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.

Antibacterial activity of clove (Syzygium aromaticum) and cinnamon (Cinnamomum burmannii) essential oil against extended-spectrum ß-lactamase-producing bacteria.

BACKGROUND AND AIM: Extended-spectrum ß-lactamase (ESBL) is an enzyme produced by the family of Enterobacteriaceae, especially Escherichia coli and Klebsiella pneumoniae, which can hydrolyzeß-lactam antibiotics, such as penicillins, cephalosporins, cephamycin, and carbapenem. ESBL-producing bacteria are widely distributed from farms to slaughterhouses until food products originating from animals are available in the market, which plays an important role as a pathway for the exposure and transmission of ESBL-producing bacteria from food products of animal origin to humans. This study aimed to determine the antibacterial activity of Syzygium aromaticum (clove) and Cinnamomum verum (cinnamon) essential oils against strains resistant to ESBL-producing E. coli and K. pneumoniae isolates. MATERIALS AND METHODS: The antibacterial activity of clove and cinnamon essential oils was tested against three strains of tested bacteria using the disk diffusion method. The minimum inhibitory concentration (MIC) of clove and cinnamon essential oils was determined using the broth microdilution method. The minimumbactericidal concentration (MBC) was determined using the MIC. Morphological changes on each tested bacteria were observed through scanning electron microscopy (SEM). RESULTS: Both essential oils exhibited inhibitory effects toward all test organisms, indicated by inhibition zones around the disk. The MIC values of clove essential oil were 0.078% (v/v) for all tested bacteria, whereas the MICs of cinnamon essential oil ranged from 0.039% (v/v) to 0.156% (v/v) for all tested bacteria. MBC values of clove and cinnamon essential oils ranged from 0.078% (v/v) to 0.156% (v/v) for all tested bacteria. There were morphological changes in each tested bacterial cell that was observed through SEM. Each tested bacteria treated with clove and cinnamon essential oils showed shrinkage and cells lysis. CONCLUSION: It was concluded that clove and cinnamon essential oils have emerged as effective antibacterial agents by showing high antibacterial activity against ESBL-producing E. coli and K. pneumoniae isolates, as evidenced by the inhibition zone diameter and MIC value.

Leptospirosis in Ruminants in Yogyakarta, Indonesia: A Serological Survey with Mixed Methods to Identify Risk Factors.

Leptospirosis is a zoonotic disease occurring worldwide with reproductive symptoms and production losses in livestock, while humans can suffer fatal renal failure. In Yogyakarta Special Province, Indonesia, there have been several outbreaks with high case fatality, demonstrating the public health importance, but there is limited understanding of the epidemiology. This study used an EcoHealth approach to ensure transdisciplinarity and community participation. Seroprevalence of Leptospira in animals was studied between October 2011 and May 2013 in 15 villages. Serum samples from 1404 cattle and 60 small ruminants were screened by a Microscopic Agglutination Test (MAT), first in pools, and then the individual positive samples were identified. Focus group discussions including farmers, village officials, and official stakeholders were used to explore knowledge and behavior of zoonotic diseases, particularly leptospirosis. Two small ruminants were seropositive for Leptospira icterohemorrhagiae. From the cattle, 3.7% were seropositive, and the most common serovars were Leptospira hardjo, followed by L. icterohemorrhagiae. Out of all farms, 5.6% had at least one positive cattle. Risk factor analyses showed that the risk of the farm being seropositive increased if the farmer used water from an open source, or if farming was not the main occupation. This study showed the presence of Leptospira spp. in ruminants in Yogyakarta and identified use of open water as a risk factor for the livestock. We also observed that the knowledge related to leptospirosis was low, and risky farm management practices were commonly employed.

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.

Detection of Escherichia coli O157:H7 and Shiga toxin 2a gene in pork, pig feces, and clean water at Jagalan slaughterhouse in Surakarta, Central Java Province, Indonesia.

BACKGROUND AND AIM: The feasibility assessment of food products on the market becomes one of the milestones of food safety. The quality of food safety of animal origin especially pork need to get attention and more real action from the parties related and concerned. Since pork is also a source of transmission for the contagion of foodborne disease so that the study of the existence of several agents in the pork and its products become the benchmark of safety level. This study aimed to isolate, identify, and detect the Shiga toxin 2a (stx2a) gene from Escherichia coli O157:H7 in pork, pig feces, and clean water in the Jagalan slaughterhouse. MATERIALS AND METHODS: A total of 70 samples consisting of 32 pork samples, 32 pig fecal samples, and 6 clean water samples were used to isolate and identify E. coli O157:H7 and the stx2a gene. Isolation and identification of E. coli O157:H7 were performed using culture on eosin methylene blue agar and Sorbitol-MacConkey agar media and confirmed molecularly with polymerase chain reaction to amplify the target genes rfbE (317 bp) and fliC (381 bp). The isolates, which were identified as E. coli O157:H7, were investigated for the stx2a gene (553 bp). RESULTS: The results of this study show that of the total collected samples, E. coli O157:H7 was 28.6% in Jagalan slaughterhouse and consisted of 25% of pork samples, 31.25% of pig fecal samples, and 33.3% of clean water samples. The isolates that were identified to be E. coli O157:H7 mostly contained the stx2a gene, which was equal to 75%, and consisted of seven isolates from pork samples, seven isolates from fecal samples, and one isolate from clean water samples. CONCLUSION: E. coli O157:H7 was found in 28.6% of pork, pig feces, and clean water in Jagalan slaughterhouse and 75% of identified E. coli O157:H7 contained the stx2a gene.

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.

Passive transfer of antibodies to Shiga toxin-producing Escherichia coli O26, O111 and O157 antigens in neonatal calves by feeding colostrum.

To study whether or not passive immunity of neonatal calves against Shiga toxin-producing Escherichia coli (STEC) O26, O111, and O157 was obtained by colostrum administration, serum antibodies in calves after the feeding were determined by enzyme-linked immunosorbent assay (ELISA) in comparison with antibodies in colostrum and sera from donor dams. The highest antibody titers to STEC in colostrum from dams were detected soon after parturition. The antibody titers were found to be elevated in sera of neonatal calves (4-9 hr after birth) orally administered with colostrum with high antibody titers, suggesting that passive immunity of neonatal calves to STEC infection may be obtained by feeding colostrum. These results suggest that colostrum administration to neonatal calves may play an important role in elevating serum antibodies against STEC in neonatal calves.