Fate Of Thermotolerant Coliform From Hospital Sewage And Antibiotic Resistane Gene In Water Environment

There has been growing concern about the highest resistance conditions of bacterium found in aquatic environment and antibiotic resistance strains existed in all manner of water environment such as sewge, mineral water, strainer water, drinking water and salt water.Bacterium in nature environment had genetic factor such as plasmid, transposon and phage which could transfer between or across species by conjugation, transformation and transduction to adapt the environment. Plasmid and phage harbored integron which could recruit and express the antibiotic resistance gene.The aquatic environment had become a major reservoir for antibiotic-resistant microbes. With global travel and widespread commerce, antibiotic-resistant microbes could spread to all parts of the world. Water-borne bacterial pathogens could lead to disease outbreaks that may have serious medical and economic implications. This problem was further serious by the increasing incidence of pathogens with antibiotic resistance.We must pay more attention to the detection of ESBLs-producing strains in aquatic environment. When producing broad-spectrum plasmid-encoded enzymes, organisms became highly effective at inactivating penicillin, most cephalosporins, and aztreonam. Sharma detected ESBLs-producing Yersinia enterocolitica from the river of Indian Madrid. Cernat isolated 7 Aerobacter cloacae which produced extended spectrumβ-lactamases from aquatic environment and separated them into two subtype by RAPD and PFGE. Xiaoyun Liu of our department detected 18.75% ESBLs-producing bacterium in aquatic environment. It suggested that the ESBLs-producing bacterium had already existed in water environment of our country. Since the water environment could provide opportunity to ESBLs-producing bacterium to live and transfer the resistance gene, once human was infected by these resistance pathogenic microbes through drink or open wound, it would be very difficult to treatment and control.Although previous researches displayed that the highest resistant rates were found in aquatic environment and the aquatic environment had become a major reservoir for antibiotic-resistant microbes, the ecological significance and importance of environmental gene transfer and survival of ESBLs-producing microorganisms have been relatively poorly understood. Knowledge about how antibiotic resistance arises, how resistant strains and resistance genes survive and spreading in water environment and the significance of this for humans and nature is far from complete. There are not enough data available to draw a final conclusion especially with respect to the input of already resistant bacterium into water environment.Thermotolerant coliform isolated from this water environment contaminated by the hospital sewage was used to investigate antibiotic resistance condition in part 1. ESBLs-producing bacterium was screened and plasmid, genome, antibiotic resistant gene and gene transfer were studied in part 2. Survival and stability of plasmid and resistant gene of ESBLs-producing bacterium in different laboratory water environment, the influence sequence of environmental parameters such as temperature, pH value and particle and the impact of Kirbychlor disinfect method were investigated in part 3.Methods1. All water samples were collected from 4 different sites situated upstream 100 meters (site A), hospital effluent export (site B), downstream 150 meters (site C) and 300 meters (site D). Water samples were diluted and then filtered through 0.45 mm filter papers. Membrane filters were sticked to m-FC plates and then incubated for 5 hours at 37℃and 19 hours at 45°C. Isolates were identified with API Lab Plus system. Antibiotic susceptibility tests (AST) were performed with Kirby-Bauer disc diffusion method recommended by CLSI (2005). The antibiotics selected were commonly used in patients such as ampicillin, gentamicin, amikacin, ciprofloxacin, chlormycetin, rifampicin, cefotaxime, ceftazidime, aztreonam, imipenem, ceftriaxone and cefpodoxime. Escherichia coli ATCC25922 used as standard strain.2. ESBLs-producing isolates were detected by the double disc test performed as a standardized disk diffusion assay. 3 primers were designed to detect the commonly antibiotic resistant gene. The plasmid DNA extraction was performed using E.Z.N.A Plasmid Mini KitⅠ(OMEGA) and products were used as templates for the amplification of various ESBLs genes. The plasmids exacted from the isolates and PCR products were all analyzed by gel electrophoresis. 12-mer random primers (CAA TCG CCG T GC) was used for RAPD. Mating experiments were performed on liquid media (Luria Bertani). The ESBLs-producing bacteria were selected to be donors (18, 33, 34, 37) and non-ESBLs -producing bacteria to be recipients (15, 20, 25, 36, 44). The acquisition of resistance by recipient strains was confirmed by colony color and antibiotic resistant difference between donors and recipients. Plasmid profiles were used to detect relocation of plasmid DNA from donor to recipient strains.3. NO.33 strain was selected to represent ESBLs-producing bacteria to investigate the ability to survive in different water environment which included autoclaved river water (AR), untreated river water(R), autoclaved tap water (AT), untreated tap water (TW) and autoclaved distilled water (AD). The cefotaxime resistance phenotypes and blue color on m-FC agar characteristic of the strain was used as selective markers for selection of indigenous bacteria. The experiments were performed using low bacterial inoculums (105 CFU/ml) with the scope to reproduce the actual conditions. At the end phase of NO.33 suvived in different water environment, plasmid, genome and antibiotic resistant gene were analyzed. Orthogonal design software was used to detect the impact of 3 factors (temperature, pH value and particles) on bacterium survive in 3 levels. Chlorine sterization was operated according to"sterilize technological specification 2002". Suspension quantitative germicidal test was applied.Results1. Escherichia coli was the prevalent species (82%). The others included Citrobacter freundii, Klebsiella pneumonia, Enterobacter cloacae, Serratia odorifera, Hafinia alvei and Morganella morganii. The quanlity of Thermotolerant coliforms increased from 0.72×109/100ml to 1.71×109/100ml(P<0.05)and percentage increased from 42% to 66%. The rate of multi-resistance was in increasing order for Ampicillin(P<0.01), Ciprofloxacin, Gentamicin, Chlormycetin, Aztreonam, Ceftriaxone, Cefotaxime (P<0.05). The rate of multi-resistant increased from 0 to 50%. When comparing the ARPs of the environmental isolates with local clinical isolates from the patients, the resistance patterns from both isolates were strongly correlated (r = 0.943, P<0.01).2. Compared with non-ESBLs bacterium, the highest resistance rate in ESBLs-producing group (22%) are found for CIP, CTX, CRO and CAZ. All strains except NO.23 strain comprised 1-5 plasmids with molecular weights ranging from 1,375 to 21,226bp. TEM was the major type ofβ-lactamase among ESBLs-producing E. coli, followed by CTX-M and SHV group. 2 strains produced not only TEM type but also CTX-M type. The distribution of resistance gene type was coincidence with this of local hospital isolates. After primers screening and condition optimization, RAPD could be used to distinguish 11 ESBLs-producing strains into 10 types. In laboratory condition, gene transfer took place between E.coli and hafnia alvei by conjugation. The transfer rate was 8.21×10-4.3. The survival time of strain in different water environment in decrease order was: AR, R, AD, AT, TW. Bacterium lived in R and AR environment displayed a characteristic four-phase pattern of growth curve while two-phase pattern in AT, T and AD. On the end phase of bacterium surviving, few changes were generated on plasmid and genome, but the resistant gene was still stability. Temperature impacted the growth of bacterium much more than pH value and particles. The multiple linear regression equation was Y=0.508+0.097X1+0.013X2(R2=0.709,P=0.000<0.05) (X1= effective chlorine concentration, X2= reaction time, Y=killing rate).Conclusion1. The quantity of Thermotolerant coliform in water environment increased significantly after being contaminated by hospital sewage.β-lactamase and multi-resistance (50%) were likely to be widely available in this area. Hospital sewage effluent might be the major reason causing the increase of antibiotic resistance in water environment.2. The rate of ESBLs-producing bacterium was higher in this area. TEM type was the main resistant gene type of ESBL-producing bacteria. RAPD method could be employed to genotyping and homology analysis. Antibiotic resisance gene could be transferred between species by conjugation.3. Strain NO.33 could survive in different water environment through accommodation of plasmid and genome. Even in the end phase of survive, antibiotic resistance gene was detected stable. The multiple linear regression equation was Y=0.508+0.097X1+0.013X2 (R2=0.709, P=0.000<0.05) (X1= effective chlorine concentration, X2= reaction time, Y=killing rate). When chlorine residual concentration was 0.05mg/L or 0.3mg/L and 30min reaction time, the killing rate was only 94.7% and 97.9% which was lower than 100% of"Drinking Water hygienic standard 2002". If we want to get the 100% killing rate, the effective chlorine concentration must be 1.05mg/L (chlorine residual concentration 0.46mg/L) in tap water and 30min reaction time.Question and Prospect1. This research only focused on one water environment contaminated by hospital sewage and the quantity of bacterium was only 50. Otherwise the sampling time was only in winter. Those were limited to study the whole condition of ESBLs ratio and genotyping.2."Drinking Water hygienic standard 2002"recommended the basic level of chlorine residual concentration in water factory and tap water was 0.3mg/L and 0.05mg/L in 30min reaction time. Our results showed the killing rate was only 97.9% and 94.7% according to the rule. If we want to get the killing rate of 100%, the chlorine residual concentration must be 1.05mg/L (0.46mg/L). These means that the current standard maybe not suitable for ESBLs-producing bacterium in this area.3. Results showed that most ESBLs-producing strains habored plasmids and antibiotic resistance gene commonly located on plasmid. We could control the antibiotic resistance gene transfer effectively if methods were found to eliminate plasmid.