A controlled in vitro study of the antimicrobial effectiveness of Colibacillinum against E. coli

Background Due to the rise of antibiotic resistant bacterial infections, alternative methods of treatment need to be explored. Homoeopathic medicine is based on the principle of ‘like cures like’ (O’Reilly, 1996) i.e. the symptoms the substance can cause, it can cure which is the underlying rationale for this study. Colibacillinum is a remedy prepared from an enteropathogenic strain of E. coli, this remedy is already used in clinical practice against chronic cystitis (Leupen, 2010) caused by E. coli, however, an extensive literature search has revealed there to be no empirical investigation into its antibiotic capacity. This study aimed to show whether or not this remedy demonstrates antimicrobial capacity against E. coli in an in vitro setting. Parallell homoeopathic potencies of this remedy and one prepared from a uropathogenic strain of E. coli were tested for antimicrobial effects against enteropathogenic and uropathogenic strains of E. coli in vitro using the disc diffusion method. Aim of the study The aim of this controlled in vitro study is to determine the antimicrobial effectiveness of parallel potencies of the homoeopathic remedy Colibacillinum (manufactured from a uropathogenic strain and enteropathogenic strains respectively) against uropathogenic and enteropathogenic cultures of E. coli in vitro by means of the disc diffusion assay method. Methodology Measurements were by means of the disc diffusion essay. For this experiment thirty Mueller-Hinton plates were prepared and inoculated with each test bacteria in turn. Fifteen plates were inoculated with Uropathogenic strain of E. coli and the remaining 15 plates were inoculated with Uropathogenic strain of E. coli. A sterile 5mm Whatman® filter paper number 4 discs were individually inoculated with test substances 3CH,9CH,30CH and 200CH potencies and the controls, negative (43% ethanol) and positive control (Ciprofloxacin) using a micropipette, before being allowed to dry in the incubator. A Ciprofloxacin antibiotic (positive control) was included in the experiment with sole purpose of accounting for plate-plate variations in the pharmacological sensitivity of the same specie of bacteria. The plates were incubated at 37°C, and the zones of inhibition measured with a pair of Vernier calipers at 24 hours. Data entry was done using the SPSS statistical package. ANOVA was used to compare the differences between the test and control groups, Mauchly’s Test of Sphericity for Uropathogenic prepared strain, Mauchly’s Test of Sphericity for Enteropathogenic prepared strain, Normality test. Results The results obtained from this study showed that the Homoeopathic remedy Colibacillinum prepared from both Uropathogenic and Enteropathogenic strains displayed inhibitory effects against Enteropathogenic and Uropathogenic strains of E. coli, and exhibited statistically significance. The control group (ciprofloxacin) had the highest inhibitory effect (42.3±0.58mm) against Enteropathogenic and Uropathogenic E. coli, while the negative control (43% ethanol) had the lowest inhibitory effect (0.67±1.15mm). Colibacillinun 200CH prepared from a Uropathogenic strain of E-coli (Coli-b_U 200CH) displayed statistically significant antimicrobial effects against uropathogenic E.coli; such antimicrobial effects were significantly greater than 43% ethanol (negative control); the antimicrobial effect was however inferior to Ciprofloxacin (positive control). Colibacillinum 9CH prepared from Enteropathogenic strain of E-coli (Coli-b_E 9CH) also displayed statistically significant antimicrobial effects against enteropathogenic E.coli which were significantly greater than 43% ethanol (negative control) but inferior to Ciprofloxacin. Conclusion This study concluded that Colibacillinum prepared from Uropathogenic and Enteropathogenic strains of E. coli, are effective in inhibiting the in vitro growth of E.coli when evaluated by means of disc diffusion. The study further confirmed that the biological (anti-microbial) activity of an ultra-high homoeopathic dilution (Coli-b_U 200CH) (1:10400) and in the case of Coli-b_U the findings support existing literature which suggests that the anti-microbial properties of homeopathic nosodes increase with potency; all hypotheses for this remedy were thus accepted. This trend was not noted for Coli-b_E in which the potency with the greatest anti-microbial effect was the 9CH, thus Colibacillinum prepared from Enteropathogenic strain (Coli-b_E) did not conform with hypothesies one, two and four that were proposed in chapter one. Despite this the confirmation of significant antimicrobial effects of a substance at this level of deconcentration (1:1018) is noteworthy.

NanR Regulates nanI Sialidase Expression by Clostridium perfringens F4969, a Human Enteropathogenic Strain

ABSTRACT Clostridium perfringens can produce up to three different sialidases, including NanI, its major exosialidase. The current study first showed that human intestinal strains of C. perfringens can grow by utilizing either glucose or sialic acids, such as N-acetylneuraminic acid (Neu5Ac), which are the end products of sialidase activity. For the human enteropathogenic strain F4969, it was then determined that culture supernatant sialidase activity and expression of exosialidase genes, particularly nanI, are influenced by the presence of Neu5Ac or glucose. Low Neu5Ac concentrations increased culture supernatant sialidase activity, largely by stimulating nanI transcription. In contrast, low glucose concentrations did not affect exosialidase activity or nanI transcription. However, either high Neu5Ac or high glucose concentrations repressed F4969 culture supernatant sialidase activity and nanI transcription levels. Furthermore, high glucose levels repressed F4969 culture sialidase activity and nanI expression even in the presence of low Neu5AC concentrations. To begin to evaluate the mechanistic basis for nanI expression, a nanR null mutant was used to demonstrate that NanR, a member of the RpiR family of regulatory proteins, decreases exosialidase activity and nanI transcription in the absence of sialic acid. The ability of C. perfringens to regulate its exosialidase activity, largely by controlling nanI expression, may affect intestinal pathogenesis by affecting the production of NanI, which may affect C. perfringens growth, adhesion, and toxin binding in vivo.

Biofilm Formation by and Multicellular Behavior of Escherichia coli O55:H7, an Atypical Enteropathogenic Strain

ABSTRACT Enteropathogenic Escherichia coli (EPEC) is an important causal agent of diarrheal illness throughout the world. Nevertheless, researchers have only recently begun to explore its capacity to form biofilms. Strain O55:H7 (DMS9) is a clinical isolate belonging to the atypical EPEC (aEPEC) group, which displays a high degree of genetic relatedness to enterohemorrhagic E. coli. Strain DMS9 formed a robust biofilm on an abiotic surface at 26�C, but not at 37�C. It also formed a dense pellicle at the air-liquid interface and developed a red, rough, and dry (RDAR) morphotype on Congo red agar. Unlike a previously described E. coli O157:H7 strain, the aEPEC strain seems to express cellulose. Transposon mutagenesis was used to identify biofilm-deficient mutants. One of the mutants was inactivated in the csgFG genes, required for assembly and secretion of curli fimbriae, while a second mutant had a mutation in crl, a thermosensitive global regulator that modulates σS activity and downstream expression of curli and cellulose. The two mutants were deficient in their biofilm formation capabilities and did not form a pellicle at the air-liquid interface. Unlike in Salmonella, the csgFG mutant in aEPEC completely lost the RDAR phenotype, while the crl mutant displayed a unique RDAR “pizza”-like morphotype. Genetic complementation of the two mutants resulted in restoration of the wild-type phenotype. This report is the first to describe and analyze a multicellular behavior in aEPEC and support a major role for curli and the crl regulator in biofilm development at low temperatures corresponding to the nonmammalian host environment.

Properties of Vero cytotoxin-producingEscherichia coliof human and animal origin belonging to serotypes other than O157: H7

SUMMARYEight non-O157: H7 Vero cytotoxin (VT)-produeingEscherichia coli(VTEC) strains isolated from ill persons and nine bovine and lamb strains of serogroups matching the human strains, were characterized for various properties known to be associated withE. colivirulence. Five different serogroups were represented: O5. O55, O103, O111 and O153. The bovine and lamb strains produced VT1, while 3 human strains produced VT1, 3 produced VT2 and 2 were positive for both VT1 and VT2. The strains were non-haemolytic on horse blood agar, did not produce either heat stable toxin A (STA) or heat labile toxin (LT), and were non-invasive. The CVD419 probe which has been proposed to identify enterohaemorrhagicE. coli(EHEC) hybridized with all of the 05 and 0103 strains, none of the 055 and 0153 strains, and 3 of the 4 0111 strains. The strains carried several different sized plasmids and hybridization of Southern blots with the CVD419 probe identified plasmids ranging in size from 42 × 10 to 90 × 10. The strains did not hybridize with the enteroadherence factor (EAF) probe derived from an enteropathogenic strain and associated with the ability to give localized adherence to HEp-2 cells. Nevertheless five of the strains adhered in a localized pattern to HEp-2 cells and Intestine 407 cells. Adhesion to either HEp-2 or Intestine 407 cells did not correlate with hybridization with the CVD419 probe or haemagglutinating properties.

Ultrastructural Changes in the Small Intestine of Neonatal Calves with Enteric Colibacillosis

The small intestines of calves inoculated orally with the enteropathogenic strain of Escherichia coli 0101:K'B41′, K99 were examined by electron microscopy at 3, 6, 12, 16, 21, 36, 69, 70 and 72 hours after inoculation. The challenge organism adhered to the mucosa of the distal small intestine from six hours post-inoculation. Bacteria were separated from the microvillous brush border by a gap of 200 to 300 nm in which bacterial fimbriae and the microvillous glycocalyx were seen. Bacteria never were found in epithelial cells but were present in macrophages in the lamina propria from 12 hours. At three and six hours, cytopathic changes were not seen in the small intestine, but from 12 hours epithelial cells on affected villi had blunt and thick microvilli and contained cytoplasmic inclusions. Epithelial cells were seen frequently in the process of extrusion from the villi, either singly, in small groups, or as ribbons of cells. Intervillous bridges, characteristic of villous fusion, were seen frequently from 69 hours.

Experimental Peracute Colibacillosis: Gastrointestinal Lesions in Gnotobiotic Piglets

Three-day-old, hysterectomy-derived germ-free piglets were challenged with an enteropathogenic strain of Escherichia coli. This particular strain (serotype 08:K87, K88ac:H19) was highly pathogenic and produced clinical signs of enteric colibacillosis. The major lesions consisted of marked gastric dilation and extensive congestion involving the greater curvature of the fundus. The gastrointestinal lesions were considered to be the direct result of the active infection by this strain. No similar condition was observed in similar experiments with 16 other E. coli serotypes in neonatal germ-free swine.