Bactericidal and morphological effects of amoxicillin on Helicobacter pylori

Abstract

The growth kinetics of Helicobacter pylori after it has been exposed to amoxicillin have been investigated in conjunction with studies of cell morphology. A potent bactericidal effect was observed at concentrations 10-fold higher than the MIC, but this was accompanied by an increase in the residual numbers of coccoid forms observed. In the presence of 10 g of amoxicillin per ml, these forms could be detected as rapidly as 6 h after exposure to the antibiotic. Although the clinical relevance of coccoid forms remains unknown, such forms should be considered when potential anti-Helicobacter agents are tested in vitro.Helicobacter pylori exists in several morphologically distinct forms, including nonculturable coccoid forms (3, 5). Coccoid formation has been attributed to environmental stress, including nutrient deprivation, extended incubation, accumulation of metabolic products, pH alteration, and exposure to antimicrobial agents (1,3,5). The last may be of major importance when therapy is considered, but the activity of agents in vitro is often determined only by susceptibility tests, e.g., agar dilution, in which the morphological status of the organism is unknown. Recent studies have demonstrated that amoxicillin at concentrations close to the MIC can result in the development of coccoid forms after 72 h of incubation (2). We describe observations on the time course of the bactericidal and morphological effects of a range of concentrations of amoxicillin on H. pylori.A broth cultivation technique similar to that described previously by Iwahi and coworkers (4) was used. Briefly, stock cultures of H. pylori NCTC 11637 were inoculated into 200 ml of Brucella broth (BBL) with 2.5% fetal calf serum (Gibco) in 500-ml sterile tissue culture flasks. After 16 h of microaerobic incubation (Gaspak jars and Campypaks; BBL) with gyration at 100 rpm, the culture (approximately 5 log 10 CFU/ml) was divided into 36-ml aliquots in 80-cm 2 tissue culture flasks, and 4-ml volumes of fresh medium or amoxicillin (SmithKline Beecham Pharmaceuticals, Worthing, England) prepared in broth to give concentrations of 0.01, 0.1, 1.0 and 10 g/ml were added. The MIC of amoxicillin for H. pylori NCTC 11637, as determined by agar dilution, was 0.015 g/ml. The cultures were reincubated as described previously, and at times from 0 to 36 h, 0.5-ml volumes were removed for the enumeration of viable bacteria, measurement of residual antibiotic concentration, and electron microscopy. The viability was determined by a modified Miles-Misra technique of preparing serial 10-fold dilutions in phosphate-buffered saline (PBS) and inoculating 20-l samples in triplicate onto Mueller-Hinton agar (Oxoid Ltd.) supplemented with 7% defibrinated horse blood. Colonies were counted after 3 days of incubation at 37ЊC under microaerophilic conditions and were expressed as CFU per milliliter. Samples, taken at random, were also plated onto nonselective media to determine purity, and organisms other than H. pylori were not detected. A large-plate agar diffusion techniq...

Pyrazolopyrimidinediones Are Selective Agents for Helicobacter pylori That Suppress Growth through Inhibition of Glutamate Racemase (MurI)

“…The rapid response was typical of the pyrazolopyrimidinediones and was not seen when cell growth was inhibited with tetracycline for the same time (data not shown). When cells were exposed to amoxicillin, a ␤-lactam that inhibits peptidoglycan biosynthesis, similar rapid morphology changes were noted (6). Thus, the morphological changes seen with the pyrazolopyrimidinediones are in agreement with inhibition of peptidoglycan biosynthesis through MurI.…”

Anti-Infective Activities of Lactobacillus Strains in the Human Intestinal Microbiota: from Probiotics to Gastrointestinal Anti-Infectious Biotherapeutic Agents

“…The molecule(s) present in L. acidophilus LB CFCS that exerts bactericidal activity against H. pylori remain to be identified. The appearance of coccoid forms after treatment with the above-mentioned Lactobacillus strains is important since it resembles the morphological changes produced in H. pylori by antibiotics (155,156) or colloidal bismuth subcitrate (157). Moreover, the transformation into coccoid forms is interesting in terms of pathogenesis, since even though these forms conserve the genes that code for virulence factors found in the spiral form, they are known to be less likely to colonize and induce inflammation than the corresponding spiral forms (158).…”

Interleukin-8 induction and adhesion of the coccoid form of Helicobacter pylori

“…Helicobacter pylori changes its morphology in vitro from a helical form to a coccoid form under various conditions such as extended cultivation [1,2], aerobic culture [3], alkaline pH [3] and antibiotic treatment [4,5]. In the human stomach, the ability of the helical form of the micro-organism to colonise gastric mucosa and to attach to mucosal epithelial cells has been well documented.…”