Rapid differentiation of Prevotella intermedia and P. nigrescens by 16S rDNA PCR-RFLP

Bacteroides spp. are opportunist pathogens that cause blood and soft tissue infections and are often resistant to antimicrobial agents. We have developed a combined PCR-restriction fragment length polymorphism (RFLP) technique to characterize the 16S rRNA gene for identification purposes and the nitroimidazole resistance (nim) gene for detection of resistance to the major antimicrobial agent used to treat Bacteroidesinfections: metronidazole (MTZ). PCR-RFLP analysis of 16S ribosomal (rDNA) with HpaII and TaqI produced profiles that enabled discrimination of type strains and identification of 70 test strains to the species level. The 16S rDNA PCR-RFLP identification results agreed with routine phenotypic testing for 62 of the strains. The discrepancies between phenotypic and PCR-RFLP methods for eight strains were resolved by 16S rDNA sequencing in three cases, but five strains remain unidentified. The presence of nim genes was indicated by PCR in 25 of 28 strains that exhibited reduced sensitivity to MTZ. PCR-RFLP of the nim gene products identified the four reported genes (nimA, -B, -C, and -D) and indicated the presence of a previously unreported nim gene in 5 strains. This novelnim gene exhibited 75% DNA sequence similarity withnimB. These rapid, accurate, and inexpensive methods should enable improved identification ofBacteroides spp. and the detection of MTZ resistance determinants.

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DETECTING MYCOBACTERIUM TUBERCULOSIS RAPIDLY AND MYCOBACTERIUM TUBERCULOSIS STRAINS WITHOUT IS6110 SEQUENCE BY PCR ASSAY

Journal of Medicine and Pharmacy ◽

10.34071/jmp.2012.1.2 ◽

2012 ◽

pp. 15-19

Author(s):

Thi Chau Anh Nguyen ◽

Hoang Bach Nguyen ◽

Hai Duong Huynh ◽

Nu Xuan Thanh Le ◽

Xuan Cuong Le ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

16S Rdna ◽

False Negative ◽

Clinical Samples ◽

Tuberculosis Complex ◽

Pcr Assay ◽

Complex Objects ◽

Method Performance ◽

Gene Coding ◽

16S Rdna Pcr

Background: The Nested IS6110 PCR is used for detecting tuberculosis, however IS6110 sequence is not present in the genome of all strains of M.tuberculosis, the result may be false negative. The gene coding 16S ribosome always contains a short sequence specific to M. tuberculosis complex. Objects: Performance of the 16S Real-time PCR to detect M. tuberculosis and combining to the nested IS6110 PCR to determine the rate of Mtb strains without IS6110 from clinical samples. Materials and method: Performance of 16S rDNA PCR by commercial kit of Viet A Inc. for all 480 samples, the samples which were positive with the 16S rDNA PCR were retested in IS6110 PCR assay by in-house kit. Results: The Realtime 16S rDNA PCR detected 258 cases (53.8%) of tuberculosis. There were 3 (1.2 %) M. tuberculosis strains which do not harbor IS6110 sequence in genome. Conclusion: The IS6110 nested PCR can be applied more widely than the 16S rDNA realtime PCR. In case of using IS6110 PCR assay, results may show a low proportion of false negative. Combining 16S rDNA PCR with the IS6110 based PCR allowed detection of deletion of IS6110 sequence in M. tuberculosis isolates.

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Isolation of Enterobacter sakazakii from Stable Flies, Stomoxys calcitrans L. (Diptera: Muscidae)†

Journal of Food Protection ◽

10.4315/0362-028x-69.3.671 ◽

2006 ◽

Vol 69 (3) ◽

pp. 671-673 ◽

Cited By ~ 22

Author(s):

F. MRAMBA ◽

A. BROCE ◽

L. ZUREK

Keyword(s):

16S Rdna ◽

Foodborne Pathogen ◽

Animal Manure ◽

Stomoxys Calcitrans ◽

Fecal Coliforms ◽

Stable Fly ◽

Enterobacter Sakazakii ◽

Stable Flies ◽

16S Rdna Pcr ◽

Glucose Agar

Enterobacter sakazakii is an opportunistic foodborne pathogen that causes meningitis, enterocolitis, and sepsis, primarily in immunocompromised infants. Previously, it was suggested that stable flies, Stomoxys calcitrans, were a vector or reservoir of this pathogen. In our study, by means of a culturing approach combined with 16S rDNA PCR–restriction fragment length polymorphism genotyping and sequencing, we screened 928 individual stable flies collected in Kansas and Florida. Two stable flies (0.2%) were positive for E. sakazakii. In addition, 411 (44%) stable flies carried bacteria-forming red colonies (presumably enterics) on a violet red bile glucose agar (mean count = 6.4 × 104 CFU per fly), and 120 (13%) stable flies carried fecal coliforms (mean count = 8.7 × 103 CFU per fly). Sequencing of 16S rDNA showed that enterics from violet red bile glucose agar were represented by several genera, including Escherichia, Shigella, Providencia, Enterobacter, Pantoea, Proteus, Serratia, and Morganella. Our study shows that stable flies carry bacteria typically present in animal manure (a developmental site of stable fly larvae), which indicates that the natural reservoir of E. sakazakii is the digestive tract or manure of domestic animals. The low prevalence of E. sakazakii associated with stable flies suggests that stable flies do not play a major role as a reservoir or vector of this pathogen.

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