| Diarrheagenic Escherichia coli(DEC)and Campylobacter are important foodborne pathogens associated with acute intestinal infection and severe diarrhea in human.Both of the two clinically important pathogens are ubiquitous in environments.Thus,it is significant to get better understanding of their epidemiology and antimicrobial resistant patterns to construct framework for food safety control.In this study,we investigated the epidemiology,antimicrobial susceptibility and virulence of DEC and Campylobacter isolated from patients with diarrhea in Shanghai,China.In addition,multidrug resistant Campylobacter were selected to conduct phylogenetic analysis and molecular identification of antibiotic resistant genes to determine their genetic relatednesses and antimicrobial resistance mechanisms.1.Epidemiology and antimicrobial resistance of DEC from patients with diarrheaDiarrheagenic Escherichia coli(DEC)are major causes of bacterial gastroenteritis worldwide.This study was conducted in four hospitals in Shanghai,China during the period from June 2012 to October 2013.A total of 735(10.2%)DEC were identified from 7,204 stool samples of patients with diarrhea,including 594(9.7%)isolated from 6,122 stool samples of Chinese patients and 141(13.1%)isolated from 1,082 stool samples of foreigners living in Shanghai.A total of 374 enteropathogenic Escherichia coli(EPEC),318 enterotoxigenic Escherichia coli(ETEC),36 Shigella/enteroinvasive Escherichia coli(EIEC)and 7 Shiga toxin-producing Escherichia coli(STEC)were identified.EPEC and ETEC were the major subtypes among children younger than 5 and adults aged 19-60,respectively.High resistance rates among the 735 isolates were observed to streptomycin(90.7%),ampicillin(63.4%),nalidixic acid(61.1%),sulfisoxazole(49.1%),tetracycline(41.2%),trimethoprim(35.6%),trimethoprim-sulfamethoxazole(35.4%),followed by amoxicillin/clavulanic acid(27.2%),cefotaxime(24.5%),cefepime(23.5%),gentamicin(16.7%),ceftazidime(12.4%),chloramphenicol(10.6%),ciprofloxacin(7.2%)and ofloxacin(3.4%).All the isolates were susceptible to imipenem.In addition,potential virulence genes were screened by PCR.Our findings highlighted the need of active surveillance programs for DEC in order to better understand the epidemiology and antimicrobial resistance trends,and to rapidly detect high-risk lineage of these significant pathogens.2.Molecular analysis of ETEC from patients with diarrheaEnterotoxigenic Escherichia coli(ETEC)is frequently associated with travelers' diarrhea in developed world,and is a major cause of childhood diarrhea in developing countries.A total of 123 ETEC isolated from patients with diarrhea from June to December 2012 in Shanghai,China were examined to determine their genetic relatedness using multi-locus sequence typing(MLST)and pulsed field gel electrophoresis(PFGE)and for the presence of virulence genes and antimicrobial susceptibility.Twenty-nine sequence types(STs)and 63 PFGE patterns were identified,and results from the two subtyping methods correlated well.The 12 isolates of PFGE cluster B all belonged to ST 2332 and were associated with nosocomial neonatal diarrhea.Isolates of a cluster usually had the same set of virulence factors,whereas isolates of different clusters carried diverse combinations of virulence determinants.Isolates belonging to ST 2332 and ST 182(n=9)were resistant to at least six antimicrobials.These results highlighted the need of active surveillance programs for infectious diseases collecting data at both epidemiological and genetic levels that can detect high-risk lineages of pathogens in order to rapidly identify disease outbreaks.3.Molecular characterization,antimicrobial resistance and virulence of Campylobacter jejuni/coli from young children with diarrheaCampylobacter is a major cause of bacterial gastroenteritis worldwide.Young children represent a particular age group of population in Campylobacter infection because of their limited diets and weak immune systems.In this study,a total of 110 Campylobacter(80 C.jejuni and 30 C.coli)isolated from children of less than five years of age with diarrhea in Shanghai,China in 2011 were examined for their genetic relationship and antimicrobial susceptibility.The presence of virulence genes and its association with invasion potential to Caco-2 cell were also determined.MLST revealed 62 sequence types under 14 clonal complexes from C.jejuni and 15 STs under 2 clonal complexes from C.coli.ST 464 and ST 574 were the predominant clones in C.jejuni,and ST 828 was the prodominant one in C.coli.High resistance rates among the 110 isolates were observed to nalidixic acid(88.2%),ciprofloxacin(87.3%)and tetracycline(87.3%),followed by ampicillin(30.9%),gentamicin(28.2%),clindamycin(21.8%),erythromycin(21.8%),and chloramphenicol(8.2%).Compared with that of C.jejuni(32.5%),a larger proportion of C.coli(83.3%)were resistant to multiple antimicrobials,including 16 isolates belonging to the ST 828 clonal complex resistanted to a panel of six antimicrobials(ciprofloxacin,clindamycin,erythromycin,gentamicin,nalidixic acid,tetracycline).Virulenc genes cad F(100%),ceu E(99.1%),cia B(98.2%),fla A(98.2%),ans B(97.3%),cdt B(96.4%),cdt C(96.4%)and cdt A(94.6%)were detected in most isolates,while fuc P,vir B11 and ggt were only detected in 31.8%?1.8% and 1.8% isolates,respectively.Furthermore,57 Campylobacter isolates were selected based on their distinct STs and the presence of virulence genes to determine their abilities to adhere to and invade Caco-2 cells.The level of invasion varied widely among isolates,and had relatively weak correlation with the genotype data.Twelve hyper invasive isolates were identified,such as ST 21,ST 3930 and ST 1953.These multidrug resistant or hyper-invasive lineages can be recognized as potential high-risk clones that are able to cause severe diarrhea among young children,and should draw our particular attention in clinical settings.4.Emergence and antimicrobial resistance mechanisms of multidrug resistant CampylobacterAnitibiotic resistance has been a serious problem over recent years.Multidrug resistant Campylobacter that resist to most of the commonly used antibiotics in clinic has emerged,which can be a potential threat to public health.In this study,548 Campylobacter isoaltes(372 Campylobacter jejuni and 176 Campylobacter coli)from stool samples of diarrheal patients and chicken,duck and pigeon meat were subjected for antibiotic susceptibility analysis to ciprofloxacin,tetracycline,gentamicin,erythromycin and clindamycin,in order to select multidrug resistant isoaltes.A total of 32(8.6%)Campylobacter jejuni and 119(67.6%)Campylobacter coli were respectively identified as multidrug reisistant isoaltes.PFGE analysis was performed on them to determine their genetic relatenessesses.A total of 26 and 77 different PFGE patterns were determined among the 32 Campylobacter jejuni and 119 Campylobacter coli multidrug resistant isolates,respectively.An 85% similarity threshold defines seven major clusters(A to G)in the 77 PFGE patterns of Campylobacter coli.The cluster A(n=16)and G(n=16)were the major lineages,containing 11 and 8 isolates of the same PFGE patterns,respectively,indicating these two lineages were potiental high risk clones that might be able to spread among patients.In order to investigate the molecular machnisms of multidrug resistant patterns,antibiotic resistant genes of the the 151 isolates were amplified and sequenced.Almost all(150,99.3%)had gyr A mutantion at codon 86 that causes ciprofloxacin resistance;tet(O),the tetracycline resistance associated gene,was identifed in 148(98%)isolates;23s r RNA A2075 G,which is regarded to be associated with erythromycin,was found in 56(37.1%)isolates;and 89(58.9%)and 10(6.6%)isolates carried aad E and aad E-sat4-aph A cluster respetively,and were regarded associated with both gentamicin and clindamycin resistance.In addition,the cme R-cme ABC intergenic regions were investigated.Gene mutations were identified at the inverted repeat region and its flanking regions in both C.jejuni and C.coli strains,and 9 and 15 differents sequence types were determined,respectively.Among them,23 novel ones were identified.The epidemiological and molecular data in this study revealed the molecular mechanisms of antibiotic resistance phenotypes among the multidrug resistant Camplobacter isolates,which may help better understand the situation of antibiotic resistance trends,and provide guidelines of antibiotic use for treatment of Camplobacter infection in clinical settings. |
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