S217

Document heading doi: 10.1016/S1995-7645(14)60235-2

Molecular characterization and in vitro susceptibilities of β-lactamase producing Escherichia coli, Klebsiella species, Acinetobacter baumannii, Pseudomonas aeruginosa and Staphylococcus aureus to CSE1034 and other β-lactams Manu Chaudhary, Anurag Payasi*

Venus Medicine Research Centre, Hill Top Industrial Estate, Bhatoli Kalan, Baddi, H.P. -173205 India

Asian Pac J Trop Med 2014; 7(Suppl 1): S217-S223

Asian Pacific Journal of Tropical Medicine

journal homepage:www.elsevier.com/locate/apjtm

*Corresponding author: Dr. Anurag Payasi, Venus Medicine Research Centre, Hill Top Industrial Estate, Bhatoli Kalan, Baddi, H.P.-173205, India. Tel: 91-1795-302072 Fax: 91-1795-302133 E-mail: ccmb@vmrcindia.com Foundation Project: Supported by Venus Medicine Research Centre, Werne, Germany and BFMC, Faridkot, Punjab, India (Grant No. VPG-011-2011).

1. Introduction

Enterobacteriaceae are a family of Gram-negative bacilli that includes Escherichia coli (E. coli), Klebsiella pneumoniae (K. pneumoniae) and other clinically important species of the pathogenic bacteria. These organisms cause serious infections such as bacteraemia, pneumonia, urinary tract and wound infections[1]. According to the World

Health Organization (WHO), every year approximately 4.5 billion cases of infections by these organisms have been reported of which 1.9 million end in death[2]. In addition to Enterobacteriaceae, Pseudomonas species, Acinetobacter species and Staphylococcus species also cause various types of infections in human[3-9]. It is increasingly being reported that β-lactam antimicrobial agents are widely used to treat the infections caused by these organisms accounting for over 50% of all antibiotics use[10]. However, in recent years, treatment of Enterobacteriaceae, Pseudomonas spp. Staphylococcus spp. and Acinetobacter spp. is becoming tough because of increasing antibiotic resistance[11,12]. There are many factors involved in the development of antibiotic resistance including over expression of efflux pump[13], acquisition of extended-spectrum β-lactamases

ARTICLE INFO ABSTRACT

KeywordsClinical isolatesCSE1034 Extended-spectrum-β-lactamases.

Objective: To study the prevalence of extended-spectrum β-lactamases (ESBLs) among 663 clinical isolates obtained from various parts of India and to study the occurrence of different variants of ESBLs among these isolates.Methods: Phenotypic characterization and susceptibility studies were performed according to the methods described in Clinical and Laboratory Standards Institute guidelines. The occurrence of ESBL variants was analyzed with PCR using the previously reported primers.Results: Among the six hundred sixty three isolates, the identified isolates were Acinetobacter baumannii (72), Escherichia coli (218), Klebsiella pneumoniae (30), Klebsiella oxytoca (63), Pseudomonas aeruginosa (264) and Staphylococcus aureus (16). PCR results revealed that approximately 89.0% of Pseudomonas aeruginosa isolates were positive for ESBL followed by Escherichia coli (85.3%), Klebsiella pneumoniae (76.6%), Klebsiella oxytoca (73.0%), Acinetobacter baumannii (72.2%) and Staphylococcus aureus (31.2%). The overall prevalence of ESBL was 82.5%. The presence of TEM type ESBLs were the predominant (in 186 isolates), followed by SHV (138), OXA (92), CTX-M (65), AmpC (33), KPC (28) and blaZ (5). Of the drugs involved in the study, CSE1034 was found to be the most efficacious against all of ESBL positive clinical isolates showing susceptibility approximately 95.7% with minimal inhibitory concentration values between 0.125 and 8.000 µg/mL for all strains tested. The susceptibilities of penems (meropenem and imipenem and cilastatin) ranged between 83% and 93% for all the isolates. The susceptibilities of other drugs like piperacillin and tazobactam, amoxicillin and clavulanic acid, cefoperazone and sulbactam were <45% for all the isolates. Conclusions: Results of the present study indicated that majority of the isolates was susceptible to CSE1034 and it could be a potent antibacterial agent for the treatment of severe bacterial infections caused by such organisms.

Article history:Received 23 Jun 2014Received in revised form 29 Aug 2014Accepted 4 Sep 2014Available online 23 Sep 2014

Contents lists available at ScienceDirect

Manu Chaudhary and Anurag Payasi /Asian Pac J Trop Med 2014; 7(Suppl 1): S217-S223S218

(ESBLs) and metallo-β-lactamases[14,15], target site or outer membrane modification[16]. Among these factors, prevalence of antibiotic resistance to beta-lactam antibiotics among members of Enterobacteriaceae, Pseudomonas spp. and Acinetobacter spp. because of ESBL has been steadily increasing across the world over the past few years, resulting in the limitation of therapeutic options[11,12,14,15,17]. The overall prevalence of ESBL producers is found to vary greatly in different geographical areas such as from 66.7% to 73.5% in India[9,18], 54.7% to 59.2% in Iran, 41% in United Arab Emirates[19,20]. The ESBL enzyme has resistance not only to broad-spectrum cephalosporins, including oxymino-β-lactam antibiotics but also to other commonly used antibiotics including aminoglycosides and quinolones[21,22]. A number of studies have demonstrated the decreased susceptibility of Enterobacteriaceae to cephalosporins and other drugs[23-27]. Similarly, the decreased susceptibility rates of Pseudomonas aeruginosa (P. aeruginosa) and Acinetobacter baumannii (A. baumannii) to β-lactams, carbapenems, quinolones and aminoglycosides have been reported in various countries[11,28-30]. The increasing rate of the antibiotic resistance and its impact on treatment failure encouraged us to study newly reported concept of antibiotic adjuvant entity by which the increasing failure rate of antibiotics in treatment can be controlled. Information regarding the prevalence of antimicrobial resistance in pathogens can be used for selecting an alternative option. Data on the prevalence of TEM, SHV, Amp-C, KPC and CTX-M variants in Enterobacteriaceae (E. coli and K. pneumoniae) and non-fermenting Gram-negative bacilli (A. baumannii and P. aeruginosa) from Indian hospitals are needed. Therefore, the current study was undertaken to study the molecular characterisation of the selected ESBL producing isolates. The study also analysed the susceptibility behaviour of these isolates to different antibiotics including CSE1034, a new antibiotic adjuvant entity which is a combination of β-lactam, β-lactamase inhibitor and nonantibiotic adjuvant altogether termed as CSE1034 with other antibiotics against these isolates.

2. Materials and methods

2.1. Clinical specimen collection and their identification

All the specimens were collected from different hospitals of India. The name of hospitals can not be disclosed due to confidential agreement. This study was conducted from January 2013 to March 2014. All the specimens including blood, urine, sputum and pus were isolated and identified isolates according to the standard microbiological techniques and VITEK-2 (BioMérieux, Marcy l’Etoile, France)[31]. Before use, all the specimens were inoculated in Soyabean-Casein Digest Agar (Hi-Media, Mumbai, India), and incubated at 37 °C for overnight. At least three to five colonies of each specimen were selected from the Soyabean-Casein Digest Agar plate and were transferred into a tube containing 10 mL of sterile Mueller-Hinton broth (Hi-Media, Mumbai, India) to produce a suspension which matched the

turbidity standard of 0.5 McFarland standard.

2.2. Collection procedure of clinical isolates and quality control points

The clinical isolates were collected using the procedures mentioned in the manual for the laboratory identification and antimicrobial susceptibility testing of bacterial pathogens of public health importance in the developing world. Quality control points were also followed while collecting the strains as mentioned in the above guidelines.

2.3. Antimicrobial agents

CSE1034 containing ceftriaxone sodium 1 g plus sulbactam sodium 0.5 g along with 37 mg adjuvant disodium edetate (CSE1034 of Venus Remedies Limited, India), amoxicillin plus potassium clavulanate 1.2 g (Augmentin, GlaxoSmithKline, Pharmaceuticals Limited, Mumbai, India), piperacillin plus tazobactam 4.5 g (Zosyn, Wyeth Pharmaceuticals, Mumbai, India), meropenem 1 g (Meronem, AstraZeneca Pharma India Limited, Banglore, India), imipenem plus cilastatin 500 mg (Imecila, Nicholas Piramal, Limited, India) cefoperazone plus sulbactam 1 g (Magnex, Pfizer Limited, Mumbai, India) were used in the study. All the drugs were reconstituted in sterile water for injection.

2.4. Phenotypic screening of isolates for ESBL

Screening of clinical isolates for ESBL production was performed according to the procedures as recommended by the Clinical and Laboratory Standards Institute (CLSI)[32], using indicator cephalosporins, ceftriaxone (30 µg), ceftazidime (30 µg) and cefotaxime (30 µg). The respective zone size was interpreted according to the recommendations of CLSI. Isolates exhibiting zone size ≤22 for ceftazidime and ≤27 with cefotaxime were considered as possible ESBL producers. Further, testing was done using ceftazidime-clavulanic acid (30/10 µg) and cefotaxime-clavulanic acid (30/10 µg) to confirm the ESBL production according to the CLSI[32].

2.5. Detection of varinats of ESBL genes

2.5.1. DNA isolation DNA isolation from all the clinical isolates phenotypically confirmed to be ESBL positive and was carried out according to the method of alkaline lysis[33]. Five millilitre of overnight grown culture was used for the DNA isolation.

2.5.2. PCR The detection of variants of ESBL genes, such as TEM-1, TEM-3, TEM-50, SHV-1, SHV-4, SHV-10, SHV-28, Amp-C, OXA-2, OXA-10, OXA-23, OXA-48, KPC-1, KPC-2, CTX-M-1, CTX-M-9 and Bla-Z was carried out as described previously[34-46]. The primers used in the study are shown in Table 1. All of the respective primers were obtained from Sigma Aldrich Chemicals Pvt. Ltd., Banglore, India. For PCR amplifications, about 200 pg of DNA was added to 20 µL mixture containing 0.5 mmol/L of dNTPs, 1.25 µmol/L of each primer and 3.0 µL/IU of Taq polymerase (Banglore Genei) in

Manu Chaudhary and Anurag Payasi /Asian Pac J Trop Med 2014; 7(Suppl 1): S217-S223 S219

1伊 PCR buffer. Amplification was performed in a Eppendorf thermal cycler (Germany). Table 1 Oligonucleotides used in the study for each tested genes.Primer Primer sequences (5'-3') Amplicon (base

pair)

References

TEM-1 F-5' ATG AGT ATT CAA CAT TTC CG-3' 858 [41]R-5' CCA ATG CTT ATT CAG TGA GG-3'

TEM-3 F-ATA AAA TTC TTG AAG AC-3' 652 [41]R-TTA CCA ATG CTT AAT CA-3'

TEM-50 F-AAG ACG AAA GGG CCT CGT G 1074 [38]R-GGT CTG ACA GTT ACC AAT GC

SHV-1 F-5' CTG GGA AAC GGA ACT GAA TG-3' 308 [41]R-5' GGG GTA TCC CGC AGA TAA AT-3'

SHV-4 F-5' TCA GCG AAA AAC ACC TTG C-3' 322 [34]R-5' CGA TCG TCC ACC ATC CAG TG-3'

SHV-10 F-5' CCG ATA AGA CCG GAG TTC GC-3' 248 [34]F-5' AGT CAT ATC GCC CGG CAC-3'

SHV-28 F-5' ATT TGT CGC TTC TTT ACT CGC-3' 1052 [40]R-5' GGT CAA AGG TAA CGC CAT AAA-3'

AmpC F-5 CCC CGC TTA TAG AGC AAC AA-3 634 [35]R-5 TCA ATG GTC GAC TTC ACA CC-3

OXA-2 5’-GCC AAA GGC ACG ATA GTT GT-3’ 701 [38]5’-GCG TCC GAG TTG ACT GCC GG-3’

OXA-10 5’-TCT TTC GAG TAC GGC ATT AGC-3’ 759 [38]5’-CCA ATG ATG CCC TCA CTT TCC-3’

OXA-23 5’-GAT CGG ATT GGA GAA CCA GA-3’ 501 [46]5’-ATT TCT GAC CGC ATT TCC AT-3’

OXA-48 5’-GCG TGG TTA AGG ATG AAC AC-3’ 438 [42]5’-CAT CAA GTT CAA CCC AAC CG-3’

KPC-1 F: 5'-CTT GCT GCC GCT GTG CTG-3' 489 [45]R: 5’-GCA GGT TCC GGT TTT GTC TC-3'

KPC-2 F: 5'-GCT ACA CCT AGC TCC ACC TTC-3' 989 [43]R: 5'-GCA TGG ATT ACC AAC CAC TGT-3'

CTX-M-1 F: 5' GAC GAT GTC ACT GGC TGA GC 3' 499 [41]R: 5’ AGC CGC CGA CGC TAA TAC A 3’

CTX-M-9 F: 5' TAT TGG GAG TTT GAG ATG GT 3' 932 [38]R: 5’ TCC TTC AAC TCA GCA AAA GT 3

Bla-Z F: 5' AAG AGA TTT GCC TAT GCT TC 3' 517 [36]R: 5’ GCT TGA CCA CTT TTA TCA GC3

F: Sense primer; R: Antisense primer.

2.5.3. Gel electrophoresis The amplified products were separated in 1.5% agarose gel containing 2.5 µL of 10 mg/mL ethidium bromide. The gel was run at 70 v for 1 h. The gel images were taken under ultraviolet light using gel documentation system (Bio-Rad, USA). A 100 bp ladder (Banglore Genie) was used to measure the molecular weights of amplified products. The images of ethidium bromide stained DNA bands were visualized using a gel documentation system (Bio-Rad, USA).

2.6. Antibiotic susceptibility testing

Susceptibility to various classes of antibiotics were done by two methods: minimum inhibitory concentration (MIC) and antibiotic susceptibility (AST).

2.7. MIC

MIC was determined according to the guidelines of CLSI[32]. E. coli ATCC 35218 (TEM), K. pneumoniae ATCC 700603 (SHV), Enterobacter cloacae ATCC BAA-1143 (AmpC) (E. cloacae), K. pneumoniae ATCC BAA-1705 (KPC), E. coli NCTC 13302 (OXA)

and E. coli NCTC 13353 (CTX-M-15), were procured from LGC, Promochem, Bangalore, India, which were used as positive controls. MIC value represents the lowest dilution at which bacteria fail to grow. Serial two fold dilutions of the drugs from 0.031 25 to 1 024 µg/mL were made using Cation-Adjusted Mueller-Hinton (Hi Media, Mumbai, India) broth in wells of 96-well plate.

2.8. AST

AST testing of the drugs were determined by disc diffusion method according to the CLSI. Positive controls were the same which were used in MIC study. Inoculum containing 106 CFU/mL of test strain was spread with a sterile swab on a Petri dish containing Mueller-Hinton agar and the plates were dried. Then, disc of the respective antibiotic was placed in the plate and allowed to diffuse at room temperature. The plates were incubated in the upright position at 37 °C for 18 h. After incubation the zone of inhibition around the disc was measured in millimeter, and then was averaged and the mean values were recorded. The discs of CSE1034 (30:15 µg), piperacillin plus tazobactam (100:10 µg), amoxicillin plus clavulanic acid (20:10 µg), cefoperazone plus sulbactam (75:30 µg), imipenem plus cilastatin (10 µg) and meropenem (10 µg) were obtained from HiMedia (Mumbai, India). All the test were repeated 3 times and results were expressed as mean依SD values.

3. Results

3.1. Isolation of clinical isolate from specimens

Out of 3 816 clinical specimens, 1 365 specimens were sterile; 2 451 specimens showed the growth of which 1 788 isolates were of Mycoplasma, Candida albicans, Haemophilus influenzae, Mycobacterium tuberculosis, Salmonells typhi, Staphylococcus epidermidis, Sterptococcus pyogenes, Nisseria spp., Burkholderia spp. which were not part of study. The remaining specimens showed the growth of 663 isolates which includes P. aeruginosa (264/663, 39.8%) followed by E. coli (218/663, 32.8%), A. baumannii (72/663, 10.8%), Klebsiella oxytoca (K. oxytoca) (63/663; 9.5%), K. pneumoniae (30/663; 4.5%) and Staphylococcus aureus (S. aureus) (16/663; 2.4%) (Table 2).Table 2 Isolation of target clinical isolates from different clinical specimens.Name of the

specimens

Number of

specimensA.

baumanniiE.

coliK.

pneumoniaeK.

oxytocaP.

aeruginosa S. aureus

Blood 749 16 46 6 13 41 1

Urine 1 088 8 129 4 8 34 4

Sputum 731 35 23 17 33 31 1

Pus 1 248 13 20 3 9 158 10

Total 3 816 72 218 30 63 264 16

3.2. Detection of ESBL

Among the 663 isolates, phenotypical 87.6% (n=581) isolates were found to be ESBL positive by disc diffusion method. Further, genotypic characterization of the same isolates

Manu Chaudhary and Anurag Payasi /Asian Pac J Trop Med 2014; 7(Suppl 1): S217-S223S220

with PCR revealed approximately 82.5% (n=547) isolates were ESBL positive, and there was 5.1% decrease in the ESBL positive isolates when analyzed with genotypic method. PCR results indicated that approximately 89.0% of P. aeruginosa isolates were positive for ESBL followed by E. coli (85.3%), K. pneumoniae (76.6%), K. oxytoca (73.0%), A. baumannii (72.2%) and S. aureus (31.2%) (Table 3). For further study only genotypically characterized ESBL isolates were used.Table 3 Detection of ESBLs among clinical isolates by disc diffusion and PCR methods.Name of species (total)

No. of isolates positive for ESBL by disc diffusion (%)

No. of isolates positive for ESBL by PCR (%)

A. baumannii (72) 54 (75.0) 52 (72.2)

E. coli (218) 196 (89.9) 186 (85.3)

K. pneumoniae (30) 24 (80.0) 23 (76.6)

K. oxytoca (63) 54 (85.7) 46 (73.0)

P. aeruginosa (264) 245 (92.8) 235 (89.0)

S. aureus (16) 8 (50.0) 5 (31.3)

Total (663) 581 (87.6) 547 (82.5)

3.3. Diversity of ESBLs

PCR analyses using specific primers for class A, C and D β-lactamase genes revealed the following frequencies: TEM type ESBLs (blaTEM-1, blaTEM-3, blaTEM-50) were the most frequently detected, followed by SHV type ESBLs (SHV-1, SHV-4, SHV-10, SHV-28), OXA type ESBLs (OXA-2, OXA-10, OXA-23, OXA-48) , CTX-M type ESBLs (CTX-M-1, CTX-M-9), AmpC , KPC type ESBLs (KPC-1, KPC-2) and blaZ (Table 4).

3.4. MIC

The MIC values of CSE1034, piperacillin plus tazobactam, amoxicillin plus clavulanic acid, cefoperazone plus sulbactam, imipenem plus cilastatin and meropenem were determined against all of the selected clinical isolates and presented in Table 5. MIC values of CSE1034 for 95.7%, 92.3%, 91.3%, 93.5%, 94.9%, and 93.3% isolates of E. coli, A. baumannii, K. pneumoniae, K. oxytoca, P. aeruginosa and S. aureus, respectively were between 0.125 and 8.000 µg/mL whereas penems (meropenem and imipenem plus cilastatin) MIC ranged from 0.125 to 1.000 µg/mL for 83.9% to 93.3% isolates for the same isolates. Piperacillin plus tazobactam exhibited MIC value of 4.000-16.000 µg/mL for 45.7% isolates of E. coli while 38.5%, 34.8%, 41.3%, 38.7% and 36.0% isolates of A. baumannii, K. pneumoniae, K. oxytoca, P. aeruginosa and S. aureus respectively with MIC values of 8.000 to 16.000

µg/mL, respectively. Amoxycillin plus clavulanic acid exhibited MIC values of 4.000-8.000 µg/mL against 35.5%, 28.8%, 26.0%, 26.0%, 25.9% and 25.3% isolates of E. coli, A. baumannii, K. pneumoniae, K. oxytoca, P. aeruginosa and S. aureus, respectively. Cefoperazone plus sulbactam produced MIC values 8.000-16.000 µg/mL against 31.2%, 25.0%, 21.7%, 21.7%, 23.8%, and 22.7% isolates of E. coli, A. baumannii, K. pneumoniae, K. oxytoca, P. aeruginosa and S. aureus, respectively. The detailed results are shown in the Table 5.

3.5. AST

The antibiotic susceptibility profile was determined by disc diffusion method and results are presented in Table 6. Of the drugs included in the study, CSE1034 seems to be more efficacious. Approximately, more than 90% isolates were susceptible to CSE1034 with AST values between 23 and 29 mm. Imipenem plus cilastatin was found second most active agent with approximately 90% isolates of E. coli, A. baumannii, K. pneumoniae, K. oxytoca, P. aeruginosa and S. aureus were susceptible with AST values 16-35 mm whereas >80% isolates showed susceptibility to meropenem with 16-29 mm. Piperacillin plus tazobactam was susceptible in <45% isolates with AST values 22-35 mm. Amoxycillin plus clavulanic acid found to be susceptible to <35% isolates with AST values 18-31 mm. Cefoperazone plus sulbactam found to be susceptible to <31% with AST values 21-30 mm. The detailed results are shown in the Table 6.

4. Discussion

ESBLs continue to be the leading cause of resistance to β-lactam antibiotics among Gram-negative bacteria. There has been increased incidence and prevalence of ESBLs which show a wide spread in hospital settings worldwide[47]. In this study, 663 clinical isolates of Gram-negative and Gram-positive isolates were collected from various clinical specimens and were subjected to screening for ESBLs. In our study, approximately 87.6% of the isolates were observed to be ESBL positive by disc diffusion methods. However, when the same strains were processed to check the positivity of ESBL with PCR as it is considered as gold standard only 82.5% of isolates were confirmed to be ESBL positive. These results suggest that disc diffusion method may have some false detection. Previous studies also demonstrated the steadily increasing frequency of ESBLs 50% to 85% in A. baumannii[48,49], 4% to 78% in Klebsiella species[50-52], 46% to 79% in E. coli[53,54], 45% to 53.9% in P. aeruginosa[55,56].

Table 4 Distribution of variants of ESBL enzymes in different bacterial species.Species (no. of ESBL positive

isolates by PCR, n=547)TEM-1 TEM-3 TEM-50 SHV-1 SHV-4 SHV-10 SHV-28 AmpC OXA-2 OXA-10 OXA-23 OXA-48 KPC-1 KPC-2 CTX-M-1 CTX-M-9 Bla-Z

A. baumannii (52) 5 5 6 10 7 - - 3 4 - - 12 - - - - -E. coli (186) 37 32 - 24 20 10 5 14 - - - 23 - - - 21 -K. pneumoniae (23) 5 5 - 1 - - - - - - - - 6 6 - - -K. oxytoca (46) 10 - - 10 - - - - - - - 6 8 8 - 4 -P. aeruginosa (235) 34 33 14 31 - 20 - 16 5 - - 42 - - 15 25 -S. aureus (5) - - - - - - - - - - - - - - - - 5

Most of the isolates contain more than one ESBL genes.

Manu Chaudhary and Anurag Payasi /Asian Pac J Trop Med 2014; 7(Suppl 1): S217-S223 S221

Increased prevalence of ESBL in our community is suggested to be due to intense prescription of the third generation cephalosporins in hospitals and the dissemination of these organisms by inappropriate hygienic measures. The results of the antibiotic susceptibility testing performed on the all chosen clinical isolates showed that CSE1034 was the most active agent and susceptibility rates of it against the studied isolates were very close to the penems (meropenem and imipenem plus cilastatin). CSE1034 however was observed to show an intermediate and resistant trend against higher OXA types and a few Amp-C types. Earlier studies also supports the higher susceptibilities of CSE1034 against these isolates[56,57]. Less susceptibility of piperacillin plus tazobactam in Enterobacteriaceae (E. coli and K. pneumoniae) and non-Enterobacteriaceae (A. baumannii and P. aeruginosa) has been reported in India[58-60], which is in line with current findings with susceptibility <50% against most pathogens. Several studies have also reported meropenem resistance in Enterobacteriaceae and non-Enterobacteriaceae isolated from India and abroad[59,61]. In the current study, resistance to meropenem was 18.7% for P. aerugionsa which are in line with the result of an earlier study where resistance to meropenem for P. aeruginosa was 20.3%[49]. The resistance to meropenem in

A. baumannii was 17.3% in our study which was observed to be in accordance with a previous report[49]. Furthermore, our data shows 17.4% of K. pneumoniae and 9.3% of E. coli isolates were resistant to meropenem. The resistance to meropenem in these isolates probably results from reduced accumulation of drug or over activation of efflux pump[62]. A very high susceptibility of CSE1034 against these isolates is likely to be associated with synergistic activity of components. Results of this study demonstrate that the frequency of ESBL mediated resistance among the clinical isolates has been increasing and the drugs commonly used for the treatment of infections appear to be resistant caused by ESBL producing organisms. Data obtained from this investigation clearly demonstrate the potent in vitro activity of CSE1034 against ESBL producing organisms. Hence, in the case of infection with ESBL producing organisms, CSE1034 can be a drug to be chosen for the treatment.

Conflict of interest statement

We declare that we have no conflict of interest.

Table 5 Comparative MIC values of ESBL producing strains.

Name of micro-organisms

MIC values of drugs (µg/mL)

Total No. of

strains (547

clinical

isolates)

Ceftriaxone plus sulbactam

(CSE1034)

Piperacillin plus

tazobactam

Amoxicillin plus

clavulanic acid

Cefoperazone plus

sulbactam

Imipenem plus

cilastatinMeropenem

S I R S I R S I R S I R S I R S I RE. coli 186 0.125-8.000 16-32 64-128 4-16 32-64 128-256 4-8 16 32-512 8-16 32 64-512 0.125-1.000 2 4-32 0.50-1.00 2 4-32

A. baumannii 52 0.250-8.000 16-32 64-128 8-16 32-64 128-512 4-8 16 32-512 8-16 32 64-512 0.125-1.000 2 4-16 0.50-1.00 2 4-64

K. pneumoniae 23 0.250-8.000 16-32 64-128 8-16 32-64 128-256 4-8 16 32-512 8-16 32 64-512 0.125-1.000 2 4-16 0.25-1.00 2 4-32

K. oxytocca 46 0.500-8.000 16-32 64-128 8-16 32-64 128-256 4-8 16 32-512 8-16 32 64-512 0.125-1.000 2 4-32 0.50-1.00 2 4-32

P. aeruginosa 235 1.000-8.000 16-32 64-128 8-16 32-64 128-512 4-8 16 32-512 8-16 32 64-512 0.125-1.000 2 4-16 0.5-1.00 2 8-64

S. aureus 5 0.125-8.000 16-32 64-128 8-16 32-64 128-256 4-8 16 32-512 8-16 32 64-512 0.125-1.000 2 4-16 0.25-1.00 2 4-16

E. coli ATCC 35218 (TEM) 1 0.500 4 8 8 0.250 0.25

K. pneumoniae ATCC 700603 (SHV) 1 0.500 4 8 8 0.250 0.25

Enterobacter cloacae ATCC BAA-1143 (AmpC) 1 2.000 8 8 8 0.500 0.25

K. pneumoniae ATCC BAA-1705 (KPC) 1 8.000 16 32 16 1.000 2.00

E. coli NCTC 13302 (OXA) 1 8.000 32 16 32 2.000 2.00

E. coli NCTC 13353 (CTX-M-15) 1 1.000 8 8 8 0.500 0.50

S: Susceptible; I: Intermediate; R: Resistance. E. coli ATCC 25922 was used as a quality control strain.

Table 6 Comparative antimicrobial susceptibility of ESBL producing strains.

Name of micro-organismsTotal No.

of strains

Inhibition zone of drugs (mm)

Ceftriaxone plus

sulbactam (CSE1034)

Piperacillin plus

tazobactam

Amoxicillin plus

clavulanic acid

Cefoperazone plus

sulbactam

Imipenem plus

cilastatinMeropenem

S I R S I R S I R S I R S I R S I RE. coli 186 25-29 20-22 10-19 22-29 18-20 7-17 19-22 15-18 7-14 21-25 16-20 10-15 23-33 20-22 11-19 23-29 20-22 11-19

A. baumannii 52 23-28 14-20 7-13 22-25 18-20 11-17 18-24 15-17 8-14 21-28 18-20 13-17 16-27 14-15 10-13 16-27 14-15 10-13

K. pneumoniae 23 23-29 20-22 8-19 22-28 18-20 9-17 19-24 15-18 11-14 21-29 16-20 10-15 23-29 20-22 15-19 23-29 20-22 13-19

K. oxytocca 46 24-29 20-22 11-19 22-30 18-20 8-17 19-25 15-18 9-14 21-30 16-20 12-15 23-35 20-22 9-19 23-29 20-22 9-19

P. aeruginosa 235 23-29 14-20 10-13 22-35 15-20 11-14 18-27 15-17 11-14 22-29 18-20 13-17 19-33 16-18 10-15 19-24 16-18 7-15

S. aureus 5 23-30 14-20 10-13 22-25 15-20 11-14 21-31 15-20 13-16 21-25 16-20 12-15 19-30 14-15 7-13 19-33 14-15 10-13

E. coli ATCC 35218 (TEM) 1 29 22 21 21 27 26

K. pneumoniae ATCC 700603 (SHV) 1 31 23 22 22 29 29

E. cloacae ATCC BAA-1143 (AmpC) 1 28 15 20 19 30 27

K. pneumoniae ATCC BAA-1705 (KPC) 1 25 23 17 18 25 22

E. coli NCTC 13302 (OXA) 1 26 19 13 17 24 23

E. coli NCTC 13353 (CTX-M-15) 1 27 20 22 21 28 24

S: Susceptible; I: Intermediate; R: Resistance. E. coli ATCC 25922 was used as a quality control strain.

Manu Chaudhary and Anurag Payasi /Asian Pac J Trop Med 2014; 7(Suppl 1): S217-S223S222

Acknowledgements

Researchers are thankful to Venus Medicine Research Centre, Werne, Germany for providing assistance (Grant No. VPG-011-2011) to carry out this study.

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