Comparison of the Multiple Platforms to Identify Various Aeromonas Species.

We compared several identification methods for Aeromonas genus members, including traditional biochemical testing, multiplex-PCR amplification, mass spectrometry identification, whole-genome sequencing, multilocus phylogenetic analysis (MLPA), and rpoD, gyrA, and rpoD-gyrA gene sequencing. Isolates (n = 62) belonging to the Aeromonas genus, which were came from the bacterial bank in the laboratory, were used to assess the identification accuracy of the different methods. Whole-genome sequencing showed that the Aeromonas spp. isolates comprised A. caviae (n = 21), A. veronii (n = 18), A. dhakensis (n = 8), A. hydrophila (n = 7), A. jandaei (n = 5), A. enteropelogenes (n = 2), and A. media (n = 1). Using the whole-genome sequencing results as the standard, the consistency of the other methods was compared with them. The results were 46.77% (29/62) for biochemical identification, 83.87% (52/62) for mass spectrometric identification, 67.74% (42/62) for multiplex-PCR, 100% (62/62) for MLPA typing, 72.58% for gyrA, and 59.68% for rpoD and gyrA-rpoD. MLPA was the most consistent, followed by mass spectrometry. Therefore, in the public health laboratory, both MLPA and whole-genome sequencing methods can be used to identify various Aeromonas species. However, rapid and relatively accurate mass spectrometry is recommended for clinical lab.

Genetic Diversity, Multidrug Resistance, and Virulence of Citrobacter freundii From Diarrheal Patients and Healthy Individuals.

Objectives:Citrobacter freundii is a frequent cause of nosocomial infections and a known cause of diarrheal infections, and has increasingly become multidrug resistant (MDR). In this study, we aimed to determine the genetic diversity, the antimicrobial resistance profiles and in vitro virulence properties of C. freundii from diarrheal patients and healthy individuals. Methods: 82 C. freundii isolates were obtained from human diarrheal outpatients and healthy individuals. Multilocus Sequence Typing (MLST) of seven housekeeping genes was performed. Antimicrobial susceptibility testing was carried out using the disk diffusion method according to the Clinical and Laboratory Standards Institute (CLSI) recommendations. Adhesion and cytotoxicity to HEp-2 cells were assessed. PCR and sequencing were used to identify blaCTX-M, blaSHV, blaTEM, qnrA, qnrB, qnrS, qnrC, qnrD, aac(6')-Ib-cr, and qepA genes. Results: The 82 C. freundii isolates were divided into 76 sequence types (STs) with 65 STs being novel, displaying high genetic diversity. Phylogenetic analysis divided the 82 isolates into 5 clusters. All 82 isolates were sensitive to imipenem (IPM), but resistant to one or more other 16 antibiotics tested. Twenty-six isolates (31.7%) were multidrug resistant to three or more antibiotic classes out of the 10 distinct antibiotic classes tested. Five MDR isolates, all of which were isolated from 2014, harbored one or more of the resistance genes, blaTEM-1, blaCTX-M-9, aac(6')-Ib-cr, qnrS1, qnrB9, and qnrB13. All 11 qnrB-carrying C. freundii isolates belonged to cluster 1, and one C. freundii isolate carried a new qnrB gene (qnrB92). Six isolates showed strong cytotoxicity to HEp-2 cells, one of which was multidrug resistant. Conclusions:C. freundii isolates from human diarrheal outpatients and healthy individuals were diverse with variation in sequence types, antibiotic resistance profiles and virulence properties.

[Development of a singleplex PCR assay targeting O-antigen modification genes for molecular serotyping of Shigella flexneri].

OBJECTIVE: To develop a singleplex PCR assay targeting O-antigen modification genes for molecular serotyping of Shigella (S.) flexneri. METHODS: Eight pairs of primer for O-antigen synthesis and modification genes of S. flexneri were designed and used for developing an O-antigen modification gene-specific singleplex PCR assay to serotype 14 most common S. flexneri serotypes (1a, 1b, 1c, 2a, 2b, 3a, 3b, 4a, 4b, 5a, Y, X, Xv and F6). Bacterial pathogens which causing diarrheal disease were used for specificity detection. 106 S. flexneri clinical isolates were serotyped by this method and compared with the slide agglutination method. RESULTS: An O-antigen modification, gene-specific singleplex PCR was developed. When six singleplex PCR reactions were performed, 14 of the 15 recognized S. flexneri serotypes were identified, except for serotype Xv. The detection threshold ranged from 10 pg to 1 ng DNA in a 20 µl reaction system. A high concordance between the singleplex PCR assay and slide agglutination were observed when 106 S. flexneri strains of various serotypes were analyzed with an exception that 1 serotype Y strain showed that it was carrying the additional defective gtr II genes. CONCLUSION: This method showed advantages over the traditional slide agglutination methods, and was promising when under application in the following situations as clinical diagnosis.

[Molecular analysis on non-O1 and non-O139 Vibrio cholerae isolates].

OBJECTIVE: According to results from the two-month consecutive surveillance program in Maanshan, six suspected cases of non-O1 non-O139 Vibrio (V.) cholerae infection, were found that called for identification of pathogens as well as molecular-epidemiological analysis to determine the aggregation of the epidemic situation. METHODS: Biochemical and serotype identification, hemolysis test, and drug sensitive test were used to detect the drug resistance spectrum. Real-time PCR and conventional PCR were used to detect the presence of V. cholerae specific genes, virulent genes and its related genes, including ompW, ctx, tcpA, toxR, hlyA, zot, ace, rstR and gIII(CTX). Pulsed-field gel electrophoresis (PFGE) was used to analyze the molecular type of strains. RESULTS: All the six isolates of non-O1 non-O139 V. cholerae were identified by biochemical and serologic tests, and appeared to be ß hemolytic. Twelve out of the 14 kinds of drugs showed 100% sensitive. All isolates were positive of ompW gene by real-time PCR, but negative for ctx, tcpA, zot, ace, rstR and gIII(CTX). Five of the six isolates were positive for toxR and hlyA, except for strain 1001434446. All strains had different PFGE types, but two strains had similar types. All strains had a low similarity compared to the toxigenic V. cholerae. CONCLUSION: Six cases of non-O1 and non-O139 nontoxigenic V. cholerae infection appeared in the same period. Along with epidemiological information, we noticed that these cases had a sporadic nature, but frequently appeared in the same area. We got the impression that public health measurements should be strengthened, with special attention paid to those diarrhea outbreaks caused by non-O1/non-O139 strains since V. cholerae had appeared in low incidence.

[Primary investigation on variable but nonculturable-state of enterotoxigenic Escherichia coli in vitro].

OBJECTIVE: 7 variable but nonculturable-state strains of Enterotoxigenic Escherichia coli (ETEC) during the routine bacterial subculture were found in our lab and their morphology and antigen studied. Biological features, antigens and pathogenicity of the revertants were also tested and compared to that of the initial strains in order to detect their variations. METHODS: Biological variations between the variable but nonculturable-state and the revertant of every strain were detected, using the routine gram-staining, reverting the isolates in animal intestinal, reverting their pathogenicity, serological agglutination, biochemical identifications and antibiotic resistance tests. RESULTS: For the 7 variable but nonculturable-state strains of ETEC,other than the trains that had changed into sphero vegetale cells, there were no other obvious variations found. However, high pathogenicity of these strains still remained. CONCLUSION: The presence of variable but nonculturable-state strains suggested that the routine method of bacteria storage should be changed and more attention should be paid to realize the existence of this kind of bacteria during the routine surveillance of the communicable diseases.