Genotype variation of ACE and ACE2 genes affects the severity of COVID-19 patients.

OBJECTIVE: Genetic polymorphisms in ACE and ACE2 genes are involved in the RAS regulation of blood pressure and their activity may confer susceptibility to hypertension. In addition, they may play a role in SARS-CoV-2 pathogenesis and the severity of COVID-19. This study aims to determine the effect of genetic variations in the ACE (rs4331) and ACE2 (rs2074192) genes with hypertension comorbidity on the severity of COVID-19 in the Indonesian population. RESULT: 186 patients were enrolled and assigned into the COVID-19 group (n = 95) and non-COVID-19 group (n = 91) in this cross-sectional study. GG genotype frequency was dominant in ACE gene, but there were no significant differences between the groups (p = 0.163). The two groups had a significant difference (p = 0.000) for the CC genotype frequency (0,37 vs. 0.01) in the ACE2 gene. The proportion of women with COVID-19 is higher (51%), but men with hypertension had more severe symptoms (44%). Men with hypertension comorbidity, GG (ACE), and TT (ACE2) genotypes tended to have moderate-to-severe symptoms (25%). Similarly, women with hypertension as well as GG and CT genotypes tended to have moderate-to-severe symptoms (21%). We conclude that hypertension and mutations in the ACE (rs4331) and ACE2 (rs2074192) genes affect the severity of COVID-19.

Metagenomic profiles of the early life microbiome of Indonesian inpatient neonates and their influence on clinical characteristics.

Determining the initial normal neonatal gut microbiome is challenging. The debate regarding the sterile fetal environment is still ongoing. Therefore, studying and comparing normal and dysbiotic microbiomes requires the elucidation of both the fetal and infant microbiomes. Factors influencing the normal microbiome also include regional and genetic factors specific to different countries. Determining the normal microbiome population in our center and their association with the clinical conditions of infants is helpful as a tool for both the prevention and treatment of related diseases during neonatal care. Here, we employed metagenomic sequencing to characterize meconium and the subsequent early-life gut microbiome of preterm neonates in Jakarta, Indonesia. Microbiome diversity and complexity was higher in the meconium and on day 4 than on day 7. At the genus level, the most abundant genus overall was unidentified Enterobacteriaceae, with meconium samples dominated by Ureaplasma, day 4 fecal samples dominated by Staphylococcus, and day 7 samples dominated by Clostridiales, while at the phylum level the most abundant was Proteobacteria and Firmicutes. Perinatal factors of PROM and mother's diet influenced the meconium microbiome, while day 4 and day 7 microbiome was associated with bacteremia and early administration of antibiotics. One of our sample sets was derived from triplets, and they had varying diversity despite being triplets. These data are valuable for understanding the formation of a healthy microbiome specific to neonates and devising a strategy to improve both the gut health and related clinical outcomes of the neonate.

New robustaflavone from Garcinia latissima Miq. leave and Its antibacterial activity.

Isolation and determination of antibacterial compounds from plants are essential to obtain a new antibacterial as a substitute for conventional resistant antibiotics. This study aims to isolate and identify a new robustaflavone as antibacterial activity from Garcinia latissima Miq. leave. In this study, the isolation process was carried out using column chromatography followed by preparative thin layer chromatography (TLC) based on the TLC profile. The fraction D was tested for anti-bacterial Bacillus subtilis using the TLC bioautography method. The isolates obtained were then identified using 1H-NMR, 13C-NMR, distortionless enhancement by polarization transfer, heteronuclear single quantum coherence, and heteronuclear multiple bond coherence. The Activity assay of the isolate was performed using the microdilution method. A pure compound obtained the result of the separation process with eluent n-hexane: Ethyl acetate (3:2) with Rf 0.6. This spot follows the spot in the contact bioautographic result of fraction D, the spot with Rf 0.6 gives an inhibition zone. After identifying and purifying the isolate were known as Robustaflavone, this compound has activity against B. subtilis with a (minimum inhibitory concentration) value of 2500 ppm. Robustaflavone successfully isolated and identified from G. latissima leave and its antibacterial activity.

Clinical profile, treatment, and outcomes of patients with COVID-19 in a tertiary referral hospital in South Sumatera, Indonesia: A retrospective single-center study.

Background: Although there are fewer COVID-19 cases in Indonesia, the pandemic is still ongoing. COVID-19 has a significant death rate in Indonesia, but lack of information on the effect of different clinical and demographic factors on COVID-19-related grimness and mortality in Indonesia. Objective: This study examined the clinical profile, treatment, and outcomes of patients with COVID-19 at Lahat Regency Hospital in South Sumatera, Indonesia, to find relevant markers that might be utilized to predict the prognosis of these patients. Methods: This was a retrospective single-center study of all medical record files of confirmed patients with COVID-19 admitted to Lahat Hospital from September 2020 to August 2021 (n = 285). Descriptive statistics, Chi-square, Mann-Whitney, Multiple Logistic Regression, and Cox's proportional hazards model were used for data analyses. Results: This study included 65 non-hospitalized and 220 hospitalized patients. Hospitalized patients were divided into dead and alive groups. The median age was lower in the non-hospitalized group without gender discrimination, and most hospitalized patients had comorbidities. Vital signs and clinical features were significantly different in hospitalized patients compared to non-hospitalized. The survival patients in the hospitalized group showed lower white blood cell (WBC), neutrophil percentages, and neutrophil-lymphocyte ratio (NLR) but higher lymphocyte and eosinophil. Non-survival patients had elevated alanine aminotransferase (ALT), blood urea nitrogen (BUN), creatinine, blood glucose, and potassium. The use of Favipiravir and Remdesivir was significant between the alive and dead groups. The mean hospital stay for all patients was 9.49 ± 4.77 days, while the median duration of hospital time was 10.73 ± 4.33 days in the survival group and 5.39 ± 3.78 days in the non-survival group. Multiple logistic regression analysis determined respiration rate, WBC, and BUN as predictors of survival. Conclusions: Age and comorbidities are significant elements impacting the seriousness of COVID-19. Abnormal signs in laboratory markers can be used as early warning and prognostic signs to prevent severity and death. Potential biomarkers at various degrees in patients with COVID-19 may also aid healthcare professionals in providing precision medicine and nursing.

New approach for the identification of potentially toxigenic Corynebacterium sp. using a multiplex PCR assay.

In diphtheria laboratory examinations, the PCR test can be applied to isolates and clinical specimens. This study aimed to develop a PCR assay to identify the species and toxigenicity of diphtheria-causing bacteria, including the prediction of some NTTB types. Seven reference isolates, four synthetic DNA samples, 36 stored isolates, and 487 clinical samples used for PCR optimization. The PCR results was confirmed by DNA sequence analysis. The results of the PCR examination of the 7 reference isolates and 36 stored isolates were similar to the results obtained using conventional methods as gold standard, both for diphtheria-causing and non-diphtheria-causing bacteria. The validation of the PCR results using DNA sequence analysis showed that there was no mispriming or misamplification. The multiplex PCR assay developed in this study could correctly identify the species and toxigenicity of diphtheria-causing bacteria, including the prediction of some NTTB types not yet covered by established PCR methods.

Construction and Functional Analysis of the Recombinant Bacteriocins Weissellicin-MBF from Weissella confusa MBF8-1.

BACKGROUND: Bacteriocins (Bac1, Bac2, and Bac3) from Weissella confusa MBF8-1, weissellicin- MBF, have been reported as potential alternative substances as well as complements to the existing antibiotics against many antimicrobial-resistant pathogens. Previously, the genes encoded in the large plasmid, pWcMBF8-1, and the spermicidal activity of their synthetic peptides, originally discovered Indonesia, have been studied. Three synthetic bacteriocins peptides of this weissellicin-MBF have been reported for their potential activities, i.e. antibacterial and spermicidal. OBJECTIVE: The aim of this study was to construct the recombinant Bacteriocin (r-Bac) genes, as well as to investigate the gene expressions and their functional analysis. METHODS: Here, the recombinant Bacteriocin (r-Bac) genes were constructed and the recombinant peptides (r-Bac1, r-Bac2, and r-Bac3) in B. subtilis DB403 cells were produced on a large scale. After purification, using the His-tag affinity column, their potential bioactivities were measured as well as their antibacterial minimum inhibitory concentrations against Leuconostoc mesenteroides and Micrococcus luteus, were determined. RESULTS: Pure His-tag-recombinant Bac1, Bac2, and Bac3 were obtained and they could inhibit the growth of L. mesenteroides and M. luteus. CONCLUSION: The recombinant bacteriocin could be obtained although with weak activity in inhibiting gram-positive bacterial growth.

Clinical characteristics influence cultivable-bacteria composition in the meconium of Indonesian neonates.

BACKGROUND: Microbial colonization of a neonate's gastrointestinal tract has significant perinatal and lifetime health consequences. However, information regarding the profile of meconium microbiota in neonates and the influence of clinical parameters are lacking in the Indonesian population. This study aimed to preliminary investigate the profile of cultivable bacterial diversity of meconium isolated from neonates born at Cipto Mangunkusumo Hospital (CMH), Jakarta. The cultivable bacteria were isolated from meconium samples and were then processed for cultivation and molecular identification. RESULTS: Fourteen neonates were enrolled as described, i.e., seven hyperbilirubinemia (Hyp) and seven non-Hyp with ten neonates delivered by cesarean section (CS) and four others by vaginal route (VR), and with five exclusive breastfeeding (Ebf), four formula milk, and five combinations. Microbiological identification, molecular 16S rDNA PCR-Sanger sequencing, and PCA analysis of cultivable bacteria isolated from meconium showed Firmicutes' predominance (84.41%), with an abundant population of Staphylococcus, which consist of S. hominis, S. epidermidis, and S. haemolyticus. The influence of mode of delivery showed a lower diversity than the CS populates the VR, but their composition was similar. Concurrently, between feeding patterns, the genera profile did not show much difference; in the non-Ebf group, the total amount of Staphylococcus and Bacillus showed a higher amount but a less diverse. Interestingly, the non-Hyp group showed more abundant and diverse Staphylococcus than that of the Hyp group. In contrast, neonates diagnosed with NEC and proven sepsis showed the same pattern of Staphylococcus domination. CONCLUSION: Staphylococcus predominated the composition of cultivable bacteria in neonates meconium. Due to the small sample size, only the hyperbilirubinemia parameter significantly influenced the profile, i.e., Staphylococcus's proportion (p = 0.037).

Simultaneous profiling and cultivation of the skin microbiome of healthy young adult skin for the development of therapeutic agents.

BACKGROUND: Studies on the impact of the skin microbiota on human health have been gaining more attention. Bacteria are associated with various diseases, although certain strains of bacteria, which are known as probiotics, are considered beneficial. Mixtures of several bacteria (bacterial cocktail) isolated from targeted organs have shown promising modulatory activities for use in skin therapeutics. The objectives of this study were to determine and identify the microbial communities on the skin that can potentially be used as probiotics, as determined by bacterial isolation and cultivation, followed by next-generation sequencing (NGS). RESULTS: Samples were collected by swabbing on forehead and cheek skin. Genomic DNA from bacterial swab samples were directly extracted to be further processed into NGS. Cultivation of skin bacteria was carried out in subsequent medium. Thus, around twenty bacterial isolates with different characteristics were selected and identified by both culture-based method and 16sRNA sequencing. We found that Actinobacteria and Firmicutes are the most abundant phylum present on the skin as presented by NGS data, which constitute to 67% and 28.59% of the whole bacterial population, consecutively. However, Staphylococcus hominis, Staphylococcus warneri, and Micrococcus luteus (AN MK968325.1; AN MK968315.1; and MK968318.1 respectively) were able to be obtained in the samples of cultivable, and could be potentially developed as probiotics in skin microbiome therapeutic as well as for postbiotic formulation. CONCLUSION: Skin microbiome is considered to provide several probiotics for skin therapeutic. However, some opportunistic pathogens were discovered in this study population. Thus, the promising formula of bacterial cocktail for skin microbiome therapeutic must be thoroughly elucidated to avoid unwanted species. Our study is the first human skin microbiome profile of Indonesia resulted from a Next Generation Sequencing as an effort to show a representative of tropical country profile.

Profiling of UGT1A1*6, UGT1A1*60, UGT1A1*93, and UGT1A1*28 Polymorphisms in Indonesian Neonates With Hyperbilirubinemia Using Multiplex PCR Sequencing.

Background: Single nucleotide polymorphism (SNP) variants of the uridine diphosphate glucuronosyltransferase 1A1 (UGT1A1) gene have been studied as an important factor in neonatal hyperbilirubinemia (jaundice) severity. Specific ethnicities, including Asians, have certain SNPs that appear more frequently than others. Aim: To identify the most common SNPs in Indonesian neonates and their association with the severity of neonatal hyperbilirubinemia. Methods: Eighty-eight inborn and outborn jaundiced infants from three different hospitals (Bengkulu, Jakarta, Biak Papua) across Indonesia were enrolled in this cross-sectional study and their peak total serum bilirubin (TSB) levels assessed. SNP variant analyses of the TATAA box, promoter, and exon 1 regions of UGT1A1 gene from 78 of the 88 infants were carried out using the SNaPshotR Multiplex Polymerase Chain Reaction (PCR) System followed by DNA sequencing. Results: We detected SNP variants UGT1A1 * 28, UGT1A1 * 60, UGT1A1 * 93, and UGT1A1 * 6 in our population. Mean total serum bilirubin (TSB) was 14.59 ± 5.57 mg/dL. Bivariate analyses using delivery location, gestational age, birth weight, mother's age, and ethnicity were shown to be associated with moderate-to-severe hyperbilirubinemia (p < 0.05). None of the four SNPs appeared to be associated with moderate-to-severe hyperbilirubinemia. In multivariate analysis, however, only the "other ethnic group" (e.g., Chinese, Bengkulu, Papua, Bima) category showed an association with moderate-to-severe hyperbilirubinemia, with an odds ratio of 6.49 (95% CI 1.01-41.67; p < 0.05). Conclusions: We found that the UGT1A1 * 60 is the most common SNP detected in neonates with hyperbilirubinemia in the Indonesian population. Interestingly, in Indonesia, UGT1A1 polymorphisms do not appear to be associated with differences in the severity of hyperbilirubinemia.

Three Bacteriocin Peptides from a Lactic Acid Bacterium Weissella confusa MBF8-1 with Spermicidal Activity.

BACKGROUND: The development of antibiotic resistance amongst bacterial pathogens and a population explosion, e.g. in countries such as Indonesia, are two issues the world is facing today. These issues have stimulated interest in the development of new antimicrobial therapeutic agents and contraceptive strategies, such as novel spermicides. Bacteriocins, which are bacterially-derived antimicrobial peptides, may fulfill some of the criteria for these new agents. METHODS: Weissella confusa MBF8-1, originally isolated from a homemade soy product, exhibits antibacterial activity that was subsequently found to be plasmid-encoded, presumably by three peptides Bac1, Bac2 and Bac3. In the present study, we tested cell-free MBF8-1 bacteriocin preparations and chemically-synthesized versions of Bac1, Bac2 and Bac3 peptides for (i) its antibacterial activity against the indicator bacterium Leuconostoc mesenteroides and (ii) its ability to affect the motility of spermatozoa. Nisin, a known lantibiotic bacteriocin, was used as the control. RESULTS: Here, we demonstrate that synthetic Bac1, in combination with synthetic Bac2, was sufficient to inhibit the growth of L. mesenteroides and affect sperm motility. However, the presence of all three synthetic peptides, s-Bac1, s-Bac2 and s-Bac3, was required for full potency. CONCLUSION: In summary, the bacteriocin-like peptides of W. confusa MBF8-1 have the potential to be developed as a narrow-spectrum antimicrobial agent and a novel spermicidal agent.

SLCO1B1 c.388A > G variant incidence and the severity of hyperbilirubinemia in Indonesian neonates.

OBJECTIVE: It has been established that genetic factors play a substantial role in the development of neonatal hyperbilirubinemia. The population of Indonesia and other Southeast Asian countries has similar, yet different genetic makeup compared to the rest of Asia. Aside from UGT1A1, variants of SLCO1B1 have also been known to contribute to the severity of neonatal hyperbilirubinemia in Asian populations. Since there has been no report on SLCO1B1 polymorphism in relation with hyperbilirubinemia in Indonesia, this study aims to explore incidence of SLCO1B1*1B polymorphism in Indonesia based on 3 hospitals from different provinces and population, and their association with hyperbilirubinemia severity. METHODS: Our study included 88 neonates with mild and moderate-severe hyperbilirubinemia from 3 NICU in hospitals representing homogenous and heterogenous populations: Biak General Hospital Papua, Cipto Mangunkusumo Hospital (Jakarta), and M Yunus Hospital (Bengkulu). We collected samples between November 2016 and September 2017. DNA was obtained from existing samples of the patients from previous studies and were subjected to Polymerase Chain Reaction - Restriction Fragment Length Polymorphism (PCR-RFLP). We analyzed the *1B variant located in exon 5 of SLCO1B1 with TaqI restriction endonuclease. Clinical, demographic, and laboratory data was also collected from medical records and parents' interviews. RESULTS: The most dominant variant of SLCO1B1*1B in our population is the homozygous G/G (68.18%), followed by heterozygous A/G (26.14%), and wild type A/A (5.68%). The heterozygous A/G had an Odds Ratio (OR) of 0.73 (95% CI 0.10-5.2) and homozygous G/G with OR of 0.51 (95%CI 0.08-3.27), both were not significant. Genotypic distribution across the different centers were also similar and not significant. The significant risk factors for moderate-severe hyperbilirubinemia were the population the neonate originated from (p = < 0.001) and the delivery location (p = 0.001), while SLCO1B1*1B was not associated with the different severity of hyperbilirubinemia. CONCLUSIONS: SLCO1B1*1B is not associated with higher bilirubin levels among neonates with hyperbilirubinemia in Indonesia. Further study is needed to find other potentially important genetic polymorphisms in the development of severe hyperbilirubinemia in Indonesia.

UGT1A1 gene and neonatal hyperbilirubinemia: a preliminary study from Bengkulu, Indonesia.

OBJECTIVE: The genetic involvement in unconjugated neonatal hyperbilirubinemia has been extensively studied. Despite the high incidence of hyperbilirubinemia in Indonesia, studies are lacking. The objective of this study is to elucidate the role of polymorphism in the UGT1A1 in Neonatal Hyperbilirubinemia in Bengkulu, Indonesia. RESULTS: There were 41 neonates enrolled in the study; 30 had a total serum bilirubin level ≥ 15 mg/dL (hyperbilirubinemia neonates) while 11 has < 15 mg/dL (control neonates). Genetic mutations in Exon 1, UGT1A1*6 (c211g > a) and one in promoter region, UGT1A1*60 (c3279t > g) were determined by polymerase chain reaction-restriction fragment length polymorphism. We found 18 (60%) mutation in exon 1 in hyperbilirubinemia group and 7 (64%) in the control group with an identical allele frequency of 0.3 in both groups. We found heterozygous UGT1A1*60 4 times (13.3%) and homozygous 26 times (86.7%) in the hyperbilirubinemia group, with an identical allele frequency of 0.935 in hyperbilirubinemia and 1 in control group. This study supports the involvement of genetic factors in the development of unconjugated hyperbilirubinemia in Bengkulu population.

Bacteriocin-like inhibitory substance (BLIS) activity of Streptococcus macedonicus MBF10-2 and its synergistic action in combination with antibiotics.

OBJECTIVE: To characterize the bacteriocin-like inhibitory-substances (BLIS) activity of Streptococcus macedonicus MBF10-2 [named BLIS(MBF10-2)], a bacteriocinogenic strain isolated from an Indonesian tofu byproduct. METHODS: BLIS(MBF10-2) was obtained by culturing the bacterium, and standard deferred antagonism assays were used to demonstrate its activity. The antibacterial testing of fractions collected by filtration using 3-30 kDa cut-off membrane sizes were carried out by performing well diffusion method. RESULTS: The growth of Micrococcus luteus, Streptococcus pyogenes, Lactococcus lactis, Leuconostoc mesenteroides and Weissella confusa were inhibited by BLIS(MBF10-2). Interestingly, BLIS-containing fractions obtained from sequential application on ultrafiltration membranes indicated that this bacterium Streptococcus macedonicus MBF10-2 could produces at least two antimicrobial peptides activities, one of which is likely to be a lantibiotic peptide. Potential synergistic activity against certain Gram-positive (but not Gram-negative) species when partnered with antibiotics (ampicillin, tetracycline or kanamycin) were observed. CONCLUSION: Combination of some BLIS(MBF10-2) active fractions with antibiotics (ampicillin, tetracycline or kanamycin) could demonstrate synergistic activities against certain Gram-positive species.

Draft Genome Sequence of Weissella confusa MBF8-1, a Glucansucrase- and Bacteriocin-Producing Strain Isolated from a Homemade Soy Product.

We report here the draft genome sequence of Weissella confusa MBF8-1, an isolate from a homemade fermented soybean product that produces sucrases and exhibits antibacterial (bacteriocin) activity. The draft genome of W. confusa MBF8-1 comprises a 2.2-Mbp chromosome and a 17.8-kbp bacteriocin-encoding plasmid. Two putative glucansucrase genes were also identified.

Identification and sequence analysis of pWcMBF8-1, a bacteriocin-encoding plasmid from the lactic acid bacterium Weissella confusa.

Members of the Gram-positive lactic acid bacteria (LAB) are well-known for their beneficial properties as starter cultures and probiotics. Many LAB species produce ribosomally synthesized proteinaceous antibiotics (bacteriocins). Weissella confusa MBF8-1 is a strain isolated from a fermented soybean product that not only produces useful exopolysaccharides but also exhibits bacteriocin activity, which we call weissellicin MBF. Here, we show that bacteriocin production by W. confusa MBF8-1 is specified by a large plasmid, pWcMBF8-1. Plasmid pWcMBF8-1 (GenBank accession number KR350502), which was identified from the W. confusa MBF8-1 draft genome sequence, is 17 643 bp in length with a G + C content of 34.8% and contains 25 open reading frames (ORFs). Six ORFs constitute the weissellicin MBF locus, encoding three putative double-glycine-motif peptides (Bac1, Bac2, Bac3), an ABC transporter complex (BacTE) and a putative immunity protein (BacI). Two ORFs encode plasmid partitioning and mobilization proteins, suggesting that pWcMBF8-1 is transferable to other hosts. To the best of our knowledge, plasmid pWcMBF8-1 not only represents the first large Weissella plasmid to be sequenced but also the first to be associated with bacteriocin production in W. confusa.

Cloning and heterologous expression of the ftfCNC-2(1) gene from Weissella confusa MBFCNC-2(1) as an extracellular active fructansucrase in Bacillus subtilis.

Fructan-exopolysaccharides (fructan-EPS) (inulin and levan) and their oligosaccharides (fructooligosaccharides, FOS) have drawn considerable interest in the food and pharmaceutical industries. EPS-producing lactic acid bacteria have been reported to produce ß-fructans (inulin and levan), as well as α-glucans, by the function of sucrase enzymes, i.e., fructansucrase and glucansucrase. A fructansucrase ftfCNC-2(1) gene from Weissella confusa strain MBFCNC-2(1) was previously cloned in Escherichia coli. In this study, we aimed to express the ftf[CNC-2(1)] gene in Bacillus subtilis to obtain the active form of the extracellular recombinant protein FTF[CNC-2(1)]. This cloning was achieved by inserting the gene in-fusion with the signal sequence of the B. subtilis subtilisin E. SDS-polyacrylamide gel electrophoresis analysis and in situ activity assay with Periodic Acid-Schiff staining revealed that the recombinant FTF[CNC-2(1)] was successfully expressed as an extracellular protein from B. subtilis DB403 in its active form, which was confirmed using sucrose and raffinose.

4,6-α-glucanotransferase, a novel enzyme that structurally and functionally provides an evolutionary link between glycoside hydrolase enzyme families 13 and 70.

Lactobacillus reuteri 121 uses the glucosyltransferase A (GTFA) enzyme to convert sucrose into large amounts of the α-D-glucan reuteran, an exopolysaccharide. Upstream of gtfA lies another putative glucansucrase gene, designated gtfB. Previously, we have shown that the purified recombinant GTFB protein/enzyme is inactive with sucrose. Various homologs of gtfB are present in other Lactobacillus strains, including the L. reuteri type strain, DSM 20016, the genome sequence of which is available. Here we report that GTFB is a novel α-glucanotransferase enzyme with disproportionating (cleaving α1→4 and synthesizing α1→6 and α1→4 glycosidic linkages) and α1→6 polymerizing types of activity on maltotetraose and larger maltooligosaccharide substrates (in short, it is a 4,6-α-glucanotransferase). Characterization of the types of compounds synthesized from maltoheptaose by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS), methylation analysis, and 1-dimensional ¹H nuclear magnetic resonance (NMR) spectroscopy revealed that only linear products were made and that with increasing degrees of polymerization (DP), more α1→6 glycosidic linkages were introduced into the final products, ranging from 18% in the incubation mixture to 33% in an enriched fraction. In view of its primary structure, GTFB clearly is a member of the glycoside hydrolase 70 (GH70) family, comprising enzymes with a permuted (ß/α)8 barrel that use sucrose to synthesize α-D-glucan polymers. The GTFB enzyme reaction and product specificities, however, are novel for the GH70 family, resembling those of the GH13 α-amylase type of enzymes in using maltooligosaccharides as substrates but differing in introducing a series of α1→6 glycosidic linkages into linear oligosaccharide products. We conclude that GTFB represents a novel evolutionary intermediate between the GH13 and GH70 enzyme families, and we speculate about its origin.

Molecular characterization of human group A rotavirus from stool samples in young children with diarrhea in Indonesia.

Detection and genotyping of group A rotavirus strains from stool samples in young children with diarrhea in Indonesia were examined using reverse transcription-nested multiplex PCR. Of 421 stool specimens, 257 samples was rotavirus positive. G1 type was the most common G-type (54%), followed by G2 (6%) and G9 (3%). P[8] was the most common P-type (39%), followed by P[6] (19%), P[4] (10%) and P[11] 1%. Eighteen percent of the samples had mixed G genotype infection and 5% had mixed P genotype infection. The prevalence of G-P combination type was genotype G1P[8] (24%), followed by G1P[6] (7%), G2P[4] (3%), and G1P[4] (2%). A total of 118 specimens could not be assigned as a G and/or P type suggesting the presence of new circulating genotypes in Indonesia.

Screening of lactic acid bacteria from Indonesia reveals glucansucrase and fructansucrase genes in two different Weissella confusa strains from soya.

Homopolysaccharide (glucan and fructan) synthesis from sucrose by sucrase enzymes in lactic acid bacteria (LAB) has been well studied in the genera Leuconostoc, Streptococcus and Lactobacillus. This study aimed to identify and characterize genes encoding glucansucrase/glucosyltransferase (GTF) and fructansucrases/fructosyltransferase (FTF) enzymes from genomic DNA of 'rare' Indonesian exopolysaccharide-producing LAB. From a total of 63 exopolysaccharide-producing LAB isolates obtained from foods, beverages and environmental samples, 18 isolates showing the most slimy and mucoid colony morphologies on sucrose were chosen for further study. By comparing bacterial growth on De Man, Rogosa and Sharpe (MRS)-sucrose with that on MRS-raffinose, and using the results of a previous PCR screening study with degenerate primer pairs targeting the conserved catalytic domain of GTFs, various strains were identified as producers of fructan (13), of glucan only (five) or as potential producers of both glucan and fructan (nine). Here, we report the characteristics of three gtf genes and one ftf gene obtained from Weissella confusa strains MBF8-1 and MBF8-2. Strain MBF8-1 harbored two putative gtf genes with high sequence similarity to GTFB of Lactobacillus reuteri 121 and GTF180 of L. reuteri 180, respectively. Strain MBF8-2 possessed single gtf and ftf genes with high sequence similarity to GTFKg3 of Lactobacillus fermentum Kg3 and DSRWC of Weissella cibaria, and FTF levansucrase of L. reuteri 121, respectively.

Cloning, DNA sequence, and expression of Aeromonas caviae WS7b chitinase gene.

A chitinase-producing bacterium, designated WS7b, was isolated from a soil sample obtained from a black-pepper plantation on Bangka Island, Indonesia. Fatty-acid methyl-ester analysis indicated that the isolate was Aeromonas caviae. A chitinase gene from WS7b was cloned in a pUC19-based plasmid vector, but without its natural promoter. The complete nucleotide sequence of the gene was determined, and the structural gene consisted of a 2748-bp region encoding 864 amino acids. DNA sequence analysis indicated that the gene had been cloned without its promoter, and this was confirmed by chitinase-plate assay of the truncated version of the gene in Escherichia coli. The chitinase gene product showed amino-acid sequence similarity to chiA from A. caviae. Chitinase enzyme activity was determined spectrophotometrically, using colloidal chitin azure as substrate for extracellular and intracellular fractions. The ability of the chitinase cloned in E. coli to hydrolyze chitin was less than that of the enzyme in its indigenous host.