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Frequency of rapid growing mycobacteria among tuberculosis suspected patients in Basra-Iraq.

Introduction

Non-tuberculousis mycobacteria (NTM) especially rapid growing mycobacteria (RGM) are environmental opportunistic pathogens and their role in human disease is increasingly recognized. In addition, several studies indicate that humans can be infected by NTM from various environmental sources, especially soil and water [1,2].

Runyon classified these organisms as rapidly growing mycobacteria as they can produce mature colonies on agar plates within 7 days, while slowly growing mycobacteria need several more days to grow [3,4].

Identification of rapidly growing mycobacteria is important for clinical and epidemiological studies because of their spread worldwide [2].

Although a system of national disease surveillance, as exists for Mycobacterium tuberculosis in developed countries, has not been implemented, infections caused by rapidly growing mycobacteria have been increasingly reported in recent years [5-7].

These rapidly growing organisms have been associated with a wide range of clinical syndromes in both immunocompetent and immunocompromised hosts, ranging from mild, such as infections of skin and soft tissue, to more serious disorders, including osteomyelitis, and lymph node, respiratory tract, bloodstream infections and disseminated infection [6,8].

The differences in susceptibility patterns of species and resistance to first line antituberculosis drugs create challenges in the approach to treatment of these organisms [9].

This study is focused on the isolation and identification of rapidly growing mycobacteria from tuberculosis suspected patients, and testing their drug susceptibility because of their emerging importance in both sporadic infection and outbreak settings.

Materials and Methods

Sample collection

A total of 150 sputum samples were obtained from 150 patients admitted to the ACCDR, during a one year period "March 2013 to February 2014"

All sputa were collected in sterile, screw cap containers. The expectorated sputum was taken by asking the patient to cough deeply into the container, followed by immediate screwing off the cap. Samples were transported to the laboratory within two hours and processed immediately or refrigerated at 4[degrees]C as soon as possible [10].

Microbiological examination

The specimens were processed on the same day for microscopy and culture using standard procedures [11]. Smears were stained with the Ziehl Neelsen (ZN) technique. Specimens were inoculated onto Lowenstein Jensen (LJ) medium, after decontamination procedures and concentration, and then incubated at 37[degrees]C. Cultures were examined every day for a week and then once a week for eight weeks. Isolates obtained weekly were confirmed as acid-fast bacilli by ZN staining.

Identification to species level was achieved on the basis of the growth characteristics, including growth in less than 7 days, growth at 37[degrees]C, growth in the presence of NaCl 5%, pigment production, Niacin production, pyrazinamidase, urease, nitrate reduction test, catalase test, heat-stable catalase (pH 7, 68[degrees]C), Tween 80 hydrolysis, growth on MacConkey agar, arylsulfatase test, and colony morphology.

Genetic identification

DNA was extracted by DNA PrepMate-M (Bioneer) according to the manufacture's instructions and DNA was detected on agarose gel electrophoresis [12]. 16S rDNA sequencing was done in Nicem company (USA) and Macrogen company (Korea).

Duplex PCR: RNA polymerase gene (rpoB) primers

The isolates were subjected to identification by using Duplex PCR (two pairs of primers) in order to amplify rpoB gene which is listed in Table 1.

Thermal cycling condition

The PCR method for amplifying the rpoB gene was conducted as reported by Singh et al. [13] (Table 2).

Drug susceptibility tests

The in vitro antimicrobial susceptibility of these RGM was performed according to the proportional method [14]. Five antibiotics were used as rifampicin 1 [micro]g/ml, ethambutol 2 [micro]g/ml, pyrazinamide 0.25 [micro]g/ml, isoniazid 0.2 [micro]g/ml, streptomycin 2 [micro]g/ml.

The results were read after three weeks of incubation at 35[degrees]C. The isolate was recorded as resistant when growth in the critical concentration of the drug is more than 1% the growth of the Middlebrook 7H10 medium without antibiotic. When the case is opposite, the isolate is considered as susceptible. The resistance percent was calculated by the following Equation 1 [15]:

Number of colonies on the drug/Number of colonies on the control * 100 = % resistance (1)

Results

Of one hundred fifty tuberculosis suspected patients, attended the ACCDR who were suffering from upper respiratory tract infections, twenty three isolates were MTB (15.33%) and sixteen (10.66%) were NTM, seven of them (43.75%) were rapid growing mycobacteria, which included 4 (25%) as M. chelonae, 2 (12.5%) M. abscessus and 1 (6.2%) M. smegmatis (Figures 1 and 2) (Table 3).

[FIGURE 1 OMITTED]

[FIGURE 2 OMITTED]

Genetic identification

Amplifying universal 16S rDNA: The extracted DNAs for the isolates were subjected to PCR for amplifying universal 16S rDNA gene. PCR products for the universal 16S rDNA primers gave bands on an agarose gel at the position 1500bp when compared with standard molecular DNA ladder (Figure 3).

[FIGURE 3 OMITTED]

DNA amplification and duplex PCR: The isolates were identified by D-PCR method for differentiating between Mycobacterium tuberculosis (MTB) and NTM based on amplification of rpoB gene sequences. The results showed two bands, 235 bp band for MTB and 136 bp band for NTM (Figure 4).

[FIGURE 4 OMITTED]

Sequencing for Universal 16S rDNA Gene: The isolates have been identified using 16S rDNA sequencing, the results showed that 6 RGM matched with biochemical results, as follows: M. chelonae (n=3), M abscessus (n=2) and M. smegmatis (n=1), while one M. chelonae did not match, and gave M. chitae.

Antibiotic susceptibility: The drug susceptibility results showed that, One M. chelonae isolate was resistant to EMB and one was MDR (RIF and EMB). M. smegmatis isolate was MDR (RIF and PZA), while one isolate of M. abscessus appeared to be resistant to all antibiotics tested. It was from a thirty year male, non smoker, with no previously documented lung disease, and lives in the poor socio-economic district. The rest isolates were sensitive (Table 4).

Discussion

Non-tuberculous mycobacterial infections are becoming increasingly common. Among them, the rapidly growing organisms such as Mycobacterium chelonae, Mycobacterium fortuitum and Mycobacterium abscessus are widespread in nature and in hospital environments [16].

The results showed that the rates of infection in Iraq are considered lower than the rates in the world based on previous studies [17-19].

To the best of our knowledge, this is the first study conducted in Iraq for the isolation and identification of NTM with human infection, a previous study was on environmental sites [1].

The results of the present study showed that, the prevalence of M chelonae was 42.8%, M. abscessus 28.5% and M. smegmatis 14.2% of RGM for all isolates from pulmonary samples. M. chelonae and M abscessus have similar phenotypes and antimicrobial susceptibility profiles [20,21]. The profile of antibiotics of the RGM isolates appeared that, most isolates have the ability to resist one or more antimicrobial drugs (Table 4), One isolate of M. abscessus was resistant to all antimycobaterial drugs and this bacterium is deemed to be one of the most virulent and resistant species of rapidly growing mycobacteria. Many researchers found that, this bacterium is usually resistant to imipenem, ciprofloxacin, cotrimoxazole, linezolid, and doxycycline, but is susceptible only in vitro to amikacin and clarithromycin [22-25].

Depending on sputum smear only for diagnosis is not decisive as positive sputum smear may, in fact, due to an NTM that is then erroneously treated with standard anti-TB medications. As many NTM is resistant to first-line anti-TB medications, most of these cases would be considered failures, and subsequently treated with the second line regimen. On failure of the latter regimen, the patients are reported as chronic cases [26].

The mycobacterial cell wall functions as an efficient protective barrier and limits the entry of drug molecules to their cellular targets [27]. A major porin of M. smegmatis, MspA, forms a tetrameric complex with a single central pore [28]. Deletion of the porins MspA and MspC raised the resistance to [beta]-lactam antibiotics without changing its [beta]-lactamase activity. Hydrophilic fluoroquinolones such as norfloxacin, and chloramphenicol, diffuse through porins in mycobacteria [29].

The cell wall barrier alone is not enough to explain the intrinsic drug resistance of these bacteria. Drug efflux is a drug resistance mechanism contributed to intrinsic or acquired resistance in a wide range of bacteria [30]. M. smegmatis LfrA was the first multidrug efflux pump confirmed in mycobacteria [31]. It produces low-level resistance to fluoroquinolones and other toxic compounds such as ethidium bromide [31,32]. EfpA, Tap, and P55 are the three other major facilitator super family (MFS) pumps found in several mycobacterial species, and of these pumps, Tap and P55 are known to give low-level resistance to aminoglycosides and tetracyclines [33]. Mmr (a small multidrug resistance family (SMR) pump) and DrrAB (an ATP-binding cassette super family (ABC) exporter) were reported in MTB [34]. These exporters produce low-level resistance to certain antimicrobial agents [35]. Physiological adaptations appearing in the host can also lead to antibiotic tolerance [36].

Conclusion

An early identification of MTB and NTM is highly recommended, so that a correct antibiotic treatment can be initiated. The Duplex PCR proved to be the right differential technique. Concurrently, emerging drug resistance is indicated here by the appearance of one M. abscessus isolate resistant to all antimycobacterials pressing for more effective drugs.

Also, it is worthwhile to suggest the implementation of the monophasic--biphasic culture setup of tuberculosis broth and LJ agar for rapid recovery of MTB [1].

References

[1.] Al-Sulami AA, Al-Taee AM, Widaa Q (2012) Isolation and Identification of Mycobacterium avium complex and other non-tuberculosis mycobacteria from drinking water in Basra government, Iraq. East Mediter Health J 18: 274-278.

[2.] Rahideh S, Farnia P, Darbouy M (2014) Isolation and identification of rapidly growing mycobacteria from water and soil by PCR-RFLP method in Robat Karim. J Heal Hug 4: 321-329.

[3.] RUNYON EH (1959) Anonymous mycobacteria in pulmonary disease. Med Clin North Am 43: 273-290.

[4.] Runyon EH (1970) Identification of mycobacterial pathogens utilizing colony characteristics. Am J Clin Pathol 54: 578-586.

[5.] Sungkanuparph S, Sathapatayavongs B, Pracharktam R (2003) Infections with rapidly growing mycobacteria: report of 20 cases. Int J Infect Dis 7: 198-205.

[6.] De Groote MA, Huitt G (2006) Infections due to rapidly growing mycobacteria. Clin Infect Dis 42: 1756-1763.

[7.] Chang CY, Tsay RW, Lin LC, Liu CE (2009) Venous catheter-associated bacteremia caused by rapidly growing mycobacteria at a medical center in central Taiwan. J Microbiol Immunol Infect 42: 343-350.

[8.] Redelman-Sidi G, Sepkowitz KA (2010) Rapidly growing mycobacteria infection in patients with cancer. Clin Infect Dis 51: 422-434.

[9.] Martm-Casabona N, Bahrmand AR, Bennedsen J, Thomsen VO, Curcio M, et al. (2004) Non-tuberculous mycobacteria: patterns of isolation. A multi-country retrospective survey. Int J Tuberc Lung Dis 8: 1186-1193.

[10.] SIREVA: Pan America health organization regional systems for vaccines (1998) Streptococcus pneumoniae and Haemophilus influenzae identification and susceptibility testing. Proceedings of the Institute Nacional de Saluda, Santa Fe de Bogota.

[11.] Koneman EW, Allen SD, Janda WM, Schreckenberger PC, Winn WC (1997) Colour atlas and textbook of diagnostic Microbiology. (5thedn) JB Lippincot Company, pp. 897-906.

[12.] Sambrook J, Russell DW (2001) Molecular cloning--A laboratory manual. (3rdedn) Cold spring harbor laboratory press, Cold Spring Harbor, NY, USA.

[13.] Singh P, Sharma V, Sharma N, Singh D, Kandpal J (2013) Evaluation of DPCR using the RNA polymerase gene (rpoB) primers for rapid differential identification of Mycobacterium tuberculosis complex and Nontuberculous Mycobacteria. Ann Bio Res 4: 45-48.

[14.] Canetti G, Froman S, Grosset J, Hauduroy P, Langerova M, et al. (1963) Mycobacteria: Laboratory Methods for Testing Drug Sensitivity and Resistance. Bull World Health Organ 29: 565-578.

[15.] Vestal AL (1975) Procedures for the isolation and identification of mycobacteria. U.S. Department of Health, Education and Welfare publication no. (CDC) 76-8230: 97-115, Center for Disease Control, Atlanta.

[16.] Sethi NK, Aggarwal PK, Duggal L, Sachar VP (2003) Mycobaterium chelonae infection following laparoscopic inguinal herniorrhaphy. JAPI 51: 81.

[17.] Wallace RJ, Swenson JM, Silcox VA, Good RC, Tschen JA, et al. (1983) Spectrum of Disease Due to Rapidly Growing Mycobacteria. Clin Infect Dis 5: 657-679.

[18.] Jarand J, Levin A, Zhang L, Huitt G, Mitchell JD, et al. (2011) Clinical and Microbiologic Outcomes in Patients Receiving Treatment for Mycobacterium abscessus Pulmonary Disease. Clin Infect Dis 52: 565-571.

[19.] O'Driscoll C, Konjek J, Heym B, Fitzgibbon MM, Plant BJ, et al. (2016) Molecular epidemiology of Mycobacterium abscessus complex isolates in Ireland. J Cyst Fibros 15: 179-185.

[20.] El Helou G, Viola GM, Hachem R, Han XY, Raad II (2013) Rapidly growing mycobacterial bloodstream infections. Lancet Infect Dis 13: 166-174.

[21.] O'Driscoll C, Konjek J, Heym B, Fitzgibbon MM, Plant BJ, et al. (2016) Molecular epidemiology of Mycobacterium abscessus complex isolates in Ireland. J Cyst Fibros 15: 179-185.

[22.] Wallace RJ, Cook JL, Glassroth J, Griffith DE, Olivier KN, et al. (1997) The American Thoracic Society statement: diagnosis and treatment of disease caused by nontuberculous mycobacteria. Am J Respir Crit Care Med 156: S1-S25.

[23.] Griffith DE, Aksamit T, Brown-Elliott BA, Catanzaro A, Daley C, et al. (2007) An official ATS/IDSA statement: diagnosis, treatment, and prevention of nontuberculous mycobacterial diseases. Am J Respir Crit Care Med 175: 367-416.

[24.] Han XY, De I, Jacobson KL (2007) Rapidly growing mycobacteria: clinical and microbiologic studies of 115 cases. Am J Clin Pathol 128: 612-621.

[25.] Esteban J, Martm-de-Hijas NZ, Fernandez AI, Fernandez-Roblas R, Gadea I; Madrid Study Group of Mycobacteria (2008) Epidemiology of infections due to nonpigmented rapidly growing mycobacteria diagnosed in an urban area. Eur J Clin Microbiol Infect Dis 27: 951-957.

[26.] Tabarsi P, Baghaei P, Farnia P, Mansouri N, Chitsaz E, et al. (2009) Nontuberculous mycobacteria among patients who are suspected for multidrug-resistant tuberculosis-need for earlier identification of nontuberculosis mycobacteria. Am J Med Sci 337: 182-184.

[27.] Brennan PJ, Nikaido H (1995) The envelope of mycobacteria. Annu Rev Biochem 64: 29-63.

[28.] Engelhardt H, Heinz C, Niederweis M (2002) A tetrameric porin limits the cell wall permeability of Mycobacterium smegmatis. J Biol Chem 277: 37567-37572.

[29.] Danilchanka O, Pavlenok M, Niederweis M (2008) Role of porins for uptake of antibiotics by Mycobacterium smegmatis. Antimicrob Agents Chemother 52: 3127-3134.

[30.] Li XZ, Nikaido H (2004) Efflux-mediated drug resistance in bacteria. Drugs 64: 159-204.

[31.] Takiff HE, Cimino M, Musso MC, Weisbrod T, Martinez R, et al. (1996) Efflux pump of the proton antiporter family confers low-level fluoroquinolone resistance in Mycobacterium smegmatis. Proc Natl Acad Sci USA 93: 362-366.

[32.] Liu J Takiff HE, Nikaido H (1996) Active efflux of fluoroquinolones in Mycobacterium smegmatis mediated by LfrA, a multidrug efflux pump. J Bacteriol 178: 3791-3795.

[33.] Silva PE, Bigi F, Santangelo MP, Romano MI, Martin, C, et al. (2001) Characterization of P55, a multidrug efflux pump in Mycobacterium bovis and Mycobacterium tuberculosis. Antimicrob. Agents Chemother 45: 800-804.

[34.] Choudhuri BS, Bhakta S, Barik R, Basu J, Kundu M, et al. (2002) Over expression and functional characterization of an ABC (ATP-binding cassette) transporter encoded by the genes drrA and drrB of Mycobacterium tuberculosis. Biochem J 367: 279-285.

[35.] De Rossi E, Branzoni M, Cantoni R, Milano A, Riccardi G, et al. (1998) mmr, a Mycobacterium tuberculosis gene conferring resistance to small cationic dyes and inhibitors. J Bacteriol 180: 6068-6071.

[36.] Nguyen L, Pieters J (2009) Mycobacterial subversion of chemotherapeutic reagents and host defense tactics: challenges in tuberculosis drug development. Annu Rev Pharmacol Toxicol 49: 427-453.

Amin A. Al-Sulami (1), Asaad Al-Taee (2) * and Zainab A. Hasan (1)

(1) College Education for Pure Science, University of Basra, Basra, Iraq

(2) Marine Science Center, University of Basra, Basra, Iraq

* Corresponding author: Asaad Al-Taee, College Education for Pure Science, University of Basrah, Basra, 61001, Iraq, Tel: 009647801405715; E-mail: amraltaee@yahoo.com

Received date: February 28, 2016; Accepted date: April 13, 2016; Published date: April 18, 2016

doi: 10.4172/0974-8369.1000297
Table 1: rpoB gene primers.

Gene        Primer    Primer sequence (5'-3')            Size of
            type                                         product

rpoB gene   Tbc 1F    5'-CGTACGGTCGGCGAGCTGATCCAA-3'     235 bp
            TbcR5 R   5'-CCACCAGTCGGCGCTTGTGGGTCAA-3'
            M5 F      5'-GGAGCGGATGACCACCCAGGACGTC-3'    136 bp
            RM3 R     5'-CAGCGGGTTGTTCTGGTCCATGAAC-3'

Table 2: Program used in PCR amplification for rpoB gene.

Steps                  Temperature    Time     No. of cycles

Initial denaturation   95[degrees]C   5 min    1
Denaturation           94[degrees]C   20 Sec
Annealing              55[degrees]C   20 Sec   40
Extension              72[degrees]C   40 Sec
Final extension        72[degrees]C   5 min    1

TM = Melting temperature, TA = Annealing temperature

Table 3: Biochemical tests of rapid growing mycobacteria.

Tests                      M. chelonae   M. abscessus   M. smegmatis
                           4 isolates    2 isolates     1 isolate

Growth in less than        +             +              +
  7 days
Growth at 37[degrees]C     +             +              +
Photo reactive pigment     -             +              -
Pigment in dark            -             -              +
Growth in presence         +             +              +
  of NaCl 5%
Growth on MacConkey agar   +             -              -
Niacin production          -             -              -
Nitrate reduction          +             -              +
Arylsulfatase (3 days)     +             +              -
Tween 80 hydrolysis        +             -              +
Heat stable catalase       -             -              -
Pyrazinamidase             -             +              +
Urease                     -             +              +

Table 4: Resistance percentage of the RGM isolates
against some antibiotics.

Isolate   Mycobacterial   Antibiotics   Resistant
No.       species                       percent

48        M. chelonae     RIF           95.70%
                          EMB           4.20%
114       M. chelonae     EMB           2%
50        M. abscessus    RIF           94%
                          STR           24%
                          EMB           4%
                          INH           10%
                          PZA           8%
110       M. smegmatis    RIF           100%
                          PZA           14.20%
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Article Details
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Title Annotation:Research Article
Author:Sulami, Amin A. Al-; Taee, Asaad Al-; Hasan, Zainab A.
Publication:Biology and Medicine
Article Type:Report
Geographic Code:7IRAQ
Date:Jul 1, 2016
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