Background & objective: Enzymatic modifications of aminoglycosides result in high-level resistance in numerous bacterial species. However, the data on this aspect are elementary in our country. The present study was therefore designed to determine resistance rates and patterns, and to find out the prevalent aminoglycoside modifying enzymes (AMEs) in clinical isolates of Pseudomonas aeruginosa from hospitalized burn patients.
Methods: Forty two, non-repeat, clinical isolates of P. aeruginosa obtained during a period from February to July 2003, were analysed for the presence of antibiotics resistance. On the basis of aminoglycoside susceptibility patterns, resistance phenotypes and possible AMEs were inferred according to interpretative reading. Seven isolates collected during the same period and previously characterized to harbour a 48.5 kb plasniid, encoding multiple drug resistance, were also analysed for aminoglycoside susceptibility patterns, and AMEs encoded by the plasniid were inferred.
Results: Ninety six per cent of the isolates were multi drug-resistant and majority (71.4%) were resistant to 5 or more antibiotics. Markedly high resistance to tobramycin (83.6%) and amikacin (55.1%) was noted, whereas gentamicin resistance was present in 32.6 per cent isolates. The enzyme N-acetyl transferases (AAC) viz. AAC(6')-I was the most common isolated AME followed by AAC(3)-II in 42.8 and 20.4 per cent of isolates respectively. The plasniid harbouring isolates belonged to AAC(6') phenotype and the enzyme encoded was inferred to be AAC(6')-I.
Interpretation & conclusion: Markedly high resistance to tobramycin and amikacin was noted in the present study. AAC(6')-I was the most common AME and was inferred to be plasniid encoded in R-plasmid harbouring isolates. This is among the premier reports regarding the aminoglycoside resistance due to AMEs especially plasmid encoded, in P. aeruginosa from India. Further studies are required from different parts of the country to findout the prevalence of aminoglycoside resistance due to AMEs in P. aeroginosa isolates.
Key words Aminoglycoside - aminoglycoside resistance - modifying enzymes - Pseudomonas aeruginosa
The emergence and spread of antimicrobial resistance determinants are problems of increasing importance worldwide, particularly among nosocomial bacterial pathogens1. There are several factors influencing such increase in bacterial resistance1. The increased use of a variety of antimicrobials including aminoglycosides and the clinical introduction of numerous closely related compounds are known to be related to the emergence and dissemination of resistant bacterial strains1. Aminoglycoside resistance in all types of bacteria, including Pseudomonas, has become more complex with the increased time of aminoglycoside usage. Further, combinations of mechanisms have occurred which have broadened the spectrum of aminoglycoside resistance2.
In the last two decades, P. aeruginosa has been increasingly recognised as an aetiological agent in a variety of serious infections especially with impaired immune defences3. Enzymatic modifications, the most common type of aminoglycoside resistance, results in high level resistance in P. aeruginosa4. Though, information on aminoglycoside resistance in P. aeruginosa due to aminoglycoside modifying enzymes (AMEs) is available from many developed countries25, reports from our country are scanty. The present preliminary study was therefore designed to determine the resistance rates and patterns, and to find out the prevalent aminoglycoside modifying enzyme(s) responsible for aminoglycoside resistance in clinical isolates of P. aeruginosa obtained from hospitalized patients with burns in a hospital in north India.
Material & Methods
Bacterial isolates: Forty nine (42+7 plasmid harbouring), non-repeat isolates of P. aeruginosa obtained from the hospitalized burn patients during February to July 2003 in J.N. Medical College and Hospital, Aligarh, India, were included in the study. The isolates were screened for the presence of aminoglycoside modifying enzymes responsible for aminoglycoside resistance by performing phenotypic characterization6. Seven isolates of P. aeruginosa collected during the same study period and, in which we have previously7 isolated and characterized a 48.5 kb plasmid, responsible for amikacin resistance. were also characterized by interpretative reading6 to find out the possible aminoglycoside modifying enzyme encoded by these plasmids.
Antibiotics susceptibility testing: Antibiotics susceptibility patterns of the isolates were determined by the method of Bauer et al8. On Mueller Hinton agar (Hi-Media, Mumbai) using commercially available paper discs (Hi-Media, Mumbai). The antibiotics were used in the following concentrations (in µg/disc): amikacin (A), 30; gentamicin (G), 10; tobramycin (To), 10; netilmicin (Nt), 30; neomycin (ne), 30; kanamycin (Kn), 30; ciprofloxacin (C), 5; ceftazidime (Cz), 30; carbenicillin (Cr), 100. Experiments were performed in triplicate and mean of zones of inhibition was derived. Results were interpreted as per the guidelines of National Committee for Clinical Laboratory Standards (NCCLS)9.
Phenotypic characterization by interpretative reading: On the basis of susceptibility of isolates against a range of aminoglycosides, resistance phenotypes were determined and possible aminoglycoside modifying enzymes unique to a specific resistance pattern were inferred according to the interpretative reading6 (Table I).
Results & Discussion
Antibiotics resistance patterns: Ninety six per cent of the isolates were multidrug-resistant (resistant to three or more drugs) and majority (71.4%) of isolates were resistant to 5 or more antibiotics. The most common resistance patterns were GCNtToKn an ANtToKnNe, noticed in 12.2 per cent of isolates each. The resistance pattern of plasmid harbouring isolates was ANtToCrKnNe (TableII). Markedly high resistance to tobramycin (83.6%) and amikacin (55.1%) was noticed in the present study (Fig.).
Phenotypic characterization of the aminoglycoside resistance due to AMEs: There are three types of aminoglycoside modifying enzymes, viz., N-acetyl transferases (AAC), catalyses acetyl Co-A-dependent acetylation of an amino group; O-adenyl transferases (ANT), catalyses ATP-dependent adenylation of hydroxyl group, and O-phospho transferases (APH) which catalyses ATP-dependant phosphorylation of a hydroxyl group. Of these, the enzymatic modification is the most common type of aminoglycoside resistance4. On the basis of interpretative reading, the phenotypes isolated were AAC(6') 42.8 per cent; AAC(3)II, 20.4 per cent, APH(3')/classical, 16.3 per cent; AAC(6')II, 8.1 per cent while 12.2 per cent isolated could not be characterised (Table-II). Since a resistance pattern is unique to a specific enzyme10, the possible aminoglycoside modifying enzymes responsible for aminoglycoside resistance in the present study were inferred to be AAC(6')-I, AAC(3)-II, APH(3')-VI and AAC(6')-II, respectively. The 48.5 kb plasmid harbouring isolates of P. aeruginosa belonged to AAC(6') phenotype and enzyme encoded by the plasmid was inferred to be AAC(6')-I as evident by the loss of the aminoglycoside resistance concomitant with the loss of plasmid content after curing and transformation experiments7 (data not shown).
The aminoglycosides, in combination with beta-lactam antibiotics, are commonly used empirically in the treatment of pseudomonal infections. Though the resistance to aminoglycosides is increasing, these continue to play an important role in the treatment of these infections2,11. The excessive morbidity and mortality associated with ineffective empirical therapy in pseudomnal infections underscores the need of reliable data12. Substantial regional variation in resistance patterns has also been observed, and is probably related to antibiotic treatment regimens13. Periodic monitoring of resistance rates and patterns is mandatory, with special reference to aminoglycoside antibiotics, as the emergence of markedly high resistance to amikacin has already being noticed7.
Though P. aeruginosa isolates in the present study showed better susceptibility to ceftazidime, carbenicillin and ciprofloxacin, they were invariably found resistant to commonly prescribed aminoglycosides (amikacin and tobramycin) in the burn unit of our hospital. Compared to the international scenario2, we found markedly high resistance to amikacin and tobramycin, in our isolates, however, gentamicin resistance was comparable. Resistance to amikacin was similar to other studies from India11,14, though gentamicin resistance was comparatively lower.
In epidemiological surveys, the mechanism of aminoglycoside resistance is first ascertained by examining the susceptibility of the isolates to a panel of clinically used and experimental aminoglycosides, called as phenotypic characterization15. Such studies quickly led to the recognition of a large diversity of phenotypes. In the present study, based on phenotypic characterization, the isolated phenotypes [AAC(6'), AAC(3)II, APH(3') or classical and AAC(6')II] were similar to previous reports16,17. AAC(6')-I was the most common AME, which was also present in the isolates harbouring a 48.5 kb plasmid. Though, AAC(3)-II has been reported to be very rare enzyme in P. aeruginosa6, this was present in (20.4%) of the isolates in the present study. Similarly AAC(6')-II, reported as a frequent enzyme, occurred only in 8.1 per cent of our isolates. This might be due to the variations in the usage of aminoglycosides in different geographical areas.
In this study 12.2 per cent of isolates could not be characterized due to complex resistance pattern and remained undetermined. However, possibility of combinations of AMEs may not be excluded. The possible combinations of aminoglycoside enzymes for the resistance phenotypes (AToKnNe) could be AAC(6')-I plus APH(3') class of AMEs and for phenotypes (GAToKnNe) it could be AAC(6')-I plus gentamicin modifying enzymes; APH (2'')-I plus APH (3') class of AMEs or APH (3') plus bifunctional AAC(6')-APH(2'') (though bifunctional enzyme is reported mainly in Gram positive organisms).
Susceptibility test results are normally recorded and categorised individually a susceptible or resistant to that drug. However, this strategy underutilizes the data, since it ignores the fact that resistance to related antibiotics often depends on single mechanism6,18,19. Interpretative reading aims to analyse the susceptibility pattern, not just the results for individual antibiotics, and to predict the underlying mechanisms. Based on this interpretation, susceptibilities that appear tentative can be identified and reviewed, and further drugs that merit testing can be identified6,18,19. Further, in vitro susceptibility interpretation as intermediate susceptible may create confusion to clinicians, therefore, phenotypic mechanism(s) should be interpreted by the microbiologists and should edit the susceptibility result of the drug from intermediate susceptible to resistant on the basis of interpretative reading. For example, in AAC(6') phenotypes, one component of gentamicin remains active against the organism, but in vivo use is best avoided6.
Interpretative reading is an advance on current diagnostic laboratory practice, but is no substitute for identifying resistance mechanism by genetic and biochemical investigations6. We could not characterize our isolates genetically due to lack of molecular diagnostic facilities and hence suggest that the diagnostic laboratories lacking molecular facilities should at least characterize the aminoglycoside resistant clinical isolates by interpretative reading. Practical use of these methods for clinicians would be referring to the institution's antibiogram when selecting aminoglycosides for empiric use and to patient specific culture and susceptibility data for definitive use.
In conclusion, among the commonly prescribed aminoglycosides at our hospital, markedly high resistance to tobramycin and amikacin was noted in P. aeruginosa. AAC(6)-I. was the most common AME followed by AAC(3)-II. AAC(6')-I was inferred to be plasmid encoded in R-plasmid harbouring isolates. This is among the premier reports from India regarding the aminoglycoside resistance due to AMEs in clinical isolates of P. aeruginosa from burn patients. However, studies from various geographical regions are required to find out the exact prevalence of aminoglycosides resistance due to AMEs and frequency of occurrence of AMEs in P. aeruginosa isolates from India.
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M. Shahid & Abida Malik
Department of Microbiology, J.N. Medical College & Hospital, Aligarh Muslim University
Received February 9, 2004
Reprint requests: Dr M. Shahid, Lecturer, Department of Microbiology, J.N. Medical College, Aligarh Muslim University
Aligarh 202002, India
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