Metagenome and Resistome Analysis of Beta-Lactam-Resistant Bacteria Isolated from River Waters in Surabaya, Indonesia.

Antimicrobial agents are administered to humans and livestock, and bacterial antimicrobial resistance (AMR) and antimicrobial agents are released into the environment. In this study, to investigate the trend of AMR in humans, livestock, and the environment, we performed a metagenomic analysis of multidrug-resistant bacteria with CHROMagar ESBL in environmental river water samples, which were collected using syringe filter units from waters near hospitals, downtown areas, residential areas, and water treatment plants in Surabaya, Indonesia. Our results showed that Acinetobacter, Pseudomonas, Aeromonas, Enterobacter, Escherichia, and Klebsiella grew in CHROMagar ESBL; they were most frequently detected in water samples from rivers surrounding hospitals contaminated with various AMR genes (ARGs) in high levels. These results identified bacteria as ARG reservoirs and revealed that hospitals could be sources for various ARGs disseminated into the environment. In conclusion, this study details a novel metagenomic analysis of collected bacteria in environmental water samples using a syringe filter unit for an AMR epidemiological study based on the One Health approach.

Molecular identification of blaTEM and blaCTX-M genes in multidrug-resistant Escherichia coli found in milk samples from dairy cattle farms in Tulungagung, Indonesia.

Introduction: Escherichia coli is an opportunistic bacteria that can grow easily, produce toxins, and resist antibiotics. The phenomenon of E. coli developing multidrug resistance is currently the subject of extensive research. The objective of this study was to molecularly identify blaTEM and blaCTX-M genes in multidrug-resistant E. coli found in milk samples from dairy cattle farms in Tulungagung, Indonesia. Material and Methods: One hundred and ten milk samples were collected from 45 dairy cattle farms in Tulungagung, Indonesia. Indole, methyl red, Voges-Proskauer and in citrate tests and triple iron sugar agar tests were used to identify E. coli. Multidrug resistance was determined in isolates through antibiotic sensitivity tests using tetracycline, streptomycin, trimethoprim, chloramphenicol and aztreonam. Extended-spectrum beta lactamase enzyme production was confirmed by double-disc synergy test (DDST). Molecular identification was performed to confirm the blaTEM and blaCTX-M genes. Results: One hundred and one (91.82%) E. coli strains were isolated from the samples. The antibiotic sensitivity test showed four (3.96%) multidrug-resistant (MDR) and one (0.99%) ESBL-positive E. coli by DDST confirmation. There were three (77.78%) blaTEM genes and one (0.99%) blaCTX-M gene discovered in the MDR E. coli isolates using PCR for molecular identification. Conclusion: The findings of the blaTEM and blaCTX-M genes encoding ESBL E. coli in dairy cattle milk in Tulungagung, Indonesia is concerning and argues for prompt action to stop the emergence of antibiotic resistance which has an impact on public health.

Antibiotic Resistance in Non-Typhoidal Salmonella enterica Strains Isolated from Chicken Meat in Indonesia.

The increase in antibiotic resistance in non-typhoidal Salmonella enterica (NTS) has been confirmed in Indonesia by this study. We confirmed the virulence genes and antimicrobial susceptibilities of clinical NTS (n = 50) isolated from chicken meat in Indonesia and also detected antimicrobial resistance genes. Of 50 strains, 30 (60%) were non-susceptible to nalidixic acid (NA) and all of them had amino acid mutations in gyrA. Among 27 tetracycline (TC) non-susceptible strains, 22 (81.5%) had tetA and/or tetB. The non-susceptibility rates to ampicillin, gentamicin or kanamycin were lower than that of NA or TC, but the prevalence of blaTEM or aadA was high. Non-susceptible strains showed a high prevalence of virulence genes compared with the susceptible strains (tcfA, p = 0.014; cdtB, p < 0.001; sfbA, p < 0.001; fimA, p = 0.002). S. Schwarzengrund was the most prevalent serotype (23 strains, 46%) and the most frequently detected as multi-antimicrobial resistant. The prevalence of virulence genes in S. Schwarzengrund was significantly higher than other serotypes in hlyE (p = 0.011) and phoP/Q (p = 0.011) in addition to the genes above. In conclusion, NTS strains isolated from Indonesian chicken had a high resistance to antibiotics and many virulence factors. In particular, S. Schwarzengrund strains were most frequently detected as multi-antimicrobial resistant and had a high prevalence of virulence genes.

Molecular detection of a new pathotype enteroaggregative haemorrhagic Escherichia coli (EAHEC) in Indonesia, 2015.

Enteroaggregative haemorrhagic Escherichia coli (E. Coli, EAHEC) has been identified as the agent responsible for one of the largest outbreaks of gastroenteritis and Haemolytic-uremic syndrome (HUS) that is transmitted through food in Germany in 2011. The hypervirulent pathotype has a unique combination of two pathogens namely enterohemorrhagic E.coli strain (EHEC) which produces shiga/verotoxin and enteroaggregative E.coli toxins (EAEC) which produces toxins similar to ST and hemolysin. The toxin produced by the EAHEC strain is a hybrid pathotype that combines the virulence potential of the EAEC and EHEC strains that will damage the microcirculation, cause vasculitis and other toxic effects. The purpose of this study was to determine the percentage of samples infected with enteroaggregative hemorrhagic E. coli bacteria (EAHEC) in pediatric diarrhea patients at DR. Soetomo Hospital, Surabaya, Indonesia, 2015. This study used PCR (Polymerase Chain Reaction) method to detect enteroaggregative E. coli strains (CVD432 and aaic genes) and enterohemorrhagic E.coli (eae gene).The results showed that 33 out of 40 (82,5%) stool samples examined were detected enteroaggregative E. coli (EAEC), 4 out of 40 (10%) enterohemorrhagic E. coli (EHEC) and 3 out of 40 (7,5%) enteroaggregative haemorrhagic E. coli bacteria (EAHEC), which caused diarrhea in pediatric diarrhea patients at Dr. Soetomo General Hospital. The unique combination of genomic features of the Surabaya outbreak strain, containing characteristics from pathotypes EAEC and EHEC, suggested that it represents a new pathotype enteroaggregative haemorrhagic E. coli (EAHEC). It is expected that development of specific primer design and sequencing are needed to continue in this research.

Molecular Epidemiology and Clinical Features of Rotavirus Infection Among Pediatric Patients in East Java, Indonesia During 2015-2018: Dynamic Changes in Rotavirus Genotypes From Equine-Like G3 to Typical Human G1/G3.

Group A rotavirus (RVA) is the most important cause of severe gastroenteritis among children worldwide, and effective RVA vaccines have been introduced in many countries. Here we performed a molecular epidemiological analysis of RVA infection among pediatric patients in East Java, Indonesia, during 2015-2018. A total of 432 stool samples were collected from hospitalized pediatric patients with acute gastroenteritis. None of the patients in this cohort had been immunized with an RVA vaccine. The overall prevalence of RVA infection was 31.7% (137/432), and RVA infection was significantly more prevalent in the 6- to 11-month age group than in the other age groups (P < 0.05). Multiplex reverse transcription-PCR (RT-PCR) revealed that the most common G-P combination was equine-like G3P[8] (70.8%), followed by equine-like G3P[6] (12.4%), human G1P[8] (8.8%), G3P[6] (1.5%), and G1P[6] (0.7%). Interestingly, the equine-like strains were exclusively detected until May 2017, but in July 2017 they were completely replaced by a typical human genotype (G1 and G3), suggesting that the dynamic changes in RVA genotypes from equine-like G3 to typical human G1/G3 in Indonesia can occur even in the country with low RVA vaccine coverage rate. The mechanism of the dynamic changes in RVA genotypes needs to be explored. Infants and children with RVA-associated gastroenteritis presented more frequently with some dehydration, vomiting, and watery diarrhea, indicating a greater severity of RVA infection compared to those with non-RVA gastroenteritis. In conclusion, a dynamic change was found in the RVA genotype from equine-like G3 to a typical human genotype. Since severe cases of RVA infection were prevalent, especially in children aged 6 to 11 months or more generally in those less than 2 years old, RVA vaccination should be included in Indonesia's national immunization program.

Clinical manifestation of norovirus infection in children aged less than five years old admitted with acute diarrhea in Surabaya, Indonesia: a cross-sectional study.

Background: The objective of this study was to investigate the clinical manifestation of norovirus infection between norovirus genogroup and severity of acute diarrhea in pediatric patients at the Dr. Soetomo Hospital, Surabaya, Indonesia. Methods: This cross-sectional study involved 31 participants aged 1-60 months admitted to the hospital with acute diarrhea from April 2012 to March 2013. Norovirus genogroups (GI and II) were identified from patient stool using reverse transcription polymerase chain reaction (RT-PCR). Severity was measured using the Ruuska and Vesikari scoring system. Results: In total, 94 stool samples were obtained, of which 31 (19%) were norovirus positive. Norovirus GI was found in one sample with mild diarrhea. Norovirus GII was found in 30 samples (96.8%); one sample with mild diarrhea (3.3%), 20 samples with moderate diarrhea (66.7%), and nine samples with severe diarrhea (30%). Conclusion: Norovirus GII was the most prevalent cause of acute diarrhea and 30% of the cases manifested as severe diarrhea.

Antibiotic Susceptibilities and Genetic Characteristics of Extended-Spectrum Beta-Lactamase-Producing Escherichia coli Isolates from Stools of Pediatric Diarrhea Patients in Surabaya, Indonesia.

The purpose of this study was to investigate extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli isolates from pediatric (aged 0 to 3 years) diarrhea patients in Surabaya, Indonesia, where this kind of survey is rare; our study included assessment of their antibiotic susceptibilities, as well as ESBL typing, multilocus sequence typing (MLST), and diarrheagenic E. coli (DEC)-typing. ESBL-producing E. coli were detected in 18.8% of all the samples. Many ESBL-producing E. coli had significantly lower susceptibility to gentamicin (p ＜ 0.0001) and the quinolones nalidixic acid (p＝0.004) and ciprofloxacin (p ＜ 0.0001) than non-producers. In ESBL-producing E. coli, 84.0% of strains expressed CTX-M-15 alone or in combination with other ESBL types. MLST revealed that 24.0% of ESBL-producers had sequence type 617, all of which expressed the CTX-M-15 gene; we also detected expression of 3 DEC-related genes: 2 enteroaggregative E. coli genes and 1 enteropathogenic E. coli gene. In conclusion, CTX-M-15-type ESBL-producing E. coli ST617 appear to have spread to Indonesia.

Genotyping and clinical factors in pediatric diarrhea caused by rotaviruses: one-year surveillance in Surabaya, Indonesia.

BACKGROUND: Rotavirus infections are a major cause of diarrhea in children in both developed and developing countries. Rotavirus genetics, patient immunity, and environmental factors are thought to be related to the severity of acute diarrhea due to rotavirus in infants and young children. The objective of this study was to provide a correlation between rotavirus genotypes, clinical factors and degree of severity of acute diarrhea in children under 5 years old in Surabaya, Indonesia. METHODS: A cross-sectional study was conducted in children aged 1-60 months with acute diarrhea hospitalized in Soetomo Hospital, Surabaya, Indonesia from April to December 2013. Rotavirus in stool specimens was identified by ELISA and genotyping (G-type and P-type) using multiplex reverse transcription PCR. Severity was measured using the Ruuska and Vesikari scoring system. The clinical factors were investigated included patient's age (months), hydration, antibiotic administration, nutritional state, co-bacterial infection and co-viral infection. RESULTS: A total of 88 children met the criteria; 80.7% were aged 6-24 months, watery diarrhea was the most common type (77.3%) and 73.6% of the subjects were co-infected with bacteria, of which pathogenic Escherichia coli was the most common (42.5%). The predominant VP7 genotyping (G-type) was G2 (31.8%) and that of VP4 genotyping (P-type) was P[4] (31.8%). The predominant rotavirus genotype was G2P[4] (19.3%); G1P[4] and G9P[4] were uncommon with a prevalence of 4.5%. There were significant differences between the common genotype and uncommon genotype with respect to the total severity score of diarrhea (p <0.05). G3, G4 and G9 were significantly correlated with severe diarrhea (p = 0.009) in multivariate analyses and with frequency of diarrhea (>10 times a day) (p = 0.045) in univariate analyses, but there was no significant correlation between P typing and severity of diarrhea. For combination genotyping of G and P, G2P[4] was significantly correlated with severe diarrhea in multivariate analyses (p = 0.029). CONCLUSIONS: There is a correlation between rotavirus genotype and severity of acute diarrhea in children. Genotype G2P[4] has the highest prevalence. G3, G4, G9 and G2P[4] combination genotype were found to be associated with severe diarrhea.

Frequency of diarrheagenic Escherichia coli among children in Surabaya, Indonesia.

Diarrheagenic Escherichia coli (DEC) is a major etiologic agent of childhood diarrhea in developing countries. We investigated the frequency of DEC in stool samples from 125 diarrheal children (age, 1-10 years) and 92 non-diarrheal children in Surabaya, Indonesia. The non-diarrheal children served as healthy controls. DEC was detected in 23 of 125 (18.4%) and 47 of 92 (51.1%) samples in the diarrheal and non-diarrheal children, respectively. Enteropathogenic E. coli was the most prevalent in the non-diarrheal children (25.0%), and its prevalence was significantly higher than that in the diarrheal children (0.8%) (P < 0.0001). Interestingly, Shiga toxin-producing E. coli (4.3%) was detected only in the non-diarrheal children (P = 0.031). This is the first study comparing between diarrheal children with non-diarrheal or healthy children to investigate the role of DEC in pediatric diarrheal diseases in Indonesia.

Modulation of O-antigen chain length by the wzz gene in Escherichia coli O157 influences its sensitivities to serum complement.

Escherichia coli O157 strains belonging to a distinct lineage and expressing different O-antigen (Oag) lengths were isolated. Although the function of wzz in E. coli has not been adequately investigated, this gene is known to be associated with regulation of Oag length. Using E. coli O157:H7 ATCC43888 (wild-type), several wzz mutants of E. coli O157, including a wzz deletion mutant, were generated and the relationship between the length of Oag modulated by the wzz gene and sensitivities to serum complement investigated. SDS-PAGE, immunoblot analyses and sensitivity tests to human serum complement were performed on these strains. The lengths of the O157-antigen could be modulated by the wzz gene mutations and were classified into long, intermediate and short groups. The short chain mutant was more serum sensitive than the wild-type strain and the other wzz mutants (P < 0.001). In conclusion, Oag chain length modulated by the wzz gene in E. coli O157 influences its sensitivities to serum complement. The present findings suggest that E. coli O157 strains with intermediate or long length Oag chains might show greater resistance to serum complement than those with short chains.