Prevalence and molecular characterization of Vibrio cholerae from fruits and salad vegetables sold in Jakarta, Indonesia, using most probable number and PCR.

OBJECTIVE: Cholera is an intestinal infection caused by Vibrio cholerae, it is usually occurs in developing countries that lack of sanitation. In developing country including Indonesia, awareness importance of sanitation is still low. Unfortunately, research related to the detection of V. cholerae from fruit and vegetables in Indonesia is still rare. In this study, MPN method was used to determine the prevalence of V. cholerae followed by single and multiplex PCR to detect virulence genes, including toxR, ctxA, tcpA, hlyA, ace, ompU, and zot. RESULTS: We found 3 fruits and 2 vegetables positive for toxR gene. Fruit samples which were showed toxR positive found from East Jakarta while for vegetables, it was recovered from West Jakarta and Central Jakarta. Twenty-three isolates were recovered from toxR positive samples. The result of antibiotic resistance analysis showed that 4.35% of the isolates resistant to gentamicin, streptomycin (17.39%), trimethoprim (52.17%), ciprofloxacin (30.43%), ampicillin (13.04%), nalidixic acid (82.61%), and polymyxin B (91.30%). None of these isolates were resistant to kanamycin. Combination of MPN and Multiplex PCR method can be used to detect the prevalence and characterize the virulence properties of V. cholerae.

Maternal Multiple Micronutrient Supplementation Stabilizes Mitochondrial DNA Copy Number in Pregnant Women in Lombok, Indonesia.

BACKGROUND: The Supplementation with Multiple Micronutrients Intervention Trial (SUMMIT) in Lombok, Indonesia showed that maternal multiple micronutrients (MMN), as compared with iron and folic acid (IFA), reduced fetal loss, early infant mortality, and low birth weight. Mitochondria play a key role during pregnancy by providing maternal metabolic energy for fetal development, but the effects of maternal supplementation during pregnancy on mitochondria are not fully understood. OBJECTIVE: The aim of this study was to assess the impact of MMN supplementation on maternal mitochondrial DNA copy number (mtDNA-CN). METHODS: We used archived venous blood specimens from pregnant women enrolled in the SUMMIT study. SUMMIT was a cluster-randomized double-blind controlled trial in which midwives were randomly assigned to distribute MMN or IFA to pregnant women. In this study, we selected 108 sets of paired baseline and postsupplementation samples (MMN = 54 and IFA = 54). Maternal mtDNA-CN was determined by real-time quantitative polymerase chain reaction in baseline and postsupplementation specimens. The association between supplementation type and change in mtDNA-CN was performed using rank-based estimation for linear models. RESULTS: In both groups, maternal mtDNA-CN at postsupplementation was significantly elevated compared with baseline (P < 0.001). The regression revealed that the MMN group had lower postsupplementation mtDNA-CN than the IFA group (ß = -4.63, P = 0.003), especially for women with mtDNA-CN levels above the median at baseline (ß = -7.49, P = 0.007). This effect was rapid, and observed within 33 d of initiation of supplementation (ß = -7.39, P = 0.017). CONCLUSION: Maternal MMN supplementation rapidly stabilized mtDNA-CN in pregnant women who participated in SUMMIT, indicating improved mitochondrial efficiency. The data provide a mechanistic basis for the beneficial effects of MMN on fetal growth and survival, and support the transition from routine IFA to MMN supplementation.This trial was registered at www.isrctn.com as ISRCTN34151616.

Prevalence of pathogenic Escherichia coli from salad vegetable and fruits sold in Jakarta.

OBJECTIVE: Escherichia coli is a normal inhabitant of mammalian's gut, but some strains acquired virulence factor and became pathogenic. Enterotoxigenic E. coli (ETEC), enterohemorrhagic E. coli (EHEC), enteropathogenic E. coli (EPEC), enteroinvasive E. coli (EIEC), and enteroaggregative E. coli (EAEC) are among pathogenic strains of E. coli. Vegetables and fruits could be sources of transmission. Samples were collected and subjected to three-tubes Most Probable Number (MPN) analysis followed by Multiplex PCR. Six sets of primer encoding virulence genes were used: stx, ipah, aggr, eae, elt and est. RESULTS: From this study we found, the highest maximum number for the MPN result reached > 1100 MPN/mL and the lowest is 3 MPN/mL. From first multiplex PCR showed 65 salad vegetable samples, 7.69% were positive and from the 63 fruit samples, 11.11% were positive. From second multiplex PCR for 76 isolates, 55 (72.37%) isolates were aggR positive (EAEC), 12 (15.79%) isolates were eae positive (EPEC), and 9 (11.84%) were elt positive (ETEC). Antimicrobial resistance assay showed that 83.33% of the isolates were multi resistant. Resistances are observed to 10 µg Ampicillin (22.22%), 5 µg Ciprofloxacin (11.11%), 10 µg Gentamycin (33.33%), 30 µg Kanamycin (38.89%), 10 µg Streptomycin (55.56%), 5 µg Trimethoprim (16.67%), and 300 U Polymyxin B (61.11%).

Correction to: Prevalence and molecular characterization of Salmonella enterica serovar Typhimurium from ice and beverages in Jakarta, Indonesia.

Following publication of the original article [1], the authors reported that the family name of one of the authors was omitted. In this Correction the incorrect and correct author name are shown. The original publication of this article has been corrected.

Prevalence and molecular characterization of Salmonella enterica serovar Typhimurium from ice and beverages in Jakarta, Indonesia.

OBJECTIVE: The presence of Salmonella enterica serovar Typhimurium becomes a concern in relation to the safety of drinking water and ice. We detected and enumerated the bacteria from ice and beverages collected from several areas in Jakarta. Most Probable Number (MPN) and multiplex PCR method were used. Three sets of primers were used rfbJ, fliC, and fljB. Two Multiplex PCR's were performed, the first is to detect the presence of Salmonella and the second is to confirm the positive isolate of S. enterica serovar Typhimurium. RESULTS: A total of 50 beverages and 50 ices were collected MPN result ranged from < 3 to > 11,000 MPN/ml. The highest MPN value > 11,000 MPN/ml. The first Multiplex PCR result from beverages, 58% positively contained Salmonella spp. with amplification of fliB gene and no amplification of rfbJ and fliC genes. For ice samples, 2% positively contained Salmonella spp. with rfbJ gene amplification, 62% fliB gene and no amplification of fliC gene. The second Multiplex PCR results from beverages identified 21 positive isolates of S. enterica serovar Typhimurium. In which, 17 isolates contained fljB gene and 4 isolates contained both fljB and rfbJ genes. From ice, 17 isolates having both rfbJ and fljB genes.

Genetic diversity of Escherichia coli isolated from ice cube production sites.

OBJECTIVE: The prevalence of Escherichia coli including from ice cubes in Indonesia is quite high. Unfortunately, little is known about the genetic diversity of E. coli from ice cube production site. Genotypic variation in E. coli populations is a major barrier to control public health risk associated with foodborne pathogen. The aims of this study were to analyze the genotypic diversity of E. coli strains isolated from various samples in order to determine the genetic relationship between those strains. This study is also important to understand the occurrence, prevalence and profile picture of different pathogenic E. coli in various sources which potentially cause disease. RESULTS: Enterobacterial repetitive intergenic consensus (ERIC) and repetitive extragenic palindromic polymerase chain reaction (REP-PCR) dendrogram showed high genetic diversity of 120 E. coli isolates in majority of sampling sites. DNA fingerprint patterns showed 26 and 21 clusters with 11 and 3 fingerprints individual lineages for ERIC and REP-PCR respectively. There was no correlation observed between phylogenetic relationship and virulence genes. The result indicated a variation of E. coli isolates in ice cube manufacturers. ERIC-PCR method is more discriminative compared with REP-PCR to analyze the genetic diversity of E. coli from ice cubes production sites.

Edible ice in Jakarta, Indonesia, is contaminated with multidrug-resistant Vibrio cholerae with virulence potential.

Consumption of street food is considered a major health risk in the absence of public-health inspection programmes in Indonesia. It is hypothesized that ice used in street food could be one of the major sources of Vibrio cholerae contamination. This study documented V. cholerae contamination in edible ice from different areas of Jakarta, the capital city of Indonesia, and attempted to characterize the virulence potential of the strains. A selective medium was used to isolate 98 V. cholerae strains and their identity was confirmed using biochemical assays. Serological tests classified the majority (78%) in the non-O1 serogroup. Multiplex PCR was used to detect the presence of V. cholerae virulence genes, namely ctxA, ompU, tcpA, ace, zot and toxR. The toxR, ctxA, ompU and zot genes were detected in 75, 26, 15 and 1% of isolates, respectively. The ace and tcpA genes were not detected in any of the isolates. The ctxA gene encoding the cholera toxin subunit A, which has been associated only with clinical strains of O1, here was present in both serogroups. The antibiotic-resistance profile showed that 65, 60, 52, 39, 37, 19 and 3% of the isolates were resistant to ampicillin, streptomycin, kanamycin, sulfamethoxazole-trimethoprim, erythromycin, tetracycline and ciprofloxacin, respectively. A large proportion of V. cholerae isolates came from west and south Jakarta, and these strains exhibited multidrug resistance to ampicillin, streptomycin, tetracycline, erythromycin, kanamycin and sulfamethoxazole-trimethoprim. Many of these isolates from west and south Jakarta also harboured toxR, encoding a regulator, and ctxA. The presence of multidrug-resistant V. cholerae with virulence genes in edible ice, which could cause a severe outbreak, reflects the poor water quality in Jakarta, and indicates an urgent need for better surveillance and management.

Prevalence and molecular characterization of Vibrio cholerae from ice and beverages sold in Jakarta, Indonesia, using most probable number and multiplex PCR.

Vibrio cholerae is well recognized as the causative agent of cholera, an acute intestinal infection characterized by watery diarrhea that may lead to dehydration and death in some cases. V. cholerae is a natural inhabitant of the aquatic environment in the tropical regions. Jakarta has the highest percentage of individuals affected by sporadic diarrheal illness compared with other areas in Indonesia. Inadequate safety measures for drinking water supplies, improper sanitation, and poor hygiene can increase the risk of cholera outbreaks. Few studies have been conducted on the prevalence of these bacteria in ice and beverages that are popularly sold and consumed in Jakarta. In this study, we detected and quantified V. cholerae from ice and beverages collected from several areas in five regions of Jakarta. Levels of V. cholerae in both ice and beverages were determined with the three-tube most-probable-number (MPN) method and ranged from < 0.3 to > 110 MPN/ml. The presence of regulatory and virulence gene sequences was determined by using uniplex and multiplex PCR assays. Of 110 samples tested, 33 (30%) were positive for V. cholerae; 21 (64%) were ice samples and the remaining 12 (36%) were beverages. A total of 88 V. cholerae strains were isolated, based on the presence of the toxR gene sequence identified by PCR. Other genetic markers, such as hlyA (59%), ompU (16%), and ctxA (19%), also were found during the search for potential pathogenic strains. The detection and isolation of potentially harmful V. cholerae from ice and beverages in Jakarta indicate that these products pose a health risk from choleragenic vibrios, particularly because of the emergence of classical biotypes of V. cholerae O1 and potentially harmful non-O1 serovars of this species.

Use of REP- and ERIC-PCR to reveal genetic heterogeneity of Vibrio cholerae from edible ice in Jakarta, Indonesia.

BACKGROUND: Vibrio cholerae is the causative organism of waterborne disease, cholera. V. cholerae has caused many epidemics and pandemics of cholera for many years. In this study, V. cholerae recovered from edible ice were investigated for their genetic diversity using Enterobacterial Repetitive Intergenic Consensus (ERIC) PCR and Repetitive Extragenic Palindromic (REP) PCR. Isolation was done using selective medium and the presumptive isolates were confirmed through biochemical and serological assays. RESULTS: Seventy-five isolates of V. cholerae were recovered from ice samples collected from different locations of Jakarta. Specifically, 19 of them were identified as O1 serotype, 16 were Ogawa, 3 isolates were Inaba and the remaining isolates were non-O1. The fingerprinting profiles of V.cholerae isolated from ice samples were very diverse. CONCLUSION: This result showed that the ERIC sequence is more informative and discriminative than REP sequence for analysis of V. cholerae diversity.