INTRODUCTION:

Urinary tract infections (UTIs are the second most common bacterial infections in general practice and a frequent indication for prescription of antimicrobials [1]. Community acquired infection is caused by Escherichia coli, Klebsiella pneumoniae (K. pneumonia), Proteus mirabilis (P. mirabilis), Staphylococcus saprophyticus and Enterococcus faecalis [2]. Resistant Extended spectrum β lactamases (ESBL bacteria are increasingly reported worldwide, both in community and hospital settings [3].

They are mainly found in Escherichia coli, Klebsiella species and Proteus species but can occur in other members of Enterobacteriaceae family [4]. Indian studies have reported 26 to 48% of Enterobacteriaceae causing UTI were ESBL producers [5]. Treatment failure and clinical mortality are also more likely to occur in patients infected with ESBLs-producing P. mirabilis [6]. The emergence and global spread of carbapenemase producing Enterobacteriaceae in recent years, especially isolates carrying genes encoding K. pneumoniae carbapenemase and NDM (New Delhimetallo-β-lactamase carbapenemases, have compromised options available for treatment [7,8]. ESBL have the ability to hydrolyse and cause resistant to various type of newer β-lactam antibiotics including the extended–spectrum cephalosporins, monobactams and carbapenems [9]. Prevalence of ESBLs varies from region and region. Previous studies from India have reported ESBL production varying from 6% to 87% [10-12]. The present trends of the uropathogens and their susceptibility to various antibiotics are essential to formulate guidelines for the empirical treatment of UTIs. Hence the present study.

MATERIAL AND METHODS:

A total of 3415 urine samples clinically suspected with UTI were collected from October 2013 to May 2015. Laboratory Samples received included mid-stream clean catch urine, suprapubic aspirate and from Foley’s catheter. Samples were processed and isolates were identified as per standard methods [13].

Antimicrobial Susceptibility Testing:

Antimicrobial susceptibility was determined by Kirby-Bauer disk diffusion method as per CLSI recommendations [14]. Antimicrobial disks used were Ampicillin (10μg), Amoxycillin-clavulanic acid (20/10μg), Piperacillin (100μg), Piperacillin-tazobactam (100/10μg , Nitrofurantoin (300µg ), Ciprofloxacin (5μg), Ofloxacin (5μg ), Cefuroxime (30μg), Ceftriaxone (30μg), Ceftazidime (30μg), Gentamicin (10μg), Amikacin (30μg), Tobramycin (30μg), Co-trimoxazole (1.25/23.75 μg), Aztreonam (30μg) and Imipenem (10μg). (Hi media, Mumbai).

Screening test for ESBLs:

Screening of ESBLs was done as per CLSI guidelines, isolates showing inhibition zone size of <22 mm with Ceftazidime (30 µg , <25 mm with Ceftriaxone (30 µg , and <27 mm with Cefotaxime (30 µg were identified as potential ESBL producers and shortlisted for confirmation of ESBL production

Confirmatory Tests for ESBLs:

1. Phenotypic confirmatory test with combination disk

Disk of Ceftazidime (30µg) and a disk of Ceftazidime +Clavulanic acid (30 µg/10 µg) were used. Both the disks were placed at least 25 mm apart, center to center, on a lawn culture of the test isolate on Mueller Hinton Agar plate and incubated overnight at 37°C. Difference in zone diameters with and without clavulanic acid was measured. When there is an increase of >5 mm in inhibition zone diameter around combination disk of Ceftazidime +Clavulanic acid versus the inhibition zone diameter around Ceftazidime disk alone, it confirms ESBL production

2. MIC reduction test (E test)

The isolates positive with combination disk test were further confirmed for ESBL production by this test. Minimum inhibitory concentration of the isolates was determined by Broth dilution method. The values of range of concentration of antibiotics tested were as follows:

Ceftazidime: 0.25 µg/mL to 128 µg/mL Ceftazidime-clavulanic acid: 0.25/4 µg/mL to 128/4 µg/mL

Interpretation: A >3 two-fold decrease in MIC for Ceftazidime when tested in combination with clavulanic acid versus its MIC when tested alone indicates that the strain is an ESBL producer [14]. K. pneumoniae ATCC 700603 (an ESBL producer was used as control strain.The data obtained was analyzed using Microsoft excel (2010 version. The results are explained in frequency and percentage.

RESULTS:

Out of 3415 samples collected, 227 K. pneumoniae and 186 P. mirabilis were isolated. The age and sex distribution of K. pneumoniae cases is shown in table 1.

Table 1: Age and sex distribution of K. pneumoniae cases (n=227

Age group (years

Male

Female

Total

0-10

11-20

21-30

31-40

41-50

51-60

61-70

Total

104

123

227

Maximum number of cases were from females in the age group of 31-40 years.

Out of 227 K. pneumoniae, 85(37.4% were ESBL producers. The antibiogram of ESBL and non-ESBL producers is shown in table 2.

Table 2: Antibiogram of K. pneumoniae (Resistance pattern)

Antibiotic	ESBL producers (n=85)	Non ESBL producers (n=142)
Antibiotic	n (%)	n (%)
Ampicillin	85 (100)	39 (27.4)
Amoxycillin-clavulanic acid	55 (64.7)	22 (15.4)
Piperacillin	85 (100)	47 (33)
Piperacillin-tazobactam	22 (25.8)	4 (2.8)
Nitrofurantoin	15 (17.6)	27 (19)
Ciprofloxacin	39 (45.8)	56 (39.4)
Ofloxacin	37 (43.5)	43 (30.2)
Cefuroxime	35 (41.1)	19 (13.3)
Ceftriaxone	26 (30.5)	15 (10.5)
Ceftazidime	29 (34.1)	14 (9.8)
Gentamicin	41 (48.2)	34 (23.9)
Amikacin	39 (45.8)	29 (20.4)
Tobramycin	38 (44.7)	28 (19.7)
Co-trimoxazole	33 (38.8)	22 (15.4)
Aztreonam	0	0
Imipenem	0	0

Highest resistance was seen with Ampicillin and Piperacillin and least with Aztreonam and Imipenem. The age and sex distribution of P. mirabilis cases is shown in table 3.

Table 3: Age and sex distribution of Proteus mirabilis cases (n=186

Age group (years)	Male	Female	Total
0-10	2	1	3
11-20	5	2	7
21-30	14	18	32
31-40	24	28	52
41-50	17	16	33
51-60	22	32	54
61-70	3	2	5
Total	87	99	186

Maximum number of cases were from females in the age group of 51-60 years.

Out of 186 P. mirabilis, 59 (31.7%) were ESBL producers. The antibiogram of ESBL and non-ESBL producers is shown in table 4.

Table 4: Antibiogram of P. mirabilis (Resistance pattern)

Antibiotic	ESBL producers (n=59)	Non ESBL producers (n=127)
Antibiotic	n (%)	n (%)
Ampicillin	59 (100)	127 (100)
Amoxycillin-clavulanic acid	49 (83)	104 (81.8)
Piperacillin	54 (91.5)	96 (75.5)
Piperacillin-tazobactam	11 (18.6)	2 (1.5)
Nitrofurantoin	7 (11.8)	11 (8.6)
Ciprofloxacin	24 (40.6)	34 (26.7)
Ofloxacin	19 (32.2)	23 (18.1)
Cefuroxime	13 (22)	11(8.6)
Ceftriaxone	11 (18.6)	9 (7)
Ceftazidime	11 (18.6)	12 (9.4)
Gentamicin	12 (20.3)	12 (9.4)
Amikacin	7 (11.8)	6 (4.7)
Tobramycin	7 (11.8)	9 (7)
Co-trimoxazole	6 (10.1)	12 (9.4)
Aztreonam	0	0
Imipenem	0	0

Highest resistance was seen with Ampicillin, Amoxycillin-clavulanic acid and Piperacillin, least with Aztreonam and Imipenem

DISCUSSION:

In the present study, prevalence of ESBL producing K. pneumoniae was 37.4%. Other stduies conducted in India have reported prevalence of ESBL producing K. pneumoniae varying from 6% to 87% [10-12]. In recent years, a significant increase in ESBL producers was reported from USA, Canada, China, and Italy [15-18]. Highest resistance of K. pneumoniae was seen with Ampicillin and Piperacillin and least with Aztreonam and Imipenem (table 2. Other studies have also reported Imipenem as the most effective drug [20-21].

After Impinem and Aztreonam, the most sensitive drugs for K. pneumoniae were Piperacciilin + Tazobactum and Nitrfurantoin. Resistance to Quinolone group and Cephalosporin group is a cause of concern. Other studies support these findings [22-23]. Nitrofurantoin can be used for most non-complicated threatening urinary tract infections caused by ESBL producing isolates. This finding is also evident in other studies [24-25].

In the present study, out of 186 P. mirabilis, 59 (31.7% were ESBL producers. Infection was more in females and in the age group of 51-60 years, probably due to predisposing factors like surgery, sepsis, catheterization and diabetes mellitus. There is lack of data regarding prevalence of ESBL P. mirabilis, studies conducted outside India have reported prevalence of 3% to 20% [26-28]. Highest resistance was seen with Ampicillin, Amoxycillin-clavulanic acid and Piperacillin, least with Aztreonam and Imipenem, same as with ESBL producing K. pneumoniae (table 4). After Impinem and Aztreonam, the most sensitive drugs for P. mirabilis were Aminoglycosides and Nitrfurantoin. Similar findings were reported by other authors [29-30].

There is a wide variability of antibiotic resistance pattern among K. pneumoniae and P. mirabilis. The reasons might be, geographical variation, local antibiotic use and disease patterns. There is a paucity of data on antibiotic consumption from India, the available data suggest that it is higher than other developing nations of the world. Rates are further lower in developed nations. Use of carbapenem and piperacillin-tazobactum has increased significantly in 10 years [31-33]. The other causes for resistance against K. pneumoniae among different third generation might be due to production of Amp C beta lactamases and production of porins [34-35].

Limitations of the study:

We did not test for Amp C and metallo beta lactamse (MBL), the study was done in only one centre. Future studies should be multicentric and should include testing for Amp C and metallo beta lactamse (MBL).

CONCLUSION:

The present study showed a high percentage of ESBL producers among clinical isolates of K. pneumoniae and P. mirabilis to commonly used antibiotics. Knowledge of antibiogram studies will help physicians choosing a empirical treatment. Regular antibiotic susceptibility studies should be conducted to know the changing trends in resistance. In addition to monitoring of Imipenem sensitivity and routine testing of newer carbapenemes like Meropenem and Ertapenem should be carried out further to detect early resistance and save these powerful antibiotics for life threatening infections.

CONFLICT OF INTEREST:

None

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Received on 20.08.2015 Modified on 10.09.2015

Research J. Pharm. and Tech. 8(11): Nov., 2015; Page 1465-1468

DOI: 10.5958/0974-360X.2015.00262.0