentrations exceeding 1,000 µg kg-1. Tilmicosin was detected in three samples at a range of 49-568 µg kg-1. We observed that two of the 11 companies frequently sold antibiotic-contaminated eggs (detection rates of 56 and 60%), suggesting that a number of companies do not regulate the use of antibiotics in egg-laying hens. Our findings indicate that livestock farmers require instruction regarding antibiotic use and that continual antibiotic monitoring is essential in Vietnam.

Frequent use of colistin-based drug treatment to eliminate extended-spectrum beta-lactamase-producing Escherichia coli in backyard chicken farms in Thai Binh Province, Vietnam.

Reports of livestock infections with extended-spectrum beta-lactamase-producing Escherichia coli (ESBL-E) are increasing. Based on interviews conducted over a 6-month period, we found that veterinarians in the Vietnamese province of Thai Binh prefer to prescribe colistin-based drugs (CBD) in chicken farms. We aimed to clarify whether CBD use selects for strains of colistin-resistant ESBL-E. With the cooperation of seven local households, we detected ESBL-E in chickens' feces after treating chickens with CBD. Phylogenetic groupings and the presence of CTX-M/AmpC genes were determined, and the multi-antibiotic susceptibility of isolates was analyzed. Our results showed that ESBL-E presented in seven chickens' feces from two households. Seventy-two percent of ESBL-E isolates harbored CTX-M9 and the phylogenetic group A; the colistin minimum inhibitory concentration (MIC) of all isolated ESBL-E ranged from 0.064 to 1 µg mL-1. Moreover, ESBL-E isolates were used to experimentally select for colistin resistance, and the effect of commercial CBD on ESBL-E was investigated. The results showed that an ESBL-E strain with a colistin MIC of 4 µg mL-1 was able to grow in media with CBD. Although CBD treatment was effective, in vitro experiments demonstrated that ESBL-E can easily acquire colistin resistance. Therefore, restrictions on colistin use are necessary to prevent the emergence of colistin-resistant bacteria.

Enterococcus saigonensis sp. nov., isolated from retail chicken meat and liver.

Two Gram-stain-positive strains, VE80T and VE116, which were resistant to vancomycin, were isolated from retail chicken meat and liver in Ho Chi Minh, Vietnam, respectively. These strains were characterized by sequence analyses of 16S rRNA, RNA polymerase α-subunit (rpoA), ATP synthase α-subunit (atpA), and phenylalanyl-tRNA synthase α-subunit (pheS) genes, determination of DNA G+C content, cellular fatty acid methyl ester analysis, DNA-DNA hybridization, and conventional morphological and biochemical tests. Strains VE80T and VE116 had 99.6 % 16S rRNA gene sequence similarity with Enterococcus canintestini LMG 13590T, and 99.1 % 16S rRNA gene sequence similarity with Enterococcus dispar ATCC 51266T. However, the two isolates could be clearly differentiated from these reference strains by the low sequence similarities (86.1-86.8 %) of the atpA gene, low DNA-DNA relatedness (<22.8 %), and differences in the production of acid from melezitose and methyl α-d-glucoside. Based on the results obtained in the present study, these two isolates are considered to represent a novel species of the genus Enterococcus, for which the name Enterococcus saigonensis sp. nov., is proposed. The type strain is VE80T (=JCM 31193T=CCUG 68827T).

Limited transmission of bla(CTX-M-9)-type-positive Escherichia coli between humans and poultry in Vietnam.

We examined whether Escherichia coli isolates that produce CTX-M-9-type extended-spectrum ß-lactamases (ESBL) are transferred between humans and chickens in a Vietnamese community. The phylogenetic group compositions, sequence types, antimicrobial resistance profiles, the prevalence of plasmid antibiotic resistance genes, and the plasmid replicon types generally differed between the human and chicken E. coli isolates. Our results suggest that transmission of the bla(CTX-M-9)-positive E. coli between humans and poultry was limited.

Carriage of Escherichia coli Producing CTX-M-Type Extended-Spectrum ß-Lactamase in Healthy Vietnamese Individuals.

Healthy carriage of CTX-M-type extended-spectrum ß-lactamase (ESBL)-producing Escherichia coli was examined by thrice collecting fecal samples from the same 199 healthy Vietnamese subjects every 6 months. Using pulsed-field gel electrophoresis (PFGE), identical PFGE patterns throughout the three samplings were not observed, although prevalence of E. coli in the subjects was around 50% in the three samplings. Our results suggested a short carriage period of the CTX-M-type ESBL-producing E. coli in healthy Vietnamese subjects.

Evaluation of a Method Using Three Genomic Guided Escherichia coli Markers for Phylogenetic Typing of E. coli Isolates of Various Genetic Backgrounds.

Genotyping and characterization of bacterial isolates are essential steps in the identification and control of antibiotic-resistant bacterial infections. Recently, one novel genotyping method using three genomic guided Escherichia coli markers (GIG-EM), dinG, tonB, and dipeptide permease (DPP), was reported. Because GIG-EM has not been fully evaluated using clinical isolates, we assessed this typing method with 72 E. coli collection of reference (ECOR) environmental E. coli reference strains and 63 E. coli isolates of various genetic backgrounds. In this study, we designated 768 bp of dinG, 745 bp of tonB, and 655 bp of DPP target sequences for use in the typing method. Concatenations of the processed marker sequences were used to draw GIG-EM phylogenetic trees. E. coli isolates with identical sequence types as identified by the conventional multilocus sequence typing (MLST) method were localized to the same branch of the GIG-EM phylogenetic tree. Sixteen clinical E. coli isolates were utilized as test isolates without prior characterization by conventional MLST and phylogenetic grouping before GIG-EM typing. Of these, 14 clinical isolates were assigned to a branch including only isolates of a pandemic clone, E. coli B2-ST131-O25b, and these results were confirmed by conventional typing methods. Our results suggested that the GIG-EM typing method and its application to phylogenetic trees might be useful tools for the molecular characterization and determination of the genetic relationships among E. coli isolates.

Characteristics of Extended-Spectrum ß-Lactamase-Producing Escherichia coli in Retail Meats and Shrimp at a Local Market in Vietnam.

BACKGROUND: Contamination of food with multiantibiotic-resistant bacteria, particularly extended-spectrum ß-lactamase (ESBL)-producing Enterobacteriaceae, is considered a potential source for the wide dissemination of ESBL-producing bacteria in communities. However, little is known about the extent of contamination of food with ESBL-producing bacteria in Vietnam. OBJECTIVE: This study was conducted to assess the characteristics of ESBL-producing Escherichia coli isolated from retail meats and shrimp in Nha Trang, Vietnam. MATERIALS AND METHODS: A total of 350 food samples (poultry [n=143], pork [n=147], and shrimp [n=60]) were purchased in July and November 2013 from a local market. ESBL-producing E. coli were isolated, and ESBL genotypes, phylogenetic groups, and antibiotic resistance profiles were determined. RESULTS: The prevalence of ESBL-producing E. coli in retail foods was 40.6%. ß-Lactamase-encoding genes of the CTX-M-1 (50.7%), CTX-M-9 (41.5%), TEM (59.9%), and SHV (2.8%) groups were detected singly or in combination. The percentages of single ESBL isolates harboring CTX-M-1 or -9 plus TEM groups were 35.2% and 16.2%, respectively. B1 was the most prevalent phylogroup in ESBL isolates from pork (44.7%), poultry (55.9%), and shrimp (72.7%). B2 was the least prevalent (4.2% and 4.8% for pork and poultry isolates, respectively). The prevalence of multidrug resistance (MDR; resistance to ≥ 3 antimicrobial groups) in ESBL-producing E. coli isolated from food was 85.9%. DISCUSSION AND CONCLUSIONS: This is the first report of the characteristics of ESBL-producing E. coli in retail foods in a local city in Vietnam. Our findings indicate that retail foods are contaminated with ESBL-producing E. coli, of which many were MDR. Further monitoring and public health efforts targeting food administration are needed to control the spread of ESBL-producing bacteria in communities.

Chlamydia pneumoniae effector chlamydial outer protein N sequesters fructose bisphosphate aldolase A, providing a benefit to bacterial growth.

BACKGROUND: Pathogenic chlamydiae are obligate intracellular pathogens and have adapted successfully to human cells, causing sexually transmitted diseases or pneumonia. Chlamydial outer protein N (CopN) is likely a critical effector protein secreted by the type III secretion system in chlamydiae, which manipulates host cells. However, the mechanisms of its action remain to be clarified. In this work, we aimed to identify previously unidentified CopN effector target in host cells. RESULTS: We first performed a pull-down assay with recombinant glutathione S-transferase (GST) fusion CopN proteins (GST-CpCopN: Chlamydia pneumoniae TW183, GST-CtCopN: Chlamydia trachomatis D/UW-3/CX) as "bait" and soluble lysates obtained from human immortal epithelial HEp-2 cells as "prey", followed by SDS-PAGE with mass spectroscopy (MS). We found that a host cell protein specifically bound to GST-CpCopN, but not GST-CtCopN. MS revealed the host protein to be fructose bisphosphate aldolase A (aldolase A), which plays a key role in glycolytic metabolism. We also confirmed the role of aldolase A in chlamydia-infected HEp-2 cells by using two distinct experiments for gene knockdown with an siRNA specific to aldolase A transcripts, and for assessment of glycolytic enzyme gene expression levels. As a result, both the numbers of chlamydial inclusion-forming units and RpoD transcripts were increased in the chlamydia-infected aldolase A knockdown cells, as compared with the wild-type HEp-2 cells. Meanwhile, chlamydial infection tended to enhance expression of aldolase A. CONCLUSIONS: We discovered that one of the C. pneumoniae CopN targets is the glycolytic enzyme aldolase A. Sequestering aldolase A may be beneficial to bacterial growth in infected host cells.

Chromosomal qnrB19-carrying Escherichia coli isolated from the stool sample of a community resident in Ecuador.

We identified a chromosomal qnrB19 gene within a transposon in a colistin-resistant Escherichia coli strain isolated from the stool sample of an Ecuadorian resident. This finding suggests a more stable acquisition of quinolone resistance on chromosomes than that on plasmids and the potential for propagation to other DNA structures.

New insights into raceway cultivation of Euglena gracilis under long-term semi-continuous nitrogen starvation.

This study aimed to investigate the physiological responses of Euglena gracilis (E. gracilis) when subjected to semicontinuous N-starvation (N-) for an extended period in open ponds. The results indicated that the growth rates of E. gracilis under the N- condition (11 ± 3.3 g m-2 d-1) were higher by 23% compared to the N-sufficient (N+, 8.9 ± 2.8 g m-2 d-1) condition. Furthermore, the paramylon content of E.gracilis was above 40% (w/w) of dry biomass in N- condition compared to N+ (7%) condition. Interestingly, E. gracilis exhibited similar cell numbers regardless of nitrogen concentrations after a certain time point. Additionally, it demonstrated relatively smaller cell size over time, and unaffected photosynthetic apparatus under N- condition. These findings suggest that there is a tradeoff between cell growth and photosynthesis in E. gracilis, as it adapts to semi-continuous N- conditions without a decrease in its growth rate and paramylon productivity. Notably, to the author's knowledge, this is the only study reporting high biomass and product accumulation by a wild-type E. gracilis strain under N- conditions. This newly identified long-term adaptation ability of E. gracilis may offer a promising direction for the algal industry to achieve high productivity without relying on genetically modified organisms.

Comparative genome analysis of colistin-resistant Escherichia coli harboring mcr isolated from rural community residents in Ecuador and Vietnam.

The spread of colistin-resistant bacteria among rural community residents of low- and middle-income countries is a major threat to community health. Although the mechanism of the spread of colistin-resistant bacteria in communities is unknown, geographic and regional characteristics may influence it. To elucidate the spread mechanism of colistin-resistant bacteria, we analyzed the genomes of colistin-resistant Escherichia coli isolated from Vietnam and Ecuador residents, which are geographically and socially different. Stool specimens of 139 and 98 healthy residents from Ecuador and Vietnam rural communities, respectively, were analyzed for colistin-resistant E. coli with mcr. Its prevalence in the residents of all the communities assessed was high and approximately equal in both countries: 71.8% in Ecuador and 69.4% in Vietnam. A phylogenetic tree analysis revealed that the sequence type of colistin-resistant E. coli was diverse and the major sequence types were different between the two countries. The location of mcr in the isolates showed that the proportion of chromosomal mcr was 35.1% and 8.5% in the Vietnam and Ecuador isolates, respectively. Most of these chromosomal mcr genes (75%-76%) had an intact mcr-transposon Tn6330. Contrastingly, the replicon types of the mcr-carrying-plasmids were diverse in both countries, but almost all belonged to IncI2 in Ecuador and IncX1/X4 in Vietnam. Approximately 26%-45% of these mcr-plasmids had other resistance genes, which also varied between countries. These results suggest that although the overall profile of the colistin-resistant E. coli isolates is diverse in these countries, the phylogenesis of the isolates and mcr-carrying plasmids has regional characteristics. Although the contributing factors are not clear, it is obvious that the overall profile of colistin-resistant bacteria dissemination varies between countries. Such different epidemic patterns are important for establishing country-specific countermeasures against colistin-resistant bacteria.

Co-Harboring of Beta-Lactamases and mcr-1 Genes in Escherichia coli and Klebsiella pneumoniae from Healthy Carriers and Backyard Animals in Rural Communities in Ecuador.

Few studies have addressed drug resistance of Enterobacterales in rural communities in developing countries. This study aimed to determine the coexistence of extended-spectrum ß-lactamase (ESBL) and carbapenemase genes in Escherichia coli and Klebsiella pneumoniae strains carrying the mcr-1 gene in rural communities in Ecuador from healthy humans and their backyard animals. Sixty-two strains, thirty E. coli and thirty-two K. pneumoniae strains carrying the mcr-1 gene were selected from a previous study. PCR were performed for the presence of ESBLs and carbapenemase genes. The strains were further characterized, and the genetic relationship was studied with multi-locus sequencing typing (MLST) of seven housekeeping genes. Fifty-nine of the sixty-two mcr-1 isolates (95%) harbored at least on ß-lactam resistance gene. The most prevalent ESBL genes were the blaTEM genes (present in in 80% of the E. coli strains) and the blaSHV gene (present in 84% of the K. pneumoniae strains). MSLT analysis revealed 28 different sequence types (ST); 15 for E. coli and 12 for K. pneumoniae, with most ST never described in humans and animals. The coexistence of mcr-1 and ß-lactams resistant genes in E. coli and K. pneumoniae strains is alarming and threatens the efficacy of last-resort antibiotics. Our findings highlight backyard animals as a reservoir of mcr-1/ß-lactams resistant genes.

Prevalence of and risk factors associated with faecal carriage of CTX-M ß-lactamase-producing Enterobacteriaceae in rural Thai communities.

OBJECTIVES: To determine the prevalence of CTX-M ß-lactamase-producing Enterobacteriaceae and to study the risk factors associated with faecal carriage in asymptomatic rural Thai people. METHODS: In all, 417 stool samples were obtained from rural Thai people and screened for extended-spectrum ß-lactamases (ESBLs) using MacConkey agar supplemented with 2 mg/L cefotaxime. Results were confirmed using cefotaxime and ceftazidime with and without clavulanic acid. The bla(CTX-M) genes were identified and genotyped using PCR with bacterial DNA samples. Multivariate analysis was performed to investigate risk factors associated with the faecal carriage of CTX-M producers. RESULTS: The prevalence of CTX-M-type ESBL-producing Enterobacteriaceae was 65.7%. The CTX-M-9 group (60.6%) was dominant, followed by the CTX-M-1 group (38.7%). Most of the bacteria were Escherichia coli (85.4%) and Klebsiella pneumoniae (4.7%). Of a total of 234 E. coli strains, 48.7% belonged to phylogenetic group A, 28.6% to group B1, 15.8% to group D and 6.8% to group B2. Most CTX-M producers were susceptible to carbapenems and amikacin, but resistant to tetracycline and gentamicin. In a multivariate logistic regression model, better education status (OR 2.245; 95% CI 1.297-3.884), history of hospitalization (OR 1.643; 95% CI 1.036-2.603) and the use of antibiotics within the last 3 months (OR 1.883; 95% CI 1.221-2.903) were independently associated with faecal carriage. CONCLUSIONS: Faecal carriage of CTX-M-type ESBL-producing Enterobacteriaceae among asymptomatic individuals in rural Thailand remains alarmingly high, and previous antibiotic use and a history of hospitalization may contribute to its dissemination.