Phenotypic Variation On Bacteria L-form And Rifampin-dependent Mycobacterium Tuberculosis From Clinical Sputums

Objective:Screening L-form Mycobacterium tuberculosis (MTB) from patients' specimens from Lanzhou Pulmonary Hospital,Gansu province, and observing L-form MTB morphology.Methods:In order to screening L-form MTB,340 sputum specimens from Lanzhou Pulmonary Hospital during September 2008 to September 2009 were cultured on LJ medium, Middlebrook MGIT-960,92-3 TB liquid medium with or without sodium chloride and Middlebrook 7H9 semi-solid medium with or without sucrose, then the growth of colony was observed. We observed morphology of bacteria by Ziehl-Neelsen staining method.Genomic DNA was extracted and the DNA sequence of 16SrRNA (580bp) was amplified and sequenced to analysis the homology. M. bovis was lack (RV1970F) of RD7 area but nor was MTB. So amplifying the RD7 area (RV1970F) fragment could distinguish MTB form M. bovis.Results:The specimen numbered 1415 was cultured and the colony grew and embedded into 7H9-L semi-solid as fried egg colonies, which was acid fast negative and rod under optical microscope.16S rRNA DNA sequence analysis revealed that the strain belonged to M. tuberculosis complex. RD7 sequence analysis revealed that the strain belonged to MTB. The patient,19-year-old, had a history of tuberculosis (TB) for one and a half years.Conclusion:The L-form MTB strain was isolated on the 7H9-L semi-solid media from clinical patients. The L-form MTB may cause the lung infection of the patient. Objective:Screening L-form Gordonia bronchialis (G. bronchialis) from patients'sputum specimens in Lanzhou Pulmonary Hospital, Gansu province, and analysising L-form G. bronchialis situation and morphology preliminarily.Methods:In the process of screening for L-form G. bronchialis,340 sputum specimens from Lanzhou Pulmonary Hospital during the period from September 2008 to September 2009 were cultured on LJ medium, Middlebrook MGIT-960, 92-3 TB liquid medium with or without sodium chloride and Middlebrook 7H9 semi-solid medium with or without sucrose, then the growth condition and the morphology of colony were observed. Bacterial morphology was observed in ZN and Gram staining method before and after reculturing while bacterial morphology was observed under scanning electron microscope (SEM). Genomic DNA was extracted and amplifyed sequence of 16SrRNA (580bp). The PCR product was sequenced to analysis homologically. Then we tested drug susceptibility to some antibiotics.Results:Two bacterial strains numbered 866 and 1302 had embedded growth on 7H9-L semi-solid as fried egg colonies, which were acid fast negative and round under optical microscope. After subculture, the colony showed pinkish and reddish color, and there were a few positive rod bacteria by acid fast staining. The bacteria appeared as rod in optical and scanning electron microscopes (SEM). The DNA sequence of 16SrRNA showed the two bacteria numbered 866 and 1302 belonged to Gordonia bronchialis series of Gordona genus in Actinomycosis order. The G. bronchialis was sensitive to RIF and RFD, and resistent to INH, PZA, PAS, EMB, TH, SM. The patient numbered 866,78-year-old, had coughed and expectorated sputum intermittently for four years. The patient numbered 1302 was outpatient, male,38-year-old.Conclusion:The L-form G. bronchialis strain exists in clinical patients'sputums. And L-form G. bronchialis might cause lung infection in some patients. Objective:Screening rifampin-dependent Mycobacterium tuberculosis (MTB) from patients'sputum specimens in Lanzhou Pulmonary Hospital, Gansu province, analysising morphology and genetics variation of the rifampin-dependent MTB preliminarily.Methods:200 sputum specimens from Lanzhou pulmonary hospital of Gansu province were digested by digestive juice and centrifuged, then inoculated 0.1 ml and cultured stationarily in Middlebrook MGIT960 liquid medium, and L-J media with or without rifamcin. Then the growth of colonies was observed. If the bacteria were rifampin-dependent (RIF-D) TB, the bacteria were recultured in the L-J media with or without rifamcin. If the bacteria were positive in Middlebrook MGIT960 liquid medium, drug susceptible test was taken by absolute concentration method to identify its chemosensitivity. The bacteria were stained by Ziehl-Neelsen staining method to observe bacterial morphology. The nucleotides of 16S rRNA (580bp) and rpoB (3519 bp) were amplifyed and the PCR product were sequenced to identify the bacteria and analysis the possible mutations in rpoB.Results:Six RIF-D TB strains were obtained from the 200 sputum specimens. Among the six patients infected with RIF-D MTB, there were 3 male (50%) and 3 female (50%), and the average age was 37.8 years old (range of 25-55 years old). Drug susceptible test(DST) showed that 4 RIF-D MTB were SM resistent strains,1 RIF-D MTB was INH resistent strain,4 RIF-D MTB were PZA resistent strains,1 RIF-D MTB was TH resistent strain. The bacteria were shorter in the L-J media without RIF than with RIF by acid fast staining. After subculturing, the bacteria growed shorter in the L-J media without RIF, and stronger in the media with RIF by acid fast staining under microscope. By 16SrRNA DNA sequencing, the six RIF-D MTB belonged to M.tuberculosis complex.Conclusions:The RIF-D MTB strains existed in clinical patients. The RIF-D MTB strain was a special kind of RIF resistent strain.