90
Indian Journal of Medical Microbiology
Further, the sensitivity pattern of isolates revealed, 29/43 (67.4%) isolates were multiple drug resistant to more than one class of antibiotics. These isolates also exhibited high level resistance to aminoglycosides upon agar screening method. Surprisingly, all the isolates were susceptible to ampicillin on MIC; though they were resistant on disc diffusion method. Interestingly, all strains were susceptible to linezolid and teicoplanin by disc diffusion method. Although 2 VRE were obtained, they were also susceptible (≥14 mm) to teicoplanin (30 mcg Himedia disc); however, this should be further confirmed by MIC to prove its susceptibility. Since, these strains were vanA gene positive, these patients may not respond to teicoplanin. The genotype identification is more rapid than conventional methods. The antibiotic history of the patients had showed that these patients were treated with vancomycin, cloxacillin, amikacin and cephalosporin intravenously. However, the present study found a very good sensitivity for linezolid, the treatment option for VRE and MRSA. VRE is now frequently being reported in India. A recent study from Chennai[5] has reported VRE in 18 isolates (4%) which had been treated with linezolid. Considering the increasing frequency of VRE isolation from different parts of India; appropriate surveillance, continuous monitoring is very important to control the spread and cross contamination of these resistant clones.
3. 4. 5.
2.
Textbook of diagnostic microbiology. 6th ed. 2006. p. 732-3. Performance standards for antimicrobial susceptibility testing; CLSI 21st informational supplement. Vol. 31. Wayne, PA: CLSI; 2011. p. M100-S21. Bell MJ, Paton CJ, Turnidge J. Emergence of vancomycin resistant enterococci in Australia: Phenotypic and genotypic characteristics of isolates. J Clin Microbiol 1998;36:2187-90. Vidyalakshmi PR, Gopalakrishnan R, Ramasubramanian V, Abdul Ghafur K, Nambi PS, Thirunarayana MA. Clinical, epidemiological and microbiological profile of patients with vancomycin-resistant enterococci from a Tertiary Care Hospital. J Global Infect Dis 2012;4:137-8.
E Padmasini, R Padmaraj, *SS Ramesh Department of Microbiology (EP, SSR), Dr. ALM Post Graduate Institute of Basic Medical Sciences, University of Madras, Taramani, Department of Paediatric Nephrology (RP), Institute of Child Health, Egmore, Chennai, Tamil Nadu, India *Corresponding author (email: ) Received: 16-05-2013 Accepted: 23-09-2013
Access this article online Quick Response Code:
References 1.
vol. 32, No. 1
Cattoir V, Leclercq R. Twenty-five years of shared life with vancomycin resistant enterococci: Is it time to divorce? J Antimicrob Chemother 2013;68:731-42. Washnigton W, Allen S, Janda W, Koneman E, Procop G, Schreckenberger P, Woods G. Koneman’s colour atlas and
Website: www.ijmm.org PMID: *** DOI: 10.4103/0255-0857.124339
In-vitro antibacterial activity of some essential oils against clinical isolates of Acinetobacter baumannii Dear Editor, Until 1970s, Acinetobacter baumannii was considered to be a rare cause of nosocomial infections. However, the incidence of Acinetobacter infections has reached a point of concern and poses a threat to debilitated patients around the world.[1] The clinical importance of Acinetobacter has improved rapidly in the last 20 years, due to the world-wide expression of intensive care units that led to a change in the type of infections and the emergence of multi drug resistant Acinetobacter isolates.[2] Essential oils have been implicated in a wide array of applications for several 1000 years owing to their antimicrobial properties as alternative medicine.[3] Thus, the development of resistance to antibiotics is believed to be counteracted by the use of natural herbal medicine are being considered to be the need of the hour. The present study was undertaken to determine the in-vitro antibacterial
activity of clove, peppermint and eucalyptus oils against clinical isolates of A. baumannii. Essential oils were procured from Tegraj and Co (P) Ltd, India and their purity was about 99.9%. The major constituents were found to be eugenol (84.2%), eugenyl acetate (9.7%) for clove oil. P-menthone (19.5%), menthol (63.5%) and 1,8-cineole (74.3%), -terpinenol (10.3%) for peppermint oil and eucalyptus oil respectively. We tested a total of 50 isolates of A. baumannii from different clinical specimens that includes blood (15), endotracheal aspirates (14), bronchoalveolar lavage (6), pus (6), urine (5) and sputum (4). Minimum inhibitory concentrations (MICs) were determined for these oils by agar dilution method.[3] The concentrations were ranging from 0.03 to 2% (v/v). A sterile Mueller Hinton Agar without oils served as control plate for bacterial growth. Overnight culture suspensions of A. baumannii isolates were adjusted to 0.5 McFarland standard for turbidity and 10 L
www.ijmm.org
January-March 2014
91
Correspondence
of suspension was spot inoculated onto plates with afore mentioned concentrations of clove, peppermint and eucalyptus oils. Experiments were carried out in triplicate. Inoculated plates were incubated at 37°C for 24 h and MIC was determined. The lowest concentration of oils that inhibited the growth of A. baumannii isolates were considered as MIC. All the isolates were not inhibited at a concentration of 0.03% of clove oil, 0.03-0.125% of peppermint oil and 0.03-0.06% of eucalyptus oil. Strains were inhibited from 0.06 to 0.25%, 0.25-1% and 0.125-1% for clove, peppermint and eucalyptus oils respectively. In clove oil, 14/50 (28%) isolates were inhibited at 0.06%, 25/50 (50%) at 0.125% and 11/50 (22%) at 0.25% of clove oil. In peppermint oil, 34/50 (68%) isolates were inhibited at 0.25%, 12/50 (24%) and 4/50 (8%) were at 0.5% and 1% concentrations of peppermint oil respectively. In eucalyptus oils, 10/50 (20%) isolates were inhibited at 0.125%, 18/50 (36%) at 0.25%, 16/50 (32%) and 6/50 (12%) were at 0.5% and 1% respectively. Thus, the MIC of clove oil was found to be 0.06%, 0.25% for peppermint oil and 0.125% for eucalyptus oil [Table 1]. Similarly study was performed by Hammer et al. in 1999 on antibacterial activity of essential oils found that the MIC values of 0.25%, 0.5% and 1% were determined for clove, peppermint and eucalyptus oils respectively.[3] These oils have been shown to be effective against clinical isolates of A. baumannii in in-vitro condition. Carbapenem group of antibiotics are used as the most effective drug to treat infections caused by this notorious pathogen. Thus, we have also tested susceptibility of the same isolates to meropenem (Meronem, Astra Zeneca, UK) by MIC method as per Clinical Laboratory Standards Institute guidelines.[4] A standard strain of Pseudomonas aeruginosa ATCC 27853 was also included as quality control. MIC of ≥ 16 was considered as resistant to meropenem. Out of 50, 21 (42%) isolates were found to be meropenem sensitive with MIC range of ≤ 4 μg/mL, 2/50 (4%) isolates were intermediate and 27/50 (54%) were resistant with MIC ranges of 8 μg/mL and ≥ 16 μg/mL respectively. The study conducted by Taneja et al. in 2003 documented an increased percentage of carbapenem resistant (>20%) Acinetobacter isolates.[5] Similarly, Corbella et al. found > 36% of Acinetobacter spp. were appeared to be carbapenem resistant.[6] These reports are in concordant with our data as increased percentage of isolates was found to be resistant to carbapenem. Moreover, these isolates were found to be resistant to other classes of antibiotics except colistin and tigecycline by MIC. High antibiotic pressure due to indiscriminate usage of such group of drugs in the clinical settings led to the Table 1: MIC of three different essential oils against clinical isolates of Acinetobacter baumannii Essential MIC ranges (n=50) (%) oils 0.03 0.06 0.125 0.25 0.5 1 Clove 0 14 (28) 25 (50) 11 (22) 0 0 Peppermint 0 0 0 34 (68) 12 (24) 4 (8) Eucalyptus 0 0 10 (20) 18 (36) 16 (32) 6 (12)
marked resistance among A. baumannii. In some essential oils such as eucalyptus and clove oils, there has been much research and documented some toxic and irritant properties.[7] In spite of this, most of these oils are available as whole oils or as part of pharmaceutical and cosmetic products. However, the studies on toxic and irritant properties of essential oils are imperative, especially when considering any new products for human administration. Thus, we suggest that these three oils were found to have good antibacterial activity against A. baumannii. This can be used as alternative and complementary antibacterial agents for controlling the Acinetobacter infections. References 1.
2. 3. 4.
5. 6.
7.
Villers D, Espaze E, Coste-Burel M, Giauffret F, Ninin E, Nicolas F, et al. Nosocomial Acinetobacter baumannii infections: Microbiological and clinical epidemiology. Ann Intern Med 1998;129:182-9. Peterson LR. Squeezing the antibiotic balloon: The impact of antimicrobial classes on emerging resistance. Clin Microbiol Infect 2005;11 Suppl 5:4-16. Hammer KA, Carson CF, Riley TV. Antimicrobial activity of essential oils and other plant extracts. J Appl Microbiol 1999;86:985-90. Clinical Laboratory Standards Institute. Performance Standards for Antimicrobial Susceptibility Testing: Twenty Second Informational Supplement. CLSI Document M100-S22.Wayne, PA: CLSI; 2012. Taneja N, Maharwal S, Sharma M. Imipenem resistance in nonfermenters causing nosocomial urinary tract infections. Indian J Med Sci 2003;57:294-9. Corbella X, Montero A, Pujol M, Domínguez MA, Ayats J, Argerich MJ, et al. Emergence and rapid spread of carbapenem resistance during a large and sustained hospital outbreak of multiresistant Acinetobacter baumannii. J Clin Microbiol 2000;38:4086-95. Lawless J. The Illustrated Encyclopedia of Essential Oils. Shaftesbury, UK: Element Books Ltd.; 1995.
Gopinath Prakasam, Manju Bhashini, Lakshmipriya, S. Srivani Ramesh Department of Microbiology (GP, MB, LP, SSR), Dr. ALM Post Graduate Institute of Basic Medical Sciences, University of Madras, Taramani, Chennai, Tamil Nadu, India *Corresponding author (email: ) Received: 24-05-2013 Accepted: 20-09-2013
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MIC: Minimum inhibitory concentrations
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Website: www.ijmm.org PMID: *** DOI: 10.4103/0255-0857.124341
Copyright of Indian Journal of Medical Microbiology is the property of Medknow Publications & Media Pvt. Ltd. and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use.
Indian Journal of Medical Microbiology
Further, the sensitivity pattern of isolates revealed, 29/43 (67.4%) isolates were multiple drug resistant to more than one class of antibiotics. These isolates also exhibited high level resistance to aminoglycosides upon agar screening method. Surprisingly, all the isolates were susceptible to ampicillin on MIC; though they were resistant on disc diffusion method. Interestingly, all strains were susceptible to linezolid and teicoplanin by disc diffusion method. Although 2 VRE were obtained, they were also susceptible (≥14 mm) to teicoplanin (30 mcg Himedia disc); however, this should be further confirmed by MIC to prove its susceptibility. Since, these strains were vanA gene positive, these patients may not respond to teicoplanin. The genotype identification is more rapid than conventional methods. The antibiotic history of the patients had showed that these patients were treated with vancomycin, cloxacillin, amikacin and cephalosporin intravenously. However, the present study found a very good sensitivity for linezolid, the treatment option for VRE and MRSA. VRE is now frequently being reported in India. A recent study from Chennai[5] has reported VRE in 18 isolates (4%) which had been treated with linezolid. Considering the increasing frequency of VRE isolation from different parts of India; appropriate surveillance, continuous monitoring is very important to control the spread and cross contamination of these resistant clones.
3. 4. 5.
2.
Textbook of diagnostic microbiology. 6th ed. 2006. p. 732-3. Performance standards for antimicrobial susceptibility testing; CLSI 21st informational supplement. Vol. 31. Wayne, PA: CLSI; 2011. p. M100-S21. Bell MJ, Paton CJ, Turnidge J. Emergence of vancomycin resistant enterococci in Australia: Phenotypic and genotypic characteristics of isolates. J Clin Microbiol 1998;36:2187-90. Vidyalakshmi PR, Gopalakrishnan R, Ramasubramanian V, Abdul Ghafur K, Nambi PS, Thirunarayana MA. Clinical, epidemiological and microbiological profile of patients with vancomycin-resistant enterococci from a Tertiary Care Hospital. J Global Infect Dis 2012;4:137-8.
E Padmasini, R Padmaraj, *SS Ramesh Department of Microbiology (EP, SSR), Dr. ALM Post Graduate Institute of Basic Medical Sciences, University of Madras, Taramani, Department of Paediatric Nephrology (RP), Institute of Child Health, Egmore, Chennai, Tamil Nadu, India *Corresponding author (email: ) Received: 16-05-2013 Accepted: 23-09-2013
Access this article online Quick Response Code:
References 1.
vol. 32, No. 1
Cattoir V, Leclercq R. Twenty-five years of shared life with vancomycin resistant enterococci: Is it time to divorce? J Antimicrob Chemother 2013;68:731-42. Washnigton W, Allen S, Janda W, Koneman E, Procop G, Schreckenberger P, Woods G. Koneman’s colour atlas and
Website: www.ijmm.org PMID: *** DOI: 10.4103/0255-0857.124339
In-vitro antibacterial activity of some essential oils against clinical isolates of Acinetobacter baumannii Dear Editor, Until 1970s, Acinetobacter baumannii was considered to be a rare cause of nosocomial infections. However, the incidence of Acinetobacter infections has reached a point of concern and poses a threat to debilitated patients around the world.[1] The clinical importance of Acinetobacter has improved rapidly in the last 20 years, due to the world-wide expression of intensive care units that led to a change in the type of infections and the emergence of multi drug resistant Acinetobacter isolates.[2] Essential oils have been implicated in a wide array of applications for several 1000 years owing to their antimicrobial properties as alternative medicine.[3] Thus, the development of resistance to antibiotics is believed to be counteracted by the use of natural herbal medicine are being considered to be the need of the hour. The present study was undertaken to determine the in-vitro antibacterial
activity of clove, peppermint and eucalyptus oils against clinical isolates of A. baumannii. Essential oils were procured from Tegraj and Co (P) Ltd, India and their purity was about 99.9%. The major constituents were found to be eugenol (84.2%), eugenyl acetate (9.7%) for clove oil. P-menthone (19.5%), menthol (63.5%) and 1,8-cineole (74.3%), -terpinenol (10.3%) for peppermint oil and eucalyptus oil respectively. We tested a total of 50 isolates of A. baumannii from different clinical specimens that includes blood (15), endotracheal aspirates (14), bronchoalveolar lavage (6), pus (6), urine (5) and sputum (4). Minimum inhibitory concentrations (MICs) were determined for these oils by agar dilution method.[3] The concentrations were ranging from 0.03 to 2% (v/v). A sterile Mueller Hinton Agar without oils served as control plate for bacterial growth. Overnight culture suspensions of A. baumannii isolates were adjusted to 0.5 McFarland standard for turbidity and 10 L
www.ijmm.org
January-March 2014
91
Correspondence
of suspension was spot inoculated onto plates with afore mentioned concentrations of clove, peppermint and eucalyptus oils. Experiments were carried out in triplicate. Inoculated plates were incubated at 37°C for 24 h and MIC was determined. The lowest concentration of oils that inhibited the growth of A. baumannii isolates were considered as MIC. All the isolates were not inhibited at a concentration of 0.03% of clove oil, 0.03-0.125% of peppermint oil and 0.03-0.06% of eucalyptus oil. Strains were inhibited from 0.06 to 0.25%, 0.25-1% and 0.125-1% for clove, peppermint and eucalyptus oils respectively. In clove oil, 14/50 (28%) isolates were inhibited at 0.06%, 25/50 (50%) at 0.125% and 11/50 (22%) at 0.25% of clove oil. In peppermint oil, 34/50 (68%) isolates were inhibited at 0.25%, 12/50 (24%) and 4/50 (8%) were at 0.5% and 1% concentrations of peppermint oil respectively. In eucalyptus oils, 10/50 (20%) isolates were inhibited at 0.125%, 18/50 (36%) at 0.25%, 16/50 (32%) and 6/50 (12%) were at 0.5% and 1% respectively. Thus, the MIC of clove oil was found to be 0.06%, 0.25% for peppermint oil and 0.125% for eucalyptus oil [Table 1]. Similarly study was performed by Hammer et al. in 1999 on antibacterial activity of essential oils found that the MIC values of 0.25%, 0.5% and 1% were determined for clove, peppermint and eucalyptus oils respectively.[3] These oils have been shown to be effective against clinical isolates of A. baumannii in in-vitro condition. Carbapenem group of antibiotics are used as the most effective drug to treat infections caused by this notorious pathogen. Thus, we have also tested susceptibility of the same isolates to meropenem (Meronem, Astra Zeneca, UK) by MIC method as per Clinical Laboratory Standards Institute guidelines.[4] A standard strain of Pseudomonas aeruginosa ATCC 27853 was also included as quality control. MIC of ≥ 16 was considered as resistant to meropenem. Out of 50, 21 (42%) isolates were found to be meropenem sensitive with MIC range of ≤ 4 μg/mL, 2/50 (4%) isolates were intermediate and 27/50 (54%) were resistant with MIC ranges of 8 μg/mL and ≥ 16 μg/mL respectively. The study conducted by Taneja et al. in 2003 documented an increased percentage of carbapenem resistant (>20%) Acinetobacter isolates.[5] Similarly, Corbella et al. found > 36% of Acinetobacter spp. were appeared to be carbapenem resistant.[6] These reports are in concordant with our data as increased percentage of isolates was found to be resistant to carbapenem. Moreover, these isolates were found to be resistant to other classes of antibiotics except colistin and tigecycline by MIC. High antibiotic pressure due to indiscriminate usage of such group of drugs in the clinical settings led to the Table 1: MIC of three different essential oils against clinical isolates of Acinetobacter baumannii Essential MIC ranges (n=50) (%) oils 0.03 0.06 0.125 0.25 0.5 1 Clove 0 14 (28) 25 (50) 11 (22) 0 0 Peppermint 0 0 0 34 (68) 12 (24) 4 (8) Eucalyptus 0 0 10 (20) 18 (36) 16 (32) 6 (12)
marked resistance among A. baumannii. In some essential oils such as eucalyptus and clove oils, there has been much research and documented some toxic and irritant properties.[7] In spite of this, most of these oils are available as whole oils or as part of pharmaceutical and cosmetic products. However, the studies on toxic and irritant properties of essential oils are imperative, especially when considering any new products for human administration. Thus, we suggest that these three oils were found to have good antibacterial activity against A. baumannii. This can be used as alternative and complementary antibacterial agents for controlling the Acinetobacter infections. References 1.
2. 3. 4.
5. 6.
7.
Villers D, Espaze E, Coste-Burel M, Giauffret F, Ninin E, Nicolas F, et al. Nosocomial Acinetobacter baumannii infections: Microbiological and clinical epidemiology. Ann Intern Med 1998;129:182-9. Peterson LR. Squeezing the antibiotic balloon: The impact of antimicrobial classes on emerging resistance. Clin Microbiol Infect 2005;11 Suppl 5:4-16. Hammer KA, Carson CF, Riley TV. Antimicrobial activity of essential oils and other plant extracts. J Appl Microbiol 1999;86:985-90. Clinical Laboratory Standards Institute. Performance Standards for Antimicrobial Susceptibility Testing: Twenty Second Informational Supplement. CLSI Document M100-S22.Wayne, PA: CLSI; 2012. Taneja N, Maharwal S, Sharma M. Imipenem resistance in nonfermenters causing nosocomial urinary tract infections. Indian J Med Sci 2003;57:294-9. Corbella X, Montero A, Pujol M, Domínguez MA, Ayats J, Argerich MJ, et al. Emergence and rapid spread of carbapenem resistance during a large and sustained hospital outbreak of multiresistant Acinetobacter baumannii. J Clin Microbiol 2000;38:4086-95. Lawless J. The Illustrated Encyclopedia of Essential Oils. Shaftesbury, UK: Element Books Ltd.; 1995.
Gopinath Prakasam, Manju Bhashini, Lakshmipriya, S. Srivani Ramesh Department of Microbiology (GP, MB, LP, SSR), Dr. ALM Post Graduate Institute of Basic Medical Sciences, University of Madras, Taramani, Chennai, Tamil Nadu, India *Corresponding author (email: ) Received: 24-05-2013 Accepted: 20-09-2013
Access this article online Quick Response Code:
2 0 0 0
MIC: Minimum inhibitory concentrations
www.ijmm.org
Website: www.ijmm.org PMID: *** DOI: 10.4103/0255-0857.124341
Copyright of Indian Journal of Medical Microbiology is the property of Medknow Publications & Media Pvt. Ltd. and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use.
