A pncA polymorphism to differentiate between Mycobacterium bovis and Mycobacterium tuberculosis

The pyrazinamidase gene coding for the enzyme that activates the bactericidal drug pyrazinamide contains a polymorphic site that is preserved in Mycobacterium bovis. We synthesized two sets of primers, one encompassing a 180 bp fragment, and the second spanning a 726 bp fragment including the full pncA gene. Following PCR of Mycobacterium tuberculosis and M. bovis samples, it is possible to discriminate by this polymorphism between these species by digestion with Eco065 I. Digestion of the 180 bp fragment results in two fragments of 101 and 79 bp, specific for M. tuberculosis. Alternatively, digestion of the 726 bp fragment yields three fragments of 452, 165 and 109 bp for M. tuberculosis, but only two fragments of 561 and 165 bp for M. bovis.     

Identification of Mycobacterium tuberculosis in sputum by direct inoculation of the BACTEC NAP vial

The Mycobacterium tuberculosis complex (M. tuberculosis, M. bovis, and M. africanum) can be differentiated from mycobacteria other than M. tuberculosis (MOTT bacilli) with the BACTEC NAP test (Johnston Laboratories, Becton, Dickinson & Co., Towson, MD), by selectively inhibiting their growth with p-nitro-alpha-acetyl-amino-beta-hydroxypropriophenone (NAP). The BACTEC NAP test is recommended for isolated mycobacterial cultures. In this report, a direct NAP test is performed by immediate inoculation of BACTEC NAP vials with processed sputum specimens that stain acid-fast positive. Seventy-six of 80 M. tuberculosis were correctly identified and all of the MOTT bacilli (nine M. avium complex and one M. kanasii) were correctly classified. The average time required for identification of M. tuberculosis with the direct BACTEC NAP test described here is more convenient than the recommended indirect test, and it is an accurate and rapid method to differentiate the M. tuberculosis complex from MOTT bacilli.     

多位点PCR技术用于四川267株分枝杆菌临床分离株的鉴定

目的 初步了解四川省肺结核患者分枝杆菌菌种类型。 方法 收集267株分枝杆菌临床菌株,经PNB/TCH鉴别培养基进行培养鉴定后,采用聚合酶链反应(PCR)对16S rRNA、Rv0577、IS1561、Rv1510、Rv1970、Rv3877/8和Rv3120 基因位点进行扩增,鉴定至种,再经PRA-rpoB、hsp65基因测序进行验证。 结果 267株分枝杆菌临床分离株多位点PCR结果显示结核分枝杆菌262株,非洲分枝杆菌Ⅰ型3株,非结核分枝杆菌2株。PNB/TCH鉴别培养基培养鉴定结果为结核分枝杆菌复合群266株,非结核分枝杆菌1株。2株非结核分枝杆菌分别为鸟分枝杆菌(M. avium)和脓毒分枝杆菌(M. septicum)。多位点PCR结果与rpoB-PRA、hsp65基因测序结果一致。 结论 多位点PCR技术鉴定分枝杆菌菌种结果准确可靠,且具有简便和快速等优点,有较大的分子流行病学应用价值,且对于临床诊断和治疗都具有重要意义。     

Bovine variants of Mycobacterium tuberculosis isolated in Liverpool during the period 1969 to 1983: an epidemiological survey

Between 1969 and 1983 inclusive, the bovine variants of Mycobacterium tuberculosis (M. bovis and M. africanum) were isolated from 75 patients with tuberculosis. This represented 2.9 per cent of all significant mycobacteria identified at the Liverpool Public Health Laboratory during this period. The clinical and radiological features of infection did not differ from those found with M. tuberculosis. There was an association between M. bovis infection, extrapulmonary disease and lifelong United Kingdom residency, and between M. africanum infection, pulmonary disease and immigrant status. Correlations between the present incidence of reactivated M. bovis disease and past prevalence of bovine tuberculosis in man and cattle, and between the isolation rate of M. africanum and the size of the immigrant community served by the laboratory, were also demonstrated. All the patients with M. bovis infection were considered to have a reactivated or slowly progressive primary infection. It is proposed that M. bovis only accounts for a small proportion of isolates from adults with tuberculosis because the organism displays a low tendency to reactivate.     

In vitro susceptibility of Mycobacterium tuberculosis, Mycobacterium africanum, Mycobacterium bovis, Mycobacterium avium, Mycobacterium fortuitum, and Mycobacterium chelonae to ticarcillin in combination with clavulanic acid.

The in vitro susceptibility of Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium africanum, Mycobacterium avium, Mycobacterium fortuitum, and Mycobacterium chelonae (M. chelonei) to ticarcillin in combination with calvulanic acid (CA) was studied by the agar dilution method. All the M. tuberculosis, M. bovis, and M. africanum strains were inhibited at a ticarcillin concentration of 32 micrograms/ml or lower in combination with 5 micrograms of CA. M. chelonae and M. avium strains proved resistant to more than 128 micrograms of ticarcillin plus 5 micrograms of CA per ml. M. fortuitum strains needed 128 micrograms of ticarcillin plus 5 micrograms of CA to inhibit approximately 30% of the isolates.     

Mycobacterium tuberculosis detection by polymerase chain reaction and identification of M. tuberculosis strain

A rapid multiprimer PCR method for detection of Mycobacterium tuberculosis complex (MTC) and simultaneous identification of M. tuberculosis in clinical samples has been developed. The method is based on simultaneous amplification of two targets: a 401 bp region from the mtp40 species-specific gene sequence of M. tuberculosis and a 544 bp fragment from the RD1 genome region which is specific for MTC but absent in BCG strains. Polymerase inhibitors in this study were detected by internal control in each test. Detection sensitivity was 25 copies of M. tuberculosis genomic DNA. Seven methods for isolation of mycobacterial DNA were compared and the technique with chloroform extraction was selected as the most efficient. The proposed method was used for analysis of 37 clinical samples and the results were compared with the results of culturing, acid-fast bacilli staining, and clinical diagnosis. The method proved to be sufficiently sensitive and specific for detection of mycobacterial DNA. Moreover, in countries with only two main pathogenic species of MTC circulating (M. tuberculosis and M. bovis) this method can be used for differentiation of these two species.     

Rapid differential diagnosis of Mycoplasma agalactiae and Mycoplasma bovis based on a multiplex-PCR and a PCR-RFLP

The membrane-protein 81 gene (mb-mp81) of Mycoplasma bovis was cloned, sequenced and compared to membrane-protein 81 gene (ma-mp81) of Mycoplasma agalactiae. After alignment of both sequences, specific primers pairs were designed from variable or unchanging nucleotide segments. In this study, we describe the development and optimization of a multiplex-PCR (MPCR) for the rapid detection of M. agalactiae and M. bovis strains. In addition, a simple and rapid PCR-restriction fragment length polymorphism (RFLP) assay, using the restriction enzymes AluI, DraI, RsaI and XbaI, is described to distinguish between both species. The results suggest that MPCR and PCR-RFLP assays could be used as an alternative method in routine diagnosis for rapid and specific simultaneous detection of M. agalactiae and M. bovis.     

Study of the gyrB gene polymorphism as a tool to differentiate among Mycobacterium tuberculosis complex subspecies further underlines the older evolutionary age of 'Mycobacterium canettii'

The present investigation evaluated the PCR-restriction fragment length polymorphism (RFLP) analysis of hsp65 and gyrB targets for differentiation of the species within the Mycobacterium tuberculosis complex (MTC) both by including new restriction enzymes and previously unstudied species. The hsp65 restriction analysis using HhaI resulted in a characteristic 'Mycobacterium canettii' pattern. A study of the gyrB gene polymorphism using TaqIalpha and HinfI allowed the initial division of MTC into two major groups, one consisting of M. tuberculosis and 'M. canettii' as opposed to another single group with other species. Three different patterns were observed with RsaI, the first characteristic of Mycobacterium microti, the second with Mycobacterium bovis, M. bovis BCG and Mycobacterium caprae (M. caprae was easily separated from M. bovis, and M. bovis BCG by SacII digestion), and the third with M. tuberculosis, 'M. canettii', Mycobacterium africanum, Mycobacterium pinnipedii, and the dassie bacillus. Although further discrimination within the last group was not obtained using additional restriction enzymes, the HaeIII and RsaI digestions highlighted an important gyrB polymorphism among 'M. canettii' strains. A study of the single nucleotide polymorphisms (SNP) within the gyrB by sequence analysis not only confirmed the results of the restriction analysis, but showed further differences among 'M. canettii' isolates that were not picked up using the existing battery of restriction enzymes. As many as 11 different SNPs were identified in the collection of eight 'M. canettii' isolates studied. Considering that gyrB variability among MTC member species other than 'M. canettii' is as restricted as hsp65 variability among MTC, our data corroborate a recent proposition that the 'M. canettii' group is evolutionary much older than the other MTC members. In conclusion, gyrB PCR-RFLP is a simple and rapid low-cost method that combined with phenotypic characteristics, may be helpful to differentiate most of the subspecies within the MTC.     

In vitro activities of levofloxacin used alone and in combination with first- and second-line antituberculous drugs against Mycobacterium tuberculosis.

By using the radiometric BACTEC 460-TB methodology, the inhibitory and bactericidal activity of the optically active L-isomer of ofloxacin (levofloxacin) was compared with those of the D-isomer and the commercially available mixture containing equal amounts of DL-isomers (ofloxacin) against the Mycobacterium tuberculosis complex (type strain H37Rv, a panel of drug-susceptible and -resistant clinical isolates including multidrug-resistant isolates of M. tuberculosis, as well as M. africanum, M. bovis, and M. bovis BCG). Levofloxacin MICs (range 0.50 to 0.75 microgram/ml) were about 1 dilution lower than those of ofloxacin (MIC range, 0.75 to 1.00 microgram/ml) and 5 to 6 dilutions lower than those of the D-isomer (MIC range, 32 to 60 micrograms/ml). The MICs of levofloxacin, ofloxacin, and D-ofloxacin at which 90% of the strains are inhibited were 0.50, 1.00, and 64 micrograms/ml, respectively. The multidrug-resistant M. tuberculosis strains resistant to first-line drugs were as susceptible to quinolones as the wild-type drug-susceptible isolates. Levofloxacin at 0.5 microgram/ml showed bactericidal activity comparable to the activities of 1.0 microgram of ofloxacin per ml and 64 micrograms of D-ofloxacin per ml, with MBCs within the range of 0.5 to 2.0 micrograms/ml, compared with MBCs of 0.75 to 4.0 micrograms of ofloxacin per ml for M. tuberculosis, M. africanum, M. bovis BCG. Combination testing of sub-MICs of levolofoxacin with other first-line (isoniazid, rifampin, and ethambutol) and second-line (amikacin and clofazimine) antituberculous drugs was evaluated with various two-, three-, and four-drug combinations; enhanced drug activity was observed in 8 of 25, 12 of 20, and 8 of 15 tests, respectively, indicating that levofloxacin acts in synergy with other antituberculous drugs.     

IS6110: conservation of sequence in the Mycobacterium tuberculosis complex and its utilization in DNA fingerprinting.

Multiple copies of an insertion sequence, IS6110, were shown to be present in the genome of members of the Mycobacterium tuberculosis complex (M. tuberculosis and M. bovis). Ten to 12 copies are present in various strains of M. tuberculosis, while strains of M. bovis contain only one to three copies. IS6110 was not detected in the DNA of other species of mycobacteria. Restriction endonuclease analysis indicated that the sequence of IS6110 is conserved across strain and species lines. Hybridization to the insertion sequence can be used to detect restriction fragment length polymorphism reflecting divergence in the sequence of regions flanking the various copies of IS6110. These differences were used to fingerprint various strains of the M. tuberculosis complex.     

聚合酶链反应-反向斑点杂交鉴定分枝杆菌菌种

目的 建立一种简便、快速、敏感和特异的分枝杆菌菌种鉴定方法。方法以１６ＳｒＤＮＡ为靶序列,采用聚合酶链反应（ＰＣＲ）－反向斑点杂交检测２５种分枝杆菌１１种分枝杆菌标准株、１２０株分枝杆菌临床分离株和２６份痰标本。结果 分枝杆菌、非分枝杆菌标准株经ＤＮＡ扩增,分枝杆菌均出现５７８ｂｐＤＮＡ片段,非分枝杆菌除假白喉棒状杆菌可见同样片段外,其余菌种均未见扩增。敏感性试验可检测出１００ｆｇ结核分枝杆菌ＤＮ     

Antimicrobial susceptibility of Enterobacteriaceae, including molecular characterization of extended-spectrum beta-lactamase-producing species, in urinary tract isolates from hospitalized patients in North America and Europe: results from the SMART study 2009-2010

A real-time quantitative polymerase chain reaction (qPCR) was developed for detection and discrimination of Mycobacterium tuberculosis (H37Rv and H37Ra) and M. bovis bacillus Calmette-Guérin (BCG) of the Mycobacterium tuberculosis complex (MTBC) from mycobacterial other than tuberculosis (MOTT). It was based on the melting curve (Tm) analysis of the gyrB gene using SYBR^® Green I detection dye and the LightCycler 1.5 system. The optimal conditions for the assay were 0.25 μmol/L of primers with 3.1 mmol/L of MgCl₂ and 45 cycles of amplification. For M. tuberculosis (H37Rv and H37Ra) and M. bovis BCG of the MTBC, we detected the crossing points (Cp) at cycles of 16.96 ± 0.07, 18.02 ± 0.14, and 18.62 ± 0.09, respectively, while the Tm values were 90.19 ± 0.06 °C, 90.27 ± 0.09 °C, and 89.81 ± 0.04 °C, respectively. The assay was sensitive and rapid with a detection limit of 10 pg of the DNA template within 35 min. In this study, the Tm analysis of the qPCR assay was applied for the detection and discrimination of MTBC from MOTT.     

Rapid and specific detection of the Mycobacterium tuberculosis complex using fluorogenic probes andreal-time PCR

A rapid and sensitive strategy for the specific identification of Mycobacterium tuberculosis (TB) was designed and evaluated using crude mycobacterial lysates. The speed of real-time polymerase chain reaction (PCR) was combined with the sensitivity of fluorogenic probes to confirm the presence of mycobacteria as well as specifically identify the presence of members of the mycobacteria tuberculosis complex (MTC) in a single-tube assay. Oligonucleotides were designed to amplify the internal transcribed spacer (ITS) from several mycobacterial species. Specific fluorogenic probes were included in the PCR reaction for the identification of TB as well as Mycobacterium bovia and Mycobacterium africanum in bacterial lysates. The combination of TB-specific fluorogenic probes with real-time PCR formed an approach determined to be fast (less than 40 min), sensitive (less than 800 copies of DNA) and reliable for the specific detection of the MTC. Our data demonstrate the use of real-time PCR and fluorogenic probes in a rapid and sensitive assay to distinguish members of the MTC from other mycobacterial species.     

结核分枝杆菌利福平耐药基因的检测

以药物敏感试验为标准，应用聚合酶链反应－单链构象多态性分析方法对６８例利福平耐药菌株和２０例利福平敏感菌株进行测定，结果８８例结核分枝杆菌均扩增出目标ＤＮＡ条带，１０例非结核分枝杆菌和８例非分枝杆菌未出现目标ＤＮＡ条带，所用引物扩增ｒｐｏＢ基因２１５ｂｐ片段为结核分枝杆菌复合群异性的。     

Characterization of rifampin-resistance in pathogenic mycobacteria.

The emergence of rifampin-resistant strains of pathogenic mycobacteria has threatened the usefulness of this drug in treating mycobacterial diseases. Critical to the treatment of individuals infected with resistant strains is the rapid identification of these strains directly from clinical specimens. It has been shown that resistance to rifampin in Mycobacterium tuberculosis and Mycobacterium leprae apparently involves mutations in the rpoB gene encoding the beta-subunit of the RNA polymerases of these species. DNA sequences were obtained from a 305-bp fragment of the rpoB gene from 110 rifampin-resistant and 10 rifampin-susceptible strains of M. tuberculosis from diverse geographical regions throughout the world. In 102 of 110 rifampin-resistant strains 16 mutations affecting 13 amino acids were observed. No mutations were observed in rifampin-susceptible strains. No association was found between particular mutations in the rpoB gene and drug susceptibility patterns of multidrug-resistant M. tuberculosis strains. Drug-resistant M. tuberculosis strains from the same outbreak and exhibiting the same IS6110 DNA fingerprint and drug susceptibility pattern contained the same mutation in the rpoB gene. However, mutations are not correlated with IS6110 profiling outside of epidemics. The evolution of rifampin resistance as a consequence of mutations in the rpoB gene was documented in a patient who developed rifampin resistance during the course of treatment. Rifampin-resistant strains of M. leprae, Mycobacterium avium, and Mycobacterium africanum contained mutations in the rpoB gene similar to that documented for M. tuberculosis. This information served as the basis for developing a rapid DNA diagnostic assay (PCR-heteroduplex formation) for the detection of rifampin susceptibility of M. tuberculosis.     

Comparison of the in vitro activities of rifapentine and rifampicin against Mycobacterium tuberculosis complex

The in vitro activity of rifapentine for 44 clinical isolates of Mycobacterium tuberculosis complex was compared with that of rifampicin using the Bactec radiometric method and the absolute concentration method for susceptibility testing. Twenty-nine M. tuberculosis, 11 Mycobacterium bovis and four Mycobacterium africanum strains were studied. Control tests showed that rifapentine was stable for 14 days in 7H9 broth and for 3 weeks in 7H10 agar medium. The 44 M. tuberculosis complex strains were more susceptible to rifapentine than to rifampicin, irrespective of the testing method. In the radiometric system, the MIC50 and MIC90 of rifapentine for M. tuberculosis complex strains were one or two two-fold dilutions lower than those of rifampicin (0.06-0.125 mg/L versus 0.25 mg/L, respectively). By the absolute concentration method, the MIC50 and MIC90 of rifapentine for M. tuberculosis complex strains were two two-fold dilutions lower than those of rifampicin (0.125-0.25 mg/L versus 0.5-1 mg/L, respectively). The MIC90 of rifapentine for the 44 M. tuberculosis complex strains was always 0.25 mg/L, irrespective of the method used, but the radiometric method was more reliable and more reproducible than the agar 7H10 method.     

DNA probes for mycobacteria. I. Isolation of DNA probes for the identification of Mycobacterium tuberculosis complex and for mycobacteria other than tuberculosis (MOTT)

Traditional methods used in identifying mycobacteria such as acid-fast bacillus stains and culture are often time-consuming, insensitive and non-specific. The isolation of DNA probes, coupled to a non-radioactive, e.g. biotin-based detection system, have the potential to foster the development of clinical assays for Mycobacterium tuberculosis and mycobacteria other than tuberculosis (MOTT) that are rapid, sensitive and specific. To this end, we have isolated two different probes: one which is specific for the Mtb complex and one which recognizes all other potentially pathogenic mycobacteria. The use of these probes in combination should allow the detection and differentiation of M. tuberculosis from MOTT. To isolate the first probe, we prepared a library of M. tuberculosis DNA fragments in a lambda EMBL phage vector. Recombinant phage were screened by plaque-lift hybridization procedures using nick-translated mycobacterial genomic DNA to identify sequences specific to the Mtb complex. Inserts from candidate recombinant phage were purified, nick-translated and hybridized against a wide variety of filter-bound mycobacterial and non-mycobacterial DNAs. Two clones were identified which hybridized to the closely related M. tuberculosis, M. bovis and M. microti but not to other species of mycobacteria. The second probe was isolated by preparing a library of M. malmoense DNA fragments in lambda EMBL and screening by plaque-lift hybridization. One clone was identified which, in addition to recognizing members of the Mtb complex, also hybridized to M. intracellulare, M. malmoense, M. scrofulaceum, M. simiae, M. xenopi, M. avium, M. szulgai, M. kansasii and M. haemophilum. None of the three clones hybridized to DNA from non-mycobacterial species.     

Activity of rifapentine and its metabolite 25-O-desacetylrifapentine compared with rifampicin and rifabutin against Mycobacterium tuberculosis, Mycobacterium africanum, Mycobacterium bovis and M. bovis BCG

The in vitro activity of rifapentine and its metabolite, 25-O:-desacetylrifapentine, as compared with that of rifampicin and rifabutin, was determined against Mycobacterium tuberculosis, Mycobacterium africanum, Mycobacterium bovis and M. bovis BCG. MICs were determined radiometrically and by the 1% proportional method using Middlebrook 7H11 agar. The bactericidal effect of the drugs was determined in parallel at selected concentrations. For drugsusceptible isolates of M. tuberculosis, the Bactec MICs of rifapentine and 25-O:-desacetylrifapentine were 0.03-0.06 mg/L and 0. 125-0.25 mg/L, respectively. Similar MICs were obtained for M. africanum (0.03-0.125 and 0.125-0.50 mg/L, respectively), and M. bovis (0.063-0.25 and 0.125-1.0 mg/L, respectively), but MICs were considerably lower for M. bovis BCG (0.008-0.063 mg/L for rifapentine and 0.016-0.125 mg/L for its metabolite). In general, MICs determined using 7H11 agar medium were usually one or two dilutions higher than those obtained using Bactec broth. When compared with rifampicin and rifabutin, the inhibitory activity of rifapentine for drug-susceptible isolates was roughly equal to that of rifabutin, and the inhibitory activity of 25-O:-desacetylrifapentine was comparable to that of rifampicin; however, rifapentine was somewhat more bactericidal than rifabutin at equal concentrations. Clinical isolates of M. tuberculosis with a high degree of resistance to rifampicin (MIC >/= 32 mg/L) were also highly resistant to rifabutin, rifapentine and 25-O:-desacetylrifapentine, although the MICs of rifabutin in this case were somewhat lower than the MICs of rifapentine.     

In vitro activities of the ketolides telithromycin (HMR 3647) and HMR 3004 compared to those of clarithromycin against slowly growing mycobacteria at pHs 6.8 and 7.4

The in vitro activities of HMR 3647 (telithromycin) and HMR 3004, two novel semisynthetic ketolides, were investigated and compared with that of the reference macrolide drug, clarithromycin, against 34 strains of slowly growing mycobacteria at pHs 6.8 and 7.4, as determined radiometrically. The MICs at pH 7.4 were about 1 to 2 dilutions lower than those observed at pH 6.8. In terms of the highest to the lowest activity, the three antibiotics could be classified as follows: clarithromycin > HMR 3004 > HMR 3647. Among the species tested, Mycobacterium bovis BCG, M. ulcerans, M. avium, and M. paratuberculosis were moderately susceptible to HMR 3004 and HMR 3647 (MICs at pH 7.4, < or =5.0 and < or =20.0 microg/ml, respectively, versus < or =1.25 microg/ml for clarithromycin), whereas M. tuberculosis, M. africanum, M. bovis, and M. simiae were resistant (MICs, > or =10.0 and > or =40.0 microg/ml, respectively, at pH 7.4). Although not more active than clarithromycin in vitro, the high level of intracellular accumulation of the two ketolides inside phagocytes warrants further screening in experimental animal models.     

2011-2018年四川省自贡市分枝杆菌临床分离株鉴定分析

目的了解自贡市肺部感染分枝杆菌的菌种菌型特征,为临床分枝杆菌病的诊断和治疗及制定有效防控策略提供科学依据。方法收集2011－2018年临床疑似结核病患者分离到的抗酸染色阳性培养物,经对硝基苯甲酸（PNB）和噻吩-2-羧酸肼（TCH）鉴别培养和多位点PCR鉴定为结核分枝杆菌和非结核分枝杆菌（NTM）,再经rpoB和hsp65序列分析对NTM进行种的鉴定。结果2 751株分枝杆菌临床分离株经PNB/TCH鉴别培养基和多位点PCR鉴定,结核分枝杆菌2 647株,占96.22%,非洲分枝杆菌Ⅰ型32株,占1.16%,NTM 72株,占2.62%。 72株NTM鉴定出11个种。结论自贡市分枝杆菌肺部感染主要为结核分枝杆菌所致,感染致病的NTM种类较多,以脓肿分枝杆菌、胞内分枝杆菌、堪萨斯分枝杆菌和鸟分枝杆菌为主。 外来分枝杆菌和奥巴涅分枝杆菌在国内为首次报道。