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Elaboration of culture media for the bacteriologic diagnosis of Campylobacter infections


Author: Valentina Melnic
Degree:doctor of medicine
Speciality: 03.00.07 - Microbiology
Year:2005
Scientific adviser: Liviu Sicinschi
doctor habilitat
Institution: Nicolae Testemitanu State University of Medicine and Pharmacy of the Republic of Moldova
Scientific council:

Status

The thesis was presented on the 22 April, 2005
Approved by NCAA on the 23 June, 2005

Abstract

Adobe PDF document0.38 Mb / in romanian

Keywords

thermophilic Campylobacter, isolation, cultivation, identification, culture medium

Summary

The goal of the study was the improvement of the bacteriological diagnosis of acute enteric infections, caused by thermophylic Campylobacter. Experiments in the area of cultivation and identification of Campylobacter were combined with diagnostic investigations using materials from children patients with acute diarrheic syndrome.

Theoretical knowledge (predominance of glutamic cycle in the Campylobacter’s metabolism, importance of the presence of potassium ions in the bacterium environment) and our experimental tests (different nutrients) allowed us to propose two new culture media for the cultivation of the thermophylic campylobacteria species.

One culture medium, with blood, is designated for the simultaneous isolation and identification of thermophylic campylobacteria species. It consists of casein hydrolyzate, fermentative peptone, yeast extract, agar, potassium chloride, donor blood, and antibiotic selective supplement (cefazolin, rifampicin, polymixin M sulfate, nizoral). The medium assures the growth of Campylobacter in 48 hours in microaerophilic conditions at +420C, and it discriminates C.jejuni and C.coli as morphologically distinguishable colonies. As a result, identification data could be provided one day earlier than the standard incubation period. Additionally, the method is more economical and does not require any additional biochemical assays for identification.

The second culture medium is identical to the first, except blood is omitted and L glutamic acid is added. It allows the growth of non-coccoid Campylobacter strains with typical morphology 48 hours after incubation at +420C in microaerophilic conditions. This medium is useful when blood is unavailable or inconvenient for any reason. During the process of testing of the new culture media, 182 children (0-3 years) with acute diarrhea syndrome were examined. The data confirmed the prevalence of thermophylic Campylobacter (18,1%) in the etiology of the acute diarrhea, compared to Salmonella (13,7%) and Shigella (1,6%). The cultural and biochemical properties of Campylobacters were studied, and their genus, species and subspecies were identified. They were distributed as C.jejuni species (43,8%; biovariant 1 - 64,3% and biovariant 2 - 35,7%) and C.coli species (56,2%; biovariant 1 - 72,2% and biovariant 2 – 27,8%). The designation of C.jejuni and C.coli according to their colony morphology was confirmed by enzymatic (hippurate hydrolysis) and genetic tests (PCR).

The susceptibility of clinical Campylobacter strains to antimicrobial preparations was analyzed. They were susceptible to erythromycin, levomycetin, gentamycin and furagine. All clinical strains were resistant to cephalosporins (100%). 31,3% of the strains were resistant to polymixin B, 18,8% were resistant to ampicilin, 15,6% were resistant to ciprofloxacin, and 9,4% were resistant to nalidixic acid. Simultaneous resistance to 2 antibiotics was found in 40,6% strains, simultaneous resistance to 3 and 4 antibiotics was found in 9,4% and 6,3% of strains respectively.

The two proposed new culture media allow faster isolation and identification of C.jejuni and C.coli and significant improvement of the scheme of the bacteriological diagnosis of infections caused by thermophylic Campylobacter.

Additionally the storage conditions for C.jejuni and C.coli strains on the “MTC— Campylobacter” medium were studied; the scheme of the prolonged storage of the Campylobacters on this culture medium was proposed for the use in the conditions of a practical laboratory of bacteriology.