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P1249 Virulence, pathogenicity, antibiotic resistance and plasmid profile of Escherichia coli strains isolated from drinking and recreational waters
Community-acquired bacterial infections Methods: S. aureus strains were isolated from 337 military persons dislocated either in Latvia (Alˆuksne, 126) or being on Peacekeeping Missions in Iraq (125) and Kosovo (86). S. aureus strains isolated from nasal swabs were tested antimicrobial susceptibility (PEN, FOX, GEN, CIP, CHL, CLI, ERY, RIF, STX, TET, VAN) according to CLSI standard. The presence of the mecA and toxin (PVL, HLG, TSST) genes was determined by PCR. Strain typing was performed by repetitive extragenic palindromic sequence PCR (rep-PCR). Results: While the rates of asymptomatic S. aureus carriers in Alˆuksne and Kosovo contingents were similar (30.9% and 34.8%, respectively) the percentage of carriers in Iraq unit was almost doubled (57.6%). From 141 S. aureus isolates obtained none was methicillin-resistant. 104 isolates were resistant to penicillin, and 5 to erythromycin. As MSSA was found more often among members of Iraq contingent these isolates were subjected to more detailed investigation. Molecular analysis revealed 46 strains harbouring no toxin genes, 11 were hlg positive, 7 were hlg-v positive and two strains produced PVL. Rep-PCR revealed three major groups of strains with identical chromosomal background. This strongly points toward clonal spread which may explain high carrier rate in this group. Conclusions: Antimicrobial resistance levels of analysed strains were low. Our study revealed clonal spread of S. aureus among members of closed community. Personal hygiene is of great importance to reduce dissemination of S. aureus. P1248 Outbreaks of shigellosis in England and Wales, 2004: use of phenotypic and molecular typing for strain differentiation T. Cheasty, J. Threlfall (London, UK) Objectives: In the summer of 2004 a number of apparently unrelated outbreaks of Shigella sonnei infection were observed amongst patients throughout England and Wales. Infections were identified throughout the country but were concentrated in the London area, and the northwest and north-east of England. Outbreaks were in both religious communities and in homosexual men. The outbreak among homosexual men was centred in the London area but outbreaks in the religious community occurred in London, the north-west and the north-east. For meaningful epidemiological investigations in real-time discrimination within the serovar was essential. With this in mind a hierarchical approach based on phenotypic subdivision by phage typing and antibiogram, supplemented by molecular typing using plasmid profile and macrorestriction fingerprinting by pulsed-field gel electrophoresis (PFGE) has been adopted. Methods: Isolates of Sh. sonnei from infections in England and Wales in 2004 have been phage typed using the scheme of Hammerstrom, Kallings and Sjoberg. Isolates have been tested for resistance to a range of antimicrobials, plasmids identified and sized following extraction of plasmid DNA according to Kado and Liu, and PFGE performed by standard protocols according to Pulse-Net USA. Results: In all outbreaks the only resistance pattern (R-type) identified was that of ASSuSpTTm (A, ampicillin; S, streptomycin; Su, sulfonamides, Sp, spectinomycin; T, tetracyclines; Tm, trimethoprim). The outbreak among homosexual men was characterised by a new Sh. sonnei phage type (PT), designated PT Q. The isolates from this outbreak were further characterised by a distinctive plasmid-and pulsed-field profile. Isolates from outbreaks among members of a religious community were characterised by three other phage types distinct from PT Q, and by different plasmid- and PFGE profiles, indicating the presence of temporally-associated but independent incidents. Conclusions: These results indicate that outbreaks of Sh. sonnei can be subdivided by a combination of phenotypic and molecular typing. Results can be obtained rapidly, and can be used in real-time for epidemiological investigations.
S343 P1249 Virulence, pathogenicity, antibiotic resistance and plasmid profile of Escherichia coli strains isolated from drinking and recreational waters C. Balotescu, E. Panus, M. Bucur, R. Cernat, D. Nedelcu, C. Bleotu, D. Valeanu, V. Lazar (Bucharest, Constanta, RO) The aim of this study was to investigate the antibioresistance profile and the virulence and pathogenicity hallmarks of Escherichia coli acquatic strains. Material and Methods: 116 environmental Escherichia coli were isolated from drinking water and marine, salmaster water in Constanta, Romania. They were identified both by biochemical and serological tests. Both disc diffusion susceptibility test and microplate dilution technique were used to investigate the antibiotic resistance profiles. The rapid test to nitrocephine and isoelectrofocusing techniques were used for the confirmation of the presence and type of b-lactamases. The analysis of plasmid DNA was performed using Wizard extraction kit. The virulence tested features were: adherence and invasion of HeLa cells, adherence on inert substrata quantified by slime test, production of extracellular enzymes and exotoxins (haemolysins and other pore-forming toxins, amylase, mucinase, gelatinase, caseinase, aesculin hydrolysis). Results and Discussion: The tested strains, irrespective to their source of isolation exhibited resistance to ampicillin (28% vs 23%), ticarcillin (7.7% vs 19.4%), tetracyclines (33.3% vs 37.6%) and sulphametoxazole (2.5% vs 18.2%) and were susceptible to all other tested antibiotics. 21 strains exhibiting resistance to b-lactam antibiotics (7 isolated from drinking and 14 from marine waters) proved to be positive for the presence of b-lactamases when tested by nitrocephine rapid test. The b-lactamases of the periplasmic extract exhibited an isoelectric point ranging from 7.5 to 9.6. 10% of the drinking water strains exhibited 1 to 3 plasmids, while 5% of marine strains only 1 plasmid. As concerning the virulence hallmarks, 90% of the strains isolated from drinking water exhibited high capacity of adherence to the cellular substrate (adherence indexes of 85–100% with localised, aggregative and diffuse patterns) demonstrating the potential of these strains to colonise the animal and human tissues and to initiate an infectious process as compared to the marine strains showing low adherence potential. All strains showed colonisation ability of the inert substrate as demonstrated by the high positivity rate of slime test. All tested strains produced lipase, which could act as pore-forming toxin in case of tissue colonisation. Our results are pointing out the importance of detecting specific virulence factors before incriminating water as a source of human diseases.
P1250 Influence of Lactobacilli probiotic strains on apoptosis of colon cancer cells lines A.K. Gonet-Sur´owka, M. Strus, P.B. Heczko (Cracow, PL) Objectives: Cancerogenesis is often associated with disregulation of the apoptosis process. Many external stimuli like cytokines, viruses or bacteria can regulate this process, during which activation of several different proteins takes place within the cell. The central component of apoptosis is a cascade of proteolytic enzymes called caspases. The aim of this study was to investigate how different species of Lactobacilli influence this process on human colon cancer cell lines. Methods: We studied the effect of live or heat-killed Lactobacilli strains on apoptosis by the pan-caspases activation. We used ESP’s green fluorescent-labeled inhibitor, FAM-VAD-FMK, to detect active caspase in living cells. To distinguish apoptosis from necrosis we stained the cells with propidium iodide. As a positive control we used staurosporine, a well-known caspase activator. The apoptosis profiles were determined by FACS analysis. Results: Our findings indicate that ability to induce apoptosis or necrosis in cancer cell lines vary between different species of Lactobacilli. Many of them have positive effect on cell survival even in serum-starved conditions. On the other hand cell incubation with other Lactobacilli species strongly activate pan-caspases.
S343 P1249 Virulence, pathogenicity, antibiotic resistance and plasmid profile of Escherichia coli strains isolated from drinking and recreational waters C. Balotescu, E. Panus, M. Bucur, R. Cernat, D. Nedelcu, C. Bleotu, D. Valeanu, V. Lazar (Bucharest, Constanta, RO) The aim of this study was to investigate the antibioresistance profile and the virulence and pathogenicity hallmarks of Escherichia coli acquatic strains. Material and Methods: 116 environmental Escherichia coli were isolated from drinking water and marine, salmaster water in Constanta, Romania. They were identified both by biochemical and serological tests. Both disc diffusion susceptibility test and microplate dilution technique were used to investigate the antibiotic resistance profiles. The rapid test to nitrocephine and isoelectrofocusing techniques were used for the confirmation of the presence and type of b-lactamases. The analysis of plasmid DNA was performed using Wizard extraction kit. The virulence tested features were: adherence and invasion of HeLa cells, adherence on inert substrata quantified by slime test, production of extracellular enzymes and exotoxins (haemolysins and other pore-forming toxins, amylase, mucinase, gelatinase, caseinase, aesculin hydrolysis). Results and Discussion: The tested strains, irrespective to their source of isolation exhibited resistance to ampicillin (28% vs 23%), ticarcillin (7.7% vs 19.4%), tetracyclines (33.3% vs 37.6%) and sulphametoxazole (2.5% vs 18.2%) and were susceptible to all other tested antibiotics. 21 strains exhibiting resistance to b-lactam antibiotics (7 isolated from drinking and 14 from marine waters) proved to be positive for the presence of b-lactamases when tested by nitrocephine rapid test. The b-lactamases of the periplasmic extract exhibited an isoelectric point ranging from 7.5 to 9.6. 10% of the drinking water strains exhibited 1 to 3 plasmids, while 5% of marine strains only 1 plasmid. As concerning the virulence hallmarks, 90% of the strains isolated from drinking water exhibited high capacity of adherence to the cellular substrate (adherence indexes of 85–100% with localised, aggregative and diffuse patterns) demonstrating the potential of these strains to colonise the animal and human tissues and to initiate an infectious process as compared to the marine strains showing low adherence potential. All strains showed colonisation ability of the inert substrate as demonstrated by the high positivity rate of slime test. All tested strains produced lipase, which could act as pore-forming toxin in case of tissue colonisation. Our results are pointing out the importance of detecting specific virulence factors before incriminating water as a source of human diseases.
P1250 Influence of Lactobacilli probiotic strains on apoptosis of colon cancer cells lines A.K. Gonet-Sur´owka, M. Strus, P.B. Heczko (Cracow, PL) Objectives: Cancerogenesis is often associated with disregulation of the apoptosis process. Many external stimuli like cytokines, viruses or bacteria can regulate this process, during which activation of several different proteins takes place within the cell. The central component of apoptosis is a cascade of proteolytic enzymes called caspases. The aim of this study was to investigate how different species of Lactobacilli influence this process on human colon cancer cell lines. Methods: We studied the effect of live or heat-killed Lactobacilli strains on apoptosis by the pan-caspases activation. We used ESP’s green fluorescent-labeled inhibitor, FAM-VAD-FMK, to detect active caspase in living cells. To distinguish apoptosis from necrosis we stained the cells with propidium iodide. As a positive control we used staurosporine, a well-known caspase activator. The apoptosis profiles were determined by FACS analysis. Results: Our findings indicate that ability to induce apoptosis or necrosis in cancer cell lines vary between different species of Lactobacilli. Many of them have positive effect on cell survival even in serum-starved conditions. On the other hand cell incubation with other Lactobacilli species strongly activate pan-caspases.