By Varaiya, Ami Kulkarni, Nikhil; Kulkarni, Manasi; Bhalekar, Pallavi; Dogra, Jyotsana
Background & objectives: Metallo beta lactamase (MBL) producing Pseudomonas aeruginosa have been reported to be important cause of nosocomial infections. The appearance of MBL genes and their spread among bacterial pathogens is a matter of concern with regard to the future of antimicrobial therapy. The present study was undertaken to determine the incidence of MBL producing P. aeruginosa in patients with diabetes and cancer admitted to the intensive care unit of a tertiary care hospital in western India and to assess the clinical outcome after antimicrobial treatment. Methods: A total of 240 isolates of P. aeruginosa from various specimens between January and December 2005 were subjected to susceptibility testing against various antibiotics by disc diffusion test as per the Clinical and Laboratory Standards Institute (CLSI) guidelines. Imipenem and meropenem resistant isolates were selected for the detection of MBL production by disc potentiation test. Enhancement of inhibition zone around imipenem and meropenem discs impregnated with EDTA as compared to those without EDTA confirmed MBL production.
Results: Of the 240 P. aeruginosa isolates, 60 (25%) were found to be carbapenem resistant and 50 (20.8%) were found to be MBL producers. Of the 50 MBL producing isolates, 38 (76%) were from diabetes patients and 12 (24%) from cancer patients. Overall, 36 per cent patients responded to gatifloxacin, 42 per cent responded to piperacillin/tazobactam while 14 per cent responded to combination of gatifloxacin and piperacillin/tazobactum. Due to this nosocomial pathogen, the average hospital stay was 32 days and was associated with 20 per cent mortality due to septicaemia.
Interpretation & conclusions: Our findings showed that there is a need to do surveillance to detect MBL producers, judiciously use carbapenems to prevent their spread and use effective antibiotics, such as gatifloxacin and piperacillin-tazobactum, after sensitivity testing for treatment.
Key words Carbapenem resistance – metallo beta lactamase – Pseudomonas aeruginosa
The introduction of carbapenems into clinical practice represented a great advance for the treatment of serious bacterial infections caused by beta-lactam resistant bacteria. Due to their broad spectrum of activity and stability to hydrolysis by most beta lactamases, the carbapenems have been the drug of choice for treatment of infections caused by penicillin-or cephalosporin- resistant Gram-negative bacilli especially, extended spectrum beta- lactamase (ESBL) producing Gram-negative infections1. The carbapenems available for use in India are imipenem and meropenem2. However, carbapenem resistance has been observed frequently in non fermenting bacilli Pseudomonas aeruginosa and Acinetobacter spp. Resistance to carbapenem is due to decreased outer membrane permeability, increased efflux systems, alteration of penicillin binding proteins and carbapenem hydrolyzing enzymes-carbapenemase3. These carbapenemase are class B metallo beta-lactamases (IMP, VIM) or class D-oxacillinases (OXA 23 to OXA 27) or class A – clavulanic acid inhibitory enzymes (SME, NMC, IMI, KPC)3.
Metallo beta lactamase (MBL) belongs to a group beta-lactamase which requires divalent cations of zinc as cofactors for enzyme activity. These have potent hydrolyzing activity not only against carbapenem but also against other beta-lactam antibiotics4. The IMP and VIM genes responsible for MBL production are horizontally transferable via plasmids and can rapidly spread to other bacteria5. The genes responsible for MBL production may be chromosomally or plasmid mediated and hence pose a threat of spread of resistance by gene transfer among the Gram-negative bacteria3.
Thus, MBL-producing Pseudomonas aeruginosa isolates have been reported to be important causes of nosocomial infections associated with clonal spread6. These constitute 20-42 per cent of all nosocomial isolates7,8. The appearance of MBL genes and their spread among bacterial pathogens is a matter of concern with regard to the future of antimicrobial chemotherapy6.
We undertook this study to determine incidence of MBL producing P. aeruginosa in diabetes and cancer patients admitted to the intensive care unit of a tertiary care hospital in western India over a period of one year from January to December 2005 and to observe the clinical outcome in these patients after treatment.
Material & Methods
Two hundred and forty isolates of P.aeruginosa were obtained during a one year period from January to December 2005 in the Department of Microbiology, S.L. Raheja Hospital, Mumbai, Maharashtra. The specimens processed were: respiratory secretions (61), tissue (58), swabs pus/ wound (55), urine (52), blood culture (10) and bile (4). With Universal safety precautions, samples were collected from patients in ICU, transported and processed in the laboratory without delay. Blood cultures were processed using automated method with Versa Trek (Tri Vitron, India). Samples were cultured on brain heart infusion (BHI) blood agar and MacConkey’s agar. Identification of organisms was done by the standard laboratory technique9. Antimicrobial sensitivity testing was performed on Mueller – Hinton (MH) agar plates with commercially available discs (Hi-media, Mumbai) by Kirby Bauer disc diffusion method and interpreted as per CLSI recommendations10. P. aeruginosa ATCC 27853 (beta-lactamase negative) strain was used as control.
The routine antibiotic sensitivity tests were put up for aminoglycosides [amikacin (30 [mu]g), gentamicin (10 [mu]g), netilmicin (30 [mu]g), tobramicin (10 [mu]g)], cephalosporin’s [cefoperazone (75 [mu]g), cefepime (30 [mu]g), ceftazidime (30 [mu]g), ceftriaxone (30 [mu]g), ceftizoxime (30 [mu]g)], floroquinolones [ciprofloxacin (5 [mu]g), gatifloxacin (5 [mu]g), lomefloxacin (10 [mu]g)], carbapenems [imipenem (10 [mu]g), meropenem (10 [mu]g)], chloramphenicol (30 [mu]g) and piperacillin/ tazobactum (100/10 [mu]g).
MBL producing P. aeruginosa was suspected when the isolate was resistant to meropenem and imipenem.
Various methods have been recommended for screening MBL. These include the modified Hodge test, double disc synergy test using imipenem and EDTA discs or ceftazidime and EDTA discs, EDTA impregnated imipenem discs11 and EDTA impregnated meropenem discs7. For MIC detection of imipenem, the E -test strip7 and microdilution (microtitre) plate method12 is recommended.
We used disc potentiation test with EDTA impregnated imipenem and meropenem discs. However, MIC was not detected in this study as E- test strips were very expensive and a simple microdilution plate method is time consuming.
Disc potentiation test: A 0.5 M EDTA solution (pH 8.0) was prepared and was sterilized by autoclaving. Test organisms were inoculated onto plates of MH agar (Opacity adjusted to 0.5 McFarland opacity standards). Two 10 [mu]g imipenem discs and two 10 [mu]g meropenem discs were placed on inoculated plates and 5 [mu]l of EDTA solution was added to one imipenem and one meropenem disc. The zone of inhibition around imipenem and meropenem discs alone and those with EDTA was recorded and compared after 16-18 h incubation at 35[degrees]C. An increase in zone size of at least 7 mm around the imipenem-EDTA disc and meropenemEDTA discs was recorded as a positive result. The difference in the resistance pattern of MBL- positive and MBL-negative was considered to be statistically significant if the P value was
Results
Of the 240 isolates of P. aeruginosa, 60 (25%) were found resistant to carbapenems (both imipenem and meropenem) and 50 (20.8%) were found to be MBL producers confirmed by disc potentiation method. The ATCC 27853 P. aeruginosa did not exhibit any zone size enhancement with EDTA impregnated imipenem discs.
Of the 50 MBL producing isolates, 30 (60%) were from diabetes patients and 20 (40%) from cancer patients [acute lymphoblastic leukaemia (5), chronic myeloid leukaemia (4), carcinoma of gallbladder (3), carcinoma of oesophagus (3), prostatic adenocarcinoma (1), carcinoma of sigmoid colon (1), non-Hodgkin’s lymphoma (1), mesothelioma (1) and Hodgkin’s disease (1)]. Of the 50 patients, 38 (76%) were males and 12 (24%) were females; the average age being 63 yr (50-75 yr).
Patients were treated with gatifloxacin, piperacillin/ tazobactum, and combination of gatifloxacin and piperacillin/ tazobactum. Antibiotic sensitivity pattern of MBL positive and negative isolates is shown Table I. Antibiotic sensitivity pattern of MBL positive and negative isolates among cancer and diabetes patients is presented in Table II.
Statistically significant difference was found in the resistance pattern of MBL positive and negative isolates for amikacin, netilmicin, cefepime, ceftazidime, ceftriaxone, ciprofloxacin and lomefloxacin (P
The average hospital stay of patients with MBL producers was 32 days (range 4 days-2 months). Of the 50 patients, 10 had P. aeruginosa as the sole isolate from blood culture. All these patients died due to Pseudomonas septicaemia. Thus, the mortality was 20 per cent. Among the 40 patients with MBL producing P. aeruginosa who survived, 10 needed re-admissions to the hospital because of deterioration in their clinical condition due to progressive disease. All these patients were cancer patients.
Discussion
P. aeruginosa is a pathogen associated with numerous nosocomial infections in immunocompromised patients13. Carbapenems are the drugs of choice for multidrug resistant P. aeruginosa and ESBL producing organisms. However, resistance to carbapenems due to reduced uptake of drug leads to imipenem/meropenem resistant isolates14. In various studies across the world, varying resistance (4-60%) has been seen towards imipenem and meropenem15,16. We found 25 per cent resistance to imipenem and meropenem. P. aeruginosa producing MBL was first reported from Japan in 1991 “. In 2002 from India, Navneeth et al18 first reported MBL production in P. aeruginosa to be 12 per cent. Since then, the incidence of MBL production in P. aeruginosa has been reported to be 10-30 per cent from various clinical specimens across the country8. We found 20.8 per cent MBL production in P. aeruginosa of which 30 per cent were obtained from respiratory specimens in our study. Another study conducted by Shashikala et al14 reported 20.7 per cent carbopenem resistant P. aeruginosa isolates from endotracheal aspirates showing indwelling devices as major risk factors for the development of resistance.
Amongst the MBL positive isolates from diabetes and cancer patients admitted to ICU in this study, maximum sensitivity was observed for piperacillin/ tazobactum followed by gatifloxacin. Amongst the MBL negative isolates maximum sensitivity was observed for piperacillin/tazobactum followed by ciprofloxacin, ceftazidime, gatifloxacin, cefepime, netilmicin, ceftriaxone, amikacin, and lomefloxacin. In the study conducted by Taneja et al8, piperacillin and amikacin had the best in vitro susceptibility. In our study, 18 patients responded to gatifloxacin, 24 to piperacillin/tazobactum while 7 patients responded to combination of gatifloxacin and piperacillin/tazobactum. Amongst the 20 cancer patients in whom MBL producers were isolated, 9 showed sensitivity to gatifloxacin and 7 showed sensitivity to piperacillin/tazobactum. Amongst the 30 diabetes patients in whom MBL producers were isolated, 7 showed sensitivity to gatifloxacin and 14 showed sensitivity to piperacillin/tazobactum.
P. aeruginosa are responsible for 3-7 per cent bloodstream infections and high mortality rates (27-48%) in critically ill patients18. We observed 20 per cent mortality due to P. aeruginosa septicaemia in our patients.
In conclusion, our findings showed that there is a need to do surveillance to detect MBL producers, judiciously use carbapenems to prevent their spread and use effective antibiotics, such as gatifloxacin and piperacillin-tazobactum, after sensitivity testing for treatment.
References
1. Mendiratta DK, Deotale V, Narang P. Metallo beta lactamase producing Pseudomonas aeruginosa in a hospital from rural area. Indian J Med Res 2005; 121 : 701-3.
2. Gupta E, Mohanty S, Sood S, Dhawan B, Das BK, Kapil A. Emerging resistance to carbapenems in a tertiary care hospital in north India. Indian J Med Res 2006; 124 : 95-8.
3. Gladstone P, Rajendran P, Brahmadathan KN. Incidence of carbapenem resistant nonfermenting Gram negative bacilli from patients with respiratory -infections in the intensive care unit. Indian J Med Microbiol 2005; 23 : 189-91.
4. Bush K. Metallo beta-lactamase: a class apart. Clin infect Dis 1998; 27(Suppl 1): S48-53.
5. Bennett PM. Integrons and gene cassettes; A genetic construction kit for bacteria. Antimicrob Agents Chemother 1999; 43 : 1-4.
6. Bush K, Jacoby GA, Medeiros A. A functional classification scheme for beta lactamase and its correlation with molecular structure. Antimicrob Agents Chemother 1995; 39 : 1211-33.
7. Walsh TR, Toleman MA, Poirel L, Nordmann P. Metallo-beta- lactamase: the Quiet before the Storm? Clin Microbiol Rev 2005; 18 : 306-25.
8. Taneja N, Aharwal SM, Sharma M. Imipenem resistance in non fermenters causing nosocomial urinary tract infection. Indian J Med Sci 2003; 57 : 294.
9. Forbes BA, Sham DF, Weissfeld AS. Bailey and Scott’s diagnostic microbiology, 10th ed. New York: Mosby; 1998. p. 167-87.
10. Clinical and Laboratory Standards Institute. Performance standards for antimicrobial disk tests; Approved Standards, 9th ed. CLSI Document M2- A9, Vol. 26 No 1. Wayne PA 2006.
11. Hemalatha V, Sekar U, Kamat V. Detection of metallo betalactamase producing Pseudomonas aeruginosa in hospitalized patients. Indian J Med Res 2005; 122 : 148-52.
12. Migliavacca R, Docquire JD, Mugnaioli C, Amicosante G, Daturi R, Lee K, et al. Simple Microdilution test for detection of metallo- beta-lactamase production in Pseudomonas aeruginosa. J Clin Microbiol 2002; 40 : 4388-90.
13. Bonfiglio G, Laksai Y, Franchino L, Amicosante G, Nicoletti G. Mechanisms of beta-lactam resistance amongst Pseudomonas aeruginosa isolated in an Italian survey. J Antimicrob Chemother 1998; 42 : 697-702.
14. Shashikala, Kanungo R, Srinivasan S, Devi S. Emerging resistance to carbapenem in hospital acquired Pseudomonas infection: A cause of concern. Indian J Pharmacol 2006; 38 : 287-8.
15. Forster DH, Daschner FD. Acinetobacter species as nosocomial pathogens. Eur J Clin Microbial Infect Dis 1998; 17 : 73-7.
16. Gonlugur U, Bakiri MZ, Akkurt I, Efeoglu T. Antibiotic susceptibility patterns among respiratory isolates of Gram negative bacilli in a Turkish University Hospital. BMC Microbiol 2004; 4 : 32- 6.
17. Yano H, Kuga A, Okamota R, Kitasato H, Kobayashi T, Inon M. Plasmid coded metallo beta lactamase (imp 6) conferring resistance to carbapenems, especially meropenam. Antimicrob Agents Chemother 2001; 45 : 1343-8.
18. Navneeth BV, Sridaran D, Sahay D, Belwadi MR. A preliminary study on metallo betalactamse producing Pseudomonas aeruginosa in hospitalized patients. Indian J Med Res 2002; 116 : 264-7.
19. Endimiani A, Luzzaro F, Pini B, Amicosante G, Rossolini GM, Toniolo AQ. Pseudomonas aeruginosa bloodstream infections: risk factors and treatment outcome related to expression of the PER-1 extended-spectrum beta-lactamase. BMC Infect Dis 2006; 6 : 52; available from www.biomedcentral.com/1471-23 34/6/52.
Ami Varaiya, Nikhil Kulkarni, Manasi Kulkarni, Pallavi Bhalekar & Jyotsana Dogra
Department of Microbiology, S.L.Raheja Hospital, Mumbai, India
Received November 7, 2007
Reprint requests: Dr Ami Varaiya, Consulting Microbiologist, Department of Laboratory Medicine
S.L. Raheja Hospital, Mahim West, Mumbai 400 016, India
e-mail: [email protected]
Copyright Indian Council of Medical Research Apr 2008
(c) 2008 Indian Journal of Medical Research. Provided by ProQuest LLC. All rights Reserved.
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