To the Editor—Staphylococcus aureus is the one of the most commonly isolated human bacterial pathogens causing skin and soft-tissue infections, endovascular infections, osteomyelitis, pneumonia, endocarditis, septic arthritis, and sepsis. Methicillin-resistant S. aureus (MRSA) isolates have developed resistance to all available penicillins and other β-lactam antimicrobial drugs.Reference David and Daum 1 A few drugs, such as vancomycin (glycopeptide), daptomycin (lipopeptide), and linezolid (oxazolidinone), have been approved for the treatment of serious infections caused by MRSA. However, different MRSA strains have already been emerging with resistance to these last-resort antimicrobial drugs.Reference Weigel, Clewell and Gill 2 – Reference Tsiodras, Gold and Sakoulas 4 These resistance trends for newer drugs emphasize the ongoing need for new and more potent antimicrobial drugs. Successful pathogenic bacteria may have to outcompete other coinfecting bacteria to stay in their eukaryotic host, such as humans. This interplay between pathogenic bacteria may lead to development of new antimicrobials.
In the present study, 39 Pseudomonas aeruginosa isolates were screened against MRSA. P. aeruginosa were isolated from various patients admitted in different Indian health centers. Four different strains of MRSA were used for susceptibility assays. S. aureus ATCC 25923 was used as a control strain. S. aureus cultures, grown overnight in Mueller-Hinton broth, were diluted to 0.5 OD600 (optical density at wavelength of 600 nm) and uniformly spread on Mueller-Hinton agar plate by using a sterilized cotton swab. Similarly, P. aeruginosa cultures were grown in Mueller-Hinton broth and centrifuged (10,000 g, 10 minutes) to collect the supernatant that was then filter sterilized. An aliquot (25 µL) was added to the S. aureus lawn followed by incubation at 37°C. The level of inhibition based on the presence of halo formation around the supernatant spot was defined as follows: “no inhibition” indicated as a halo less than 8 mm; “weak inhibition,” 8–15 mm; “strong inhibition,” 16–25 mm; “very strong inhibition,” greater than 25 mm. Of the 39 Pseudomonas isolates, 28 (72%) failed to inhibit the growth of S. aureus whereas 7 (18%) and 3 (8%) showed weak and strong inhibition, respectively. Only 1 isolate (3%) was able to show very strong inhibition of S. aureus (>30 mm halo). The isolate P-149 that showed maximum inhibition was selected for further analysis. Supernatant aliquots of varying volumes from P-149 (0–20 µL) were spotted on S. aureus lawns. After 24 h of incubation, an inhibition halo was visible around the spot of supernatant (5 µL) with profound growth of S. aureus on the edges compared with the control (Figure 1). However, with further increase in the supernatant volume (10–15 µL), only discrete colonies were visible on the edges of the inhibition zone. Growth of S. aureus was completely inhibited with 20 µL aliquot (Figure 1). Even after 72 h of incubation, growth of S. aureus remained inhibited. The same trend of inhibition was observed with other S. aureus strains including the ATCC control strain. The study clearly suggests that P-149 supernatant could be a potential source for the development of an antimicrobial drug against S. aureus. It should be further noted that crude supernatant was used without any purification step. Extraction of the active antimicrobial drug with organic solvents (acetone, hexane, and dichloromethane; 1:1) was unsuccessful. Treatment with nucleases did not alter the antimicrobial activity either. Certain pathogenic bacteria have been reported to secrete proteins to outgrow or outcompete other pathogens in the human body.Reference Fialho, Stevens and Das Gupta 5 However, even after treatment with proteinase K, the P-149 supernatant still retained its activity. Cytotoxicity assays revealed that the P-149 supernatant did not affect the growth of Saccharomyces cerevisiae.
FIGURE 1 Antimicrobial activity of Pseudomonas aeruginosa P-149 supernatant (5–20 µL) against methicillin-resistant Staphylococcus aureus.
Evidence demonstrates the increasing ineffectiveness of methicillin and newer agents, such as vancomycin and linezolid. The decrease in drug efficacy for S. aureus represents a looming threat to patient health with the fear of returning to the morbidity and mortality levels present before antibiotics were developed. Strategies adopted by pathogenic bacteria to keep their enemies at bay may provide an insight into the production of smart weapons.
ACKNOWLEDGMENTS
I thank E. Subudhi, Siksha ‘O’ Anusandhan University, Bhubaneswar, Odisha, India, and Dinesh Goyal, Shiv Astha Clinic, Haryana, India, for kindly providing the samples.
Financial support. Science and Engineering Research Board, Department of Science & Technology, New Delhi, India.
Potential conflicts of interest. The author reports no conflicts of interest relevant to this article.
