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Cite this article: Sujakhu C, Prajapati KG, Amatya J (2018) METALLO-β-LACTAMASE Production and Antibiotic Susceptibility Pattern of Pseudomonas aeruginosa Isolated from Clinical Samples. JSM Microbiology 6(1): 1050.

*Corresponding authorCarrol Sujakhu, Department of Microbiology, St. Xavier’s College, Kathmandu, Nepal, Tel: 977-9843025606; Email:

Submitted: 02 October 2018

Accepted: 17 November 2018

Published: 20 November 2018

Copyright© 2018 Sujakhu et al.

OPEN ACCESS

Keywords•Pseudomonasaeruginosa; Carbapenems; Metallo

beta lacatamases; Imipenem-EDTA; Combined disc test (CDT); Double disc synergy test (DDST)

Research Article

METALLO-β-LACTAMASE Production and Antibiotic Susceptibility Pattern of Pseudomonas aeruginosa Isolated from Clinical SamplesCarrol Sujakhu1*, Krishna Govinda Prajapati2, and Jeena Amatya1

1Department of Microbiology, St. Xavier’s College, Nepal2Department of Microbiology, B & B Hospital, Nepal

Abstract

Background/Purpose: Pseudomonas aeruginosa is one of the most common gram-negative bacterium identified in the clinical specimens of hospital admitted patients. Carbapenems are used as the last resort for the treatment of MDR gram-negative bacterial infection. Resistance to this life saving drug has been increasingly reported in Pseudomonas which is mainly due to the production of MBL. Therefore, this study was conducted with an aim to assess the prevalance of MBL producing strains among MDR P.aeruginosa.

Methods: Total of 5016 samples were analysed at the microbiology department of B and B hospital, Gwarko, Lalitpur from June 2017 to December 2017 for routine culture and antibiotic susceptibility testing. Organisms were identified by conventional microbiological method and antibiotic susceptibility test was performed by modified Kirby-Bauer disc diffusion (CLSI 2014) guidelines. Imipenem resistant strains were then subjected to screening for MBL production by Imipenem- EDTA CDT and Imipenem-EDTA DDST.

Results: 1426 (28.43%) showed significant growth and among 142 (9.96%) were P. aeruginosa isolates. A total of 44 (30.99%) isolates of P. aeruginosa showed resistance to Imipenem out of which 34 (77.27%) were screened as MBL producers by CDT whereas 32 (72.72%) isolates gave positive result by DDST. Prevalence of metallo β-lactamases producing P. aeruginosa strains was 23.94%.

Conclusion: Thus, the study showed increasing trend of metallo β- lactamase producing isolates indicating the need for routine surveillance and timely control of the spread of these isolates.

ABBREVIATIONSA/A: Acid/ Acid; c Susceptibility Test; BA: Blood Agar;

CAZ: Ceftazidime; CCU: Critical Deficient Agar; CVP: Central Venous Catheter Tip; DDST: Double Disc Synergy Test; DNA: Deoxy Ribonucleic Acid; EDTA: Ethylene Diamine Tetra Acetic Acid; ESBL: Extended Spectrum B eta Lactamase; Gm: Gram; GS: Gram’s Stain; HAI: Hospital Acquired Infection; H2S: Hydrogen Sulphide; ICU: Intensive Care Unit; IMP: Imipenemase; PD: Inpatient Department; IPM: Imipenem; ITCU: Intensive Trauma Care Unit; LF: Lactose fermenting; LPS: Lipopolysaccharide; MA: Mac Conkey Agar; MBL: Metallo β- lactamase; MDR: Multi Drug Resistance; MHA: Mueller Hinton Agar; µg: Microgram; ml: Milliliter; MR: Methyl Red; MRSA: Methicillin-Resistant Staphylococcus aureus; NA: Nutrient Agar; NLF: Non-Lactose fermenting; No.: Number; OPD: Outpatient Department; PBP: Penicillin Binding Proteins; POW: Post-Operative Ward; PCR: Polymerase Chain Reaction; RNA: Ribonucleic Acid; SIM Media: Sulfide Indole Motility; SPSS: Statistical Package for Social

Science; TSI: Triple Sugar Iron; TUTH: Tribhuvan University Teaching Hospital; VIM: Veronese Imipenemase; VP: Voges Proskauer; WHO: World Health Organization; W/P: Wound/Pus; %: Percentage; °C: Degree Celsius

INTRODUCTIONPseudomonas aeruginosa is a Gram negative, non-sporing,

non-capsulated, straight rod shaped bacterium occurring singly, in pairs or in short chains. P.aeruginosa is an increasing prevalent opportunistic human pathogen found in nosocomial infections. P.aeruginosa is an increasing prevalent opportunistic human pathogen found in nosocomial infections. MBL producing P.aeruginosa isolates have been responsible for serious infections, treatment failure and several nosocomial outbreaks in different parts of the world resulting in high morbidity and mortality, increased economic burden and an urgent need to establish a strong infection control protocol. P. aeruginosa is also the second leading cause of nosocomial pneumonia (14 to 16%), third most common cause of urinary tract infections (7 to 11%), fourth most

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frequently isolated pathogen in surgical site infections (8%), and seventh leading contributor to bloodstream infections 2 to 6% [1].

The beta-lactam antibiotics are the largest and most commonly used group of antibiotics globally. Bacteria often develop resistance to β-lactam antibiotics by synthesizing β- lactamases, an enzyme that attacks the β-lactam ring. There are four classes of beta -lactamases (A–D) based upon DNA sequence similarity. Classes A, C, and D utilize serine on the active site, while the class-B enzymes (MBLs) usually utilize zinc [2]. Metallo β-lactamases belong to Amber class B type of β- lactamase [3]. The updated classification system includes group 1 (class C) cephalosporinases; group 2 (classes A and D) broad-spectrum, inhibitor-resistant beta lactamases, ESBLs and serine carbapenemases; and group 3 (class B) MBLs [4]. Excessive use of broad spectrum antibiotics in hospitals has led to the emergence of resistance strain of P. aeruginosa. To reduce the selection pressure for resistance, it is important to determine antibiotic susceptibility pattern of organism which helps in appropriate treatment with narrow spectrum and target specific antibiotics [5]. Carbapenems have been the predominant antibiotic class for treatment of P. aeruginosa infection because of their stability against most β-lactamases. However, Pseudomonas aeruginosa can develop resistance to carbapenems through diminished permeability, stable depression of chromosomal AmpC β-lactamases, or over-expression of the up-regulating efflux system. Metallo-β-lactamase (MBL) mediated resistance to carbapenem is an emerging threat in Pseudomonas isolates. The MBLs are plasmid mediated, so the resistance can be spread among hospital pathogens and will cause problem in treating infection namely wounds and burns. Therefore, it is important to know the status of metallo β- lactamase producing P. aeruginosa in the hospital to prevent the spread of resistant bacteria.

Metallo beta lactamase producing P. aeruginosa strains are responsible for several nosocomial outbreaks in the hospitals in the world. Therefore, detection of these MBL producing P. aeruginosa is crucial for proper treatment of critically ill patients and to prevent the spread of resistance bacteria. The aim of this study was to detect metallo beta lactamase producing Pseudomonas aeruginosa from different clinical samples.

METHODS

Study site

This study was conducted at B and B Hospital Pvt. Ltd, Gwarko, Lalitpur.

Study design and setting

This research was hospital based cross-sectional study conducted in the Department of Microbiology of the B and B Hospital Pvt. Ltd.

Study population

The study populations were patients who attended B & B Hospital, either as inpatients or outpatients. The collection of specimens was performed by laboratory staffs in case of outpatients and by duty doctors/nurses of concerned ward in case of inpatients.

Study duration

The study period for this entire research project was 6 months from15th June 2017 to 15th December 2017.

Ethical approval

This study was ethically approved by B and B Review Committee (BBRC).

Sample size and sample types

A total of 5016 different samples were included under study. The specimens included urine, wound/pus, sputum, tips, bronchial secrections and different body fluids like CSF, peritoneal fluid, pleural fluid, joint/synovial fluid, ascitic fliud which were sent to laboratory for routine culture and antibiotic susceptibility testing were processed during the study period.

Sample processing

The samples were collected and proceeded in the lab with specific treatment for each specimen.

Culture

The collected and processed samples were inoculated on blood agar, chocolate agar and Mac Conkey agar respectively. Blood agar plates and Mac Conkey agar plates were incubated aerobically at 37°C for 24 hours whereas chocolate agar plates were incubated anaerobically at 37°C for 24 hours.

On the following day, colonial morphology was noted for the isolated colonies. Gram’s staining was performed for isolated colonies and sub-cultured on nutrient agar.

Biochemical tests

The bacterial isolates were characterized and identified on the basis of their colonial, morphological study and biochemical characteristics. Coagulase, Catalase, Motility, Vogesproskauer, Indole production, Citrate utilization, Oxidase, Methyl red and Sugar fermentation tests were performed.

Antibiotic susceptibility testing

Antibiotic susceptibility tests of all the clinical isolates towards various antibiotics were performed by Kirby-Bauer disk diffusion method as recommended by Clinical Laboratory Standard Institute (CLSI) 2014.

Detection of Metallo-β-lactamase producing strain

In this study, isolates showing resistance to Imipenem were subjected for the detection of possible MBL production. Test organisms were inoculated onto plates of Mueller-Hinton agar. Detection of MBLs was done by two phenotypic methods CDT and DDST.

Combined Disc Test (CDT)

Two 10 µg Imipenem discs were placed on the Mueller Hinton agar (MH) media 30 mm apart inoculated with test organism (maintaining 0.5 McFarland standards). To one of the Imipenem discs, 0.5 M 10 µL EDTA solution was added. After 16 h of incubation at 35°C, the zone of inhibition around Imipenem and Imipenem + EDTA discs were compared. If the zone of inhibition

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of Imipenem + EDTA discs compared to Imipenem alone is greater than 7 mm, then the test organism can be considered as MBL positive.

Double Disc Synergy Test (DDST)

Imipenem discs (10 µg) was placed on the Mueller Hinton agar media inoculated with test organism and 10 mm apart a blank filter study disc was placed to which 0.5 M 10 µL EDTA solution was added. Enhancement of zone of inhibition between IPM and EDTA disk was considered as MBL positive.

Data analysis

All findings were entered in an MS Access data sheet and on completion of the study; data was statistically analyzed in computer software program SPSS version 23.0. Statistical data analysis was performed for descriptive statistics, including frequencies and cross tabulation of microbiological characteristics. Data was also recorded manually.

RESULTSThe clinical specimens included were urine 615/1426

(43.13%), wound/pus 535/1426 (37.52%), sputum 126/1426 (8.84%), tips 94/1426 (6.73%), blood 23/1426 (1.61%), other (synovial fluid, chest tube fluid, E.T tube, throat swab, perianal swab, vaginal swab, drain fluid) 17/1426 (1.19%) and different samples as shown in Table 1.

Out of the 5016 specimens, 1426 (28.43%) showed positive growth among which isolates, Escherichia coli was found to be the most predominant constituting 538/1426 (37.72%). Other most frequently isolated organisms were Klebsiellapneumoniae 219/1426 (15.36%), Pseudomonas aeruginosa 142/1426(9.96%), Coagulase negative Staphylococci 113/1426 (7.92%) and other pathogens as shown in Table 2.

Among the 142 isolated Pseudomonas aeruginosa, 80.98% (n=115) were isolated from male patients and remaining 19.02% (n=27) were from female patients. Sex-wise, male patients constituted a larger group in this study. The result showed that male individuals were much prone to Pseudomonas aeruginosa infection than female individual. There was significant association (p-value < 0.05) between sex and growth of organisms (Table 3).

Out of 142 Pseudomonas aeruginosa isolates, highest growth was obtained from the age group 30-40 (25.4%), of which 88.88% (n=32) were male and 2.81% (n=4) were female. There was significant association (p-value < 0.05) between age group and growth of organisms (Table 4).

P. aeruginosa isolates exhibited maximum resistance towards Ceftriaxone (59.16%) followed by Ofloxacin (55.64%), Ciprofloxacin (49.3%) and Gentamicin (49.3%). Only 30.99% of organisms showed resistance towards Imipenem. Most of the isolates were susceptible towards Imipenem (66.90%) followed by Meropenem (61.98%), Piepracillin/Tazobactam (56.34%) and Amikacin (50.70%). The organism showed least susceptibility towards Ceftriaxone (39.44%). All the isolates showed sensitivity towards Colistin (100%) (Table 5).

Table 1: Distribution of various specimens with significant growth.

Name of Specimen Total no. of specimen( n )

Percentage( % )

Urine 615 43.13

Wound/pus 535 37.52

Sputum 126 8.84

Catheter tip 65 4.56

Blood 23 1.61

CVP tip 23 1.61

Drain tip 8 0.56

Bile 7 0.49

Semen 7 0.49

Others 17 1.19

Total 1426 100

Table 2: Isolated organisms with their frequency and percentage.

Name of organism No. of organism (n) Percentage (%)

E. coli 538 37.72

Klebsiellapneumoniae 219 15.36

Pseudomonas aeruginosa 142 9.96

CoNS 113 7.92

Acinetobacterspp. 102 7.15

Enterococcus spp. 72 5.06

MRSA 56 3.93

S. aureus 36 2.52

Enterobacterspp 35 2.45

Salmonella Typhi 8 0.56

Others 105 7.36

Total 1426 100

Table 3: Distribution of P. aeruginosa according to gender.

Sex of the patientPseudomonas aeruginosa isolated

P-valueNumber (n) Percentage (%)

Male 115 81.0%

0.0001Female 27 19.0%

Total 142 100%

Table 4: Distribution of P. aeruginosa according to age.

Age interval of patient (yrs.)

Pseudomonas aeruginosa isolated P-value

Number Percentage (%)0-10 7 4.9%

0.000

10-20 12 8.5%20-30 32 22.5%30-40 36 25.4%40-50 12 8.5%50-60 14 9.9%60-70 14 9.9%70-80 12 8.5%80-90 3 2.1%

90-100 0 0.0%Total 142 100%

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Among the total 142 P. aeruginosa isolates, only 90 (63.38%) were MDR. Isolates from wound/pus sample showed maximum percentage 55 (61.11%) MDR followed by urine 17 (18.88%), sputum 8 (8.88%), catheter tip 8 (8.88%) and other samples as shown in Table 6.

Out of total 90 MDR P. aeruginosa isolates, 51 (56.66%) of the isolates were from IPD that is followed by OPD 13 (9.15%) and POW 13(9.15%). Only 6.33% of the isolated multidrug resistant P. aeruginosa were from CCU. 2.81% MDR were isolated from other departments (ANNX-A, ANNX-B). Highest numbers of MDR isolates were isolated from inpatient department. The result is as shown in the Table 7.

Total of 34 MBL positive isolates shown by CDT, 12/34 (35.29%) were from IPD, 9/34 (26.47%) from OPD, 5/34 (14.70%) from POW, 6/34 (17.65%) from CCU and 2/34 (5.88%) from others (EMR, ANNX-A, ANNX-B). Similarly, out of total 32 MBL positive isolates shown by DDST, 12/32 (37.50%) were from IPD, 7/32 (21.87%) from OPD, 5/32 (15.63%) from POW and 2/32 (6.25%) from others (EMR, ANNX-A, ANNX-B). The result is as shown in Table 8.

Pseudomonas aeruginosa were mostly isolated from the wound/pus sample than other samples processed in the laboratory. Out of total 142 Pseudomonas aeruginosa isolates, maximum numbers of organisms were isolated from wound/

pus 54.22% (n=77) followed by sputum 16.19% (n=23), urine 14.79% (n=21), catheter tip 10.56% (n=15), bile fluid 2.81% (n=4) and from other samples as shown in Figure 1.

Among 142 positive cases, 53.52% (n=76) specimens were from IPD, 19.71% (n=28) specimens were from OPD, 14.08% (n=20) specimens were from POW and 9.85% (n=14) specimens were from CCU units. Highest numbers of P. aeruginosa were isolated from Inpatient Department (53.52%) which was followed by Outpatient Department (19.71%) (Figure 2).

Out of total 142 P. aeruginosa isolates, 90 isolates were found to be MDR. Out of which 42/90 (46.67%) were imipenem resistant as well as MDR and 48/90 (53.33%) were imipenem sensitive but MDR isolates. But, only the imipenem resistant P. aeruginosa (n=44) including 42 MDR isolates and 2 non-MDR isolates were further proceeded for the detection of MBL production by CDT and DDST method (Figure 3).

Out of 90 MDR Pseudomonas aeruginosa, 37.77 % (n=34) showed the production of Metallo β-lactamase enzyme where as in absence of MDR no isolates showed the production of Metallo β-lactamase enzyme (Figure 4).

Total 44/142 (30.98%) isolates were found to be imipenem resistant. And the imipenem resistant isolates were further processed for the detection of MBL production by two methods

Table 5: Antibiotic Susceptibility pattern of Pseudomonas aeruginosa isolates (n = 142).

Name of AntibioticsSensitive Intermediate Resistance

No. of isolates (n) Percentage (%) No. of isolates

(n) Percentage (%) No. of isolates (n) Percentage (%)

Amikacin 72 50.70 11 7.75 59 41.55

Cefepime 69 48.59 10 7.04 63 44.37

Ceftazidime 65 45.78 8 5.63 69 48.59

Ceftriaxone 56 39.44 2 1.40 84 59.16

Cipfofloxacin 70 49.30 2 1.40 70 49.30

Gentamicin 68 47.90 4 2.80 70 49.30

Imipenem 95 66.90 3 2.11 44 30.99

Meropenem 88 61.98 2 1.40 52 36.62

Ofloxacin 62 43.66 1 0.70 79 55.64Piepracillin/Tazobactam 80 56.34 19 13.38 43 30.28

Colistin 142 100 0 0 0 0

Table 6: Distribution of MDR Pseudomonas aeruginosa isolates on the basis of specimen.

Name of specimen Total no. of specimen Percentage (%)

Wound/pus 55 61.11

Urine 17 18.89

Sputum 8 8.89

Catheter tip 8 8.89

Suction tip 1 1.11

Bile fluid 1 1.11

CVP tip 0 0

Total 90 100

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Table 7: MDR distribution of Pseudomonas aeruginosa isolates on the basis of patient department.

Patient DepartmentTotal no. of isolated MDR Pseudomonas

aeruginosaPercentage %

OPD 13 9.15

IPD 51 56.66

POW 13 9.15

CCU 9 6.33

Others 4 2.81

Total 90 100

Table 8: Distribution of MBL producing Pseudomonas aeruginosa isolates among different wards.

Type of specimen

MBL production by CDST

MBL production by DDST

IPD 12 12

OPD 9 7

POW 5 5

CCU 6 6

Other 2 2

Total 34 32

Figure 1 Distribution of P. aeruginosa in various samples.

Figure 2 Distribution of P. aeruginosa according to different wards.

CDT and DDST. The MBL producing isolates were found as 34/44 (77.27%) by CDT and 32/44 (72.72%) by DDST method. Those isolates which were found to be MBL positive by DDST (n=32) were also found to be MBL positive by CDT (Figure 5).

Out of total 142 P. aeruginosa isolated form overall specimen, only 23.94% (n=34) were Metallo β- lactamases producer. And remaining 76.06% (n=108) of isolates did not produce the Metallo β-lactamase enzyme. The non MBL producer P. aeruginosa were isolated more than the MBL producer from the total no. of P. aeruginosa isolates (Figure 6).

DISCUSSSIONPseudomonas aeruginosa is a pervasive pathogen in hospital

acquired infections, especially among critically ill patients. Multidrug resistance in P. aeruginosa has appeared as an issue of great concern with emergence of MBL P. aeruginosa.

Out of the 5016 specimens, 28.43% of the sample showed significant growth. Among 1426 positive isolates, 9.96% were P. aeruginosa. The proportion of isolation of P. aeruginosa among various bacterial pathogens isolated ranges in various studies: 29.6%, 8.7%, and 19.26% [6]. The difference in the result may be due to the other variables that influence the outcome of results such as clinical specimens received for examination, study

Figure 3 Distribution of imipenem resistance Pseudomonas aeruginosa isolates among MDR Pseudomonas aeruginosa.

Figure 4 Distribution of MDR and MBL P. aeruginosa isolates.

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population, type of hospitals and geographical locations. In other tertiary care hospital of Nepal, the proportion of isolation of P. aeruginosa was 13% [7].

In this study the highest percentage of P. aeruginosa isolates were observed in wound/pus sample which yields 54.22% of the total isolates followed by sputum 16.19%, urine 14.79%, and catheter tip 10.56%. In a similar type of study maximum number of P. aeruginosa (17%) were isolated from wound/pus specimen [8]. In this study 53.52% of the total P. aeruginosa isolates were obtain from IPD followed by OPD (19.71%), POW (14.08%) and ICU (9.85%).

Among P. aeruginosa, 80.98% were isolated from male patients and remaining 19.02% were from female patients in this study. Sex-wise, male patients constituted a larger group in this study. The result showed that male individuals were much prone to Pseudomonas aeruginosa infection than female individual. This may be due to the presence of some protective factors in female or presence of some aggressive factors in males following easy infection by these organisms.

The antibiotic susceptibility test of P. aeruginosa isolated in this study, showed higher sensitivity towards Imipenem (66.90%) followed by Meropenem (61.98%) whereas highest resistance was observed towards Ceftriazone (59.16%) followed by Ofloxacin (55.64%). Resistance to Ciprofloxacin (49.30%), Gentamicin (49.30%), Ceftazidime (48.59%), Amikacin (41.55%) and Piperacillin+ Tazobactam (30.28%) were shown by the isolates. Imipenem was consistently the most active agent (90.6%) against most of the Pseudomonas isolates. This study showed

30.99% and 36.62% resistance to Imipenem and Meropenem respectively. The resistance toward the Carbapenem shown by P. aeruginosa is mainly due to the development of impermeability due to loss of OprDporin, upregulation of an active efflux system and production of MBLs [9]. The result from the research also emphasize towards the use of combined antibiotics in treatment of pseudomonal infection as Piperacillin/Tazobactam showed sensitivity of 56.34% and the previous studies also showed the same outcome. All the P. aeruginosa isolates were resistance to Colistin (100%).The use of Polymyxins (Polymyxin B and Colistin) was limited due to their nephrotoxicity.

In this study, 63.38% of the isolates were multi drug resistant and most of the multi-drug resistant isolates were isolated from wound/pus sample followed by urine. In a study done in hospitals of Nepal, higher prevalence of MDR P. aeruginosa was observed. Gupta et al (2012) found 65.9% MDR P. aeruginosa [7].Comparing the multidrug resistance pattern of the P. aeruginosa isolates maximum MDR were isolated in IPD (56.66%) from wound/pus (61.11%) sample. The finding was different to the findings, where ICU P. aeruginosa isolates were highly MDR compared to other wards. The high percentage of resistance to antibiotics in the hospital indicates the improper use of antibiotics and commonly used drugs can no more be used as empirical therapy for suspected pseudomonad infection.

The prevalence of MBL producing P. aeruginosa strains was found to be 23.94% in this study. In another study conducted by Kumar et al, (2012) [10]. In a study conducted at Tribhuvan University Teaching Hospital (TUTH) by Mishra et al (2012) prevalence of MBLs producing P. aeruginosa was found to be

Figure 5 Comparison of MBL production among IMP resistance P. aeruginosa isolates by CDT and DDST.

Figure 6 Distribution of MBL in total no. of P. aeruginosa isolates.

Photograph 1 Growth of Pseudomonas aeruginosa on Cetrimide Agar.

Photograph 2 Biochemical test of Pseudomonas aeruginosa.Left to right (Indole -ve, MR -ve, VP –ve, Citrate +ve, TSIA Alk/Alk G, Urease –ve and OF oxidative)

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phenotypic techniques for detecting MBL. In contrast to this result, the study of John et al, (2011), [14] and Khosravi et al, (2012), [15] showed that DDST more specific in detecting MBL in comparison to CDT. CDT and DDST both are limited by factors like temperature, aeration, pH, and thickness of media. However, the synergy between IPM and IPM+EDTA disc is influenced by diffusion. EDTA must diffuse close to IPM disc and achieve a concentration with effective chelating activity to demonstrate a synergy. Since, there are no standard guidelines for the detection of MBL, and different studies have reported the use of different methods. PCR analysis is the gold standard method for the detection of MBL production, but it is not feasible in the routine microbiology laboratory.

Therefore, early detection of infection due to these microorganisms is necessary as the appropriate treatment might reduce the spread of these resistant strains as well as the morbidity and mortality in hospitalized patients.

ACKNOWLEDGEMENTAuthor would like to acknowledge Mr. SanjitShrestha, BMLT,

MSc. Medical Microbiology, Incharge, Pathology Department B & B Hospital for his advice and support throughout my study.

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Photograph 3 Antibiotic susceptibility test of Pseudomonas aeruginosa 1=Ceftazidime (Resistant), 2=Imipenem (Resistant), 3=Piperacillin/Tazobactum (Intermediate), 4=Meropenem (Resistant), 5=Cefepime (Resistant).

Photograph 4 Detection of MBL by Imipenem- EDTA method.(1=Blank disc with 10μl EDTA, 2=Imipenem Disc and 3=Imipenem disc with 10μl EDTA).

3.3% [11]. Finding of this study was slightly higher than the previous study done in Nepal showing increasing trend of the MBL producing isolates due to the increased β- lactam usage and emergence of resistant bacteria under antibiotic pressure.

All of metallo β- lactamase producing isolates in this study were MDR and resistant to most of the antibiotics used; third generation carbapenem, cephalosporin and aminoglycosides. 37.77 % MDR showed the production of MBL enzyme where as in absence of MDR no isolates showed the production of MBL enzyme. Studies have shown higher prevalence of MDR among MBL producing strains, as compared to that in non-MBL producing strains. Imipenem resistant isolates were further proceeded for the detection of MBL production by CDT and DDST method. Some researchers recommended ceftazidime disc instead of imipenem disc in CDT/DDST [12,13]. But as there may be other CAZ resistance mechanism in MBL producers, CDT/DDST using CAZ disc will not detect MBL production and hence IPM disc is better option for screening MBL [7,9].

In this study, 30.98% isolates were IPM resistant and among them 77.27% were MBL positive by CDT and 72.72% by DDST. In the previous study, among the IMP resistant isolates 94.52% were MBL positive by CDT and 89.04% by DDST. These studies showed CDT to be superior to DDST and one of the most sensitive

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Cite this article

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15. Khosravi Y, Loke MF, Chua EG, Tay ST, Vadivelu J. Phenotypic Detection of Metallo-β-Lactamase in Imipenem-Resistant Pseudomonas aeruginosa. The Scientific World Journal. 2012.