Skip to main content
Download PDF
  • Research article
  • Open access
  • Published:

Detection of a streptomycin-resistant Mycobacterium bovis strain through antitubercular drug susceptibility testing of Tunisian Mycobacterium tuberculosis complex isolates from cattle

BMC Veterinary Research volume 14, Article number: 296 (2018) Cite this article

Abstract

Background

A rising isolation trend of drug-resistant M. bovis from human clinical cases is documented in the literature. Here we assessed Mycobacterium tuberculosis complex isolates from cattle for drug susceptibility by the gold standard agar proportion method and a simplified resazurin microtitre assay (d-REMA). A total of 38 M. tuberculosis complex strains, including M. bovis (n = 36) and M. caprae (n = 2) isolates, from cattle in Tunisia were tested against isoniazid, rifampin, streptomycin, ethambutol, kanamycin and pyrazinamide.

Results

M. caprae isolates were found to be susceptible to all test drugs. All M. bovis strains were resistant to pyrazinamide, as expected. In addition, one M. bovis isolate showed high-level resistance to streptomycin (MIC > 500.0 μg/ml). Concordant results with the two methods were found. The most common target genes associated with streptomycin resistance, namely the rrs, rpsL and gidB genes, were DNA sequenced. A non-synonymous mutation at codon 43 (K43R) was found in the rpsL gene. To the best of our knowledge, this is the first report describing the isolation of a streptomycin-resistant M. bovis isolate from animal origin.

Conclusions

Antitubercular drug susceptibility testing of M. bovis isolates from animals should be performed in settings where bTB is endemic in order to estimate the magnitude of the risk of drug-resistant tuberculosis transmission to humans.

Background

The World Organization for Animal Health (OIE) has recognized bovine tuberculosis (bTB) as an important animal disease and zoonosis [1]. bTB causes significant economic losses to farmers due to livestock deaths, reduced productivity and restrictions for trading animals.

The main causal agents of bTB are Mycobacterium bovis and, to a lesser extent, Mycobacterium caprae, both members of the Mycobacterium tuberculosis complex. These pathogens may also cause tuberculosis in humans (hereafter referred to as zoonotic tuberculosis, zTB) although the true incidence of this disease in human beings is unknown [2,3,4]. An extensive meta-analysis found the proportion of zTB to be ≤1.4% in countries outside Africa and 2.8% on average in African countries [3]. The vast majority of cases were due to M. bovis and the contribution of cases due to M. caprae was not quantified. Substantial evidence suggests that zTB might be underestimated because of two major issues hindering understanding of the true burden of this disease: first, the absence of systematic surveillance for M. bovis and M. caprae as cause of tuberculosis in humans in all low-income and high tuberculosis burden countries where bTB is endemic; and second, the inability of laboratory procedures most commonly used to diagnose human tuberculosis to identify and differentiate these pathogens from M. tuberculosis, with the result that all cases may be assumed to be caused by M. tuberculosis [5].

Animal test-and-slaughter schemes have successfully reduced the prevalence of bTB in most industrialized countries. The situation is profoundly different in unindustrialized countries where the WHO, in conjunction with FAO and OIE, has classified bTB as a neglected zoonosis. In South Africa, as in other regions in Africa, the lack of bTB control programmes [6] makes communities with high HIV/AIDS infection rates and those living in close contact with infected animals or animal products more vulnerable to zTB [7, 8].

M. bovis has one of the broadest host ranges of any known zoonotic pathogen and is globally distributed [9]. The phylogenomic analysis of M. bovis genomes has recently revealed large-scale polymorphisms, which may contribute to the differential adaptability of the pathogen [10]. M. bovis is naturally resistant to pyrazinamide (PZA) [11], which is one of the first-line antibiotics used to treat tuberculosis in humans. The lack of prompt identification of M. bovis human cases may result in improper treatments and have lethal consequences [12]. Several studies have documented additional drug resistances in human M. bovis isolates over the last two decades, such as toward isoniazid (INH) [13, 14], streptomycin (STR) [15] or multiple drugs [16,17,18,19].

M. caprae, on the other hand, is evolutionarily older than M. bovis and accounts for a smaller burden of zTB. Moreover, it is not globally distributed but primarily restricted to European countries [3, 20, 21]. Its resistance against first-line drugs is rarely documented [22]. Nevertheless, M. caprae has received more attention in recent years, particularly due to the increasing number of M. caprae outbreaks in wild or domestic animals which pose a threat to human health [21].

While mycobacteria isolated from human cases are generally assessed for drug susceptibility, studies on antitubercular drug susceptibility testing of M. bovis and M. caprae isolated from animals are limited [23,24,25,26,27,28,29]. So far, M. bovis isolates from cattle resistant to INH and rifampin (RIF) have been documented in Italy [23] and Brazil [29], to the best of our knowledge. Monitoring of antitubercular drug resistance of M. bovis isolated from animals may thus contribute to reducing the risk of drug-resistant M. bovis transmission from animals to humans and among human beings.

In Tunisia, bTB is enzootic and the consumption of raw milk and unpasteurized dairy products is common. A previous study demonstrated that raw milk consumers are at high risk of being infected with M. bovis [30]. Numerous clinical cases of human extrapulmonary tuberculosis due to M. bovis have been recently documented in Tunisia [31, 32] and the consumption of unpasteurized dairy products has been indicated as the most likely source of transmission [33]. Implementation of effective and comprehensive strategies to control bTB and to prevent zTB are therefore of primary importance in the country.

In this study we assessed drug susceptibility of M. bovis and M. caprae isolates from cattle in Tunisia towards six antitubercular drugs – PZA, INH, RIF, ethambutol (EMB), STR and kanamycin (KAN) – by both the gold standard agar proportion method and the simplified resazurin microtitre assay (REMA), the dichotomous REMA (d-REMA) recently proposed by Marianelli and colleagues [27].

Methods

All experimental procedures here described were carried out at the Department of Food Safety, Nutrition and Public Animal Health of the Istituto Superiore di Sanità (ISS), (Italy).

M. tuberculosis complex strains

A total of 38 M. tuberculosis complex strains, including M. bovis (n = 36) and M. caprae (n = 2) previously isolated in Tunisia and molecular typed by spoligotyping and MIRU-VNTR analysis [34], were provided by the University of Sfax (Tunisia). The isolates were analysed for susceptibility to INH, RIF, STR, EMB, KAN and PZA at ISS.

Isolates were subcultured in Middlebrook 7H9 medium (Biolife, Italy) with 10% oleic acid-albumin-dextrose-catalase (OADC) enrichment (Becton Dickinson and Company) before being tested. To aid the dispersion of bacterial clumps, 3-mm glass beads were added to the tubes and bacterial suspensions were vigorous vortexed for 15 s. Any remaining large bacterial clumps were allowed to settle. Bacterial suspensions were then adjusted to match a 1.0 McFarland turbidity standard.

All M. bovis and M. caprae strains were isolated from lymph node and tissue samples showing tuberculosis-compatible lesions. Samples were collected at the abattoir during the postmortem inspection, in accordance with national laws.

d-REMA testing

The d-REMA test, based on Palomino and colleagues [35] and slightly modified by Marianelli and colleagues [27] by testing only two concentrations per drug – the cut-off value for drug resistance, R, and the cut-off value of REMA drug susceptibility, S – was used. The R values were determined in studies where REMA was validated against the gold standard method [35,36,37,38,39,40]. The test was carried out in triplicate in 96-well plates, as previously described [27]. Briefly, drug solutions were prepared at concentrations of 20 mg/ml (PZA; Sigma-Aldrich, UK) and 2 mg/ml in distilled water (INH, STR, EMB, and KAN; Sigma-Aldrich, UK) or methanol (RIF; Sigma-Aldrich, UK), filter sterilised, and frozen until used.

One hundred microliters of Middlebrook 7H9 medium supplemented with OADC was inoculated into each well. One hundred microliters of each bacterial suspension – previously adjusted to a 1.0 McFarland standard and then diluted 1:20 in the same medium – was then inoculated. The antitubercular drugs were subsequently added at two final concentrations, R and S: 0.1 (S) and 0.25 (R) μg/ml for INH, 0.25 (S) and 0.5 (R) μg/ml for RIF, 0.5 (S) and 1.0 (R) μg/ml for STR, 2.5 (S) and 3.125 μg/ml for EMB, 2.5 (S) and 3.125 (R) μg/ml for KAN and 100.0 (S) and 800.0 (R) μg/ml for PZA.

Plates were covered with lids, placed in a plastic bag and incubated at 37 °C for 7 days. Finally, 30 μl of freshly prepared 0.01% resazurin solution (Acros Organics, USA) was added to each well. The plates were incubated overnight at 37 °C and assessed for colour development. The d-REMA testing was repeated twice for those isolates showing either resistance or ambiguous chromatic change.

The drug-sensitive M. bovis ATCC 19210 (used as negative control) and two resistant M. avium strains (used as positive controls) – one resistant to EMB and one resistant to both INH and EMB – from the Italian bacteria collection were included in the study.

Agar proportion method in Middlebrook 7H11 medium

The agar proportion method was performed in Middlebrook 7H11 agar. The test was carried out according to the approved standard (M24A) for Susceptibility Testing of Mycobacteria, Nocardiae, and Other Aerobic Actinomycetes published by the Clinical and Laboratory Standards Institute (CLSI) [41]. Briefly, the turbidity of the inoculum was adjusted to match a 1.0 McFarland standard, and diluted 1:100 and 1:10,000. One hundred microliters of these solutions was then inoculated into 35 mm plates with and without the test drug. The test was carried out in duplicate. The following final critical drug concentrations were used: 0.2 μg/ml for INH; 1.0 μg/ml for RIF; 7.5 μg/ml for EMB; 2.0 μg/ml for STR; 6.0 μg/ml for KAN; and 100 μg/ml for PZA [40, 41]. After 3 weeks of incubation at 37 °C, the number of colony forming units (CFU) growing on the drug-containing medium was compared with those growing on the drug-free medium and expressed as a percentage of the latter. The isolate was considered resistant if the number of colonies on a medium containing an antimicrobial agent, relative to the number observed on a drug-free medium was ≥1%. Negative (M. bovis ATCC 19210) and positive (M. avium strains) controls were also included in the test. The test was repeated twice for isolates showing resistance.

Streptomycin MIC determination by REMA

MIC testing was to be performed only in case of drug resistance. Since M. bovis growth was observed only in the presence of STR and the control drug PZA (as expected) in both the d-REMA assay and agar proportion method (see Results), MIC testing was carried out only for STR. The test was performed in triplicate according to Palomino and colleagues [35]. Ten dilutions were tested (2.5, 5.0, 10.0, 20.0, 50.0, 100.0, 200.0, 300.0, 400.0 and 500.0 μg/ml). The MIC was defined as the lowest drug concentration that prevented resazurin colour change from blue to pink. The M. bovis ATCC control was also tested.

DNA sequencing

Sequencing, too, was to be performed only in case of drug resistance. Since resistance was observed only in M. bovis and towards STR and the control drug PZA (see Results), the most common target genes associated with resistance to STR encoding 16S rRNA (rrs), ribosomal protein S12 (rpsL) [42] and a 7-methylguanosine methyltransferase (gidB) [43], were investigated in the resistant M. bovis isolate and in three randomly selected STR-susceptible M. bovis isolates..

DNA was extracted from cultures using a commercial kit (InstaGene Matrix; Bio-Rad Laboratories, Italy). The whole rrs, rpsL and gidB genes were PCR amplified and sequenced. The reference sequence accession number (AC) NC_002945 of M. bovis AF2122/97 available at NCBI was used to design primers for the PCR amplification and sequencing of the rrs gene. The rpsL and gidB genes were PCR amplified and sequenced according to Feuerriegel and colleagues [44].

PCR products were analysed by 2% agarose gel electrophoresis, stained with GelRed Nucleic Acid Stain (Biotium Inc., Hayward, CA), purified by ExoSAP-IT PCR Product Cleanup (Affymetrix, CA) and sequenced by using PCR and, if required, sequencing primers. Sequences were analysed using the ABI Prism SeqScape Software, version 2.0 (Applied Biosystems, Foster City, CA). All consensus sequences generated were then compared to the published, drug-sensitive M. bovis AF2122/97 reference strain, to detect genetic variation. Mutations were confirmed through resequencing. To distinguish silent from missense mutations, amino acid sequences were theoretically deduced. PCR and DNA sequencing primers used, PCR conditions followed and size of the amplicons obtained are listed in Table 1.

Table 1 PCR and DNA sequencing primers
Full size table

Results

Drug susceptibility

M. bovis and M. caprae isolates, as well as control strains, have been tested here against PZA, INH, RIF, EMB, STR and KAN by both the agar proportion method and d-REMA.

d-REMA results were obtained after 8 days of incubation. All M. bovis strains, including the M. bovis ATCC control, were resistant to PZA as expected. One out of 36 M. bovis isolates showed an additional drug resistance, the STR resistance as shown in Fig. 1, lines G–I. On the other hand, M. caprae isolates showed sensitivity against all drugs. The susceptibility of the M. bovis ATCC control to all test drugs (Fig. 1, lines J–L) was confirmed, as was the resistance of the two M. avium controls towards either INH or INH and ETB (Fig. 1, lines M–O). Results were confirmed through retesting the resistant isolate and controls by both drug susceptibility methods.

Fig. 1
figure 1

Drug susceptibility profiles. Drug susceptibility to INH, RIF, STR, EMB and KAN by d-REMA. A–C, D–F, G–I: sample triplicates. J–L: M. bovis ATCC control in triplicates; M–O: M. avium control resistant to both INH and EMB in triplicates. 1–2: 0.1 (S) and 0.25 (R) μg/ml for INH; 3–4: 0.25 (S) and 0.5 (R) μg/ml for RIF; 5–6: 0.5 (S) and 1.0 (R) μg/ml for STR; 7–8: 2.5 (S) and 3.125 (R) μg/ml for EMB: 9–10: 2.5 (S) and 3.125 (R) μg/ml for KAN; (+) positive control containing no drug; (−) negative control containing uninoculated media

Full size image

After 3 weeks of incubation, d-REMA results were confirmed by the agar proportion method.

The STR-resistant M. bovis isolate was subjected to further investigation, including STR MIC determination and DNA sequencing. The isolate showed resistance to all ten test dilutions: the blue-to-mauve colour change occurred at MIC values ranging from 2.5 to 500.0 μg/ml. We were thus unable to determine the STR MIC, as shown in Fig. 2, lines A–C. The M. bovis ATCC control, on the other hand, did not grow at any drug concentration tested (Fig. 2, lines D–F).

Fig. 2
figure 2

STR MIC results. A–C STR-resistant M. bovis isolate in triplicates. D–F: M. bovis ATCC control in triplicates. 1–10: 2.5, 5.0, 10.0, 20.0, 50.0, 100.0, 200.0, 300.0, 400.0 and 500.0 μg/ml for STR; (+) positive control containing no drug; (−) negative control containing uninoculated media

Full size image

DNA sequencing of drug target genes

We then PCR amplified and sequenced the most common target genes associated with resistance to STR, namely the rrs, rpsL and gidB genes, in the resistant M. bovis isolate and in three randomly selected STR-susceptible M. bovis isolates.

A consensus sequence for each gene was generated and compared to the reference strain M. bovis AF2122/97 available in GenBank to detect genetic variation. A non-synonymous mutation – nucleotide substitution AAG → AGG at codon 43 (mutation K43R) – was found only in the rpsL gene of the STR-resistant isolate. No mutations were detected in the other two target genes in either of the isolates.

Discussion

Tunisia is an endemic country for bTB. Despite the implementation of a national bTB control programme, many intensive farms belonging to the state or parastatal sector still do not meet sanitary standards for effective prophylaxis; others, belonging to private owners, also have scarce, if any, veterinary activity [45]. bTB therefore continues to be widespread in this country, mainly in the private sector, which owns more than 70% of the cattle livestock [46]. In Tunisia, the consumption of raw milk and unpasteurized dairy products is common. A previous study has shown that raw milk may spread M. bovis, putting consumers of raw milk or derivatives at high risk of being infected [30]. Although the exact contribution of M. bovis to the burden of zTB in Tunisia remains unknown, recent studies have indicated M. bovis as the main etiological agent of human extrapulmonary tuberculosis [31,32,33]. In consideration of the above, bTB represents a serious public health problem in Tunisia and effective disease control programmes have to be implemented urgently.

So far, no study has been conducted to estimate the risk of drug or multi-drug resistant bTB transmission to humans in Tunisia where M. bovis infection is endemic in livestock. On that account, we assessed at ISS the susceptibility of 36 M. bovis and two M. caprae isolates from Tunisia to six antibiotic drugs commonly administered to patients with tuberculosis. We detected STR resistance in one M. bovis isolate and characterized the nucleotide mutation associated with that resistance.

Drug susceptibility testing is rarely carried out on M. bovis and other Mycobacterium isolates of animal origin although bTB poses a serious threat to human health particularly in low-income and bTB endemic countries [3, 5]. Multidrug-resistant M. bovis outbreaks in humans have occurred, some with serious consequences [16, 18, 47]. M. bovis isolates from cattle with drug resistance towards either RIF or INH or towards both drugs have already been documented [23, 29]. Drug susceptibility surveillance of M. bovis from animals may therefore contribute to preventing the transmission of multidrug-resistant strains to humans and to controlling possible outbreaks.

Several techniques for testing mycobacterial drug susceptibility are available for M. tuberculosis and include the conventional assays [48] and the more rapid colorimetric [49] and molecular methods [50]. The WHO has recently recommended the use of colorimetric assays, which are highly sensitive, specific, rapid and inexpensive methods employing specific reagents to produce a change in colour [51].

Among these, REMA, an indirect method based on the reduction of the coloured dye resazurin added to liquid culture medium on a microtitre plate after exposure of mycobacterial strains to antituberculosis drugs in vitro, was successfully tested on human isolates of M. tuberculosis [35, 36, 39, 40]. The REMA plate method proposed by Palomino and colleagues [35] has recently been simplified by Marianelli and colleagues by testing only two concentrations per drug per isolate, the R and S cut-off values, the d-REMA assay [27]. Here, we used both d-REMA and the agar proportion method to assess the bovine isolates for drug susceptibility. We found agreement between the results from the two methods. M. caprae isolates were sensitive to all test drugs and M. bovis isolates were all PZA resistant, as expected. In addition, one M. bovis isolate showed high-level resistance to STR (MIC > 500.0 μg/ml). To our knowledge, this is the first study describing an STR-resistant M. bovis isolate of animal origin. We further investigated the STR resistance here observed by sequencing the most common STR target genes, namely the rrs, rpsL and gidB genes. We found a non-synonymous mutation AAG → AGG at codon 43 (K43R) in the rpsL gene.

The K43R substitution in the rpsL gene is the single most frequent mutation associated with high-level STR resistance in M. tuberculosis [42, 52]. It may explain why our M. bovis isolate still grew at the highest STR concentration tested (500.0 μg/ml), preventing us from determining the STR MIC. Although the most common target genes associated with resistance to STR have been analysed, we cannot exclude, however, that other mechanisms may be involved. The isolation from human clinical cases of highly STR-resistant M. tuberculosis strains carrying the K43R substitution is largely documented in the literature [53,54,55]. The isolation of STR monoresistant [15] and multidrug-resistant M. bovis isolates from humans [16,17,18,19] is also described. Drug- and multidrug-resistant tuberculosis is one of the major threats to human medicine. It leads to treatment failures and, in the worst cases, to untreatable infections that cause death.

A recent 15-year laboratory-based surveillance programme conducted in Mexico City on mycobacterial isolates from human clinical samples showed a rising trend of M. bovis isolates which caused a higher proportion of pulmonary tuberculosis than previously observed in that area [15]. Additionally, the authors described an increasing rate of primary STR monoresistance in M. bovis isolates from humans over time, perhaps as a result of STR usage in cattle [15].

Our results, coupled with the literature, suggest that overuse and misuse of antibiotics in cattle from bTB endemic areas may lead to the development of drug-resistant M. bovis strains which, consequently, put humans at risk for primary drug resistant zTB. More effective strategies to reduce antibiotic use in farm animals should therefore be implemented urgently.

Conclusions

We describe, for the first time, the detection of high-level STR resistance in M. bovis from animal origin. Our results suggest that antitubercular drug susceptibility testing of M. bovis isolates from animals should be performed in settings where bTB is endemic in order to estimate the magnitude of the risk of drug-resistant tuberculosis transmission to humans.

Abbreviations

bTB:

bovine tuberculosis

CFU:

Colony forming units

CLSI:

Clinical and Laboratory Standards Institute

d-REMA:

dichotomous resazurin microtitre assay

EMB:

Ethambutol

FAO:

Food and Agriculture Organization

INH:

Isoniazid

KAN:

Kanamycin

MIC:

Minimal inhibitory concentration

NCBI:

National Center for Biotechnology Information

OADC:

Oleic acid-albumin-dextrose-catalase

OIE:

World Organization for Animal Health

PZA:

Pyrazinamide

REMA:

Resazurin microtitre assay

RIF:

Rifampin

STR:

Streptomycin

WHO:

World Health Organization

zTB:

zoonotic tuberculosis

References

  1. World Organization for Animal Health (OIE). Bovine Tuberculosis. http://www.oie.int/fileadmin/Home/eng/Media_Center/docs/pdf/Disease_cards/BOVINE-TB-EN.pdf. Accessed 10 Jan 2018.

  2. Thoen CO, LoBue PA, de Kantor I. Why has zoonotic tuberculosis not received much attention? Int J Tuberc Lung Dis. 2010;14:1073–4.

    PubMed  Google Scholar 

  3. Müller B, Dürr S, Alonso S, Hattendorf J, Laisse CJ, Parsons SD, et al. Zoonotic Mycobacterium bovis-induced tuberculosis in humans. Emerg Infect Dis. 2013;19:899–908.

    Article  Google Scholar 

  4. Pérez-Lago L, Navarro Y, García-de-Viedma D. Current knowledge and pending challenges in zoonosis caused by Mycobacterium bovis: a review. Res Vet Sci. 2014;97 Suppl:S94–S100.

    Article  Google Scholar 

  5. Olea-Popelka F, Muwonge A, Perera A, Dean AS, Mumford E, Erlacher-Vindel E, et al. Zoonotic tuberculosis in human beings caused by Mycobacterium bovis - a call for action. Lancet Infect Dis. 2017;17:e21–5.

    Article  Google Scholar 

  6. Cosivi O, Grange JM, Daborn CJ, Raviglione MC, Fujikura T, Cousins D, et al. Zoonotic tuberculosis due to Mycobacterium bovis in developing countries. Emerg Infect Dis. 1998;4:59–70.

    Article  CAS  Google Scholar 

  7. Ayele WY, Neill SD, Zinsstag J, Weiss MG, Pavlik I. Bovine tuberculosis: an old disease but a new threat to Africa. Int J Tuberc Lung Dis. 2004;8:924–37.

    CAS  PubMed  Google Scholar 

  8. Michel AL, Geoghegan C, Hlokwe T, Raseleka K, Getz WM, Marcotty T. Longevity of Mycobacterium bovis in raw and traditional souring milk as a function of storage temperature and dose. PLoS One. 2015. https://doi.org/10.1371/journal.pone.0129926.

    Article  Google Scholar 

  9. O’Reilly LM, Daborn CJ. The epidemiology of Mycobacterium bovis infections in animals and man: a review. Tuber Lung Dis. 1995;76 Suppl 1:1–46.

    Article  Google Scholar 

  10. Patané JS, Martins J, Beatriz Castelão A, Nishibe C, Montera L, Bigi F, et al. Patterns and processes of Mycobacterium bovis evolution revealed by phylogenomic analyses. Genome Biol Evol. 2017. https://doi.org/10.1093/gbe/evx022.

    Article  Google Scholar 

  11. Scorpio A, Zhang Y. Mutations in pncA, a gene encoding pyrazinamidase/nicotinamidase, cause resistance to the antituberculous drug pyrazinamide in tubercle bacillus. Nat Med. 1996;2:662–7.

    Article  CAS  Google Scholar 

  12. Allix-Béguec C, Fauville-Dufaux M, Stoffels K, Ommeslag D, Walravens K, Saegerman C, et al. Importance of identifying Mycobacterium bovis as a causative agent of human tuberculosis. Eur Respir J. 2010;35:692–4.

    Article  Google Scholar 

  13. Bilal S, Iqbal M, Murphy P, Power J. Human bovine tuberculosis - remains in the differential. J Med Microbiol. 2010;59:1379–82.

    Article  Google Scholar 

  14. McLaughlin AM, Gibbons N, Fitzgibbon M, Power JT, Foley SC, Hayes JP, et al. Primary isoniazid resistance in Mycobacterium bovis disease: a prospect of concern. Am J Respir Crit Care Med. 2012;186:110–1.

    Article  Google Scholar 

  15. Bobadilla-del Valle M, Torres-González P, Cervera-Hernández ME, Martínez-Gamboa A, Crabtree-Ramirez B, Chávez-Mazari B, et al. Trends of Mycobacterium bovis isolation and first-line anti-tuberculosis drug susceptibility profile: a fifteen-year laboratory-based surveillance. PLoS Negl Trop Dis. 2015. https://doi.org/10.1371/journal.pntd.0004124.

    Article  Google Scholar 

  16. Guerrero A, Cobo J, Fortún J, Navas E, Quereda C, Asensio A, et al. Nosocomial transmission of Mycobacterium bovis resistant to 11 drugs in people with advanced HIV-1 infection. Lancet. 1997;350:1738–42.

    Article  CAS  Google Scholar 

  17. Long R, Nobert E, Chomyc S, van Embden J, McNamee C, Duran RR, et al. Transcontinental spread of multidrug-resistant Mycobacterium bovis. Am J Respir Crit Care Med. 1999;159:2014–7.

    Article  CAS  Google Scholar 

  18. Rivero A, Márquez M, Santos J, Pinedo A, Sánchez MA, Esteve A, et al. High rate of tuberculosis reinfection during a nosocomial outbreak of multidrug-resistant tuberculosis caused by Mycobacterium bovis strain B. Clin Infect Dis. 2001;32:159–61.

    Article  CAS  Google Scholar 

  19. Etchechoury I, Valencia GE, Morcillo N, Sequeira MD, Imperiale B, López M, et al. Molecular typing of Mycobacterium bovis isolates in Argentina: first description of a person-to-person transmission case. Zoonoses Public Health. 2010;57:375–81.

    Article  CAS  Google Scholar 

  20. Kubica T, Rüsch-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.

    Article  Google Scholar 

  21. Prodinger WM, Indra A, Koksalan OK, Kilicaslan Z, Richter E. Mycobacterium caprae infection in humans. Expert Rev Anti-Infect Ther. 2014;12:1501–13.

    Article  CAS  Google Scholar 

  22. Prodinger WM, Eigentler A, Allerberger F, Schönbauer M, Glawischnig W. Infection of red deer, cattle, and humans with Mycobacterium bovis subsp. caprae in western Austria. J Clin Microbiol. 2002;40:2270–2.

    Article  Google Scholar 

  23. Sechi LA, Zanetti S, Sanguinetti M, Molicotti P, Romano L, Leori G, et al. Molecular basis of rifampin and isoniazid resistance in Mycobacterium bovis strains isolated in Sardinia, Italy. Antimicrob Agents Chemother. 2001;45:1645–8.

    Article  CAS  Google Scholar 

  24. Parreiras PM, Lobato FC, Alencar AP, Figueiredo T, Gomes HM, Boéchat N, et al. Drug susceptibility of Brazilian strains of Mycobacterium bovis using traditional and molecular techniques. Mem Inst Oswaldo Cruz. 2004;99:749–52.

    Article  CAS  Google Scholar 

  25. Daly M, Diegel KL, Fitzgerald SD, Schooley A, Berry DE, Kaneene JB. Patterns of antimicrobial susceptibility in Michigan wildlife and bovine isolates of Mycobacterium bovis. J Vet Diagn Investig. 2006;18:401–4.

    Article  Google Scholar 

  26. Romero B, Aranaz A, Bezos J, Alvarez J, de Juan L, Tariq Javed M, et al. Drug susceptibility of Spanish Mycobacterium tuberculosis complex isolates from animals. Tuberculosis. 2007;87:565–71.

    Article  CAS  Google Scholar 

  27. Marianelli C, Armas F, Boniotti MB, Mazzone P, Pacciarini ML, Di Marco Lo Presti V. Multiple drug-susceptibility screening in Mycobacterium bovis: new nucleotide polymorphisms in the embB gene among ethambutol susceptible strains. Int J Infect Dis. 2015;33:39–44.

    Article  CAS  Google Scholar 

  28. Krajewska-Wedzina M, Zabost A, Augustynowicz-Kopec E, Weiner M, Szulowski K. Evaluation of susceptibility to antimycobacterial drugs in Mycobacterium tuberculosis complex strains isolated from cattle in Poland. J Vet Res. 2017;61:23–6.

    Article  CAS  Google Scholar 

  29. Franco MMJ, Ribeiro MG, Pavan FR, Miyata M, Heinemann MB, de Souza Filho AF, et al. Genotyping and rifampicin and isoniazid resistance in Mycobacterium bovis strains isolated from the lymph nodes of slaughtered cattle. Tuberculosis. 2017;104:30–7.

    Article  CAS  Google Scholar 

  30. Ben Kahla I, Boschiroli ML, Souissi F, Cherif N, Benzarti M, Boukadida J, et al. Isolation and molecular characterisation of Mycobacterium bovis from raw milk in Tunisia. Afr Health Sci. 2011;11:S1–5.

    Google Scholar 

  31. Ghariani A, Jaouadi T, Smaoui S, Mehiri E, Marouane C, Kammoun S, et al. Diagnosis of lymph node tuberculosis using the GeneXpert MTB/RIF in Tunisia. Int J Mycobacteriol. 2015;4:270–5.

    Article  Google Scholar 

  32. Siala M, Smaoui S, Taktak W, Hachicha S, Ghorbel A, Marouane C, et al. First-time detection and identification of the Mycobacterium tuberculosis complex members in extrapulmonary tuberculosis clinical samples in South Tunisia by a single tube tetraplex real-time PCR assay. PLoS Negl Trop Dis. 2017. https://doi.org/10.1371/journal.pntd.0005572.

    Article  Google Scholar 

  33. Fliss M, Meftahi N, Dekhil N, Mhenni B, Ferjaoui M, Rammeh S, et al. Epidemiological, clinical, and bacteriological findings among Tunisian patients with tuberculous cervical lymphadenitis. Int J Clin Exp Pathol. 2016;9:9602–11.

    Google Scholar 

  34. Djemal SE, Siala M, Smaoui S, Kammoun S, Marouane C, Bezos J, et al. Genetic diversity assessment of Tunisian Mycobacterium bovis population isolated from cattle. BMC Vet Res. 2017;13:393.

    Article  Google Scholar 

  35. Palomino JC, Martin A, Camacho M, Guerra H, Swings J, Portaels F. Resazurin microtiter assay plate: simple and inexpensive method for detection of drug resistance in Mycobacterium tuberculosis. Antimicrob Agents Chemother. 2002;46:2720–2.

    Article  CAS  Google Scholar 

  36. Martin A, Camacho M, Portaels F, Palomino JC. Resazurin microtiter assay plate testing of Mycobacterium tuberculosis susceptibilities to second-line drugs: rapid, simple, and inexpensive method. Antimicrob Agents Chemother. 2003;47:3616–9.

    Article  CAS  Google Scholar 

  37. Montoro E, Lemus D, Echemendia M, Martin A, Portaels F, Palomino JC. Comparative evaluation of the nitrate reduction assay, the MTT test, and the resazurin microtitre assay for drug susceptibility testing of clinical isolates of Mycobacterium tuberculosis. J Antimicrob Chemother. 2005;55:500–5.

    Article  CAS  Google Scholar 

  38. Jadaun GP, Agarwal C, Sharma H, Ahmed Z, Upadhyay P, Faujdar J, et al. Determination of ethambutol MICs for Mycobacterium tuberculosis and Mycobacterium avium isolates by resazurin microtitre assay. J Antimicrob Chemother. 2007;60:152–5.

    Article  CAS  Google Scholar 

  39. Affolabi D, Sanoussi N, Odoun M, Martin A, Koukpemedji L, Palomino JC, et al. Rapid detection of multidrug-resistant Mycobacterium tuberculosis in Cotonou (Benin) using two low-cost colorimetric methods: resazurin and nitrate reductase assays. J Med Microbiol. 2008;57:1024–7.

    Article  Google Scholar 

  40. Campanerut PA, Ghiraldi LD, Spositto FL, Sato DN, Leite CQ, Hirata MH, et al. Rapid detection of resistance to pyrazinamide in Mycobacterium tuberculosis using the resazurin microtitre assay. J Antimicrob Chemother. 2011;66:1044–6.

    Article  CAS  Google Scholar 

  41. Clinical and Laboratory Standards Institute. Susceptibility testing of mycobacteria, nocardia, and other aerobic actinomycetes; approved standard, 2nd ed document M24-A. Wayne: CLSI; 2011.

    Google Scholar 

  42. Sreevatsan S, Pan X, Stockbauer KE, Williams DL, Kreiswirth BN, Musser JM. Characterization of rpsL and rrs mutations in streptomycin-resistant Mycobacterium tuberculosis isolates from diverse geographic localities. Antimicrob Agents Chemother. 1996;40:1024–6.

    CAS  PubMed  PubMed Central  Google Scholar 

  43. Okamoto S, Tamaru A, Nakajima C, Nishimura K, Tanaka Y, Tokuyama S, et al. Loss of a conserved 7-methylguanosine modification in 16S rRNA confers low-level streptomycin resistance in bacteria. Mol Microbiol. 2007;63:1096–106.

    Article  CAS  Google Scholar 

  44. Feuerriegel S, Köser CU, Niemann S. Phylogenetic polymorphisms in antibiotic resistance genes of the Mycobacterium tuberculosis complex. J Antimicrob Chemother. 2014;69:1205–10.

    Article  CAS  Google Scholar 

  45. Lamine-Khemiri H, Martínez R, García-Jiménez WL, Benítez-Medina JM, Cortés M, Hurtado I, et al. Genotypic characterization by poligotyping and VNTR typing of Mycobacterium bovis and Mycobacterium caprae isolates from cattle of Tunisia. Trop Anim Health Prod. 2014;46:305–11.

    Article  Google Scholar 

  46. Bahri S, Kallel A, Gouia A. Lutte contre la tuberculose bovine: programme et réalization. El Baytari. 1991;3:1–3.

    Google Scholar 

  47. Hughes VM, Skuce R, Doig C, Stevenson K, Sharp JM, Watt B. Analysis of multidrug-resistant Mycobacterium bovis from three clinical samples from Scotland. Int J Tuberc Lung Dis. 2003;7:1191–8.

    CAS  PubMed  Google Scholar 

  48. Canetti G, Fox W, Khomenko A, Mahler HT, Menon NK, Mitchison DA, et al. Advances in techniques of testing mycobacterial drug sensitivity and the use of sensitivity tests in tuberculosis control programmes. Bull World Health Organ. 1969;41:21–43.

    CAS  PubMed  PubMed Central  Google Scholar 

  49. Palomino JC, Martin A, Portaels F. Rapid drug resistance detection in Mycobacterium tuberculosis: a review of colourimetric methods. Clin Microbiol Infect. 2007;13:754–62.

    Article  CAS  Google Scholar 

  50. Laurenzo D, Mousa SA. Mechanisms of drug resistance in Mycobacterium tuberculosis and current status of rapid molecular diagnostic testing. Acta Trop. 2011;119:5–10.

    Article  CAS  Google Scholar 

  51. World Health Organization. Noncommercial culture and drug-susceptibility testing methods for screening patients at risk for multidrug-resistant tuberculosis: policy statement. 2011. http://apps.who.int/iris/bitstream/10665/44601/1/9789241501620_eng.pdf?ua=1&ua=1. Accessed 10 Jan 2018.

  52. Finken M, Kirschner P, Meier A, Wrede A, Böttger EC. Molecular basis of streptomycin resistance in Mycobacterium tuberculosis: alterations of the ribosomal protein S12 gene and point mutations within a functional 16S ribosomal RNA pseudoknot. Mol Microbiol. 1993;9:1239–46.

    Article  CAS  Google Scholar 

  53. Tudó G, Rey E, Borrell S, Alcaide F, Codina G, Coll P, et al. Characterization of mutations in streptomycin-resistant Mycobacterium tuberculosis clinical isolates in the area of Barcelona. J Antimicrob Chemother. 2010;65:2341–6.

    Article  Google Scholar 

  54. Cuevas-Córdoba B, Cuellar-Sánchez A, Pasissi-Crivelli A, Santana-Álvarez CA, Hernández-Illezcas J, Zenteno-Cuevas R. rrs and rpsL mutations in streptomycin-resistant isolates of Mycobacterium tuberculosis from Mexico. J Microbiol Immunol Infect. 2013;46:30–4.

    Article  Google Scholar 

  55. Sun H, Zhang C, Xiang L, Pi R, Guo Z, Zheng C, et al. Characterization of mutations in streptomycin-resistant Mycobacterium tuberculosis isolates in Sichuan, China and the association between Beijing-lineage and dual-mutation in gidB. Tuberculosis (Edinb). 2016;96:102–6.

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We are grateful to Umberto Agrimi (Istituto Superiore di Sanità, Rome, Italy) for covering the publication fees.

Funding

This work was entirely financed by ISS, Italy. Travel and accommodation expenses for the training of the PhD student Saif Eddine Djemal at the ISS laboratories were covered by the University of Sfax, Tunisia. The authors received no specific funds for this work.

Availability of data and materials

All data generated or analysed during this study are included in this published article. The raw data is available from the corresponding author on request.

Author information

Authors and Affiliations

  1. Department of Life Sciences, Research Laboratory of Environmental Toxicology-Microbiology and Health (LR17ES06), Faculty of Sciences, University of Sfax, Sfax, Tunisia

    Saif Eddine Djemal, Mariam Siala & Radhouane Gdoura

  2. Department of Food Safety, Nutrition and Veterinary Public Health, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161, Rome, Italy

    Cristina Camperio, Federica Armas & Cinzia Marianelli

  3. Department of Biology, Preparatory Institute for Engineering Studies, University of Sfax, Sfax, Tunisia

    Mariam Siala & Feriele Messadi-Akrout

  4. Department of Microbiology, Regional Hygiene Care Mycobacteriology Laboratory, Hedi-Chaker University Hospital, Sfax, Tunisia

    Salma Smaoui & Feriele Messadi-Akrout

  5. Department of Biology B, Faculty of Pharmacy, University of Monastir, Monastir, Tunisia

    Salma Smaoui

  6. Department of Microbiology, National Reference Laboratory of Mycobacteria, Research Unit (UR12SP18), A. Mami University Hospital of Pneumology, Ariana, Tunisia

    Salma Smaoui

Authors
  1. Saif Eddine Djemal
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Cristina Camperio
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Federica Armas
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Mariam Siala
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Salma Smaoui
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Feriele Messadi-Akrout
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Radhouane Gdoura
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Cinzia Marianelli
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

SED was involved in performing d-REMA tests and supplied M. bovis animal isolates. CC and FA were both involved in the molecular gene characterization and supplied M. bovis control strains. MS, SS, FM-A, RG were involved in the conceptualization of the study and supplied M. bovis animal isolates. CM was involved in the conceptualization of the study; she also designed the study, performed drug susceptibility tests (d-REMA tests and agar proportion method) and molecular gene characterization, supplied M. bovis control strains, analysed data, wrote the manuscript, funded and supervised the entire research study. All authors read and approved the final manuscript.

Corresponding author

Correspondence to Cinzia Marianelli.

Ethics declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Djemal, S.E., Camperio, C., Armas, F. et al. Detection of a streptomycin-resistant Mycobacterium bovis strain through antitubercular drug susceptibility testing of Tunisian Mycobacterium tuberculosis complex isolates from cattle. BMC Vet Res 14, 296 (2018). https://doi.org/10.1186/s12917-018-1623-9

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1186/s12917-018-1623-9

Keywords

BMC Veterinary Research

ISSN: 1746-6148

Contact us