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Mycobacterium bovis isolates with M. tuberculosis specific characteristics.

Our study is the first report of exceptional Mycobacterium mycobacterium

Any of the rod-shaped bacteria that make up the genus Mycobacterium. The two most important species cause tuberculosis and leprosy in humans; another species causes tuberculosis in both cattle and humans.
 boris strains that have some characteristics of M. tuberculosis M. tuberculosis,
n the bacterium responsible for tuberculosis, generally a respiratory infection in man; nonrespiratory tuberculosis is considered an indicator disease for AIDS. See also tuberculosis.
. The strains were isolated from 8 patients living in Kazakhstan. While molecular markers were typical for M. bovis, growth characteristics and biochemical test results were intermediate between M. bovis and M. tuberculosis.


Mycobacterium bovis Mycobacterium bovis A mycobacterium that causes a TB-like infection in cows; before pasteurization was common, M bovis spread to humans via contaminated milk  causes tuberculosis (TB) mainly in cattle but has a broad host range and causes disease similar to that caused by M. tuberculosis in humans (1). It belongs to the M. tuberculosis complex (MTBC MTBC Metroplex Technology Business Council (Texas)
MTBC mycobacterium tuberculosis complex
MTBC Malaysian Tenpin Bowling Congress
MTBC Mitsubishi Trust and Banking Corporation
MTBC Mean Time Between Cleans
MTBC Mountain Biking Club
) that comprises the closely related human pathogens M. tuberculosis and M. africanum (2). Identification of M. bovis traditionally has been based on clear-cut differences in phenotypic characteristics and biochemical properties when compared to the other members of the MTBC (1,2). M. bovis shows a dysgonic colony shape on Lowenstein-Jensen medium Lowenstein-Jensen medium

one containing eggs for the cultivation of mycobacteria.
, is negative for niacin niacin: see coenzyme; vitamin.
 or nicotinic acid or vitamin B3

Water-soluble vitamin of the vitamin B complex, essential to growth and health in animals, including humans.
 accumulation and nitrate reduction, is susceptible to thiophene-2-carboxylic acid hydrazide hy·dra·zide  
An acyl derivative of hydrazine.


A compound formed by combining hydrazine with an acyl compound. Hydrazides are important in the manufacture of certain medicines.
 (TCH TCH Trans-Canada Highway
TCH Texas Children's Hospital (Houston, TX)
TCH The Children's Hospital
TCH Traffic Channel
TCH Threshold Crossing Height
TCH Toyota Camry Hybrid
TCH Taurocholic Acid
), and shows microaerophilic microaerophilic /mi·cro·aero·phil·ic/ (-a?er-o-fil´ik) requiring oxygen for growth but at lower concentration than is present in the atmosphere; said of bacteria.  growth on Lebek medium (1-3). A further criterion used for differentiation is the intrinsic resistance to pyrazinamide, which is found in most M. bovis isolates (1-3). In contrast, M. tuberculosis shows eugonic growth, is positive for niacin accumulation and nitrate reduction, is resistant to TCH, shows aerophilic growth on Lebek medium, and is usually not monoresistant to pyrazinamide (2,3).

More recently, several molecular methods have been developed that provide clear criteria for the identification of M. bovis. These comprise a variety of polymerase chain reaction polymerase chain reaction (pŏl`ĭmərās') (PCR), laboratory process in which a particular DNA segment from a mixture of DNA chains is rapidly replicated, producing a large, readily analyzed sample of a piece of DNA; the process is  (PCR PCR polymerase chain reaction.

polymerase chain reaction

Polymerase chain reaction (PCR) 
) methods, e.g., based on DNA sequence DNA sequence Genetics The precise order of bases–A,T,G,C–in a segment of DNA, gene, chromosome, or an entire genome. See Base pair, Base sequence analysis, Chromosome, Gene, Genome.  variations in the direct repeat region of MTBC complex strains (spoligotyping [4]) or on single nucleotide polymorphisms (SNPs) in either the oxyR gene (5) or the gyrB gene (6). Furthermore, MTBC isolates can be differentiated by large sequence polymorphisms or regions of difference (RD), and according to their distribution in the genome, a new phylogenetic phy·lo·ge·net·ic
1. Of or relating to phylogeny or phylogenetics.

2. Relating to or based on evolutionary development or history.
 scenario for the different species of the MTBC has been suggested (7-9). The presence or absence of particular deletions has been proposed as being discriminative dis·crim·i·na·tive  
1. Drawing distinctions.

2. Marked by or showing prejudice: discriminative hiring practices.
, e.g., lack of TdB1 for M. tuberculosis or lack of RD12 for M. bovis.

In routine diagnostics, the combination of phenotypic characteristics and biochemical features is sufficient to differentiate clinical M. bovis isolates, and in general, the results obtained are unambiguous. However, here we describe the characteristics of 8 strains of the MTBC that showed an unusual combination of phenotypic and biochemical attributes of both M. bovis and M. tuberculosis. Molecular analyses confirmed the strains as M. bovis, which in part have phenotypic and biochemical properties of M. tuberculosis.

The Study

During a previous investigation of 179 drug-resistant isolates from Kazakhstan (10), we determined the presence of 8 strains showing monoresistance to pyrazinamide. Kazakhstan is the largest of the central Asian republics Central Asian Republics, the countries of Kazakhstan, Kyrgyzstan, Tajikistan, Turkmenistan, and Uzbekistan. Constituent republics of the former Union of Soviet Socialist Republics, they all achieved independence in late 1991.  and is divided regionally into 14 oblasts. The investigation was performed as part of a nationwide drug resistance survey conducted by the national TB program of Kazakhstan with assistance from the World Health Organization/ International Union against Tuberculosis and Lung Disease lung disease Pulmonary disease Pulmonology Any condition causing or indicating impaired lung function Types of LD Obstructive lung disease–↓ in air flow caused by a narrowing or blockage of airways–eg, asthma, emphysema, chronic bronchitis;  Global Project in 2001. The subset of this survey investigated here (n = 158) represents 100% of strains resistant to isoniazid isoniazid (ī'sōnī`əzĭd), drug used to treat tuberculosis. Also known as isonicotinic acid hydrazide, isoniazid is the most effective antituberculosis drug currently available. , rifampin rifampin (rĭfăm`pĭn), antibiotic used in the treatment of tuberculosis. It is also used to eliminate the meningococcus microorganism from carriers and to treat leprosy, or Hansen's disease. , ethambutol ethambutol /etham·bu·tol/ (e-tham´bu-tol) an antibacterial, specifically effective against Mycobacterium; used with one or more other antituberculous drugs in the treatment of pulmonary tuberculosis, administered as the , or streptomycin streptomycin (strĕp'tōmī`sĭn), antibiotic produced by soil bacteria of the genus Streptomyces and active against both gram-positive and gram-negative bacteria (see Gram's stain), including species resistant to other  isolated in 9 of the 14 Kazakhstan oblasts during the study period; 21 samples had fungal contamination or showed no growth (10). All strains were isolated from sputum sputum /spu·tum/ (spu´tum) [L.] expectoration; matter ejected from the trachea, bronchi, and lungs through the mouth.

sputum cruen´tum  bloody sputum.

To further clarify if these strains were monoresistant M. tuberculosis or M. bovis isolates intrinsically resistant to pyrazinamide, we performed several routine diagnostic tests traditionally used for species differentiation (6). All strains showed eugonic growth characteristics on Lowenstein-Jensen slants and on bromcresol purple medium (Figure), which in general is typical for M. tuberculosis. However, on bromcresol purple medium, classic M. tuberculosis isolates induce a pH-dependent change of color Noun 1. change of color - an act that changes the light that something reflects
change of state - the act of changing something into something different in essential characteristics

whitening, lightening - changing to a lighter color
 from blue to yellow, which was not observed in these cases (Figure). Furthermore, all 8 isolates were positive for niacin accumulation, negative for nitrate reduction, susceptible to TCH, and showed aerophilic growth on Lebek medium. Considering all results, the 8 strains showed a combination of test results that did not allow a clear differentiation as M. bovis or as M. tuberculosis (Table 1). Such a combination of test results would apply best to M. africanum, a species from which more variable test results have been reported (3). However, this species was probably was not isolated because M. africanum strains are usually not monoresistant to pyrazinamide (3).


Therefore, we investigated all strains with several molecular techniques previously used for differentiation within the MTBC (Table 2). They all had identical spoligotype patterns (hexcode 6B-57-5F-7F-FF-60, performed according to the methods of Kamerbeek et al. [4]), that lacked spacers 3943 and identical IS6110 DNA fingerprint DNA fingerprint
An individual's unique sequence of DNA base pairs. Also called genetic fingerprint.
 patterns with 2 IS6110 copies (data not shown, performed according to the methods of van Embden et al. [11]). All isolates carried the M. boris-specific polymorphism in the oxyR gene (5), and none of them had the M. boris BCG--specific deletion in the RD1 region (12). PCR analysis of other RDs (RD3, RD4, RD5, RD9, RD10, RD12, TbD1, and IS1541) showed results typical for M. bovis when compared with the RD signatures of the American Type Culture Collection American Type Culture Collection (ATCC) is a private, not-for-profit biological resource center whose mission focuses on the acquisition, authentication, production, preservation, development and distribution of standard reference microorganisms, cell lines and other materials for  strains of M. tuberculosis, M. africanum, and M. bovis (Table 2) and with previously published data (7,8). The intrinsic resistance to pyrazinamide was confirmed by DNA sequence analysis as all strains carried the M. bovis--specific single point mutation point mutation
A mutation that involves a single nucleotide and may consist of loss of a nucleotide, substitution of one nucleotide for another, or the insertion of an additional nucleotide.
 at nucleotide position 169 of the pncA gene. Seven of the 8 strains were isolated from 30- to 55-year-old men, and 1 strain was from a 72-year-old woman. All but 1 patient had a history of previous antituberculosis treatment, but none of the strains showed any further resistance (data not shown). The patients originated from the oblast oblast (ō`bläst, ŏ`–, Rus. ô`bləstyə) [Rus.,=region], administrative and territorial division in Russia, Ukraine, Belarus, and the former USSR.  of Kostanajskaya in north Kazakhstan. Among all patients, no direct epidemiologic links could be established. However, 3 of the patients lived in the city of Kostanaj, while 5 came from rural areas. Before 1950, the Kazakh Steppe was a broad, continuous belt of grassland that stretched from the Ural River to the Altai foothills, covering large parts of Kostanajskaya; after the 1950s, the region was used extensively for agriculture. Information on contact with animals is not available, since cattle herds are only kept privately.


We describe 8 strains of M. boris with exceptional phenotypic characteristics that are intermediate between M. tuberculosis and M. bovis. This fact initially complicated a clear species differentiation; however, the battery of molecular tests performed clearly confirmed all strains as M. bovis. These tests included the presence of characteristic single nucleotide polymorphisms as well as an RD profile that is typical for the M. boris lineage of the MTBC (6, 7). To our knowledge, this is the first report describing M. bovis isolates with phenotypic characteristics and biochemical properties of M. tuberculosis. In our previous investigation of 176 M. bovis strains from Germany, all strains had phenotypic characteristics typical of M. bovis, and no strains similar to the isolates from Kazakhstan could be identified (13). The same result applies for the spoligotype patterns, as none of the strains in our database had an identical spoligotype pattern (data not shown). A further comparison with the international M. bovis spoligotype database (available from spoligodatabase) identified 1 strain isolated in Argentina with an identical spoligotype pattern; however, no further information about his strain is available.

Whether the 8 strains analyzed represent strains of an ancestral phylogenetic lineage of M. bovis that might have been conserved because of the geographic isolation of that region of Kazakhstan or whether they gained their special characteristics by new mutations is a question that cannot be answered by the data obtained in this study. All strains have been isolated from humans. We cannot say if we have found an exceptional outbreak of a particular M. bovis strain or if the patients were infected directly by wildlife, livestock, or food, and the disease developed by chance during the study. However, an overall percentage of [approximately equal to] 5% of all resistant strains investigated in this study indicates that these isolates may be important in Kazakhstan. This also poses the question of whether these strains might become more virulent in humans if they acquired phenotypic/biochemical characteristics usually observed exclusively in M. tuberculosis. However, to address this question more precisely, longitudinal studies longitudinal studies, the epidemiologic studies that record data from a respresentative sample at repeated intervals over an extended span of time rather than at a single or limited number over a short period.
 on the population structure of MTBC isolates in Kazakhstan obtained from humans and animals, in combination with experiments in virulence model systems, will be necessary. In any case, these strains represent ideal model organisms for analyzing the nature of the biologic differences observed between M. bovis and M. tuberculosis. To ensure a clear differentiation from other M. bovis strains, we suggest the name M. bovis subtype (programming) subtype - If S is a subtype of T then an expression of type S may be used anywhere that one of type T can and an implicit type conversion will be applied to convert it to type T.  "Almaty" for this genotype. Almaty is the former capital and largest city of Kazakhstan.

Dr Kubica is a physician and working in a postdoctoral position at the German National Research Center for Mycobacteria mycobacteria

members of the genus Mycobacterium.

anonymous mycobacteria
see opportunist (atypical) mycobacteria (below).

nontubercular mycobacteria
see opportunist (atypical) mycobacteria (below).
, Research Center Borstel, Borstel, Germany. Her major research interests include molecular epidemiology molecular epidemiology Molecular medicine An evolving field that combines the tools of standard epidemiology–case studies, questionnaires and monitoring of exposure to external factors with the tools of molecular biology–eg, restriction endonucleases,  and the diagnosis and treatment of mycobacterial mycobacterial

emanating from or pertaining to mycobacterium.

mycobacterial granuloma
may be caused by Mycobacterium tuberculosis (see cutaneous tuberculosis), M.


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(2.) Wayne LG, Kubica GP. The mycobacteria. In: Sneath PHA PHA


phytohemagglutinin, a plant lectin.
 and Holt JG, editors. Bergeys manual of systematic bacteriology bacteriology

Study of bacteria. Modern understanding of bacterial forms dates from Ferdinand Cohn's classifications. Other researchers, such as Louis Pasteur, established the connection between bacteria and fermentation and disease.
, vol. 2. Baltimore: Williams & Wilkins Co.; 1986. p. 1435-57.

(3.) Niemann S, Richter E, Rusch-Gerdes S. Differentiation among members of the Mycobacterium tuberculosis complex by molecular and biochemical features: evidence for two pyrazinamide-susceptible subtypes of M. bovis. J Clin Microbiol. 2000;38:152-7.

(4.) Kamerbeek J, Schouls L, Kolk A, van Agterveld M, van Soolingen D, Kuijper S, et al. Simultaneous detection and strains differentiation of Mycobacterium tuberculosis for diagnosis and epidemiology. J Clin Microbiol. 1997;35:907-14.

(5.) Sreevatsan S, Escalante P, Pan X, Gillies DA 2nd, Siddiqui S, Khalaf CN, et al. Identification of a polymorphic nucleotide in oxy R specific for Mycobaeterium bovis. J Clin Microbiol. 1996;34:2007-10.

(6.) Niemann S, Harmsen D, Rusch-Gerdes S, Richter E. Differentiation of clinical Mycobacterium tuberculosis complex strains by gyrB DNA sequence polymorphism analysis. J Clin Microbiol. 2000;38:3231-4.

(7.) Brosch R, Gordon SV, Marmiesse M, Brodin P, Buehrieser C, Eiglmeier K, et al. A new evolutionary scenario for the Myeobacterium tuberculosis complex. Proe Natl Acad Sci U S A. 2002;99:3684-9.

(8.) Huard RC, de Oliveira Lazzarini LC, Butler WR, van Soolingen D, Ho JL. PCR-based method to differentiate the subspecies subspecies, also called race, a genetically distinct geographical subunit of a species. See also classification.  of the Mycobacterium tuberculosis complex on the basis of genomic deletions. J Clin Microbiol. 2003;41:1637-50.

(9.) Mostowy S, Cousins D, Brinkman J, Aranaz A, Behr MA. Genomic deletions suggest a phylogeny for the Mycobaeterium tuberculosis complex. J Infect Dis. 2002;186:74-80.

(10.) Kubica T, Agzamova R, Wright A, Aziz MS, Rakishev G, Bismilda V, et al. The Beijing genotype is a major cause of drug resistant tuberculosis in Kazakhstan. Int J Tubere Lung Dis. 2005; 9:646-53.

(11.) van Embden JD, Cave MD, Crawford JT, Dale JW, Eisenaeh KD, Gicquel B, et al. Strain identification of Mycobacterium tuberculosis by DNA fingerprinting: recommendations for a standardized methodology. J Clin Microbiol. 1993;31:406-9.

(12.) Talbot EA, Williams DL, Frothingham R. PCR identification of Mycobacterium bovis BCG BCG bacille Calmette-Guérin.

1. bacillus Calmette-Guérin

2. ballistocardiogram

BCG, See bacille Calmette-Guórin.
. J Clin Microbiol. 1997;35:566-9.

(13.) Kubica T, Rusch-Gerdes S, Niemann S. Mycobacterium bovis subsp. caprae caused one-third of human M. bovis-associated tuberculosis cases reported in Germany between 1999 and 2001. J Clin Microbiol. 2003;41:3070-7.

Address for correspondence: Stefan Niemann, Forschungszentrum Borstel, National Reference Center for Mycobacteria, Parkallee 18, D-23845 Borstel, Germany; email:

Tanja Kubica, * Rimma Agzamova, ([dagger]) Abigail Wright, ([double dagger]) Galimzhan Rakishev, ([dagger]) Sabine Rusch-Gerdes, * and Stefan Niemann *

* National Reference Center for Mycobacteria, Borstel, Germany; ([dagger]) National Center for Tuberculosis Problems, Almaty, Kazakhstan; and ([double dagger]) World Health Organization, Geneva Geneva, canton and city, Switzerland
Geneva (jənē`və), Fr. Genève, canton (1990 pop. 373,019), 109 sq mi (282 sq km), SW Switzerland, surrounding the southwest tip of the Lake of Geneva.
, Switzerland
Table 1. Phenotypic characteristics of type strains
Mycobacterium tuberculosis H37 (ATCC 27294), M. bovis
(ATCC19210), M. africanum (ATCC25420), and the strains
analyzed *

                                   Test result

                     Colony            TCH
                     morphology      ([double
Strains              ([dagger])      dagger])

Kazakhstan (n = 8)
  ([section])        Eugonic            S
ATCC M. bovis        Dysgonic           S
ATCC H37             Eugonic            R
ATCC M. africanum    Dysgonic           S

Strains                  PZA       accumulation

Kazakhstan (n = 8)
  ([section])             R             +
ATCC M. bovis             R             -
ATCC H37                  S             +
ATCC M. africanum         S             +

                                    Test result
                                    Change of
                                     color of       Growth on
                       Nitrate      bromcresol         Lebek
Strains               reduction       medium          medium

Kazakhstan (n = 8)
  ([section])             -             -           Aerophilic
ATCC M. bovis             -             -         Microaerophilic
ATCC H37                  +             +           Aerophilic
ATCC M. africanum         +             -         Microaerophilic

* TCH, thiophene-2-carboxylic acid hydrazide; PZA, pyrazinamide;
+, positive test result; -, negative test result; S, susceptible;
R, resistant.

([dagger]) Growth characteristics on Lowenstein-Jensen and
bromcresol purple slants.

([double dagger]) Growth in presence of TCH.

([section]) All 8 strains showed identical test results.

Table 2. Genetic characteristics of type strains Mycobacterium
tuberculosis H37 (ATCC 27294), M. bovis (ATCC19210),
M. africanum (ATCC25420), and the strains analyzed *

                                 Test result

Strains                 TbD1   RD1    RD3     RD4     RD5

   (n = 8) ([dagger])    1      1      1       0       0
M. bovis ATCC            1      1      1       0       0
M. tuberculosis H37      0      1      1       1       1
M. africanum ATCC        1      1      0       1       1

                                Test result

Strains                 RD9    RD10   RD12   IS1561

   (n = 8) ([dagger])    0      0      0       1
M. bovis ATCC            0      0      0       1
M. tuberculosis H37      1      1      1       1
M. africanum ATCC        0      0      1       1

                               oxyR           gyrB
Strains                     ([dagger])     ([dagger])

   (n = 8) ([dagger])           1           M. bovis
M. bovis ATCC                   1           M. bovis
M. tuberculosis H37             0        M. tuberculosis
M. africanum ATCC               0         M. africanum

* RD, region of difference; 0, region deleted; 1, region present.

([dagger]) Presence of oxyR mutation G to A at position 285; 1,
polymorphism present; 0, polymorphism not present.

* Classification according to gyrb-polymerase chain reaction
restriction fragment length polymorphism analysis
Niemann et al. [3]).

([section]) All 8 strains showed identical test results.
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Author:Niemann, Stefan
Publication:Emerging Infectious Diseases
Geographic Code:9KAZA
Date:May 1, 2006
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