CHARACTERIZATION OF PATHOGENIC FUNGI GENOMES USING PULSED FIELD GEL ELECTROPHORESIS

Pulsed field gel electrophoresis(PFGE) has been firstly introduced in characterization of the pathogenic fungi Penicillium marne f fei and Exophiala dermatitidis genomes.The numbers and sizes of their chromosomes have been detected.Polymorphism was identified on the smallest chromosome of E.dermatitidis.The result shows that PFGE for characterization of large molecular DNA pathogenic fungi is very suitable,it is more simple and more efficacy.The result also shows the diversity of pathogenic fungi is relative common even in rare occurred pathogenic fungi such as E.dermatitidis.

Phylogenetically clustering of rhizobia by genome structure:application to unclassified Rhizobium

Previous research reveals that the genome structures of rhizobial type strains and reference strains can reflect their phylogenetic relationships. In order to further explore the potential application of genome structure as a phylogenetic marker in rhizobial natural taxonomy, this study analyzed the genome structures of 29 unclassified nodule bacteria isolated from the root nodules of leguminous trees, Robinia sp., Dalbergia spp., and A lbizia spp. and 7 rhizobial reference strains by I-CeuI cleavage, then clustered these bacteria phylogenetically based on their genome structures and compared these clusters with those based on numerical taxonomy and 16S rDNA PCR-RFLP. Eleven phylogenetic clusters were obtained, The clusters were in large part consistent with those based on numerical taxonomy and 16S rDNA PCR-RFLP. Also there are inconsistent clusters based on the above three methods. But results are completely consistent with 16S rRNA clusters. This suggested that the genome structure clustering method can be used to lastly identify root nodule isolates and detect their phylogenetic relationships. The credibility and repeatability of the results, together with the simplicity and possibility to analyze a large number of strains in a short time of the method, indicates the broad potential application of genome structure as phylogenetic marker to categorize rhizobial isolates and should in the future facilitate biodiversity studies.

Genetic diversity of Listeria monocytogenes isolates from farm products in Shanghai,China

Farm products are considered important vehicles for the transmission of Listeria monocytogenes(L.monocytogenes).The typing of L.monocytogenes from farm products contributes to the surveillance and source tracing of the pathogen.In this study,77 L.monocytogenes strains from seven farm product categories in Shanghai were analyzed by serological typing,multilocus sequence typing(MLST),pulsed-field gel electrophoresis(PFGE),and whole-genome nucleotide polymorphism(wgSNP).The results showed that the 77 isolates were classified into four serovars(1/2a,1/2c,1/2b,and 4b),and of these,1/2a(n=47)was the most prevalent.Seventeen sequence types(STs)were generated by MLST with two novel STs(ST1402 and ST1403),and 20 of 77 L.monocytogenes isolates had high genetic identity with previously documented outbreak isolates according to the minimum spanning tree from the MLST results.Moreover,34 PFGE patterns(PF1-PF34)were differentiated,and based on a similarity value higher than 80%by the unweighted pair group method dendrogram,the discriminatory index was relatively low(equal to 0.775).Furthermore,14 isolates were chosen and further analyzed by wgsNP based on the previous typing results,which demonstrated that wgSNP and MLST yielded mostly consistent typing results but higher resolution than PFGE.In conclusion,77 L.monocytogenes isolates from farm products collected in nine districts in Shanghai were highly genetically diverse,and 20 of these isolates had high relatedness with previously documented outbreak strains worldwide.The results indicate a possible cross-contamination risk of L.monocytogenes and a potential public health concern resulting from farm products during the supply chain in Shanghai,China.

PulseNet China,a model for future laboratory-based bacterial infectious disease surveillance in China

Surveillance is critical for the prevention and control of infectious disease.China’s real-time web-based infectious disease reporting system is a distinguished achievement.However,many aspects of the current China Infectious Disease Surveillance System do not yet meet the demand for timely outbreak detection and identification of emerging infectious disease.PulseNet,the national molecular typing network for foodborne disease surveillance was first established by the Centers for Disease Control and Prevention of the United States in 1995 and has proven valuable in the early detection of outbreaks and tracing the pathogen source.Since 2001,the China CDC laboratory for bacterial pathogen analysis has been a member of the PulseNet International family;and has been adapting the idea and methodology of PulseNet to develop a model for a future national laboratory-based surveillance system for all bacterial infectious disease.We summarized the development progress for the PulseNet China system and discussed it as a model for the future of China’s national laboratory-based surveillance system.

Multidrug resistance problems targeting piglets and environmental health by Escherichia coli in intensive swine farms

Multidrug resistance(MDR)problems become critical concerns in animal production through food chains and the environment.Thus,the cross-sectional study of E.coli in swine feces,drinking water,and wastewater collected on four medium and four large swine farms in Thailand was studied.The orders of antibiotics used were AMX-COL-GM-TIL and AMX-COL-GM-CER on medium-sized and large swine farms,respectively.Hemolytic E.coli(HEC)was more frequently isolated from all samples than Non-hemolytic E.coli(NHEC).The medium-sized farms were resistant to AMP,AMX,TE,and C,while large farms were resistant to AMP,AMX,TE,and PIP.Only two sensitive antibiotics,namely AK and TE,were available on both farms,whereas,banned NT was still detected in the sludge.The MDR of E.coli isolates was 95.7%and 87.7%on medium-sized and large swine farms,while the highest resistance found was 15 drugs used for NHEC on medium-sized swine farms.The PFGE molecular typing revealed the largest cluster containing five isolations,correlated with drinking water,sow,and fattening isolates;however,the phenotypic and genotypic were not in a similar pattern.The most seriously MDR related to HEC was found in the piglets,effluent,and sludge.Thus,the AMR and MDR of E.coli existence in medium-sized and large swine farms in this study.MDR of E.coli was substantially found in the environmental,including effluent and sludge.Therefore,it inevitably operates the long-term monitoring of AMR,especially targeted piglets and its environment as the main focus of critical antibiotics use in swine farms.

Environmental risk quotient of the antibiotic,phenotypic,and genotypic profiles for antibiotic resistance of Escherichia coli collected from manure and wastewater at swine farms in Prachinburi Province,Thailand

Currently,antimicrobial drug resistance is a problem that deserves thoughtful consideration.Especially in the swine production industry,many pig farms tend to release wastewater into natural water sources,which villagers use for their livelihood.This study aimed to demonstrate the occurrence of phenotypic and genotypic resistance characteristics of E.coli,as well as antibiotic residues in manure and wastewater on swine farms in Prachinburi Province,Thailand.Samples were collected in 2020 and 2021 during the rainy and summer seasons.The results of a questionnaire showed that farmers used antibiotics for disease treatment and prevention at a ratio of 2/4(50%).The most common antibiotic used was amoxicillin(4/4,100%),followed by Enrofloxacin(75%)and colistin(25%).The analysis of antibiotic residue in wastewater by Liquid Chromatography-Mass Spectrometry(LCMS,LC-MS)during the rainy season revealed that amoxicillin(54%)was the highest,followed by florfenicol(14%)and tiamulin(13%).While oxytetracycline(59%),amoxicillin(20%),and florfenicol(11%)were the highest in the summer.The study of Risk Quotient(RQ)indicated that water resources present a risk of antibiotic contamination by sulfonamides,trimethoprim,b-lactam,quinolones,and tetracycline at high levels(SRQ>1)during both seasons.The minimal inhibitory concentrations(MICs)were determined by an antibiotic susceptibility test(AST),analyzed by microdilution technique,and VITEK®2 Compact.The test result indicated that a total of 143 isolates were found in manure(45/143),pre-treatment(51/143),and final effluent(posttreatment,47/143).ESBL-positive resistance was detected at 13%.However,an immensely high percentage of antimicrobial resistance was found for ampicillin(90%),whereas imipenem did not show any drug resistance(0%).The average MIC value towards colistin in all samples was 2.71 mg/ml,while mcr1 was not found in any samples.Furthermore,MDR was expressed in E.coli at as high as 76.22%(109/143).The highest MDR pattern detected was AMP-TETeFFCeSXT.The PCR technique detected that the diversity and abundance of AMR genes were not significantly different from animal manure and wastewater at the swine farms.The bla-TEM was found more frequently than bla-PS.While the tetracycline group(tetA,tetB)and aminoglycoside(aadA1,aadA2,aadB)represented a major proportion,respectively.The PFGE study revealed the possibility of similar genetic morphology from the different isolates found in this study.