Year : 2008 | Volume
: 51 | Issue : 2 | Page : 200--203
Incidence of metallo-beta-lactamase-producing Pseudomonas aeruginosa in diabetes and cancer patients
Ami Varaiya, Manasi Kulkarni, Pallavi Bhalekar, Jyotsana Dogra
Department of Microbiology, S.L. Raheja Hospital, Mumbai, Maharashatra, India
Department of Laboratory Medicine, S.L. Raheja Hospital, Mahim West, Mumbai - 400 016, Maharashatra
Metallo-beta-lactamase (MBL)-producing Pseudomonas aeruginosa strains have been reported to be an important cause of nosocomial infections. There is not enough information from India regarding their prevalence in diabetic and cancer patients. The present study was undertaken over a period of one year from January to December 2006 to study the incidence of MBL P. aeruginosa and the clinical outcome in diabetes and cancer patients admitted to S.L. Raheja Hospital, Mumbai. Two hundred and thirty isolates of P. aeruginosa were obtained from different samples of patients. These isolates were subjected to susceptibility testing to anti-pseudomonal drugs as per CLSI guidelines. They were further screened for the production of MBL by disc potentiation testing using EDTA-impregnated imipenem and meropenem discs. Of the 230 isolates of P. aeruginosa, 60 (26%) isolates were found resistant to carbapenems (both imipenem and meropenem) and 33 (14.3%) were found to be MBL producers. Of the 33 MBL-producing isolates, 24 (72.7%) were diabetic patients, six (18.1%) were cancer patients and three (9%) patients had both diabetes and cancer. Five (15.1%) patients responded to the combination therapy of colistin, piperacillin with tazobactam and amikacin, while 28 (84.8%) patients responded to the combination therapy of amikacin, piperacillin with tazobactam and gatifloxacin. Thus, the rapid dissemination of MBL producers is worrisome and necessitates the implementation of not just surveillance studies but also proper and judicious selection of antibiotics, especially carbapenems.
|How to cite this article:|
Varaiya A, Kulkarni M, Bhalekar P, Dogra J. Incidence of metallo-beta-lactamase-producing Pseudomonas aeruginosa in diabetes and cancer patients.Indian J Pathol Microbiol 2008;51:200-203
|How to cite this URL:|
Varaiya A, Kulkarni M, Bhalekar P, Dogra J. Incidence of metallo-beta-lactamase-producing Pseudomonas aeruginosa in diabetes and cancer patients. Indian J Pathol Microbiol [serial online] 2008 [cited 2020 Jun 6 ];51:200-203
Available from: http://www.ijpmonline.org/text.asp?2008/51/2/200/41683
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 activities and stability to hydrolysis by most beta-lactamase, the carbapenems have been the drug of choice for the treatment of infections caused by penicillin or cephalosporin-resistant Gram-negative bacilli, especially ESBL Gram-negative infections.  The carbapenems available for use in India are imipenem and meropenem.  However, carbapenem resistance has been observed frequently in non-fermenting bacilli Pseudomonas aeruginosa and Acinetobacer spp. Resistance to carbapenem is due to decreased outer membrane permeability, increased efflux systems, alteration of penicillin-binding proteins and carbapenem hydrolyzing enzymes-carbapenemase. These carbapenemase are class B metallo-β-lactamases (MBLs; IMP, VIM) or class D oxacillinases (OXA 23 to OXA 27) or class A clavulanic acid inhibitory enzymes (SME, NMC, IMI, KPC). 
Metallo-β-lactamase belongs to class B, which requires divalent cations of zinc as cofactors for enzyme activity. They have potent hydrolyzing activity not only against carbapenem but also against other β-lactam antibiotics.  The IMP and VIM genes responsible for MBL production are horizontally transferable via plasmids and can rapidly spread to other bacteria.  Thus, MBL-producing P. aeruginosa strains have been reported to be important causes of nosocomial infections associated with clonal spread. 
Our institute, S.L. Raheja Hospital, is a tertiary care hospital for diabetes and cancer patients. There is not enough information from the Indian subcontinent regarding the prevalence of MBL-producing P. aeruginosa among these patients. The present study was undertaken over a period of one year from January to December 2006 to study the incidence of MBL-producing P. aeruginosa and the clinical outcome in Diabetes and Cancer patients admitted to our hospital.
Materials And Methods
Over the one-year period from January to December 2006, 230 isolates of P. aeruginosa were obtained. With Universal safety precautions, samples were collected from patients admitted to hospital. They were transported and processed in the laboratory without delay. The specimens processed were: tissue (78), respiratory secretions (73), urine (34), swabs pus/wound (32), blood culture (5), bile (4) and body fluids (4).
Samples were processed and identified by standard laboratory technique.  Blood cultures were processed using automated method with VersaTrek (Trivitron). Antimicrobial sensitivity testing was performed on Mueller Hinton Agar plates with commercially available discs (Hi-Media, Mumbai) by Kirby Bauer disc diffusion method. The results were recorded and interpreted as per CLSI recommendations.  Pseudomonas aeruginosa isolated from different clinical specimens were identified by oxidase test and biochemical reactions. Pseudomonas aeruginosa ATCC 27853 was used as a negative control.
The routine antibiotic sensitivity tests were put up for aminoglycosides [amikacin (30 µg), gentamicin (10 µg), netilmicin (30 µg) and tobramycin (10 µg)], cephalosporins [cefoperazone (75 µg), cefepime (30 µg), ceftazidime (30 µg), ceftriaxone (30 µg) and ceftizoxime (30 µg)], fluoroquinolones [ciprofloxacin (5 µg), gatifloxacin (5 µg) and lomefloxacin (10 µg)], carbapenems [imipenem (10 µg) and meropenem (10 µg)], chloramphenicol (30 µg), piperacillin/tazobactam (100/10 µg), aztreonam (30 µg) and colistin (10 µg). Isolates were considered to be carbapenem resistant when the zone size around imipenem and meropenem was ≤13 mm, intermediate 14-15 mm and sensitive ≥16 mm.
MBL-producing P. aeruginosa was suspected when the isolate was resistant to meropenem and imipenem.
Screening and confirmation for the detection of MBL was done by disc potentiation test with EDTA-impregnated imipenem discs and EDTA-impregnated meropenem discs. 
Disc potentiation test
Test organism was inoculated onto plates of Mueller-Hinton agar plate (opacity adjusted to 0.5 McFarland opacity standards).A 0.5-m EDTA solution was prepared by dissolving 186.1 g of disodium EDTA 2H 2 O in 1000 ml of distilled water and adjusting it to pH 8.0 by using NaOH. The mixture was sterilized by autoclaving.Two 10-µg imipenem discs and meropenem discs were placed on the plate; 5 µl of EDTA solution was added to one of the disc each.The inhibition zones of the imipenem and imipenem-EDTA discs and meropenem and meropenem-EDTA discs were compared after 16-18 h of incubation at 35°C.An increase in the zone size of at least 7 mm around the imipenem-EDTA disc and meropenem-EDTA discs was recorded as an MBL-positive strain.
Of the 230 isolates of P. aeruginosa , 60 (26%) were found resistant to carbapenems (both imipenem and meropenem) and 33 (14.3%) were found to be MBL producers confirmed by the disc potentiation method. The ATCC 27853 P. aeruginosa did not exhibit any zone size enhancement with EDTA-impregnated imipenem discs.
Of the 33 MBL-producing isolates, 24 (72.7%) were diabetic patients, 6 (18.1%) were cancer patients and 3 (9%) patients had both diabetes and cancer.
Among the cancer patients - carcinoma of esophagus (3), haematolymphoid malignancies with febrile neutropenia (2), carcinoma of lung (2), carcinoma of rectum (1) and carcinoma of prostrate (1).
Other co-morbid conditions - Parkinson's syndrome (1), Alzheimer's disease (1), hypothyroidism (3), leptospirosis (1), tuberculosis (4), chronic obstructive pulmonary disease (1) and one patient each who were HIV and hepatitis B positive.
Of the 33 patients, 25 (75.7%) were males and eight (24.2%) were females; median age of these patients was 68.5 years (50-87 years).
Overall, the antibiotic sensitivity pattern of carbapenem-resistant strains is reflected in [Table 1].
The antibiotic sensitivity pattern of MBL-positive and -negative strains have been detailed in [Table 2].
Among diabetic patients, colistin showed maximum sensitivity followed by aztreonam, gatifloxacin, amikacin, piperacillin/tazobactam and piperacillin. Amongst cancer patients, piperacillin showed maximum sensitivity followed by aztreonam, piperacillin/tazobactam and colistin. Among patients with both diabetes and cancer, amikacin showed maximum sensitivity followed by aztreonam [Table 3].
Among the 33 patients with MBL-positive isolates, seven patients expired; mortality rate being 21.2%. The predominant causes of death were chronic renal failure due to septicemia, terminal respiratory arrest due to septicemia and septicemia with progressive metastatic carcinoma.
Among the 26 patients with MBL-producing P. aeruginosa who survived, 8 (30.7%) needed readmissions to the hospital of which seven were diabetic patients admitted with either non-healing wound or urinary tract infections and one was a cancer patient admitted because of deterioration in clinical condition due to progressive disease. The mean hospital stay of patients in whom MBL producers were isolated was 25 days (range 6-44 days).
Pseudomonas aeruginosa producing MBLs was first reported from Japan in 1991. Since then, they have been described from various parts of the world including Asia, Europe, Australia, South America and North America. 
In various studies across the world, varying resistance (4-60%) has been seen towards imipenem and meropenem. , In 2002 from India, Navneeth et al . first reported MBL production by P. aeruginosa to be 12%.  Since then, the incidence of MBL production by P. aeruginosa has been reported to be 10-30% from various clinical specimens across the country.  A study conducted by Mary et al , reported 42% MBL production by Pseudomonas .  Another study conducted by Sarkar et al , reported 54.54% MBL production by Pseudomonas .  We report 14.3% MBL production by P. aeruginosa of the 60 carbapenem-resistant isolates.
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 discs  and EDTA-impregnated meropenem discs. 
For MIC detection of imipenem, the E -test strip is recommended where one-half of the strip is impregnated with an imipenem gradient across seven dilutions and the other half with another imipenem gradient overlaid with a constant concentration of EDTA. 
We used disc potentiation test with EDTA-impregnated imipenem and meropenem discs. E -test strips were not used in this study as they are very expensive. Data extrapolated from in vitro studies suggest that polymyxin B or colistin represent the best treatment options for MBL-producing P. aeruginosa . treatment options.  In the present study, sensitivity to colistin was 57.5%, aztreonam 21.2%, gatifloxacin 12.1%, piperacillin/tazobactam 9% and piperacillin 9%. However, colistin being very expensive, limits its use. In our study among patients with MBL-positive isolates, five (15.1%) patients responded to combination therapy of colistin, piperacillin with tazobactam and amikacin and while 28 (84.8%) patients responded to combination therapy of amikacin, piperacillin with tazobactam and gatifloxacin.
Among the diabetic patients, foot infections being polymicrobial, there is a high incidence of multidrug-resistant P. aeruginosa . Gadepalli et al , have reported P. aeruginosa strains showing 100% sensitivity to carbapenems, followed by 88.8% sensitivity to cefoperazone-sulbactam and ticarcillin-clavum combinations.  Among the 24 diabetic patients in whom MBL producers were isolated, we report 66.6% sensitivity to colistin, 16.6% to aztreonam and gatifloxacin each, 8.3% to amikacin and piperacillin/tazobactam each and 4.1% to piperacillin.
Amongst the cancer patients, Maschmeyer et al , have reported that 5-12% patients had infection with P. aeruginosa , while the incidence of infections due to P. aeruginosa was 1-2.5% in patients with febrile neutropenia. There were no differences of the incidence of Pseudomonas infections between neutropenic and non-neutropenic cancer patients, between patients with solid tumors and those with haematolymphoid malignancies.  There is paucity of data for multidrug-resistant P. aeruginosa infections among Indian cancer patients. In our study, we found that 2 out of 12 cancer patients had haematolymphoid malignancy with febrile neutropenia.
Among the 26 patients with MBL-producing P. aeruginosa who survived, eight (30.7%) needed readmissions to the hospital of which seven were diabetic patients admitted with either non-healing wound or urinary tract infections and one was a cancer patient admitted because of deterioration in clinical condition due to progressive disease.
Pseudomonas aeruginosa are responsible for 3-7% bloodstream infections and high mortality rates (27-48%) in critically ill patients.  We report five (15.1%) of 33 bloodstream infections due to P. aeruginosa and seven (21.2%) of 33 mortality cases in ICU patients. The predominant causes of death were chronic renal failure due to septicemia, terminal respiratory arrest due to septicemia and septicemia with progressive metastatic carcinoma.
Thus, the rapid dissemination of MBL producers is worrisome and necessitates the implementation of not just surveillance studies but also proper and judicious selection of antibiotics, especially carbapenems.
We thank Dr. K.D. Nihalani, Medical Superintendent, S.L. Raheja Hospital for allowing us to publish this data
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