Abstract

Mecillinam is one of the very few oral antibacterial agents used against extended spectrum b-lactamase (ESBL) producing Escherichia coli (E. coli) causing urinary tract infection (UTI)). It is reported that, resistance to mecillinam can be reversed to some extent by adding beta lactamase inhibitor like clavulanic acid. The present study was aimed to determine in-vitro activity of mecillinam and mecillinam-clavulanic acid combination on the susceptibility of ESBL producing and non-ESBL producing E. coli. Total 124 E. coli (78 ESBL positive and 46 ESBL negative) isolates from urine samples of patients with UTI were included in the study. Organisms were isolated from patients attending BIRDEM General Hospital from July 2012 to December 2012. ESBL production was tested by double disc synergy test. Minimum inhibitory concentration (MIC) of mecillinam and clavulanic acid against E. coli was determined by agar dilution method. Of the total E. coli isolates, 62.9% was ESBL positive and 37.1% was negative for ESBL. Out of ESBL positive isolates, 75.6% was sensitive to mecillinam while ESBL negative isolates showed the sensitivity as 67.4%. The sensitivity to mecillinam of ESBL positive and negative isolates increased to 85.9% and 86.9% respectively by addition of clavulanic acid with mecillinam. The MIC values of intermediate and resistant isolates converted to sensitive MIC range after addition of clavulanic acid with mecillinam. Conversion of resistance of ESBL producing isolates by adding clavulanic acid was also evident by the reduction of MIC50 and MIC90 from 4µg/ml to £1 µg/ml and from 128 µg/ml to 64 µg/ml respectively. Similar trend of reduction of MICs was also observed in non-ESBLs.

In conclusion, both ESBL positive and negative E. coli demonstrated considerable sensitivity to mecillinam and the sensitivity increased significantly (p<0.05) by adding clavulanic acid with mecillinam.

Ibrahim Med. Coll. J. 2014; 8(2): 56-60

Address for Correspondence:Dr. Khandaker Shadia, Assistant Professor, Department of Microbiology, Ibrahim Medical College, 122 Kazi Nazrul Islam Avenue, Shahbag, Dhaka-1000.

Introduction

Escherichia coli (E. coli) remains an important cause of urinary tract infections (UTIs). UTI by extended-spectrum β-lactamase (ESBL) producing strains of E. coli is difficult to treat. Concomitant resistance to other non β-lactam antibiotics like aminoglycosides and fluoroquinolones has further complicated the situation and left very limited treatment options, especially per oral regimes.1 Mecillinam is an oral amidinopenicillin which acts by binding with penicillin binding protein-2 (PBP-2). It is relatively stable to β-lactamase enzymes and reaches very high concentration in urine.2 Thus mecillinam is proven to be a suitable antimicrobial agent against ESBL producing uropathogens like E. coli and Klebsiella spp. But, chromosomal mutation and few beta-lactamase induced mechanism results in mecillinam resistance in clinical isolates.3-5 Though mecillinam resistance has been reported from various part of the world it is less pronounced than other beta-lactams.6,7 In vitro studies have shown that mecillinam resistance conferred by higher inoculum can be reversed by addition of beta-lactamase inhibitors like clavulanic acid.8 Clavulanic acid is a beta lactam compound that has weak intrinsic antibacterial activity. When used in combination with other β-lactam drugs it exerts synergistic effect by inhibiting beta lactamase enzymes.9,10

The aim of the present study was to assess in vitro activity of mecillinam alone and in combination with clavulanic acid against ESBL producing and ESBL non-producing uropathogenic E. coli.

Material and Method

Bacterial strains

Total 124 E. coli strains isolated from urine samples were included in the study. Urine was collected from both indoor and outdoor patients attending BIRDEM General Hospital during the period of July 2012 to December 2012. Isolation and identification of the species was done according to standard laboratory methods.11

Detection of ESBL

Double disc synergy test was employed to detect ESBL production.12 Bacterial suspension of 0.5 McFarland standard was plated in Muller-Hinton agar with Amoxycillin-clavulanic acid (30 µg) disc in between and 20 mm apart from Ceftazidime (30 µg) and Ceftriaxone (30 µg) discs. Expansion of the zone of inhibition around Ceftriaxone and/or ceftazidime disc towards the amoxycillin-clavulanic acid disc was considered ESBL production.

Determination of MIC

Antimicrobial susceptibility of E. coli strains was tested by agar dilution method as described in CLSI guideline.11 Minimum inhibitory concentrations (MICs) of mecillinam was determined in Muller-Hinton agar plate containing two fold (Log2) serial dilutions of mecillinam (from 1024 µg/ml to 1 µg/ml) using standard inoculum of 1x104 cfu/spot. MICs of mecillinam were also determined in presence of clavulanic acid at a concentration of 0.04µg/ml in each plate. MIC values (µg/ml) were interpreted as: sensitive: £ 8, Intermediate: 16 and Resistant: ³ 32. E. coli ATCC 25922 was used as negative control and a mecillinam resistant E. coli isolates having MIC of >1024 µg/ml (pre-determined) was used as positive control. Pivmecillinam and potassium clavulanate were obtained from General Pharmaceuticals Ltd. Bangladesh, and Incepta Pharmaceuticals Ltd. Bangladesh.

Results

Table-1 shows the susceptibility pattern of ESBL positive and negative E. coli isolates to mecillinam and mecillinam+clavulanic acid according to MIC values. Mecillinam sensitivity was demonstrated in 75.6% ESBL positive and 67.4% ESBL negative isolates which was augmented to 85.9% and 86.9% respectively after addition of clavulanic acid. In ESBL positive and negative strains 5.2% and 8.7% isolates were intermediately sensitive to mecillinam, whereas none of the isolates were intermediate sensitive to mecillinam+ clavulanic acid combination. Resistance to mecillinam was found in 19.2% and 23.9% ESBL positive and negative isolates that were reduced to 14.1% and 13.1% after adding clavulanic acid with mecillinam respectively.

Table-1: Susceptibility pattern of ESBL positive and ESBL negative E. coli to mecillinam and mecillinam+ clavulanic acid according to MIC values (N=124)

 All the isolates having 16 µg/ml and 32 µg/ml MIC of mecillinam converted to sensitive after addition of clavulanic acid. Out of 5 isolates having MIC of 64µg/ml, 2 (40%) were converted to sensitive. But the isolates having MIC of ³ 128 demonstrated no change in susceptibility. Total 55.9% intermediately sensitive and resistant E. coli became sensitive to mecillinam by adding clavulanic acid (Table-2).

Table-2: Change of susceptibility in relation to MIC values of intermediate sensitive and resistant isolates after adding clavulanic acid

 The MIC of mecillinam against ESBL producing E. coli ranged from £1-³1024 µg/ml. MIC50 and MIC90 were 4 and 128 µg/ml respectively. After adding clavulanic acid with mecillinam MIC50 and MIC90 reduced to £1 µg and 64 µg/ml respectively. In non-ESBL producing E. coli isolates MIC50 and MIC90 were 4 and 64 µg/ml respectively with mecillinam and £1and 32 with mecillinam+clavulanic acid (Table-3).

Table-3: Change of MIC50 and MIC90 values of ESBL producing and non-ESBL producing E. coli isolates by addition of clavulinic acid with mecillinam

Discussion

ESBL producing E. coli is isolated in very high frequency in nosocomial as well as community acquired urinary tract infections.8,13-15 In the present study, about 69% inpatient and 57% outpatient E. coli isolates were found ESBL producer. This proportion of ESBL isolation is similar to those described in several studies in home and abroad.8,13,14 A considerable numbers of isolates irrespective of ESBL production showed sensitivity to mecillinam. The high susceptibility rate of ESBL producing E. coli to mecillinam as determined by MIC method in this study is comparable to the findings of others who found 94% and 85% sensitivity respectively.13,14 But in the context of Bangladesh, mecillinam sensitivity of E. coli was reported as 43-67% in 2009.14,15 This divergence may be due to the fact that, in those studies uropathogenic E. coli irrespective of ESBL production was considered and disc diffusion method was used to determine the sensitivity instead of MIC method.

The present study also demonstrated a significant proportion of E. coli (27.4%) was mecillinam. Interestingly, we have observed higher rate of resistance in ESBL negative isolates. Arguably, ESBL positive strains should exhibit higher resistance than negative strains. This discrepancy could be due to small number of samples. However, though the percentage of resistance was more in ESBL negative isolates but the level of resistance was more pronounced in ESBL positive isolates as shown by the MIC50 and MIC90 values.

Mecillinam is one of the very few oral drug used in treating community acquired urinary tract infection. According to in-vitro findings, it is stable against beta lactamase enzymes produced by gram negative as well as gram positive bacteria. Extensive use of mecillinam because of its effectiveness in UTI has exerted selective pressure resulting in chromosomal mutation of the drug target and emergence of resistance. Further, few beta lactamases like type IIIa and IVc have activity against mecillinam causing hydrolysis of the agent.10 These factors together results in mecillinam resistance in clinical practice. Addition of any compound that has inhibitory effect on these enzymes may improve the antibacterial activity of mecillinam. In this ground, activity of mecillinam in combination with a beta lactamase inhibitor, clavulanic acid, was also evaluated in the study. Using the agar dilution method with standard inoculum of 1x104 cfu/spot, there was a marked decrease in the MIC of mecillinam when combined with clavulanate. As a result, sensitivity of ESBLs producing E. coli improved from 75.6% to 85.9% and for non-ESBLs producers from 67.4% to 86.9%. The MICs of individual isolates markedly reduced after adding clavulonic acid, but overall range of MICs was not changed (ranging from £1 - ³1024). Because MICs of some isolates were out of the test range it was not possible to determine whether there was a significant (³8 fold) decrease in MIC or not. The lowest value of MIC in our assay was 1 µg/ml; so in those isolates where MIC reduced beyond the concentration of 1 µg/ml could not be determined. Similar things happened in some highly resistant isolates having the MIC of >1024 µg/ml which was the highest MIC value tested. But the additional inhibitory effect of clavulanic acid could be predicted from the reduction of MIC50 and MIC90 of mecillinam after adding clavulonic acid (Table-3). Using the standard inoculum 1x104 cfu/spot, MIC50 of mecillinam was reduced from 4 µg/ml to £1 µg/ml and MIC90 from 128 µg/ml to 64 µg/ml. These findings were in accordance with previous studies.8,17 In the present study similar trend of reduction of MICs was also observed among ESBL negative E. coli. The observed synergistic effect of mecillinam-clavulanic acid combination on ESBL negative isolates could be due to the presence of other broad spectrum β-lactamases which were inhibitable by clavulanic acid or due to the primary affinity of clavulanic acid for PBP-2 of E. coli like mecillinam.18,9 Altogether, by adding clavulonic acid with mecillinam about 56% of resistant isolates became sensitive which were resistant or intermediately sensitive with mecillinam alone (Table-2). In the present study, we have further observed that all intermediate and resistant isolates having MIC of 16 µg/ml and 32 µg/ml became sensitive (MIC £1-8) when clavulanic acid was added with mecillinam. But, only 40% isolates having MIC of 64 µg/ml reverted to sensitive range and none of the isolates having MIC ³128 µg/ml became sensitive by adding clavulanic acid. It suggests that there is chance of treatment response with mecillinam-clavulanic acid combination in infection with resistant E. coli having such range of MICs. However, this combination may not be useful in severe infection by ESBL producers due to the inoculum effect of the offending organism on the drugs.7,8,19 

The results of our study suggest that clinical trial should be done to confirm the usefulness of the mecillinam-clavulanic acid combination therapy in UTI with intermediate and resistant strains of E. coli having such range of MICs.

Acknowledgement

This study was funded by the Research Grant of Ibrahim Medical College. We thank General Pharmaceutical Ltd, Bangladesh and Incepta Pharmaceutical Ltd, Bangladesh for kindly providing the pivmecillinam and potassium clavulanate.

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