INT J TUBERC LUNG DIS 14(11):14361441 2010 The Union

Concomitant increases in spectrum and level of drug resistance in Mycobacterium tuberculosis isolates

Z. Sun,* J. Zhang,* H. Song, X. Zhang,* Y. Li,* M. Tian,* Y. Liu,* Y. Zhao, C. Li*

* Department of Bacteriology and Immunology, Beijing Tuberculosis & Thoracic Tumour Research Institute, Beijing, Department of Functional Genomics, Qingdao Institute of Bioenergy and Bioprocess Technology, China Academy of Science, Qingdao, National Reference Laboratory for Tuberculosis, Beijing Tuberculosis & Thoracic Tumour Research Institute, Beijing, China

Correspondence to: C Li, Department of Bacteriology and Immunology, Beijing Tuberculosis & Thoracic Tumour Re-search Institute, Tongzhou District, Beijing 101149, China. Tel: (+86) 10 6954 5354. Fax: (+86) 10 6954 6819. e-mail: lichuanyou6688@hotmail.comArticle submitted 17 June 2009. Final version accepted 16 April 2010.

O B J E C T I V E : To determine the drug resistance spectrum and resistance levels of extensively drug-resistant (XDR-) and multidrug-resistant tuberculosis (MDR-TB) and TB resistant to either rifampicin (RMP, R) or isoniazid (INH, H; R/H-DR). D E S I G N : Of 142 drug-resistant clinical isolates exam-ined, 13 were XDR-TB, 66 were MDR-TB and 63 were R/H-DR. The drug resistance spectrum was tested by the absolute two-concentration method. Minimum inhibitory concentrations (MICs) were determined for the strains by agar dilution method on Lwenstein-Jensen slants.R E S U LT S : The drug resistance spectrum of XDR-TB, MDR-TB and R/H-DR TB isolates ranged from 4 to 9, 2 to 6 and 1 to 5 drugs, respectively. Over half of all XDR-TB (53.8%), MDR-TB (66.7%) and R/H-DR (54.0%)

isolates were resistant to two other anti-tuberculosis drugs; 38.5% of XDR-TB, 24.2% of MDR-TB and 28.6% of R/H-DR TB isolates were resistant to 3 ad-ditional anti-tuberculosis drugs in addition to those origi-nally defi ned, demonstrating that the MIC values and the proportions of strains with higher MICs followed a trend of XDR-TB > MDR-TB > R/H-DR for INH, RMP, ofl oxacin and ethambutol.C O N C L U S I O N : XDR-TB, MDR-TB and R/H-DR TB isolates exhibited both increasingly broader resistance spectra and a higher percentage of strains with high MICs to more frequently resistant drugs, which might be related to patterns of TB chemotherapy.K E Y W O R D S : M. tuberculosis; drug resistance; resis-tance spectrum

DRUG-RESISTANT TUBERCULOSIS (TB) is a seri-ous public health problem. During the 1990s, multi-drug-resistant TB (MDR-TB, defi ned as resistance to at least isoniazid [INH] and rifampicin [RMP]) emerged as a threat to TB control worldwide.1 Some MDR-TB patients were treated with second-line drugs; an unfortunate consequence of treating MDR-TB with second-line drugs, however, is the inevitable emergence of further drug resistance. If the factors that produce MDR-TB remain constant, then MDR-TB becomes extensively drug-resistant TB (XDR-TB).

XDR-TB is defi ned as TB caused by an MDR strain of Mycobacterium tuberculosis that is also re-sistant to a fl uoroquinolone and to at least one of the three injectable second-line drugs, kanamycin, ami-kacin (AMK) or capreomycin.2,3 XDR-TB was fi rst de-scribed in early 2006, following a joint survey by the United States Centers for Disease Control and Pre-vention, the World Health Organization (WHO) and the Supranational Reference Laboratory Network, and confi rmed by the WHO Global Task Force on XDR-TB.4 To date, XDR-TB has been reported in

more than 45 countries.5 XDR-TB is diffi cult and expensive to treat. An extremely high mortality rate (98%) was reported in patients with human immuno-defi ciency virus (HIV) co-infection in South Africa, and a 33% mortality rate was reported in those with-out HIV co-infection.69

Studies of the development of the XDR strain F15/LAM4/KZN have shown that MDR-TB isolates with resistance to other drugs were detected from 1994 onwards; the fi rst XDR-TB isolate was discovered in 2001.10 It is therefore very important to understand the transformation of the spectrum of resistance and levels of resistance of MDR-TB as it develops into XDR-TB. The aim of the present study was to ana-lyse 13 XDR-TB strains, 66 MDR-TB strains and 63 strains resistant to either RMP or INH (referred to as R/H-DR) for their resistance spectrum and level of resistance against nine currently used anti-tuberculosis drugs: INH, RMP, AMK, ofl oxacin (OFX), strepto-mycin (SM), ethambutol (EMB), pyrazinamide (PZA), ethionamide (ETH) and para-aminosalicylic acid (PAS). The results showed that over half of all XDR-TB,

S U M M A R Y

DST on polyresistant TB 1437

MDR-TB and R/H-DR isolates were resistant to 2 other anti-tuberculosis drugs in addition to their defi -nitions, and that the MIC values and the percentage of strains with higher MICs among resistant strains followed a trend of XDR-TB > MDR-TB > R/H-DR for INH, RMP, OFX and EMB.

MATERIALS AND METHODS

Anti-tuberculosis drugsINH, RMP, AMK, OFX, SM, EMB, PZA and ETH and PAS were purchased from Sigma-Aldrich (Beijing, China) for drug susceptibility testing (DST). The drugs were dissolved to 100-fold concentrated stock solu-tions according to the manufacturers instructions.

StrainsM. tuberculosis H37Rv (American Type Culture Col-lection 27294) was obtained from the National Myco bacterium Reference Laboratory of China. Thir-teen XDR-TB strains, 194 MDR-TB strains and 337 R/H-DR strains were obtained from a collection of 1926 clinical strains from HIV-negative TB patients in the Beijing Chest Hospital from January 2002 to De-cember 2005, as reported elsewhere.11 For this study, 66 MDR-TB strains and 63 R/H-DR strains were ran-domly chosen from the collection. Of the 66 MDR-TB strains, 32 were tested for both INH and RMP, and 42 (21 R-DR and 21 H-DR) of the 63 R/H-DR strains were analysed against either INH or RMP (Appen-dix).* The other 34 MDR-TB strains and 21 R/H-DR strains were included to obtain a suffi cient number of strains resistant to OFX, PZA or SM to test their MICs. The MIC of EMB was tested in 46/66 MDR-TB strains and 37/63 R/H-DR strains. The MICs of the different drugs of the XDR-TB isolates have been reported elsewhere.11

As the clinical isolates had different speeds of growth and showed a slightly different appearance on Lwenstein-Jensen (LJ) slants, only those isolates with nearly similar growth status were selected to avoid warp in the fi nal readings. The isolates were inoculated onto LJ slants, and single colonies were inoculated onto Middlebrook 7H9 plus 10% OADC enrichment (oleic acid, albumin, dextrose, catalase; Difco, De-troit, MI, USA) for DST by the absolute concentra-tion method. Clinical isolates found to be resistant by this method to INH, RMP, EMB, PZA, SM and OFX underwent further testing to determine the MICs to these drugs.

In vitro drug susceptibility testing by the absolute concentration method Drug susceptibility was determined using the abso-lute two-concentration method on LJ slants using the

M. tuberculosis H37Rv strain as control.12,13 Briefl y, 100 l of bacterial suspension (~102 mg/ml) was in-oculated onto drug-containing and drug-free LJ slants and incubated at 37C for 4 weeks. Final readings were reported in comparison with drug-free slants.

MIC determination by the agar dilution method and statisticsThe MICs of the nine anti-tuberculosis drugs tested were determined by the agar dilution method on LJ slants.13,14 To determine the MICs of these drugs, a standard bacterial suspension equivalent in turbidity to No. 1 McFarland standard was prepared and di-luted 100-fold. One hundred microlitres of this dilu-tion was inoculated. Final readings were taken after 4 weeks of incubation at 37C. The MIC of a drug was reported as the minimum concentration resulting in no visible growth on agar medium.

SPSS package for Windows Comprehensive Statis-tical Software (Statistical Package for the Social Sci-ences, Chicago, IL, USA) was used for MIC analysis, and the box and whisker plot was used to demon-strate their distribution.

RESULTS

Resistance spectra of three types of drug-resistant clinical isolatesTo determine the resistance spectra of XDR-TB, MDR-TB and R/H-DR M. tuberculosis to anti-tuberculosis drugs, 13 XDR-TB isolates and 66 MDR-TB and 63 R/H-DR clinical isolates were analysed for their re-sistance to nine anti-tuberculosis drugs by the abso-lute two-concentration method. High and low MICs, in accordance with high and low drug resistance, re-spectively (Table 1), were slightly different from the concentrations reported elsewhere.1517 A strain was resistant to an anti-tuberculosis drug if it grew on LJ containing the standard concentrations of the drug (Table 1). The results showed that R/H-DR, MDR-TB and XDR-TB strains had very broad drug resis-tance spectra. R/H-DR TB strains were resistant to as many as fi ve drugs, MDR-TB strains were resistant to six drugs, and XDR-TB could be resistant to all nine drugs tested (Figure 1).

The majority of the strains tested were resistant to other drugs in addition to those included in the original defi nition (Figure 1);2,3 88.9% of R/H-DR TB strains were resistant to drugs other than INH/RMP; 92.4% of MDR-TB isolates were resistant to additional drugs other than INH and RMP; and 92.3% of XDR-TB strains were resistant to more than the four drugs in the defi nition. More than half of the R/H-DR (54.0%), MDR-TB (66.7%) and XDR-TB (53.8%) isolates were resistant to at least two other anti-tuberculosis drugs, and 38.5% of the XDR-TB, 24.2% of the MDR-TB and 28.6% of the R/H-DR TB isolates were resistant to an additional

* The Appendix is available in the online version of this article at http://www.ingentaconnect.com/content/iuatld/ijtld/2010/00000014/ 00000011/art00013

1438 The International Journal of Tuberculosis and Lung Disease

three or more anti-tuberculosis drugs in addition to those defi ned.2,3

Resistance levels of three types of drug-resistant clinical isolatesTo understand the degree of resistance of the XDR-TB, MDR-TB and R/H-DR TB isolates to anti-t uberculosis drugs, the MICs of six anti-tuberculosis drugs (INH, RMP, OFX, SM, EMB and PZA) for the 13 XDR-TB strains and the randomly selected MDR-TB and R/H-DR M. tuberculosis isolates were deter-mined by the agar dilution method on LJ slants.

The MICs obtained for the XDR-TB, MDR-TB and R/H-DR strains were plotted in box and whisker plots (Figure 2). Overall, XDR-TB strains had the highest MIC value for almost every drug; only the MICs of OFX for MDR-TB and MICs of PZA for MDR-TB and R/H-DR reached similar levels. The highest MIC for RMP was 4000 g/ml in some XDR-TB strains, and that of INH reached 160 g/ml for the XDR-TB strains. XDR-TB isolates also had very

high MIC values for second-line anti-tuberculosis drugs (see Online Appendix).

The XDR-TB strains exhibited higher drug resis-tance than MDR-TB strains, while MDR-TB strains showed higher MICs than the R/H-DR isolates. For INH, RMP, OFX and EMB, the boxes and median MIC values for XDR-TB were higher than for the MDR-TB strains, while they were lower in the R/H-DR strains (Figure 2). Although only the differences in the MICs of RMP and OFX XDR-TB, MDR-TB and R/H-DR TB were statistically signifi cant (Table 2), a gradual increase in MIC values and in the percentage of strains with higher MICs from R/H-DR strains to MDR-TB to XDR-TB strains was observed (Figure 2). For INH, RMP, OFX and EMB, the MICs for XDR-TB were signifi cantly higher than for the MDR-TB and R/H-DR strains, except for INH (P = 0.076, XDR-TB vs. MDR-TB; Table 2). In other words, drug resistance levels conformed to the following trend for INH, RMP, OFX and EMB: XDR-TB > MDR-TB > R/H-DR.

Table 1 Drug concentrations used for determining low or highly resistant concentrations by the absolute two-concentration method on Lwenstein-Jensen slants (g/ml)

Rifampicin Isoniazid O oxacin Streptomycin Ethambutol Pyrazinamide

Low resistant concentration 50 1 2 10 2 50Highly resistant concentration 250 10 10 100 10 250

Figure 1 Drug resistance spectrum of three types of drug-resistant clinical isolates (XDR, MDR and R/H-DR) tested by the absolute con-centration method. A total of 13 XDR-TB strains (), 66 MDR-TB strains () and 63 R/H-DR strains () were tested. XDR-TB = extensively drug-resistant tuberculosis; MDR-TB = multidrug-resistant tuberculosis; R/H-DR = resistant to either rifampicin or isoniazid.

Figure 2 MIC values of drugs for XDR-TB, MDR-TB and R/H-DR strains plotted in box and whisker plots. The error bars represent the distribution of the top 25% and the lowest 25% of data points. MICs were determined by the agar dilution method. MIC = minimum inhibitory concentration; XDR-TB = extensively drug-resistant tuberculosis; MDR-TB = multidrug-resistant tuberculosis; R/H-DR = r esistance to either rifampicin or isoniazid.

DST on polyresistant TB 1439

DISCUSSION

We previously reported that some XDR M. tubercu-losis strains were resistant to certain anti-tuberculosis drugs, with MICs as high as 10100 times the cut-off value.11 In accordance with the defi nition of drug r esistance, R/H-DR, MDR-TB and XDR-TB strains are resistant to increasingly more anti-tuberculosis drugs.2,3 This study details the spectra and levels of resistance of XDR-TB, MDR-TB and R/H-DR TB clinical isolates. These three types of drug-resistant clinical isolates exhibited both increasingly broader resistance spectra and a higher percentage of strains with high MICs to more frequently resistant drugs. The WHO guidelines for the programmatic manage-ment of MDR-TB should be more strictly obeyed.

The results of this study showed that as TB strains became resistant to more and more anti-tuberculosis drugs (R/H-DR < MDR-TB < XDR-TB), the per-centage of strains with high levels of resistance to INH, RMP, OFX and EMB, but not to SM and PZA, also increased. Although indirect evidence appears to suggest a direct link between a broader resistance spectrum and higher resistance levels to certain drugs, this has not been fi rmly established.

It was reported that EMB resistance was related to INH and RMP resistance,18 and that the number of EMB-resistant isolates increased stepwise as these isolates became resistant to an increasing number of other fi rst-line anti-tuberculosis drugs, such as INH and RMP.19 Transfer of the mutation at codon 306 of embB to clinical M. tuberculosis strains altered sus-ceptibility to EMB, INH and RMP. This is the direct evidence to suggest that a single mutation may affect drug susceptibility against multiple fi rst-line anti-t uberculosis drugs.20 INH and RMP are the drugs of choice for newly identifi ed TB patients and are used widely, as recommended by the WHO.21,22 As DST is normally performed well after the initial course of treatment, MDR- and XDR-TB patients may also at fi rst receive treatment including these two drugs. The overuse and sometimes misuse of INH and RMP has exacerbated the situation, which may explain their higher resistance levels in MDR- and XDR-TB iso-lates. Fluoroquinolones are strongly recommended by the WHO for treating MDR-TB patients, and are at times used for some patients for whom the regular regimens are unsustainable.23 Unfortunately, treating MDR-TB with second-line drugs inevitably leads to the emergence of further drug resistance.

This study indicates that drug resistance spectrum and resistance levels simultaneously increased in M. tu berculosis clinical isolates. New anti-tuberculosis drugs should be used with caution as M. tuberculosis drug resistance evolves continuously from R/H-DR to MDR-TB and even to XDR-TB during treatment. Although in Eastern European countries one in fi ve cases would have MDR-TB, signalling that new drugs are urgently needed, countries with the highest TB burden and percentage of MDR-TB cases will likely not respond to the treatment they currently receive for reasons other than high burden.5,22 Recent re-ports have shown that there is a high likelihood of generating MDR- and XDR-TB even under adequate National Tuberculosis Control Programme (NTP) im-plementation.24 It was reported that in specifi c cir-cumstances, the WHO Category I regimen can am-plify resistance to RMP (in initial INH-resistant cases) or EMB + PZA (in initial MDR-TB cases), and that the WHO Category II regimen can also amplify resistance to EMB or SM. Moreover, the subsequent addition of the only second-line drugs available in many countries (fl uoroquinolones and/or injectables) can worsen the situation and generate XDR-TB.24

The evolution of the increasing resistance spec-trum of MDR-TB to XDR-TB requires more detailed analysis, for example whether other factors that have been demonstrated to affect the level of resistance of M. tuberculosis to anti-tuberculosis drugs are in-volved in this evolution. It is well known that cer-tain specifi c gene mutations result in the resistance of an M. tuberculosis strain to an anti-tuberculosis drug and that two or more resistance-related mutations would result in higher MICs.10,2527 In addition, non-mutation factors, such as effl ux pump and some other intrinsic drug resistance mechanisms, could also con-tribute signifi cantly to anti-tuberculosis drug resis-tance.11,26 Continuous use of anti-tuberculosis drugs in R/H-DR, MDR-TB or XDR-TB patients would modify the physical or biochemical properties of the strains and thus indirectly affect drug susceptibility.

In this study, the drugs selected for DST were widely used in China, especially second-line drugs such as AMK as representative of the aminoglycosides and OFX as representative of the fl uoroquinolones. As the WHO has not provided standards for DST of these second-line drugs, and the existing tests are less re-producible than tests for INH and RMP,28 standard-ised DST methods are required against second-line drugs. DST results were most likely to be affected by

Table 2 P values of the MICs of isoniazid, rifampicin, o oxacin, pyrazinamide, streptomycin and ethambutol among XDR-TB, MDR-TB and R/H-DR strains

Isoniazid Rifampicin O oxacin Pyrazinamide Streptomycin Ethambutol

XDR-TB vs. MDR-TB 0.076 0.001 0.009 0.382 0.433 0.009XDR-TB vs. R/H-DR 0.034 0.000 0.000 0.501 0.118 0.001MDR-TB vs. R/H-DR 0.256 0.011 0.020 0.587 0.031 0.273

MIC = minimum inhibitory concentration; XDR-TB = extensively drug-resistant tuberculosis; MDR-TB = multidrug-resistant tuberculosis; R/H-DR = resistant to either rifampicin or isoniazid.

1440 The International Journal of Tuberculosis and Lung Disease

bacilli growth status. The growth status of clinical isolates showed wide differences. Only those with al-most the same normal rate of growth were included in this study. Another limitation of the current meth-odology is that it was diffi cult to determine the col-lection time of the tested bacilli. The period of collec-tion was long, lasting from January 2002 to December 2005. This might have affected the resistance charac-teristics as M. tuberculosis drug resistance developed.10 Further DST results on R/H-DR, MDR-TB or XDR-TB M. tuberculosis strains by other laboratories in other regions are needed to confi rm the observed trend of concomitant increase between the drug resistance spectrum and resistance levels in M. tuberculosis clin-ical isolates.

In conclusion, increasingly broader drug resistance spectra and higher percentages of drug-resistant strains with higher MICs were found in R/H-DR, MDR-TB and XDR-TB M. tuberculosis isolates, which argues that further precautions should be taken in the chemo-therapy of drug-resistant M. tuberculosis.

AcknowledgementsThe authors thank Y Chao for his editing and comments on the manuscript. They are grateful for the M. tuberculosis clinical iso-lates from the out-patient department of the Beijing Chest Hospi-tal. This work was supported by the National High-tech R&D Programme (863) of China (2007AA02Z405), the National Natu-ral Science Foundation of China (30901283) and Chinese National Important Special Fund for Control and Therapy of Serious Dis-eases (2008ZX10003-009).

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O B J E C T I F : Dterminer les spectres de rsistance aux mdicaments et les niveaux de rsistance des tuberculo-ses ultrarsistantes (TB-XDR), multirsistantes (TB-MDR) et rsistant soit la rifampicine (R, RMP), soit lisoniazide (H, INH; R/H-DR).S C H M A : On a examin 142 isolats cliniques rsistants aux mdicaments, parmi lesquels 13 isolats TB-XDR, 66 TB-MDR et 63 R/H-DR. Les spectres de rsistance aux mdicaments ont t tudis par la mthode des doubles concentrations absolues. Les concentrations mini males inhibitrices (CMI) ont t dtermines pour les souches intressantes par la mthode de dilution en agar sur les pentes des milieux de Lwenstein-Jensen.R S U LTAT S : Le spectre des rsistances des isolats TB-XDR, TB-MDR et R/H-DR va respectivement de 4 9, de 2 6 et de 1 5 mdicaments. Il est tonnant de noter que plus de la moiti de lensemble des TB-XDR

(53,8%), TB-MDR (66,7%) et R/H-DR (54,0%) sont rsistants deux mdicaments antituberculeux suppl-mentaires et que 38,5% des isolats TB-XDR, 24,2% des TB-MDR et 26,8% des R/H-DR taient rsistants trois ou plus que trois mdicaments antituberculeux supplmentaires par rapport la dfi nition originale. Cette tude a dmontr que les valeurs de CMI et la pro-portion de souches avec des valeurs plus leves suivent une tendance o TB-XDR est suprieur TB-MDR qui est suprieur R/H-DR pour lINH, la RMP, lofl oxacine et lthambutol. C O N C L U S I O N : Les isolats TB-XDR, TB-MDR et R/H-DR dmontrent la fois des spectres dextension crois-sante de la rsistance et des pourcentages plus levs de souches CMI leve lgard des mdicaments pour lesquels la rsistance est plus frquente, ce qui pourrait tre li aux types de chimiothrapie antituberculeuse.

R S U M

R E S U M E N

O B J E T I V O : Determinar el espectro de farmacorresisten-cia y los niveles de resistencia de las cepas de Mycobac-terium tuberculosis extremadamente drogorresistentes (TB-XDR), las multidrogorresistentes (TB-MDR) y las re-sistentes a rifampicina (R, RMP) o isoniazida (H, INH; R/H-DR).M T O D O : Se examinaron 142 aislados clnicos con re-sistencia a los medicamentos antituberculosos, de los cuales 13 fueron TB-XDR, 66 TB-MDR y 63 R/H-DR. El espectro de resistencia a los medicamentos se evalu mediante el mtodo de dos concentraciones absolutas. Se determinaron las concentraciones inhibitorias mni-mas (CIM) de las cepas estudiadas con la tcnica de la dilucin en agar en tubos de Lwenstein-Jensen.R E S U LTA D O S : El espectro de farmacorresistencia en las cepas TB-XDR fue de cuatro a nueve medicamentos, de dos a seis medicamentos en las cepas TB-MDR y en las R/H-DR fue de uno a cinco medicamentos antitubercu-losos. Cabe sealar que ms de la mitad de todos los aisla-

dos clnicos TB-XDR (53,8%), los TB-MDR (66,7%) y los R/H-DR (54.0%) presentaron resistencia adicio-nal a dos medicamentos antituberculosos y 38,5% de los aislados TB-XDR, 24,2% de los TB-MDR y 28,6% de los R/H-DR fueron resistentes a tres medicamentos adicionales o ms, con respecto a las defi niciones origi-nales. Se defi nieron las CIM y se observ una tendencia decreciente de la proporcin de cepas con concentracio-nes ms altas, en el siguiente orden: TB-XDR, TB-MDR y luego los R/H-DR, ofl oxacino y etambutol.C O N C L U S I N : Las cepas de M. tuberculosis TB-XDR, TB-MDR y R/H-DR exhibieron respectivamente un es-pectro de resistencia a un nmero creciente de medica-mentos y presentaron adems, en el mismo orden, un porcentaje cada vez mayor de cepas con altas CIM de los antituberculosos cuya resistencia es ms frecuente, lo cual se podra relacionar con los tipos de tratamiento antituberculoso administrado.

DST on poly resistant TB i

APPENDIX

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