Screening of the novel colicinogenic gram-negative rods against pathogenic Escherichia coli O157:H7.

Purpose: Escherichia coli (E. coli) O157:H7 is gram‑negative enteric pathogen producing different types of Shiga toxin. This bacterium is the most corporate cause of haemorrhagic colitis in human. Administration of antibiotics (particularly sulfa drugs) against this pathogen is a debatable topic as this may increase the risk of uremic syndrome; especially in children and aged people. Around the world, microbiologists are in search of alternative therapeutic methods specially probiotics against this pathogen. In the present study, we have focused on the investigation of alternate bio‑therapeutics (probiotics) for the treatment of patients infected with E. coli O157:H7. This study is based on the identification of colicin‑producing gram‑negative bacteria (particularly enterobacteriaceae) which can competently exclude E. coli O157:H7 from the gut of the infected individual. Materials and Methods: Hundred samples from human, animal faeces and septic tank water were analysed for nonpathogenic gram‑negative rods (GNRs). Results: Out of these samples, 175 isolates of GNRs were checked for their activity against E. coli O157:H7. Only 47 isolates inhibited the growth of E. coli O157:H7, among which majority were identified as E. coli. These E. coli strains were found to be the efficient producers of colicin. Some of the closely related species i. e., Citrobacter sp, Pantoea sp. and Kluyvera sp. also showed considerable colicinogenic activity. Moreover, colicinogenic species were found to be nonhaemolytic, tolerant to acidic environment (pH 3) and sensitive to commonly used antibiotics. Conclusion: Nonhaemolytic, acid tolerant and sensitive to antibiotics suggests the possible use of these circulating endothelial cells (CEC) as inexpensive and inoffensive therapeutic agent (probiotics) in E. coli O157:H7 infections.

Isolation of colicinogenic E. coli, optimization of colicin production and transformation of Col plasmid.

An attempt was made for the isolation of colicinogenic E. coli from cow and sheep dung samples. Total of 112 E. coli isolates were collected from sheep and cow dung samples in which 63 isolates are from cow dung and 49 isolates are from sheep dung samples. In the screening for colicin production, two E. coli isolates C22 and C51 from cow dung sample and one isolate S39 from sheep dung sample showed activity against the pathogenic strain E. coli O157:H7. Colicin production by all the three E. coli isolates was found to be enhanced by 0.25mg/l of mitomycin-c and three hours of incubation at 370C. Plasmid DNA was isolated from colicinogenic E. coli strains by alkali lysis method and their molecular weight was found to be 6.5 kb. The transformation experiment carried out with the standard strain E. coli DH5 confirmed that the plasmids were col-plasmids. The three colicinogenic E. coli strains isolated in this study will be a candidate to develop as probiotic for veterinary applications.

Antagonistic activity expressed by Shigella sonnei: identification of a putative new bacteriocin

Bacteriocins are antibacterial, proteinaceous substances that mediate microbial dynamics. Bacteriocin production is a highly disseminated property among all major lineages of bacteria, including Shigella. In this paper, we addressed the purification and characterisation of a bacteriocin produced by a Shigella sonnei strain (SS9) isolated from a child with acute diarrhoea. The substance was purified through ammonium-sulphate precipitation and sequential steps of chromatography. The intracellular fraction obtained at 75% ammonium sulphate maintained activity following exposure to pH values from 1-11 and storage at -80ºC for more than two years and was inactivated by high temperatures and proteases. The molecular mass of the purified bacteriocin was determined by mass spectrometry to be 18.56 kDa. The N-terminal sequence of the bacteriocin did not match any other antibacterial proteins described. A putative new bacteriocin produced by S. sonnei has been detected. This bacteriocin may represent a newly described protein or a previously described protein with a newly detected function. Considering that SS9 expresses antagonism against other diarrhoeagenic bacteria, the bacteriocin may contribute to S. sonnei virulence and is potentially applicable to either preventing or controlling diarrhoeal disease.

Colicin type 7 produced by majority of Shigella sonnei isolated from Thai patients with diarrhoea

Thirty one out of 153 strains of Shigella sonnei isolated from Thai patients with diarrhoea showed antibacterial activity against S. sonnei by agar well diffusion method. All of them harbor plasmids with the genetic determination of colicin type 7 (Js) gene but without colicin E and colicin U gene. The PCR product obtained from strain 35/44 was shown to be the gene for colicin type 7 lytic protein (cja). The partially purified bacteriocin (PPB) containing colicin type 7 of strain 35/44 was prepared and used for characterization. The antibacterial activity of PPB against a total of 17 selected Gram-positive and Gram-negative bacteria was tested. It was found that PPB of strain 35/44 was active against E. coli O157, S. sonnei and S. boydii. The sensitivity of PPB from this strain to proteinase K, trypsin and α-chymotrypsin suggests the proteinaceous nature of these antimicrobial substances. Therefore, this isolated bacterium can be regarded as bacteriocin producing bacteria. The bacteriocin produced by this isolated S. sonnei was heat stable as evidenced by its ability to maintain the activity at 80 °C for 60 min. In addition, it was stable within a wide range of pH (3-9). The molecular weight of colicin type 7 from isolated S. sonnei strain 35/44 analyzed by SDS-PAGE was 54.4 kDa composing of at least five subunits. It is to our knowledge; the first report of Thai patients with diarrhoea that S. sonnei isolated from them contained colicin type 7.

EFFECT OF PHOSPHATE ON COLICIN V FORMATION BY STRAINS OF ESCHERICHIA COLI / 微生物学通报

ColV~+ strains of Escherichia coli produced larger inhibitory zones when these strains grown on nutrient agar containing phosphate after overlaid sensitive indicators. This appears that production of colicin V is increased by the addition of phosphate to nutrient agar. It was sure that stimulation of phosphate to colicin V formation results from its effect in reducing divalent cation levels in nutrient agar since adding EDTA to nutrient agar had the same effect as phosphate, but the addition of Mg~(2+) or Ca~(2+) had the oppsite effect. Therefore nutrient agar supplemented with phosphate can be used to isolate and identificate ColV~+ strains of E. coli.