ABSTRACT
A sample preparation method was developed to simulate the process of intracellular metabolites metabonomics analysis of Escherichia coli. The Escherichia coli cell was firstly quenched with cold sodium chloride solution ( 0. 85 %，precooled at -80℃ for 15 min).The quenched bacterial cell was treated by using the technique of vacuum freeze-drying and liquid nitrogen freezing combined with ultrasonic processing to increase cell membrane penetrability. Finally，a cold aqueous solution of methanol (MeOH:H2O, 1:1，V/ V, 4 ℃) was used as extraction solvent to extract metabolites. In the present research，flow cytometry and OD value recovery were performed to evaluate the degree of cell damage caused by quenching at single cell level and at integral level respectively. The tested results indicated that the degree of damage to cells caused by cold sodium chloride solution was less than 5%. The peak quantity and the total ion intensity detected by LC-TOF in low collision energy were used to evaluate extraction effects. Three different cell membrane penetrability modes and 4 kinds of extraction solvents were investigated and compared.The results showed that the technique of liquid nitrogen freezing combined with ultrasonic processing for cell membrane penetrability and a cold aqueous solution of methanol (MeOH/H2O，1:1，V/V, 4℃) for extraction of metabolites had the best extraction effect(peak quantity was greater than or equal to 105，and total ion intensity was in the range of 106-107).Therefore，in this work，the freeze drying，grinding with liquid nitrogen and ultrasonic extraction were combined to extract metabolites. In this way，it effectively promoted cell lysis and improved the efficiency of extraction. The result of synthetic analysis showed that the method proposed here could meet the requirements of the metabonomics analysis of Escherachaa coli.
ABSTRACT
Background: Enteropathogenic Escherichia coli( EPEC) is a major cause of diarrhoea in children below 5 years of age. Serotyping is the classical method and PCR detection of virulence factors is a rapid way of detecting diarrhoeagenic Esch.coli. Objectives : To study the role of EPEC in Paediatric diarrhoea by both Serogrouping and Multiplex PCR assay and to analyse the antibiotic susceptibililty pattern of EPEC strains in our area. Materials and Methods : Prospective study of stool samples collected from children with diarrhoea and without diarrhoea who were below 5 years of age was conducted from May to November 2011. EPEC isolates were identified by Serogrouping. Escherichia coli isolates were subjected to Serogrouping and Multiplex PCR assay and those isolates which showed pathogenic genes were further serotyped. Antibiotic susceptibility pattern of EPEC isolates was determined by Clinical and Laboratory Standards Institute guidelines. Results : Among the Escherichia coli isolates in the diarrhoeal group, 36.8% were identified as EPEC by Serogrouping and 38.8% of them were found to possess EAEC genes by molecular characterisation. In the nondiarrhoeal Esch. coli strains , none agglutinated with EPEC polyvalent sera, 46.6% showed EAEC genes out of which 85.7% were of a single serotype O153. Among the Escherichia coli isolates which agglutinated with EPEC polyvalent antisera , 33.3% were positive for Enteroaggregative genes. Conclusion : EPEC is still an important pathogen in paediatric diarrhoeas . O serogrouping can still be relied upon for detection of EPEC. EAEC are present in classical ‘ O ‘ serogroups. Serotype O 153 has an increasing potential for asymptomatic carrier state in children below 5 years of age.
ABSTRACT
Quinolones are broad-spectrum antibacterial agents used in human and veterinary medicine, and their extensive use have been associated with a rise of the quinolone resistance. In the present study, the quinolone resistance of avian E.coli and Salmonella isolates was evaluated and compared, in which 344 avian E.coli and 224 Salmonella isolates from 1990s were serogrouped with antisera and thc antimicrobial susceptibility test to 10 quinolones was carried out by using the Kirby-Bauer method recommended by Clinical and Laboratory Standards Institute (CLSI). It was demonstrated that the 344 isolates of avian E.coli distributed in 27 serogroups and 68.90% (237/344) of the isolates belonged to four O-serogroups: i.e. O1, O2, O18, O78, and the 224 isolates of avian Salmonella were all determined to be Salmonella pullorum. The drug-resistance rate of avian E. coli isolates to nalicixic acid from 1993-1999 was more than 60%(64.43%,131/181), whereas those of isolates to 9 antibiotics from 2000-2008 had a drug-resistance rates of more than 60%, namely,nalicixic acid(92.02%), fleroxacin(79.75%), pipemidic acid(79.14%), enrofloxacin(78.53%), enoxacin(76.07%), lomenfloxacin(74.85%), ciprofloxacin(69.33%), norfloxacin(63.80%) and ofloxacin(61.35%). For the 4 O-serogroups of the avian E.coli isolates, the drug-resistance rates of more than 50% to antimicrobials were as follows: O78 isolates to 7 antimicrobials;O18 isolates to 5 antimicrobials, and O1 and O2 isolates just to 3 antimicrobials. The quinolone resistance of Salmonella isolates was much lower than E.coli, in which 101 salmonella isolates from 1993-1999 were all susceptible to quinolones. Nalicixic acid resistance of salmonella isolate firstly appeared in 2000, and the drug-resistance rate of salmonella isolates from 2000-2008 was found to be more than 60% for nalicixic acid(83.74%), but those to other quinolones were comparatively lower. These results indicated that the quinolone resistance of avian E.coli and salmonella were increasing in the past two decads because of the over-use of antibiotics.