Indian Journal of Medical Microbiology, (2014) 32(1): 89-101
1
Correspondence
Detection of Vancomycin resistant Enterococci with vanA genotype in clinical isolates from a tertiary care centre Dear Editor, Vancomycin is a glycopeptide increasingly used in treatment as an alternative choice to penicillin, aminoglycoside combination for the treatment of enterococcal infections. Resistance to vancomycin is now widely reported worldwide after its first appearance in 1988. Dissemination of vancomycin resistance can occur through both clonal expansion of VRE and horizontal transfer of van genes to other bacteria. Glycopeptide-resistant genotypes in enterococci include vanA, vanB, vanC1/C2/C3, vanD, vanE, vanG, vanL, vanM and vanN. Multiple epidemics have been predominantly reported with vanA type[1]. vanA gene cluster is located within transposon Tn1546 and can be transferred through acquired resistance. A total of 43 non-identical clinical isolates of enterococci obtained from a tertiary care centre, Chennai, India. The specimen sources of isolates include blood, urine and pus. Enterococci were identified using standard identification methods[2]. The antibiotic susceptibility testing was done by Kirby-Bauer disc diffusion method. Enterococcus faecalis ATCC 29212 was used as control strain. The results were interpreted according to CLSI guidelines, 2011 [Table 1]. MIC for vancomycin was determined by both agar dilution method (CLSI guidelines, 2011)[3] and HiComb MIC strip (HiMedia Laboratories, Mumbai, India) method to screen for vancomycin resistance in enterococci. The specific Table 1: Antibiotic resistance among enterococcal isolates by disc diffusion method Antibiotics No. of isolates resistant (n=43) E. faecalis E. faecium Others* (n=33) (n=7) Gentamicin (120 mcg) 15 6 3 # Amikacin (30 mcg) 31 7 3 Erythromycin (15 mcg) 29 6 3 Tetracycline (30 mcg) 18 2 3 Ciprofloxacin (5 mcg) 19 6 2 Nitrofurantoin (300 mcg) 2 4 3 Ampicillin (10 mcg) s s s Teicoplanin (30 mcg) s s s Linezolid (30 mcg) s s s Levofloxacin (5 mcg) 15 5 3 Vancomycin (30 mcg) 9 1 Pristinomycin 34 6 3 (Quinupristin-dalfopristin) (15 mcg) *E. casseliflavus, E. avium; s: Susceptible; #Amikacin resistance was compared with Staphylococcus aureus ATCC 25923
vancomycin-resistant genotype vanA and vanB were determined by polymerase chain reaction (PCR) using primers (Sigma Aldrich, USA) and cycling conditions as described previously[4] with minor modifications in annealing condition (58°C for 1 min) and reducing the cycling conditions for 32 cycles. 38/43 (88.3%) were identified as E. faecalis (29 from blood and 3 from urine), 7/43 (16.2%) were Enterococcus faecium (from blood), 2 (4.6%) were Enterococcus casseliflavus (from blood) and 1 (2.3%) Enterococcus avium (from pus specimen). E. faecalis was the predominant isolate obtained in the present study followed by E. faecium. One E. faecalis and one E. faecium obtained from blood were detected as VRE with an MIC range of 128 g/ml and 256 g/ml [Figure 1] constituting 4.6% (2/43) and they carried vanA gene [Figure 2]. Accession number: KF311229 was obtained for our vanA gene PCR product from GenBank. This isolate (Efm090) was used as positive control for analysis of other isolates.
Figure 1: HiComb MIC Strip with MIC >256μg/ml for vancomycin in Efm090 isolate
Figure 2: Multiplex PCR analysis of vancomycin resistance genes in representative enterococcal isolates
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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
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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
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.
1
Correspondence
Detection of Vancomycin resistant Enterococci with vanA genotype in clinical isolates from a tertiary care centre Dear Editor, Vancomycin is a glycopeptide increasingly used in treatment as an alternative choice to penicillin, aminoglycoside combination for the treatment of enterococcal infections. Resistance to vancomycin is now widely reported worldwide after its first appearance in 1988. Dissemination of vancomycin resistance can occur through both clonal expansion of VRE and horizontal transfer of van genes to other bacteria. Glycopeptide-resistant genotypes in enterococci include vanA, vanB, vanC1/C2/C3, vanD, vanE, vanG, vanL, vanM and vanN. Multiple epidemics have been predominantly reported with vanA type[1]. vanA gene cluster is located within transposon Tn1546 and can be transferred through acquired resistance. A total of 43 non-identical clinical isolates of enterococci obtained from a tertiary care centre, Chennai, India. The specimen sources of isolates include blood, urine and pus. Enterococci were identified using standard identification methods[2]. The antibiotic susceptibility testing was done by Kirby-Bauer disc diffusion method. Enterococcus faecalis ATCC 29212 was used as control strain. The results were interpreted according to CLSI guidelines, 2011 [Table 1]. MIC for vancomycin was determined by both agar dilution method (CLSI guidelines, 2011)[3] and HiComb MIC strip (HiMedia Laboratories, Mumbai, India) method to screen for vancomycin resistance in enterococci. The specific Table 1: Antibiotic resistance among enterococcal isolates by disc diffusion method Antibiotics No. of isolates resistant (n=43) E. faecalis E. faecium Others* (n=33) (n=7) Gentamicin (120 mcg) 15 6 3 # Amikacin (30 mcg) 31 7 3 Erythromycin (15 mcg) 29 6 3 Tetracycline (30 mcg) 18 2 3 Ciprofloxacin (5 mcg) 19 6 2 Nitrofurantoin (300 mcg) 2 4 3 Ampicillin (10 mcg) s s s Teicoplanin (30 mcg) s s s Linezolid (30 mcg) s s s Levofloxacin (5 mcg) 15 5 3 Vancomycin (30 mcg) 9 1 Pristinomycin 34 6 3 (Quinupristin-dalfopristin) (15 mcg) *E. casseliflavus, E. avium; s: Susceptible; #Amikacin resistance was compared with Staphylococcus aureus ATCC 25923
vancomycin-resistant genotype vanA and vanB were determined by polymerase chain reaction (PCR) using primers (Sigma Aldrich, USA) and cycling conditions as described previously[4] with minor modifications in annealing condition (58°C for 1 min) and reducing the cycling conditions for 32 cycles. 38/43 (88.3%) were identified as E. faecalis (29 from blood and 3 from urine), 7/43 (16.2%) were Enterococcus faecium (from blood), 2 (4.6%) were Enterococcus casseliflavus (from blood) and 1 (2.3%) Enterococcus avium (from pus specimen). E. faecalis was the predominant isolate obtained in the present study followed by E. faecium. One E. faecalis and one E. faecium obtained from blood were detected as VRE with an MIC range of 128 g/ml and 256 g/ml [Figure 1] constituting 4.6% (2/43) and they carried vanA gene [Figure 2]. Accession number: KF311229 was obtained for our vanA gene PCR product from GenBank. This isolate (Efm090) was used as positive control for analysis of other isolates.
Figure 1: HiComb MIC Strip with MIC >256μg/ml for vancomycin in Efm090 isolate
Figure 2: Multiplex PCR analysis of vancomycin resistance genes in representative enterococcal isolates
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
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.
