Phenotype and genotype of Boerka goats raised in Bali.

Background and Aim: Boerka goats are the new breed in Bali targeted at meeting the increasing demand for lamb. They are meat-type goats created by crossing male Boer and female Kacang breeds. This new breed is expected to have good adaptation in a poor environment, such as Kacang goats and produce good quality lamb as Boer goats. Therefore, this study aimed to examine phenotypic and genotypic characteristics of Boerka goats raised in Bali. Materials and Methods: A total of 16 female Boerka goats at 2 years old collected from a group of livestock farmers in Sanda Village were used as samples. This observational study began with observations of qualitative characteristics and morphometric measurements of goats, followed by polymerase chain reaction (PCR) of the growth hormone (GH) gene using GH5F and GH5R primers. Polymerase chain reaction products were then sequenced and analyzed with the MEGA 11 program. Results: The results showed that all Boerka goats had stuck-down ears, the heads were predominantly brown in color (62.5%), the body color pattern tends to be white (87.50%), and the tail color pattern was also dominated by white (62.5%). In morphometric terms, the samples were close to Kacang goats based on the body weight, head length, head width, chest width, depth, and circumference, left front leg circumference, ear length, ear width, tail length, tail width, and flank height except for head height, body length, horn length, and shoulder height. Analysis of the GH gene showed that Boerka goats had a nucleotide composition dominated by the purine base guanine (26.5%) and pyrimidine cytosine (31.8%). Furthermore, they formed a separate cluster with a genetic distance of 14.1% to the Anhui White breed from China, the Tibetan breed, and the Swiss Saanen breed. Conclusion: Boerka goats raised in Bali have phenotypes, including qualitative characteristics and morphometric measurements close to Kacang goats. As meat-type goats, they also form a separate cluster distinct from similar types worldwide.

Assessment of Cell Cycle and Induction of Apoptosis by Shiga-like Toxin Produced by Escherichia coli O157:H7 in T47D Breast Cancer Cells Using Flow Cytometry.

BACKGROUND: The low general toxicity against tumors expressing globotriaosylceramide (Gb3) and Shiga-like toxins produced by E. coli have been proposed as an anti-cancer therapy because of their specific target. This study aimed to determine the potency of the local strains of E. coli O157:H7 isolated from humans and cattle as a new breast cancer therapy by analyzing the cell cycle's inhibition and apoptosis induction. MATERIAL AND METHODS: Approximately 10 cultured T47D cells were subjected to Shiga-like toxin produced by four local isolates of E. coli O157:H7, including KL-48 (2) from humans, and SM-25 (1), SM-7 (1), DS-21 (4) from cattle. Using ATCC 43894 as a control, the treatment was observed for 24 h by two replications. In addition, a FITC-Annexin V and PI assay were used to observe apoptosis and necrosis effect, as well as to analyze the cell cycle using propidium iodide (PI) staining. RESULTS: The results showed the toxicity effect of Shiga in the human T47 D cells line. The viability of the cells is subjected to Shiga-like toxins produced by KL-48 (2), SM7 (1), ATCC 43894, SM-25 (1), and DS-21 (4) isolates decreased with 15.20, 16.36, 22.17,  22.64, and 33.86%, in contrary to control of 94.36%. These were supported by the cells entering the late apoptosis of the cell cycle through each isolate with 67.66, 62.60, 63.68, 63.90, and 54.74%, and a control of 0.01%. Also, the necrosis cell for each treatment of 12.73, 19.3, 10.84, 10.53, and 4.86% was higher than the control of 5.51%. These were confirmed by the higher percentage of the cells treated with toxins of KL-48 (2), SM7(1), ATCC 43894, SM-25 (1), and DS-21 (4), which entered G0-G1 of the cell cycle phase with 66.41, 63.37, 61.52, 55.36, and 47.28%, respectively, than control of 40.69%. Additionally, the toxicity effect was supported by an increase in the cells entering the S and the G2-M phase of the cycle for each treatment. CONCLUSION: It is concluded that the Shiga-like toxin produced by E. coli O157:H7 local isolates can be developed as a drug against breast cancer based on its effect to arrest induction of the cell cycle and inducing apoptosis.

Molecular identification of lactic acid bacteria SR6 strain and evaluation of its activity as an anticancer in T47D cell line.

Background and Aim: Breast cancer is the most common type of cancer in women because it attacks the productive age. Preliminary studies showed that lactic acid bacteria (LAB) strain SR6 from the Bali cattle colon has the potential to act as a superior probiotic. It is also assumed that its bacteriocin structure is specific and has a strong relationship with the specificity of the ligand and its biological activity at a receptor. Therefore, this study aims to assess the use of local LAB strains, which produce bacteriocins as anticancer agents, as well as to identify the bacteria as potent producers molecularly. Materials and Methods: The study was initiated by cultivating LAB SR6 strain from stock isolates on De Man, Rogosa, and Sharpe (Oxoid, CM 0369, England) broth media. It was then confirmed molecularly through analysis of the 16S ribosomal ribonucleic acid gene. Subsequently, its anticancer activity was tested by assessing the cytotoxic activity in T47D cell culture using the 3-(4, 5 dimetiltiazol-2-yl)-2.5-diphenyl tetrazolium bromide (Invitrogen M6494, US) method. Results: The results showed that the LAB strain SR6 was identified molecularly as Pediococcus pentosaceus. Furthermore, it had a toxic effect on T47D cells, which was indicated by the number of deaths after treatment with the extracellular protein of the strain, especially at the 50% total cell volume level. Conclusion: Based on the toxic effect of the strain on human T47D cells, the LAB SR6 isolate, which was identified as P. pentosaceus has the potential to be developed as a good anticancer drug against breast cancer. However, there is a need to carry out an integrated study to fully explore the suitability of bacteriocins as in vivo therapeutics against the disease completely.

Antibiotics susceptibility of Escherichia coli isolates from clinical specimens before and during COVID-19 pandemic.

BACKGROUND AND OBJECTIVES: Escherichia coli is a Gram-negative organism causing mild to severe infections, with a wide spectrum range of organs involved. The study aimed to describe antibiotics susceptibility of E. coli from clinical specimens from October 11, 2019 to September 11, 2020. MATERIALS AND METHODS: Study was conducted retrospectively in a private microbiology laboratory in Mataram Indonesia. Period of study divided as two groups after WHO declared COVID-19 as pandemic by March 11, 2020; group A including the specimen related to September 2019 to March 11th 2020 and group B including the specimens related to March 11th 2020 to September 2020. All clinical specimens were subjected to identify E. coli isolates and their antibiotics susceptibility using WHO-NET 5.6 version. RESULTS: Totally, 148 E. coli isolates were found in group A and 62 isolates in group B. Prevalence of extended-spectrum beta lactamase (ESBL)- producing E. coli in group A was 50% and in group B was 20.9% with significantly difference (p<0.05). There was an increase in susceptibility to 10/16 antibiotics; where 3 antibiotics ofloxacin, aztreonam, and fosfomycin were significant (p<0.05). There was a significant decrease in susceptibility to the antibiotics piperacillin (p=0.012), amoxicillin (p=0.002), cefadroxil (p=0.036) and ampicillin (p=0.036). Type of infections between two groups: musculoskeletal infections, pneumonia, urinary tract infections and sepsis were not significant. CONCLUSION: Reduced number of E. coli isolates between two groups with decrease of ESBL-producing E. coli contribute in dynamics of antibiotics susceptibility. The longer period of analysis is needed to be done, due to the ongoing COVID-19 pandemic.

Shiga-Like Toxin Produced by Local Isolates of Escherichia coli O157:H7 Induces Apoptosis of the T47 Breast Cancer Cell Line.

PURPOSE: It has been suggested that Shiga-like toxins produced by Escherichia coli O157:H7 could be used as novel therapeutic agents against malignant tumors. In addition, the antitumor potency of local isolates from Indonesia, which are known to be less toxic than the control isolate ATCC 43894, has not yet been tested. The study aimed to analyze local strains of E. coli O157:H7 as a proapoptosis agent on the T47 breast cancer cell line. METHODS: As many as 30 culture cells of T47D breast cancer cell line were subjected to purified extracts of Shiga-like toxin originating from 5 local isolates of E. coli O157:H7: KL-48(2), SM-25(1), SM-7(1), DS-21(4), and 1 isolate ATCC 43894 which was used as a control. Toxin production of each isolate was detected using a sandwich enzyme-linked immunosorbent assay, and the treatment of cell lines was observed for 24 hours, with 2 replications; 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide tests and acridine orange/ethidium bromide double staining assays were used for detection and analyses of apoptosis. RESULTS: The study showed 2 local strains of E. coli O157:H7 (codes KL-48(2) and SM-25(1)) had toxins positive at titer 5 and 10 µg/100 µL. These titers were lower than the control isolate ATCC 43894, but they had a necrosis effect higher (P < .05), ie, 80.3%, than control isolate, ie, 63.3%. Other local strain SM-25(1) also had a good necrosis effect. It has a nondifferent necrosis effect (P > .05) with the control isolate ATCC 43894, ie, 13.0% from 13.3%. CONCLUSION: This study concludes that the Shiga toxin produced by E. coli O157:H7 local isolate (Indonesia) has potential as a proapoptotic and/or necrotic agent for treating T47 breast cancer cell lines, as effectively as ATCC 43894 control isolates.

Evaluation of the bacteriocin produced by strain 9 lactic acid bacteria isolate for biopreservation.

AIM: This study aimed to determine the effect of the bacteriocin produced by strain 9 lactic acid bacteria (LAB) isolate on the biopreservation of beef. MATERIALS AND METHODS: The strain 9 LAB isolate was identified conventionally by culturing with de Man, Rogosa, and Sharpe broth medium followed by Gram staining and catalase testing. The molecular confirmation of the isolate involved analyzing the 16S rRNA gene with specific primers, that is, B27F (5-AGAGTTTGATCCTGGCTCAG-3) and U1492R (5-GGTTACCTTGTTACGACTT-3). Then, the isolate was centrifuged to evaluate the bacteriocin production, and the effect of the biopreservative activity in beef was evaluated by measuring the NH3 produced with the Eber test and the organoleptic acceptance from expert panels. RESULTS: This study confirmed that the strain 9 LAB isolate was a strain of Pediococcus pentosaceus, and the bacteriocin product showed biopreservative potential. The biopreservative potential was characterized by a significant decline in the production of NH3 and the panel's acceptance of the texture and tenderness of the beef, compared with the control, after 10 days of constant treatment. CONCLUSION: This study highlighted the high biopreservative potency of pediocin produced by P. pentosaceus strain 9. This was noted by the production of NH3 and the modifications in texture and tenderness.

Regulatory elements of stx2 gene and the expression level of Shiga-like toxin 2 in Escherichia coli O157:H7.

BACKGROUND/PURPOSE: Shiga-like toxin (Stx) is an important factor in the pathogenesis of Escherichia coli O157:H7 infection and is responsible for some severe complications. Stx2 is usually associated with hemolytic uremic syndrome in humans. Its expression is regulated by elements located upstream of the stx2 gene, including stx2-promoter sequence, ribosome binding site, and the antiterminator q gene. The present study aimed to find the correlation between regulatory elements and the expression level of Stx2 in two local isolates of E. coli O157:H7. METHODS: Two local E. coli O157:H7 strains SM-25(1) and KL-48(2), originating from human and cattle feces, respectively, and an E. coli reference strain, ATCC 43894, were investigated. The complete stx2 gene covering the sequences of promoter, ribosome binding site, and open reading frame and q gene of each strain was analyzed. The magnitude of Stx2 production was detected with a reverse passive latex agglutination method and Stx mediated cellular damage was determined with the Vero cell assay. RESULTS: A comparison of the complete stx2 gene contained stx2-promoter, ribosome binding site, and q genes of two local strains KL-48(2) and SM25(1), and the E. coli ATCC 43894 showed that the amino acid sequences were identical. Both local isolates were Stx negative in the reverse passive latex agglutination test and nontoxic in the Vero cell assay. CONCLUSION: The expression level of Shiga-like toxin of the two local isolates of E. coli O157:H7 did not only depend on the regulatory elements of the stx2 gene.

Frequency and risk-factors analysis of Escherichia coli O157:H7 in Bali-cattle.

Cattle are known as the main reservoir of zoonotic agents verocytotoxin-producing Escherichia coli. These bacteria are usually isolated from calves with diarrhea and/or mucus and blood. Tolerance of these agents to the environmental conditions will strengthen of their transmission among livestock. A total of 238 cattle fecal samples from four sub-districts in Badung, Bali were used in this study. Epidemiological data observed include cattle age, sex, cattle rearing system, the source of drinking water, weather, altitude, and type of cage floor, the cleanliness of cage floor, the slope of cage floor, and the level of cattle cleanliness. The study was initiated by culturing of samples onto eosin methylene blue agar, then Gram stained, and tested for indole, methyl-red, voges proskauer, and citrate, Potential E.coli isolates were then cultured onto sorbitol MacConkey agar, and further tested using O157 latex agglutination test and H7 antisera. Molecular identification was performed by analysis of the 16S rRNA gene, and epidemiological data was analyzed using STATA 12.0 software. The results showed, the prevalence of E. coli O157:H7 in cattle at Badung regency was 6.30% (15/238) covering four sub districts i.e. Petang, Abiansemal, Mengwi, and Kuta which their prevalence was 8.62%(5/58), 10%(6/60), 3.33%(2/60), and 3.33(2/60)%, respectively. The analysis of 16S rRNA gene confirmed of isolates as an E. coli O157:H7 strain with 99% similarities. Furthermore, the risk factors analysis showed that the slope of the cage floor has a highly significant effect (P<0.05) to the distribution of infection. Consequently, implementing this factor must be concerned in order to decrease of infection.

Analysis of Nucleotide Sequences of the 16S rRNA Gene of Novel Escherichia coli Strains Isolated from Feces of Human and Bali Cattle.

Livestock especially cattle are known as a main reservoir of Escherichia coli O157:H7. This bacterium is considered as a pathogenic agent characterized by producing toxins, which are familiarly known as Shiga-like toxin-1 (Stx1) and Stx2. The aim of this work was to analyse the novel sequence of the 16S rRNA gene of strains isolated in this study in order to know the phylogenetic relationships between these sequences and those between the sequences of bacteria available in databanks. The results of this analysis showed that the strains KL-48(2) and SM25(1) that originated from human and cattle feces, respectively, are closely related among them and with respect to E. coli EDL 933, E. coli Sakai, E. coli ATCC 43894, E. coli O111:H-, E. coli O121:H19, E. coli O104:H4, and Shigella sonnei with more than 99% similarity values.

Erratum to "Analysis of Nucleotide Sequences of the 16S rRNA Gene of Novel Escherichia coli Strains Isolated from Feces of Human and Bali Cattle".

[This corrects the article DOI: 10.1155/2014/475754.].