P585 Comparative study for conjugative plasmids carrying CTX-M genes in Escherichia coli nosocomial isolates

Molecular characterisation of gastro-intestinal pathogens into six groups. A serovar-specific virulence plasmid of 38 MDa was detected in all of S. enteritidis strains (except one strain). Plasmids, with molecular masses varying between 2.5 and 89 MDa, were found in 12 of the 15 of the S. typhimurium strains and five different plasmid patterns were determined. After the XbaI macrorestriction profiles, we observed 23 subtypes which were grouped into five main patterns for S. enteritidis and 15 PFGE profiles were observed among the S. typhimurium strains and four patterns (I, II, III, IV) were found. Plasmids from resistant strains were not transferred by conjugation recipient Escherichia coli cells. Pulsed-field gel electrophoresis and restriction enzyme digestion analysis of DNA revealed different restriction profiles and sizes, indicating these strains usually were not clonaly related whereas MDRS. typhimurium isolates were clonaly related. Conclusion: Our study demonstrated the emergence of multiresistant S. typhimurium DT104 infections in our hospital. Therefore, investigation of the antimicrobial susceptibilities, the characteristics of resistant strains and the molecular epidemiology of the strains is more significant. PFGE is more discriminatory and can be used as a confirmatory method. P585 Comparative study for conjugative plasmids carrying CTX-M genes in Escherichia coli nosocomial isolates N. Fursova, I. Abaev, O. Korobova, E. Pecherskikh, N. Shishkova, S. Pryamchuk, A. Kruglov, D. Ivanov, L. Weigel, J. Rasheed (Obolensk, Moscow, RU; Atlanta, US) Objectives: In previous studies we have shown that CTX-M is the most prevalent type of extended-spectrum b-lactamase (ESBL) among recent clinical isolates of Enterobacteriaceae (2003 to 2005 isolates, 14 hospitals across Russian Federation). The blaCTX-M enzymes accounted for 75% of ESBLs. Major groups included CTX-M-1-related enzymes (91%) (in most cases CTX-M-15), CTX-M-9-like (7%), CTX-M-2-like (1%), and a combination of CTX-M-1 and -9-related genes (1%). To determine the potential for spread of CTX-M genes from isolates of Escherichia coli, CTX-M-positive strains were analysed for plasmids, location of CTX-M genes, and the ability to transfer plasmids with CTX-M by conjugation. Methods: The presence of blaCTX-M-1, blaCTX-M-2, and blaCTX-M-9 genes, as well as blaTEM and blaSHV genes, in ESBL-producing E. coli (n = 161) isolates was analysed by PCR. Conjugations were performed in broth using E. coli C600 (RifRAzR) recipient strain. Antimicrobial resistance phenotypes of donors and transconjugants were determined by disc diffusion and broth microdilution methods. Plasmids were extracted by the alkaline hydrolysis method. blaCTX-M genes were localised to plasmids by DNA-DNA hybridisation using high-sensitive kit Alk-Phos (Amersham). Probes have been generated by amplification of CTX-M genes using universal primers. Results: Transconjugants were identified for 35 (approx. 22%) of the E. coli isolates including: blaCTX-M-1 gene (27), blaCTX-M-9 gene (7), and blaCTX-M-2 gene (1). Transfer of blaCTX-M-1 was usually observed on the same plasmid with blaTEM. In contrast, the blaCTXM-9 gene was frequently on a separate replicon from blaTEM (Table 1). In addition to the ESBL genes, transconjugants acquired resistance to doxycycline, gentamicin, amikacin, ciprofloxacin, and sulfonamides (in different combinations). Clinical isolates had 1−12 plasmids of different molecular weights. blaCTX-M genes were consistently transferred on large plasmids (more than 100 Kb). Donor and transconjugant strains Total strains

Successful donors

Transconjugants

CTX-M1 (n = 128)

CTX-M1 (n = 3) CTX-M1 + TEM (n = 23)

CTX-M1 (n = 3) CTX-M1 + TEM (n = 19); CTX-M1 (n = 4) TEM + SHV (n = 1) CTX-M9 (n = 1) CTX-M9 + TEM (n = 2); CTX-M9 (n = 4) CTX-M2 (n = 1)

CTX-M9 (n = 29)

CTX-M1 + TEM + SHV (n = 1) CTX-M9 (n = 1) CTX-M9 + TEM (n = 6)

CTX-M2 (n = 4)

CTX-M2 (n = 1)

S135 Conclusion: Clinical isolates of E. coli isolated in the Russian Federation harbour CTX-M-type ESBLs that are located on high molecular weight conjugative plasmids. In many cases genes encoding resistance mechanisms to several groups of antimicrobial agents were located on the same plasmid capable of horizontal transfer between bacterial strains. These data suggest that the CTX-M-type enzymes, along with multidrug resistance, have the potential to become a widespread problem in this region. P586 Phylogenetic groups, antibiotic susceptibility, biofilm formation and PFGE in Escherichia coli from community-acquired cystitis K. Ejrnaes, A. Reisner, B. Lundgren, S. Ferry, S. Holm, T. Monsen, R. Lundholm, N. Frimodt-Moller (Copenhagen, Lyngby, Hvidovre, DK; Ume˚a, SE) Objectives: To study Escherichia coli (EC) isolates from communityacquired cystitis with respect to phylogenetic groups, antibiotic susceptibility, biofilm formation and PFGE. Methods: A subgroup of 243 out of 1162 women with communityacquired cystitis from a placebo-controlled comparative study of three different dosing regimes of mecillinam was studied. We stratified patients into two groups: 1) The mecillinam group (MG), all treated with mecillinam and all with EC at inclusion: a group of all those having EC at follow-up, plus a randomly selected group having negative culture at follow-up (N = 160); and 2) the placebo group (PG), all treated with placebo and all with EC at inclusion: a randomly selected group having EC at follow-up and a randomly selected group having negative culture at follow-up (N = 83). The primary infecting ECs were studied. Susceptibility to mecillinam was tested by agar dilution; MICs of ampicillin, cefpodoxime, chloramphenicol, ciprofloxacin, gentamicin, streptomycin, sulfamethoxazole, trimethoprim and tetracycline were tested with Sensititre. Phylogenetic grouping was determined by a triplex PCR assay. Biofilm formation was measured in 3 media (static growth, 48 hours, crystal violet staining). PFGE was done with XbaI to the primary infecting EC and EC from follow-up. Results: Only 20% of the strains were resistant to one or more antimicrobials. Susceptibility was significantly associated with phylogenetic group B2 for ampicillin (P = 0.048), chloramphenicol (P = 0.002), streptomycin (P = 0.001), sulfamethizole (P = 0.006), trimethoprim (P = 0.013) and tetracycline (P = 0.003). Resistance to 3 or more antimicrobials was significantly associated with phylogenetic group A (P = 0.033) and D (P = 0.014). Resistance to less than 3 antimicrobials was significantly associated with B2 (P < 0.0001). EC causing relapse/persistence had a higher biofilm formation than those causing reinfection/cure in 2 of 3 media (P = 0.002 and P = 0.011) in the PG, but there was no significant difference in the MG. Conclusion: Although representing a low-antibiotic-consumption area (Sweden) with low resistance rates, still, antimicrobial susceptibility was significantly associated with phylogenetic group B2 and resistance to 3 or more antimicrobials with A and D. EC causing relapse/persistence had a higher biofilm formation capacity than those causing reinfection/ cure in the PG. P587 Thermophilic helicase-dependent isothermal DNA amplification for molecular detection of Helicobacter pylori P. Gill, H. Abdul-Tehrani, A. Ghaemi, T. Hashempour, A. Alvandi, M. Noori-Daloii (Tehran, IR) Objectives: Helicobacter pylori is a Gram-negative, pathogenic bacterium, which specifically colonises in the human gastric mucosa. The infection with this microorganism is one of the most prevalent infections in humans and about 50% of the adults in the industry and more than 90% of the population in developing countries are infected. Several PCR-based methods have been described for molecular diagnosis of this bacterium in biological specimens. However, in this study, we designed and developed a novel procedure for detection of ureC