Third Generation Cephalosporins, Cefoperazone/Sulbactam, Ceftazidime/Sulbactam.

Ram Murugan Navaneethakrishnan1; Mohan Sundaram2; Mohamed Ismail3

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Year : 2016 Month : May Volume : 5 Issue : 37 Page : 2173-2177

SUSCEPTIBILITY OF BACTERIAL ISOLATES TO COMBINATION OF THIRD GENERATION CEPHALOSPORINS AND SULBACTAMS..

Ram Murugan Navaneethakrishnan1, Mohan Sundaram2, Mohamed Ismail3

1Senior Assistant Professor, Department of Microbiology, Institute of Microbiology, Madurai Medical College, Madurai, Tamilnadu.
2Assistant Professor, Department of Microbiology, Vinayaka Mission s Medical College, Karaikal.
3Research Coordinator, Department of Microbiology, Research Cell, Chennai Medical College Hospital & Research Centre, Irungalur, Trichy, Tamilnadu.

CORRESPONDING AUTHOR

Dr. Ram Murugan Navaneethakrishnan
Email : rammddo@yahoo.co.in

ABSTRACT

Corresponding Author:
Dr. Ram Murugan Navaneethakrishnan,
#14/2, Siva Sakthi Nagar,
K. Pudur,
Madurai-625007.
E-mail: rammddo@yahoo.co.in

ABSTRACT

BACKGROUND

This study was aimed to ascertain the response of microorganisms to third generation Cephalosporins.

METHODOLOGY

Blood, Urine, Pus, Sputum, Throat swabs, Broncho alveolar lavage (BAL), Endotracheal secretions (ET) and Vaginal swabs were collected and organisms were isolated from various Patients Sample with different pathological conditions and subjected to susceptibility tests for Cefoperazone/Sulbactam and Ceftazidime/Sulbactam.

RESULTS

100% of Enterobacter, 50% of Acinetobacter, 42% of Klebsiella, 34 % of Salmonella and 30% of E. coli, were resistant to Cefoperazone/Sulbactam, whereas 100% of Enterobacter, 75% of Acinetobacter, 33 % of Salmonella, 20 % of Klebsiella, and 15% of E. coli, were resistant to Ceftazidime/Sulbactam.

CONCLUSION

The combination of third generations Cephalosporins (Cefoperazone/Sulbactam and Ceftazidime/Sulbactam) was not effective drug to treat the patients.

KEYWORDS

Third Generation Cephalosporins, Cefoperazone/Sulbactam, Ceftazidime/Sulbactam.

INTRODUCTION

A WHO survey says that awareness of antibiotic resistance is meagre and misconception of use of antibiotic exists. The survey also exposed the misuse of antibiotics for viral infections. Rise of antibiotic resistance has become a global threat and there is a need to sensitize the medical and veterinary practitioners, drug manufactures, dispensaries, politicians, policymakers, health authorities and common public regarding the emergence of antibiotic resistance.¹ Treatment of tuberculosis, gonorrhoea and pneumonia has become difficult since the organisms have developed resistance to common antibiotics. The WHO survey in India says that 75% of the participants of the survey believe that Common cold and flu can be treated with antibiotics. Research is going on to find out newer molecules to compete with the resistant organisms since antibiotic resistance causes higher cost of medical treatment, longer duration of stay in the hospital and increases the morbidity and mortality.² The Gram-negative organisms are isolated with increasing trends of drug resistance.^3,4,5 Cefoperazone is available as a co-formulation with Sulbactam.

Cefoperazone exerts its bactericidal effect by inhibiting the bacterial cell wall synthesis, and Sulbactam acts as a beta-lactamase inhibitor, to increase the antibacterial activity of Cefoperazone against beta-lactamase-producing organisms.^6,7Cefoperazone/Sulbactam is a third generation Cephalosporin effective in treating resistant Pseudomonas infection. Ceftazidime is also a third generation cephalosporin. It differs from others in the presence of C=N-OCH₃in its chemical structure to give improved stability against beta-lactamase enzymes of gram negative bacteria. This drug is prescribed for Pseudomonas aeruginosa infections, Gram-negative, neutropenic fever patients, and also to treat respiratory tract, skin, urinary tract, blood-stream, joint, abdominal infections, and meningitis. This drug in combination with Sulbactam is also used to increase the bactericidal action.^8,9 Sulbactam is competitive irreversible β- lactamase inhibitor and has good inhibitor activity against the clinically important plasmid mediated β-lactamase which is most frequently responsible for transferred drug resistance. This study has been taken up to analyse the susceptibility status of various isolates to the Cefoperazone/Sulbactam [CFS] and Ceftazidime/Sulbactam [CAS] in a tertiary teaching hospital. This study was done to evaluate the current status of the drugs which will help the clinicians to prescribe the appropriate drugs to the patients to reduce their hospital stay and cost of treatment.

MATERIALS AND METHODS

Type of Study

Cross sectional, observational, prospective study.

Settings

The study was conducted in a tertiary care teaching hospital, Madurai Medical College, Madurai.

Sample Size

100 isolates received from various Patients Samples with different Pathological condition.

Study Period

From July to December 2015.

Inclusion Criteria

Newly admitted patients without any antibiotic treatment.

Exclusion Criteria

Patients already with antibiotic treatments.

METHODOLOGY

Samples such as Blood, Urine, Pus, Bal & ET, Throat Swab, and vaginal swab were collected from patients attending as Out patients and in different clinical departments, respective In patient wards and Intensive care units by observing standard specimen collection procedure and aseptic conditions. The samples were transported to the lab and streaked on MacConkey Agar and Blood Agar medium. After the incubation period at 37^oC the culture plates were examined and bacterial isolates were observed by Grams Staining and motility test. The identification of bacteria was performed by standard protocol (On observing MacConkey plates for Lactose/Non lactose fermentors/Non fermentors and Performing Oxidase test, Mannitol motility test, Triple sugar Iron agar, Lysine Arginine Ornithine test and Nitrate test). The susceptibility test was conducted by following Kirby-Bauer disk diffusion susceptibility method. Antibiotics Ceftazidime/Sulbactam (Zydotum, Venus Remedies Limited, Baddi, Himachal Pradesh, India), Cefoperazone/Sulbactam (Himedia Limited) were used in this study. The antibiotic sensitivity was done on Mueller Hinton agar according to Kirby- Bauer technique. The culture plates were incubated at 37°C±2°C. The results of the Kirby-Bauer disk diffusion susceptibility test were reported only as susceptible, intermediate, or resistant.^10,11

Determination of susceptibility or resistance of the organisms was determined by the CLSI 2015 guidelines.¹² For each drug, the zone size was labelled as susceptible (S), intermediate (I), or resistant (R) based on the zone of inhibition in mm. The test method was tabulated as given under.

For Enterobacteriaceae

(E. coli, Klebsiella, Enterobacter, Proteus, Serratia, Citrobacter)

Enterobacteriaceae
	(Zone Size in mm)
Antibiotic	Disc Content	Resistant	Intermediate	Sensitive
Cefoperazone/ Sulbactam	75/30 mcg	<15	16-20	>21
Ceftazidime/ Sulbactam	30/15 mcg	<17	18-20	>21

For Pseudomonas

For Pseudomonas (Zone Size in mm)
Antibiotic	Disc content	Resistant	Intermediate	Sensitive
Cefoperazone/ Sulbactam	75/30 mcg	<15	16-20	>21
Ceftazidime/ Sulbactam	30/15 mcg	<14	15-17	>18

Panel III–For Nonfermentor

Non fermentor (Zone Size in mm)
Antibiotic	Disc content	Resistant	Intermediate	Sensitive
Cefoperazone/ Sulbactam	75/30 mcg	<15	16-20	>21
Ceftazidime/ Sulbactam	30/15 mcg	<14	15-17	>18

Institutional Ethics Committee Certificate

The Research proposal was presented before the institutional ethics committee and got approval.

Informed Consent

Informed Consent was obtained from the Patients/Relatives for the collection of Samples.

RESULTS

A total of 100 isolates from patients attending the tertiary teaching hospital. Samples of Out patients, In patients (Ward) and patients of Intensive Care units [ICU] were 43%, 35% and 22% respectively [Fig. 1].

Out of 100 isolates isolated, 55 % isolates from male patients and 37% isolates from female patients. 4% isolates from male children and 4% isolates from female children. [Fig. 2].

Out of 100 isolates isolated, 56% from Urine sample, 23% from Pus Sample, 10% from Broncho alveolar lavage and Endotracheal Secretions, 5% from Blood, 3% from Sputum, 2% from Throat Swab and 1% from Vaginal swab [ Fig. 3].

Isolation of Microorganisms	Place	Samples Used
		Urine	Blood	Pus	Broncho alveolar lavage and Endotracheal Secretions.	Sputum	Throat swab	Vaginal Swab	Total
Acinetobacter	In Patient	-	-	-	1	-	-	-	1
Acinetobacter	Intensive Care Unit	-	-	-	3	-	-	-	3
Citrobacter	Out Patient	1	-	-	-	-	-	-	1
E. coli	Out Patient	23	-	1	-	-	-	1	25
	In Patient	9	-	5	1	-	-	-	15
	Intensive Care Unit	9	2	1	-	-	-	-	12
Enterobacter	In Patient	-	-	-	1	-	-	-	1
Klebsiella	Out Patient	4	1	3	-	1	-	-	9
	In Patient	4	-	4	2	1	-	-	11
	Intensive Care Unit	1	-	-	1	1	1	-	4
Proteus	In Patient	-	-	1	-	-	-	-	1
Proteus	Intensive Care Unit	-	-	1	-	-	-	-	1
Pseudomonas	Out Patient	2	1	4	-	-	0	-	7
	In Patient	2	-	2	-	-	1	-	5
	Intensive Care Unit	1	-	-	-	-	0	-	1
Salmonella	Out Patient	-	1	-	-	-	-	-	1
	In Patient		1	-	-	-	-	-	1
	Intensive Care Unit	-	1	-	-	-	-	-	1
Total Isolates		56	5	23	10	3	2	1	100
*Table 1: Isolation of Microorganisms from Samples*

Out of 100 isolates isolated, E. coli was 52%, Klebsiella was 24%, Pseudomonas was 13%, Acinetobacter was 4%, Salmonella was 3%, Proteus was 2%, while Citrobacter and Enterobacter were 1% each. [Table 1].

Sl. No.	Name of Organism	R Frequency	R %	S Frequency	S %	IS Frequency	IS %
1	Acinetobacter	2	50	0	0	2	50
2	Citrobacter	0	0	1	100	0	0
3	E. coli	16	30	36	70	0	0
4	Enterobacter	1	100	0	0	0	0
5	Klebsiella	10	42	14	48	0	0
6	Proteus	0	0	2	100	0	0
7	Pseudomonas	1	7	12	93	0	0
8	Salmonella	1	34	2	66	0	0
*Table 2: Susceptibility status of Microorganisms to Cefoperazone/Sulbactam (n=100)*

When the isolated organisms were tested for the susceptibility status for Cefoperazone/Sulbactam it was found out that 100% of Citrobacter and Proteus, 93% of Pseudomonas, 70% of E. coli, 66% of Salmonella, 48% of Klebsiella were Sensitive to the drug. 50% of Acinetobacter were in the intermediate susceptibility status.

Drug resistance was 100% of Enterobacter, 50% of Acinetobacter, 30% of E. coli, 42% of Klebsiella and 7% of Pseudomonas and 34% of Salmonella.

Sl. No	Name of Organism	R Frequency	R %	S Frequency	S %	IS Frequency	IS %
1	Acinetobacter	3	75	0	0	1	25
2	Citrobacter	0	0	1	100	0	0
3	E. coli	8	15	40	77	4	8
4	Enterobacter	1	100	0	0	0	0
5	Klebsiella	5	20	17	71	2	9
6	Proteus	0	0	2	100	0	0
7	Pseudomonas	1	7	11	85	1	8
8	Salmonella	1	33	2	67	0	0
*Table 3: Susceptibility Status of Microorganisms to Ceftazidime/Sulbactam (n=100)*

The other third generation Cephalosporins Ceftazidime/Sulbactam was also tested. It was observed that 100% of Citrobacter, 100% of Proteus, 85%, of Pseudomonas, 77% of E. coli, 71% of Klebsiella and 67% of Salmonella were sensitive to this drug. Moreover, 75% of Acinetobacter, 33% of Salmonella, 20% of Klebsiella, 15% of E. coli and 7% of Pseudomonas, were in the resistant status.

It was found out that 25% of Acinetobacter, 8% of E.Coli, 9% of Klebsiella and 8% Pseudomonas were intermediate sensitive to the drug.

DISCUSSION

Antibiotic resistance is being reported in almost all countries. Even though developing resistance to antibiotics is a natural phenomenon, misuse of antibiotics accelerates the mechanisms of the organisms to develop resistance as per the WHO report.² Datta et al in their study at Chandigarh have reported that resistant status of Cefoperazone/Sulbactam was 5.4% during 2006.¹³ In this study it was reported that resistant of Pseudomonas aeruginosa to Cefoperazone /Sulbactam was 4%, Acinetobacter 0.7% and Enterobacter species 0.7%. This study shows that increased resistant of all organisms to Cefoperazone/Sulbactam. Another study conducted at Turkey by Seniha Senbayrak Akcay et al from 2004 to 2011 found out that Klebsiella developed increased resistance from 31.3% in 2004 to 59.1% in 2011. Enterobacter spp showed increase in resistance from 14.7% in 2004 to 29.3% in 2011 and E. coli developed increased resistance from 3.6% in 2004 to 21.0% in 2011.¹⁴ Another study conducted by Kumar et al showed 23% resistant to Cefoperazone/Sulbactam and 64% to Ceftazidime/ Sulbactam.¹⁵ In this Study the susceptibility status of E. coli was 55%. Manu Chaudhary et al reported that 53.7% of P. aeruginosa, 49% of E. coli, were resistant to Cefoperazone /Sulbactam. On the contrary 6.5% of Pseudomonas Species and 13.7% of E. coli were resistant to Ceftazidime/Sulbactam. But in our study 7% Pseudomonas and 30% of E.coli were resistant to Cefoperazone/Sulbactam and 7% of Pseudomonas and 15% of E.coli were resistant to ceftazidime /Sulbactam.¹⁶ A study conducted by Ambekar Abdul Wahid et al reported that 10% of Pseudomonas, 10% of Acinetobacter, 45% of E.coli, and 36% of Klebsiella were resistant to Ceftazidime/Sulbactam.^17,18 Whereas in our study revealed that 75% of Acinetobacter, 15% of E.coli, 100% of Enterobacter, 20% of Klebsiella 7% of Pseudomonas and 33% of Salmonella Species were resistant to Ceftazidime/Sulbactam. This study revealed that microorganisms developed resistance to third generation cephalosporins in combination with Sulbactam. Kolayl and zhangli et al showed that when combination of sulbactam and Ceftazidime was used in the ratio of 1:1, Ceftazidime resistant isolates became susceptible to it. Shrivastava et al revealed that MIC analysis of Ceftazidime /Sulbactam in Pseudomonas aeruginosa, Acinetobacter baumannii, and Citrobacter were (0.5, 0.015625, and 0.0625 mg/L,) which were lower than ceftazidime and sulbactam individually.¹⁹A study by Fetiye Kolayl et al reported with 4 mg/L and 8 mg/L Ceftazidime/Sulbactam resistant to 22% in E. coli.²⁰

K Prabhash et al showed that the sensitivity pattern for the Enterobacteriaceae group revealed that 32.1% of isolates were resistant to Cefoperazone/sulbactam, 74.5% to ceftazidime. Among the non-lactose fermenters, 47.5% isolates were resistant to Cefoperazone/sulbactam, and 51.6% to Ceftazidime/Sulbactam. For the Pseudomonas species, Cefoperazone-sulbactam was resistant to 42.6%, and Ceftazidime/Sulbactam was resistant to 46.9 % isolates.²¹

CONCLUSION

Antibiotic resistance is a great concern throughout the world and top most priority in developing countries. The reasons are:

1. Irrational use of Antibiotics.

2. Non-existence of proper Antibiotics policy

3. Non implementing existing Antibiotics policy.

4. Self-medication and counter sale of Drugs in the Pharmacies.

5. Taking improper course of antibiotics.

6. Use of antibiotics for Veterinary purpose.

7. lake of evidence based medication for illness.

Hence in order to rational and judicial use of existing antibiotics, the Clinicians and Paramedicals are to be sensitized by suitable awareness programmes and the present scenario to be brought to the notice of politicians and policymakers to formulate the policy and legislation for rational use of Antibiotics to prevent the development of resistance by the disease causing microorganisms and to implement the evidence based medications for the treatment.

LIMITATIONS

This is a single centered study

ACKNOWLEDGEMENT

The Authors thank the Dean and Director of Department of microbiology Madurai Medical College, Madurai to conduct this Study. The authors gratefully acknowledge the technicians and other co staff who have helped to conduct this study successfully

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