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Disc Diffusion Based In Vitro Antibiotic Susceptibility of Recent Isolates of Burkholderia mallei.

Byline: M. SAQIB, G. MUHAMMAD, A. NAUREEN, A. SHAKOOR AND SAJJAD-UR-REHMAN - E-mails: profdrgm_pk@yahoo.com; drsaqib_vet@hotmail.com

ABSTRACT

In vitro susceptibilities of 43 non-archived recent isolates of Burkholderia mallei to 12 antimicrobials were determined by disc diffusion technique. All isolates were sensitive to chloramphenicol, co-trimoxazole, doxycycline and gentamicin but resistant to ampicillin and cephradine. The order of present resistance to amoxicillin, norfloxacin, oxytetracycline, ciprofloxacin, co-amoxiclav and enrofloxacin was 90.6 (n = 39), 20.9 (n = 9), 6.9 (n = 3), 6.9 (n = 3), 4.6 (n = 2) and 4.6 (n = 2), respectively. In the light of present results, chloramphenicol, co-trimoxazole, doxycycline, co-amoxiclav and enrofloxacin seem good candidate antibiotics for future planning of experimental therapeutic trials in target species (equids). Furthermore, this information can be useful in contingency disease managemental plans for incidences of deliberate release of B. mallei as terrorist attacks and in situation of laboratory associated B. mallei infections in humans. (c) 2010 Friends Science Publishers

Key Words: Burkholderia mallei; Glanders; Bio-warfare; Antibiotic susceptibility; Antibiotic resistance

INTRODUCTION

Burkholderia mallei is the etiologic agent of glanders, a disease primarily of equids, which is also communicable to man with fatal consequences. Glanders has been eradicated from most parts of the world. However, the disease still occurs in Asia, Africa and South America (OIE, 2008). During the last decade, the disease outbreaks were reported in Brazil, Eretria, Ethiopia, Iran, Iraq, India, Pakistan and United Arab Emirates (Dvorak and Spickler, 2008; Naureen et al., 2007). Burkholderia mallei is an established bioweapon that was used in different wars during the 20th century (Rotz et al., 2002). Since B. mallei is an agent of biological warfare/bio terrorism and since glanders is a reemerging disease, researchers are focusing on effective treatment protocols both in man and animals species and post exposure prophylaxis (Lopez et al., 2003).

Treatment of bacterial diseases is ideally guided by the results of in vitro antibiotic susceptibility. Much of the published work on in vitro susceptibility of B. mallei was carried out on 8 to 4 decades old archived isolates dating back mostly to pre-antibiotic era. Often times, these isolates might have been passaged in the laboratory creating prospects of loss of plasmids encoding for resistance to antibiotics (Muhammad et al., 1998a). During the last 20 years, only 2 reports on susceptibility of non-archived (recent) isolates of B. mallei that had been sub-cultured only a few times in laboratory were added to scientific literature Al-Ani et al., 1998; Muhammad et al., 1998b).

As we are moving forward with our experimental therapeutic treatment trials in the natural host, data of antibiogram of recent B. mallei isolates is obviously needed. This manuscript documents the antibiotic susceptibility of recent isolates of B. mallei determined by disc diffusion method.

MATERIALS AND METHODS

Burkholderia mallei isolates: Forty three isolates of B. mallei were recovered from glandered equines (horses, n = 67; mules, n = 16; and donkeys, n = 19) presented to Veterinary Teaching Hospital, Department of Clinical Medicine and Surgery, University of Agriculture, Faisalabad, Pakistan. Culture and identification of B. mallei was carried out following procedures described by Muhammad et al. (1998b). Briefly, nasal swab samples, mature skin nodule aspirates and venous blood samples from clinically diseased equids were cultured on brain heart infusion agar (BHI agar, Oxoid, UK) supplemented with sheep blood. Nasal swab samples were incubated for 3 h at room temperature in saline containing 3000 units of benzyl penicillin per mL before plating. When B. mallei originated from more than one type of samples from the same animal (nasal swabs, pus and blood), it was considered as one isolate. The representative colonies were screened for catalase, indole and colistin resistance.

Irregularly stained Gram To cite this paper: Saqib, M., G. Muhammad, A. Naureen, A. Shakoor and S.U. Rehman, 2010. Disc diffusion based in vitro antibiotic susceptibility of recent isolates of Burkholderia mallei. Int. J. Agric. Biol., 12: 777-780 negative rods, indole negative and resistant to colistin were presumptively identified as those of B. mallei. Non-motile, triple sugar iron negative, arginine and gelatin positive isolates were finally confirmed as B. mallei. These isolates were further confirmed in polymerase chain reaction by targeting Burkholderia intracellular motility A gene (Ulrich et al., 2006).

Antibiotic susceptibility testing: In vitro antimicrobial susceptibility to 12 antibiotics/antimicrobials (Table I) was determined by disc diffusion method according to the standards described by British Society of Antimicrobial Chemotherapy (Andrew, 2001). Sensitivity agar plates were seeded with 100 uL of 106 colony forming unit per milliliter of test isolates, which was adjusted spectrophotometrically. The plates were incubated at 37+-1degC for 30 h. The zones of inhibition were measured and compared with break points for B. pseudomallei and non-Enterobacteriaceae

Staphylococcus aureus ATCC 25923, E. coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853 isolates were used as quality control for antibiotics.

RESULTS AND DISCUSSION

Availability of antibiotic susceptibility data of B. mallei is an essential tool for antibiotic treatment and prophylaxis in the face of deliberate release of this bio-warfare agent. The results of in vitro sensitivity of B. mallei isolates are given in the Table I. The inhibitory zones of antibiotics against quality control isolates were within recommended ranges (Andrew, 2001). In the present study, all B. mallei isolates were resistant to ampicillin and cephradine, whereas 90.6 (n = 39), 6.9 (n = 3), 20.9 (n = 9), 4.6 (n = 2) and 6.9 (n = 3) percent isolates showed resistance to amoxicillin, oxytetracycline, norfloxacin, enrofloxacin and ciprofloxacin, respectively. Natural resistance of B. mallei to penicillin is well known since late 1960s (Mishankin and Goldberg, 1969). The resistance associated with alteration in bacterial penicillin binding proteins (PBP) has been reported in Burkholderia (Burns, 2006).

Beta-lactamase gene (PenA), which powers the resistance against penicillins and cephalosporins has also been identified in B. mallei ATCC 23344 strain (Tribuddharat et al., 2003). Resistance to ampicillin, cephradine and amoxicillin as observed in the present study might either be due to modification in PBP or the occurrence of PenA (b lactamase) gene. The later assumption is further substantiated when amoxicillin with b-lactamase inhibitor (clavulanate) was tested against amoxicillin resistant isolates. This combination remarkably restored the activity of amoxicillin against resistant isolates. However, 4.6% isolates were still resistant (Table I). Our strain-record indicated that only few isolates demonstrated b-lactamase activity (unpublished data) possibly due to the use of less sensitive method of determination of b-lactamase activity (nitrocephin touch stick) and/or slow growing nature of the organism.

In general, these results are in close agreement with those of previous studies (Al-Izzi and Al-Bassam, 1989; Antonov et al., 1991; Kenny et al., 1999; Thibault et al., 2004), wherein authors reported high resistance (MIC90grater than64 ug/mL) in B. mallei to ampicilin, amoxicilin and to a few cephalosporins (cefazolin, cefsulodin, cefoxitin; MIC90grater than64 - grater than 128). Contrary to our findings, American (Heine et al., 2001), British (Kenny et al., 1999) and French (Thibault et al., 2004) Burkholderia research groups reported no resistance to co-amoxiclav. However, one intermediately sensitive strain (MIC 16 ug/mL) has been observed (Thibault et al., 2004). Resistance against co-amoxiclav encountered in the present study may be attributed to a modification in chromosomally encoded b-lactamase (LiPuma, 2007).

Among tetercyclines, oxytetracycline showed good anti B. mallei activity although 6.9% (n = 3) isolates were found resistant to this antibiotic. No resistance was recorded against doxycycline. Similar results have also been reported previously (Al-Izzi and Al-Bassam, 1989; Manzeniuk et al., 1995; Heine et al., 2001; Thibault et al., 2004). The use of oxytetracycline reportedly delayed the Strauss reaction (orchitis), while chlorteteracycline was the most promising antibiotic among teteracylines in the treatment of experimental glanders (Nemato et al., 1961). About 5 decades ago, 3 cases of human glanders were effectively treated with oxytetracycline and chlortetracycline (Tezok, 1958). In a recent study, animal passage and re-isolation of B. mallei markedly altered MIC of doxycycline, that raises the concern that eventually true resistance could emerge against new tetracyclines (Heine et al., 2001).

Consistent with the previous findings (Batmanov, 1991), where 13 B. mallei isolates were highly resistant to norfloxacin but displayed susceptibility to ofloxacin and ciprofloxacin, a high proportion of resistance (20.9%) against norfloxacin noted in the present study discourages the use of this antibacterial in prospective treatment trials. Norfloxacin has also been found ineffective in experimental glanders therapeutic trial in guinea pig and hamster (Batmanov, 1991). Previously, Muhammad et al. (1998b) reported absolute sensitivity of B. mallei isolates (n= 13) to norfloxacin. These results might be due to the use of different concentration of norfloxacin and low volume of bacterial suspension used (adjusted parallel to 0.5 McFarland turbidity standards). Furthermore, mutation in gyrA gene could be another inciting factor (Burns, 2006) in isolates of present study. Iraqi workers (Al-Ani et al., 1998) reported susceptibility of 14 isolates to enrofloxacin.

In their study interpretive criterion seems to be based on larger zone of inhibition of enrofloxacin rather than susceptibility breakpoint. In the light of results of the present study, it appears that enrofloxacin and ciprofloxacin may be useful candidate antibiotics for the therapy of glanders as both antimicrobials reportedly accumulate in phagocytic cells (where B. mallei resides), achieving intracellular concentrations higher than the extracellular levels (Carlier et al., 1990; Schoevers et al., 1999). In B. mallei, mutation

Table I: Antibiogram Profile and Comparative inhibition zone* of different antimicrobials against 43 isolates of Burkholderia mallei recovered from glanderous equids

###Diameter of vone of inhibition (mm)###42 Resistance (%)###

Antimicrobials Disk potency Sensitivity###

###(ug)###break point###

###(mm) Grater than 8 10###12###14###16###18###20###22###24###26###28###30###32###34###36###38 40###42###

Ampicillin###25###18###24###-###17###2###100###

Amoxicillin###25###18###9###20###6###1###3###4###90.6###

Co-amoxiclav###30###18###2###-###2###24###3###1###3###5###1###2###4.6###

Cephradine###30###12###42###1###100###

Chloramphenicol 30###21###5###3###9###17###1###3###1###-###1 1###0###

Gentamicin###25###15###3###-###4###2###23###2###-###5###2###1###1###0###

Oxytetracycline 30###19###1###1###1###1###-###13###20###-###-###-###3###3###6.9###

Doxycycline###30###16###6###-###9###11###4###3###7###1###2###0###

Co-trimoxazole###25###16###1###8###15###1###1###3###9###2###2###0###

Norfloxacin###10###17###3###1###5###-###11###7###9###6###-###1###20.9###

Enrofloxacin###5###18###2###6###4###17###2###9###-###1###1###1###4.6###

Ciprofloxacin###5###21###2###1###5###3###21###2###7###1###-###1###6.9###

*No of susceptible isolates are given against black background

derived resistance against fluroquinolones including oxolinic acid, norfloxacin, enoxacin, ofloxacin and ciprofloxacin has been reported (Stepanshin, 1994). Therefore, a careful rational selection of antibiotics will be needed in any experimental therapeutic trial against glanders.

In line with the results of the earlier studies (Al-Izzi and Al-Bassam, 1989; Antonov et al., 1991; Kenny et al., 1999; Heine et al., 2001; Thibault et al., 2004), trimethoprim-sulphamethoxazole (co-trimoxazole), gentamicin and chloramphenicol showed excellent in vitro activity against all isolates of B. mallei. Lozovaia (1989) reported good in vitro anti- B. mallei activity of sulphonamides (sulphamonomethoxine, sulphasalazine, sulphanilamide and sulphamethoxazole) in combination with trimethoprim (TMP). Treatment with sulphadiazine alone or combinations of sulfas with TMP was reportedly successful in human and laboratory animals, respectively (Howe and Miller, 1947; Ansabi and Minou, 1951; Batmanov, 1993).

Whole genome analysis of B. pseudomallei strain K96243 and B. mallei strain ATCC 23344 exposed a close relationship between these organisms and B. mallei is a clonal derivative of B. psudomallei as evidenced by multilocus sequence typing (Godoy et al., 2003; Harland et al., 2007). A multidrug efflux system-Amr-OprA-identified in B. pseudomallei (Moore et al., 1999) is responsible for high level resistance to aminoglycosides (gentamicin for one). Re-annotation and comparison of ATP-binding cassette systems of both organisms revealed that B. mallei lacked an Amr-OprA system, which most likely contributes to its sensitivity to aminoglycosides (Harland et al., 2007). Therefore, sensitivity of B. mallei to gentamicin is an inherent characteristic and this antibiotic can be considered in future experimental treatment plans for glanders.

However, it is worth noting that B. mallei is an intracellular pathogen and use of this antibiotic may not give bacteriological cure as it would not attain desired therapeutic levels within the scavenger (macrophages/dendritic) cells.

Results of susceptibility of B. mallei to chloramphenicol have been inconsistent. Some researchers reported sensitivity (Heine et al., 2001; Thibault et al., 2004), while others documented resistance to this antibiotic (Al-Izzi and Al-Bassam, 1989; Kenny et al., 1999). Use of different formats of susceptibility and isolates might be the cause of this disparity. Chloramphenicol is well known for its bone marrow suppressive activity (Papich and Riviere, 2001) and use of this drug in glanders seems potentially dangerous, because B. mallei depletes bone marrow reserves in affected animals. Therefore, florfenicol, a fluorinated chloramphenicol derivative lacking undesirable effects needs to be investigated in future studies.

Antibiotic susceptibility by direct diffusion method (DDM) often differs from results obtained by E-test, agar and broth dilution technique, which give minimum inhibitory concentration (MIC). MIC statistics generates quantitative information rather than qualitative. DDM is used as a guideline for dosage determination as well as for antibiotic selection (Burrow et al., 1993). Discrepancies have been noted between results obtained by disc diffusion and E-test for B. pseudomallei (Wuthiekanun et al., 2005). To know more precisely about susceptibility data (MIC) and selection of antibiotic for experimental glanders treatment, the isolates of present study need to be tested by other susceptibility formats (agar dilution for one which is considered a gold standard for susceptibility testing).

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M. SAQIB, G. MUHAMMAD, A. NAUREEN, A. SHAKOOR AND SAJJAD-UR-REHMAN

Department of Clinical Medicine and Surgery, University of Agriculture, Faisalabad, Pakistan

Department of Clinical Medicine and Surgery, University of Veterinary and Animal Sciences, Lahore, Pakistan

Department of Microbiology, University of Agriculture, Faisalabad, Pakistan

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