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Synergistic Effect of Antibiotics against Bacterial Pathogens Causing Diarrhea Isolated from Children <5 Years of Age.

Byline: Rabia Irshad, Safia Bibi, Waqaruddin Ahmed, Syed Ejaz Alam and Furqan Hasan

Abstract

Background: Infant mortality rate due to diarrhea is high in Pakistan. The alarming increase in antimicrobial resistance of bacterial pathogens causing diarrhea has made the treatment more complicated. This study was designed to determine the in vitro synergistic (combined) effect of different antibiotics against these pathogens in order to determine whether the combined antibiotic therapy may be used to treat diarrhea.

Objectives: To study the antibiotic resistance pattern of bacterial pathogens causing diarrhea isolated from children 93% in 2006, 2007, 2008 and 2009 respectively.10 In the latest research, 58%, 85%, 12.6%, 3.0% and 2.4% Shigella isolates were resistant to ampicillin, trimethoprim-sulfamethoxazole, nalidixic acid, ofloxacin and ceftriaxone respectively.11

Another research of Karachi is also the evident of high rate of resistance in Shigella against Ampicillin and nalidixic Acid 55.5% and 39% respectively.12 Resistance to ampicillin and cotrimoxazole is also observed in E.coli as 72% and 78% respectively.13

This increasing resistance among diarrheal pathogens points towards the need of testing alternative options for treating diarrhea, synergism/ combined antibiotic effect is an important phenomenon through which diarrheal rate would be reduced. Synergistic effect is the activity of two drugs when they are used in combination, their effect is greater to the sum of their independent activity when studied separately.14 Hence this research aims to study the synergistic (combined) effect of antibiotics against bacterial pathogens causing diarrhea which shows resistance against existing antibiotics.

Materials and Methods

It was cross sectional study and samples (bacterial pathogens) from children less than 5 years of age were obtained from National Institute of Child Health (NICH), Karachi and other private laboratories / clinic of Karachi. The collected samples were brought and processed in PHRC, JPMC Karachi. A standardized proforma was designed to record details of isolate, sensitivity pattern, antibiotics tested and combination results.

A total of 80 bacterial isolates were collected including salmonella, shigella, vibrio, aeromonas and E.coli, 46 (57.5%) were collected during March to December-2013 and 34 (42.5%) were collected during January to November-2014. Smooth lawn was made on Muller Hinton Broth through sterile cotton swab and antibiotic discs were placed on it, at appropriate distance according to Kirby Bauer disk diffusion method as recommended by Clinical and Lab Standard Institute (CLSI) guidelines and incubated at 37AdegC for 24 hours. After incubation, the zone of inhibition was measured. The antibiotics which showed intermediate zone or no zone were considered as less effective for that pathogen, so was selected for combined effect.

Standard powder form of antibiotics (stored at 2-8AdegC) was used for combined activity by checker board method.15 The stock solutions and serial twofold dilutions of each antibiotic to at least double the MIC was prepared according to the recommendations of NCCLS guidelines,14 Mueller-Hinton broth (50ul) was distributed to each well of the micro dilution plate. Inoculum (0.5 McFarland) was prepared from each isolate in Mueller-Hinton broth. MIC of each pathogen was determined by micro dilution method. Each micro titer well was inoculated with 100ul of a bacterial inoculum of 5 x 105 CFU/ml and incubated at 37AdegC for 24 hours under aerobic conditions. Inhibitory concentration (results) at which antibiotics exhibits synergistic effect was recorded.

According to NCCLS guidelines: IPSFIC was calculated as follows:

IPSFIC = FIC A + FIC B

Where: FIC A is the MIC of drug A in the combination/MIC of drug A alone

FIC B is the MIC of drug B in the combination/MIC of drug B alone

The combination was considered synergistic when the IPSFIC is a$?0.5

The combination was considered indifferent when the IPSFIC is >0.5 to <2

The combination was considered antagonistic when the IPSFIC is [greater than or equal to]2

The data feeding and analysis was done on computer package SPSS (Statistical Packages of Social Sciences) version 11.0. Clinical characteristics was summarized in terms of frequencies and percentages for qualitative variables (physical examination, microscopic examination, etc.), mean +- S.D, for quantitative variables (zone of inhibition of existing antibiotics). To find the resistant pattern of existing antibiotics and to check the dilution at which antibiotics exhibits synergistic effect. Study was approved by ethical review board of National Institute of Child Health, Karachi.

Results

Out of 80 pathogens, 51% were salmonella, 20% were shigella, 13% were vibrio, 14% were E.coli and only 3% were Aeromonas. Existing antibiotics including Ampicillin (AMP), Cefotaxime (CTX), Cefixime (CFM), Co-trimoxazole (SXT) and Ofloxacin (OFX) were checked for sensitivity by disk diffusion method and findings are given in Table.

The isolates which gave intermediate zone or no zone were selected for MIC and combined effect. By this method, most of the combinations showed indifferent and antagonistic activity. The combination of ampicillin and cefixime showed as synergistic for a single isolate of E.coli. and ampicillin and co-trimoxazole exhibit synergistic effect against Vibrio. Similarly cefixime and ofloxacin showed synergy against Aeromonas.

Table: Sensitivity and resistance pattern.

Antibiotics###Salmonella###Shigella###Vibrio###E.coli###Aeromonas

###n=41 (%)###n=16 (%)###n=10 (%)###n=11 (%)###n=02 (%)

###S###25 (60.97)###08 (50.00)###01 (10.00)###03 (27.27)###01 (50.00)

###I###06 (14.63)###05 (31.25)###03 (30.00)###00 (00.00)###00

Ampicillin###R###10 (24.39)###03 (18.75)###06 (60.00)###08 (72.72)###01 (50.00)

###S###34 (82.92)###14 (87.5)###07 (70.00)###10 (90.90)###02 (100.0)

###I###01 (02.43)###00###00###01 (09.09)###00

Cefotaxime###R###06 (14.63)###02 (12.5)###03 (30.00)###00###00

###S###33 (80.48)###13 (81.25)###04 (40.00)###11 (100.0)###01 (50.00)

###I###00###01 (06.25)###00###00###00

Cefixime###R###08 (19.51)###02 (12.5)###06 (60.00)###00###01 (50.00)

###S###33 (80.48)###07 (43.75)###05 (50.00)###10 (90.90)###02 (100.0)

Co-trimoxazole###I###00###00###01 (10.00)###00###00

###R###08 (19.51)###09 (56.25)###04 (40.00)###01 (09.09)###00

###S###23 (56.09)###11 (68.75)###04 (40.00)###10 (90.90)###02 (100.0)

###I###02 (04.87)###01 (06.25)###00###01 (09.09)###00

Ofloxacin###R###16 (39.02)###04 (25.00)###06 (60.00)###00###00

Discussion

In fact antibiotics play an important role in preventing deaths due to bacterial diarrhea but it is reported that mortality rate due to diarrhea is continuously increasing. As research proved that in Pakistan "2.5 lac children die because of diarrhea annually."16 In routine clinical practices, Trimethoprim-Sulfamethoxazole, Cefixime, Ampicillin, Cefotaxime and Fluoroquinolones are recommended to treat diarrhea. But literature suggests that these existing antibiotics are becoming less effective or pathogens have acquired resistance.

So the goal of this in vitro study was to access synergies (combinations) among antibiotic that are commonly used clinically for treatment of diarrhea. In this study, bacterial pathogens were obtained from clinical specimen of diarrheal patients and their sensitivity pattern was performed. Existing antibiotics including Ampicillin, Cefotaxime, Cefixime, Co-trimoxazole and Ofloxacin to cure diarrhea were checked for sensitivity by disk diffusion method. And the selection of antibiotics for synergy studies was based on antibiotic resistance.

As study results reflects that more than 50% isolates were Salmonella while Aeromonas were very less prevalent which is only 2.5%, Actually Aeromonas has a number of virulence factors and isolated as a single enteropathogen from 80% diarrheal and 20% asymptomatic cases 17 so may be this was the reason of less prevalence of Aeromonas. Study results showed that most of the isolates exhibit resistance against existing antibiotics so constant antibiotic surveillance is necessary because the pathogens became extremely unaffected (resistant) to several antimicrobials. Therefore this study contributes to strengthening of the prevailing surveillance system and makes available aid for effective prevention and control strategies for antimicrobial resistance.18

In view of the emergence of multidrug-resistant strains, there is a need for therapeutic alternatives so the isolates which give intermediate zone or no zone were selected for MIC and combined effect by Checkerboard method. This assay was used to test the activities of drugs in combinations by determining the FICs of all combinations tested.

By this method, most of the combinations show indifferent and antagonistic activity. The combination AMP-SXT and CFM-OFX yielded synergistic activity against Vibrio and Aeromonas respectively. The AMP-CFM and AMP-OFX yielded synergistic activity against E. coli. In comparison to other international studies, research also suggested that combined medication positively cures the infection of H.pylori in up to 90% of individuals.19 Similarly, another study reported that combined therapy significantly reduced the mortality rate by 56% of patients 20 and can increase the single drug strength and slower the emergence of resistance.21 The combination of cryptdin 2 and ampicillin was also found as extremely effective, which indicated in vitro synergy between the two agents.22 Furthermore, as the results indicates that there is no combination of antibiotics proved as synergistic for Salmonella and Shigella. So the reason behind this may be the increasing resistance of pathogens.

This widespread resistance could be attributed to excessive or indiscriminate use of antibiotics.23 Finally emergence of pathogen's resistance to antibiotics is tending to be a serious problem. Furthermore, recurrent and lengthy use of antibiotics usually results in alteration of the intestinal flora.

One limitation of our study is that some combinations, such as CTX and CFM were not tested because these two drugs belong to same generation so have same activity.

Little synergistic activity of different antibiotic combinations was observed against diarrheal isolates, and most of the combinations exhibited indifferent and antagonistic activity. Hence we may conclude that single antibiotics are effective than combination.

Acknowledgement

I acknowledged Pakistan Health Research Council (PHRC) for funding this study and Dr. Shahid Jamal for providing diarrheal samples.

Conflict of interest: None declared.

References

1. Aurthur Diskin, Steven C Dronen. Gastroenteritis in Emergency Medicine. emedicine; 2011.

2. Pakistan amongst countries with highest child mortality rate. (Accessed on 14th October 2011) Available from URL: http://www.humanrights.asia

3. Luby SP, Agboatwalla M, Painter J, Altaf A, Billhimer WL, Hoekstra RM, Effect of Intensive Hand washing Promotion on Childhood Diarrhea in High-Risk Communities in Pakistan, A Randomized Controlled Trial. JAMA 2004; 291(21): 2547-54.

4. Health care in Pakistan, Wikipedia free encyclopedia. (Accessed on 12th December 2017) Available from URL: https://en.wikipedia.org/wiki/Healthcare_in_Pakistan

5. Child Mortality. (Accessed on 12th December 2017) Available from URL: http://globalhealth.org/?s=child +mortality

6. Soofi SB, Habib MA, von Seidlein L, Khan MJ, Muhammad S, Bhutto N, et al. A comparison of disease caused by Shigella and Campylobacter species: 24 months community based surveillance in 4 slums of Karachi Pakistan. J Infect Public Health; 2011;4(1): 12-21.

7. Alam M, Ansari A, Chaudhry FA, Bangash MA. Seasonal Variation in Bacterial Pathogens isolated from Stool Samples in Karachi, Pakistan. JPak Med Assoc 2003;53(3): 125-9.

8. Meng CY, Smith BL, Bodhidatta L, Richard SA, Vansith K, Thy B, et al. Etiology of diarrhea in young children and patterns of antibiotic resistance in Cambodia. Pediatr Infect Dis J 2011;30(4): 331-5.

9. Ali SQ, Zehra A, Naqvi BS, Shah S, Bushra R. Resistance Pattern of Ciprofloxacin Against Different Pathogens. Om journal 2010; 25(4): 294-8.

10. Abbasi S, Imtiaz A, Usman J, Kaleem F, Hassan A. Evaluation of the current trend of Nalidixic Acid succeptibility in typhoidal Salmonella; a marker of therapeutic failure for the fluoroquionolones. Ind J Microbiol 2011; 3: 80-3.

11. Khan E, Jabeen K, Ejaz M, Siddiqui J, Shezad MF, Zafar A. Trends in antimicrobial resistance in Shigella species in Karachi, Pakistan. J Infect Dev Ctries 2009;; 3(10): 798-802.

12. Zafar A, Sabir N, Bhutta ZA. Frequency of isolation of shigellaserogroups/serotypes and their antimicrobial susceptibility pattern in children from slum areas in Karachi. J Pak Med Assoc 2005;55(5): 184-8.

13. Thaver D, Ali SA, Zaidi AK, Antimicrobial resistance among neonatal pathogens in developing countries. Pediatr Infect Dis J 2009;28(1 Suppl): S19-21.

14. Mazen Kherallah. Combination Antibiotics, King Faisal Specialist Hospital and Research Center. (Accessed on 14th October 2011) Available from URL: https://www. google.com.pk/url?sa=tandrct=jandq=andesrc=sandsource=weband cd=1andcad=rjaanduact=8andved=0ahUKEwi3roi0iKfYAhUG nRQKHV9ZA9UQFggmMAAandurl=http%3A%2F%2Fww w.mecriticalcare.net%2Fdownloads%2Flectures%2FComb inationAntibiotics.pptandusg=AOvVaw1fTNPGqP3d7n-ljFrOqIYf

15. Orhan G, Bayram A, Zer Y, Balci I. Synergy Tests by E Test and Checkerboard Methods of Antimicrobial Combinations against Brucellamelitensis. J Clin Microbiol 2005; 43(1): 140-3.

16. What's Killing Children? Diarrhea - Policy, Sanitation, and Handwashing. 2008. (Accessed on 12th December 2017) Available from URL: http://www.machetemag.com/policy-global-development/global-development/whats-killing-children-diarrhea-policy-sanitation-and-handwashing/

17. Aslani MM, Alikhani MY. The Role of AeromonasHydrophilla in diarrhea. Iranian J publ Health 2004; 33(3) 54-9.

18. Mei Qu, Bing Lv, Xin Zhang, Hanqiu Y, Ying H, Haikun Q,. Prevalence and antibiotics resistance of bacterial pathogens of childhood diarrhea in Beijing, China (2010-2014). Gut Pathog 2016; 8: 31.

19. Crowe SE, Feldman M, Grover S. Patient information: Helicobacter pylori infection and treatment. 2011(Accessed on 12th December 2017) Available from URL: https://www.uptodate.com/contents/helicobacter-pylori-infection-and-treatment-beyond-the-basics

20. Karim SSA. Combined Drug Therapy to Treat TB and HIV Significantly Improves Survival. 2010 (Accessed on 12th December 2017) Available from URL: https://www.mailman.columbia.edu/public-health-now/news/combined-drug-therapy-treat-tb-and-hiv-significantly-improves-survival

21. Crofton SJ, TB combination Therapy pioneer - a long and admirable life, posted by Abel Pharmboy; 2009. (Accessed on 12th December 2017) Available from URL: http://scienceblogs.com/terrasig/2009/11/20/sir-john-crofton-tb-combinatio/

22. Rishi P, Preet S, Bharrhan S, Verma I. In vitro In vivo synergistic effects of Cryptdin 2 and Ampicillin against Salmonella. Antimicrob Chemotherapy 2011;55(9): 4176-82.

23. Saranya S, Hemashenpagam N. Antagonistic activity and antibiotic sensitivity of lactic acid bacteria from fermented dairy products, Pelagia Research Library 2011; 2(4): 528-34.
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Publication:Pakistan Journal of Medical Research
Article Type:Report
Geographic Code:9PAKI
Date:Dec 31, 2017
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