Antimicrobial substances produced by bacteria isolated from different Jordanian sources that are active against methicillin-resistant Staphylococcus aureus

We report here the preliminary antimicrobial activity of substances produced by Bacillus subtilis NB-6 (air flora isolate), Bacillus megaterium NB-3 (air flora isolate), Burkholderia mallei NB-8 (water isolate) and Corynebacterium kutscheri NB-1 (soil isolate) against a number of methicillin-resistant Staphylococcus aureus (MRSA). The MRSA were isolated from sheep, bovine, camel and poultry meat samples collected from retails shops and slaughter houses located in Amman area, Jordan. B. mallei NB-8 and C. kutscheri NB-1 were found to possess a good antimicrobial activity against MRSA strains.


INTRODUCTION
The spread of resistance to antibiotics undermines the therapeutic utility of anti-infective drugs in current clinical use (Bax et al., 2000).For example, Staphylococcus aureus, a major cause of community and hospitalacquired infections, has developed resistance to most classes of antibiotics, and isolates exhibiting such resistance is drawing great concern.Methicillin-resistant S. aureus (MRSA) strains appeared in the hospital environment after introduction of the semisynthetic penicillin, methicillin, leaving vancomycin as the last line of defense for MRSA treatment (Enright, 2003).With the appearance of vancomycin-resistant clinical isolates (Sieradzki et al., 1999), no antibiotic class is effective against multiresistant S. aureus infections.Thus, new antibiotic and therapy options are urgently needed to improve the management of bacterial infections (Saimann et al., 2001), and a major challenge is to find drugs that act against Methicillin-resistant S. aureus *Corresponding author.E-mail: nasserelbanna@yahoo.com.Fax.00962 2 6350520.

Preculture conditions
Bacteria were transferred by loop from slants to 125 ml flasks containing 50 ml medium like that used in subsequent experimental cultures.All precultures were incubated at appropriate temperature on a rotary shaker (Sanyo Gallenhamp PLC, Leicester, LE 3 2uz, UK) at 180 rpm for 24 h. 1 ml of preculture was used to inoculate all experimental cultures.

Agar diffusion test
Extraction of the active substance from cells grown in liquid cultures (100 ml) was carried out as follows.Briefly, cells were pelleted by centrifugation and extracted with acetone, and the supernatant was extracted with ethylacetate.Both extracts were evaporated by a rotary evaporator (Heidolph instruments, GmbH and Co KG Vertrieb, Kelheim, Germany) at < 50 C, and the dry substances (separately) were dissolved in 0.5 ml methanol.The antimicrobial activity of these extracts was carried against methicillin-resistant S. aureus strains (MRSA) by agar diffusion test.Filter discs containing 10 µl of the active substance dissolved in methanol (acetone extract or ethylacetate extract) were placed on the test plates.The plates were incubated at 37 C for 48 h, and the antimicrobial activity was determined by measuring zones of growth inhibition (El-Banna and Winkelmann, 1998).

Biotest plates preparation
Cell suspension of 24 h MRSA precultures were prepared (O.D578 = 1), and 0.5 ml of this suspension was used to inoculate 250 ml agar medium (20 ml per plate, Arab food and Media Applicances Company limited.Zarka industrial area, Jordan).

RESULTS AND DISSCUSION
The agar diffusion method was used to compare the antimicrobial activity of B. subtilis NB-6, B. megaterium NB-3, B. mallei NB-8 and C. kutscheri NB-1 against methicillin-resistant S. aureus (MRSA) isolated from meat samples from sheep, bovine, camel and poultry in Amman area, Jordan.As shown in Table 1, substances produced by B. mallei NB-8 showed good antimicrobial activity against MRSA 2, MRSA 3, MRSA 5 and MRSA 13, and weak antimicrobial activity against MRSA 1, MRSA 7 and MRSA 8, while no antimicrobial activity against MRSA 6. MRSA 2, MRSA 5 and MRSA 8 were inhibited by substances produced by C. kutscheri NB-1.The other MRSA strains (MRSA 1,3,6,7 and 13) were not inhibited at all.All MRSA strains were not inhibited by substances produced by B. subtilis NB-6 and B. megaterium NB-3 except MRSA strain 13 which was slightly inhibited by B. megaterium NB-3 produced substances.
S. aureus is an important cause of a variety of diseases in human and animals worldwide (Gilot and Leeuwen, 2004).Their ability to cause diseases in associated with several pathogenic factors including extracellular enzymes and toxins (Lee, 2003).Both animal and human isolates are generally resistant to penicillins (Seguin et al., 1999).Several reports suggest that the transfer of S. aureus between human and cattle is possible and that the infection of human by transmission through food products contaminated with animal MRSA is very plausible (Lee, 2003;Kaszanyitzky et al., 2003).Romero-Tabarez et al. (2006) has previously reported an antimicrobial activity of substances produced by B. subtilis (soil isolate) against multidrug-resestant bacterial pathogen including methicillin-resistant S. aureus.
In this study, we have described the antimicrobial activity of substances produced by bacteria against several methicillin-resistant S. aureus strains which may be promising for the development of new drugs against microbial pathogens.