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Short Communication FIRST GENERATION AND FOURTH GENERATION SUSCEPTIBILITY AGAINST CLINICAL ISOLATES

Adeel Arsalan*, Syed Baquir Shayam**, Rehan Pasha**, Osama Shakeel*** *Baqai Institute of Pharmaceutical Sciences, Baqai Medical University, Karachi-Pakistan **Department of Pharmaceutics, Faculty of Pharmacy, University of Karachi. Karachi-Pakistan ***Karachi Medical and Dental College, Karachi-Pakistan

ABSTRACT: PURPOSE: Curing of infections is usually on the top priority of health associated-professionals. Resistance of microbes by is worrying situation for medical professionals. are one of the most commonly used β-lactam antibiotics. and cephalexin are belonging to fourth and first generation cephalosporin respectively. The main aim of the present study is to evaluate the difference in resistance a pattern of pathogens between these two generations of anti-infective agents. METHOD: During the present study, in-vitro antibacterial activity of cefepime and cephalexin has been observed by Kirby-Bauer method (disk diffusion method). A sum of 91 clinical isolates of Staphylococcus aureus (43), Escherichia Coli (37), and Klebsiella pneumoniae (11) have been isolated from urine, stool, sputum and surgical, burn, and accidental wound pus from different hospitals at Karachi. RESULT: The present study has revealed that Staphylococcus aureus, Escherichia coli and Klebsiella pneumoniae has been shown 37.7%, 26.3%, and 34.8% resistance against cefepime, fourth generation cephalosporin respectively, while 77.4%, 91.7%, and 89.6% S. aureus, E. coli, and K. pneumoniae has been resistant against first generation cephalosporin, cephalexin. CONCLUSION: It has concluded from the present study, that cefepime 4th generation cephalosporin is far much better susceptibility as compared to cephalexin. The study has been strongly recommended the routine monitoring and observation of susceptibility of microbes against respective antibiotics before administration. KEYWORDS: Cephalosporin; cephalexin; cefepime; clinical isolates; sensitivity significant aspects to increase the resistance 9-11 INTRODUCTION: against antibiotics. Since last three Microorganisms are living in human body as decades, the use of broad spectrum commensalism and pathogens and also in antibiotics has been increased due to many food material by which the individual may factors like decreased drug interactions, and 1-4 reduced the risk for toxicity, and suffer from infections. Antibacterial activity 12 of antibiotics has been chiefly depending upon pharmacoeconomic advantages. the concentration of drug reached in systemic Cephalosporins, β-lactam antibiotics have circulation and infected tissue. better efficacy with tolerability as compared to Microorganisms have the ability to transfer other group of antibiotics. By cephalosporins, their resistance traits to the antibiotics to their patients are not as much of hypersensitive next generation.5 Resistance of pathogen has with cephalosporins as compared to other β- been developed against antibiotics due to low Corresponding Author: dose antibiotics, long course of antibiotics6, Dr. Adeel Arsalan too early and too late administration7, Baqai Institute of Pharmaceutical Sciences, suboptimal, irrational and extensive use.8 Baqai Medical University, Karachi-Pakistan Several workers have been found that low E-mail: [email protected] concentration of antibiotics is one of the

52 JUMDC Vol. 7, Issue 2, April-June 2016 ARSALAN A., BAQUIR SHAYAM S., etal. FIRST GENERATION AND FOURTH GENERATION lactams like and its derivates. pneumoniae, Serratia, Citrobacter, Proteus Cephalosporin has been generally classified mirabilis and less active against Bacillus into four generations.13 fragillis and .16,17 Cephalexin is first generation empirical oral Cefepime has been the drugs of choice in life cephalosporin. Cephalexin has been threatening infections like febrile neutropenia, prescribed as first line therapy for cellulitis, septicemia, and in severe infections of and patients sensitive to penicillin. Cephalexin intensive care units (ICU) patients. Several has been indicated in the treatment of otitis workers have reported that cefepime has media, streptococcal pharyngitis, bone and been less sensitive to extended-spectrum β- joint infections, pneumonia, and urinary tract lactamases (ESBLs) as produced by Gram- infections. It may be used to prevent bacterial negative pathogens than other generations of endocarditis and urinary tract infections cephalosporin.18 (UTIs).14 It has been mostly prescribed in USA MATERIALS AND METHODS: in 2008. Cephalexin has been provided antimicrobial activity against Gram-positive COLLECTION OF SPECIMENS bacteria like -susceptible A total of 91 clinical isolates belonging to Staphylococcus aureus (MSSA), coagulase- different genera were isolated from urine, negative Staphylococci, penicillin-susceptible stool, sputum, surgical, burn, and accidental Streptococcus pneumoniae, and Gram- wound pus either mixed or single culture. negative bacteria such as Moraxella Staphylococcus aureus (n=43), Escherichia catarrhalis, Escherichia coli, Klebsiella coli (n=37), and Klebsiella pneumoniae pneumoniae, Proteus mirabilis. It has been (n=11) were collected on sterile swabs. mainly indicated in skin and soft tissue Clinical isolates were obtained from patients infections (SSTI), UTIs, and respiratory tract who had developed sign and symptoms of infections (RTIs) during pregnancy due its infections from different hospitals at Karachi. safety profile.15 The isolates were identified based on their Cefepime, a fourth generation cephalosporin colony characteristics on different media and has definite edge over the first generation confirmed by biochemical reactions. The cephalosporin. Cefepime has not only broad isolates were inoculated in caso agar/ tryptic spectrum action but also acquired bactericidal soya agar slants. The slants had been activity against resistant Gram-positive preserved at 4oC in the refrigerator. Anti pathogens like methicillin-resistance Microbial Resistance (AMR) has been Staphylococcus aureus (MRSA), penicillin determined by Clinical and Laboratory resistance Streptococcus pneumoniae, Standard Institute (CLSI, formally NCCLS) Streptococcus pyogenes and extended- reference disk diffusion (Kirby-Bauer) spectrum β-lactamases (ESBLs), producing method19,20. Gram-negative Escherichia coli, Klebsiella Table 1: Summary of Clinical Isolates Clinical Isolates Source of Clinical Isolates Number of Clinical Isolates

Staphylococcus Surgical, Burn and Accidental wound pus (Skin and 43 aureus soft tissue infections) Escherichia coli Stool and Urine (Intra-abdominal and urinary tract 37 infections) Klebsiella Surgical wound pus (Skin and soft tissue infections), 11 pneumoniae Sputum (Respiratory tract infection)

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Table 2: Resistance Pattern of Clinical Isolates Involved in Different Infections Disc Diffusion Method† Percentage Antibiotics Clinical Isolates of Sensitive Intermediate Resistance Resistance Staphylococcus ≥ 18 15-17 ≤ 14 77.4% aureus Cephalexin Escherichia coli ≥ 18 15-17 ≤ 14 91.7% Klebsiella ≥ 18 15-17 ≤ 14 89.6% pneumoniae Staphylococcus ≥ 18 15-17 ≤ 14 37.7% aureus Cefepime Escherichia coli ≥ 18 15-17 ≤ 14 26.3% Klebsiella ≥ 18 15-17 ≤ 14 34.8% pneumoniae INCUBATION PREPARATION OF INOCULUMS The plates were overturned within 15 min of placing the disc on agar and incubated at 35- Muller-Hilton Broth (MHB) was used to 37 oC for 24 hours. After incubation the prepare inoculums and matched with diameter of the clear zones around the McFarland standard. All tubes were incubated disc were measured by using vernier at 37 oC for few hours to develop the required caliper. All the bench work was carried out turbidity as that of the McFarland standard. near a flame to create a zone of inhibition of Muller- Hilton Agar (MHA) was used to invading bacteria and maintained the determine the sensitivity of clinical isolates. integrity. Bauer, Kirby, Sherris and Tuck strongly suggested Mueller Hinton Agar for performing RESULT: antibiotic susceptibility tests using a single We have found total 91 clinical isolates from disk of high concentration.19 different hospital labs and pathological INOCULATION OF BACTERIAL CULTURE laboratories at Karachi. Staphylococcus aureus (n=43), the most isolated microbes, A sterile swab was dipped into a broth mainly from surgical, burn, and accidental suspension of bacterial culture. Excess wound pus samples, Escherichia coli (n=37) is inoculum was removed by rotating the swab the second competitor clinical isolate, mainly against the inside wall of the tube with slim isolated from urine, sputum, and wound pus pressure. The whole surface of MHA plate was samples, while Klebsiella pneumoniae (n=11) then streaked uniformly in three directions has been least isolated pathogen, mostly approximately at 60o angle from each other. isolated from sputum and urine samples. The The lid was then replaced and the plates were summary of the source of clinical isolates has allowed to dry for 10-15 min. been given in table 1. PLACEMENT OF ANTIBIOTIC DISC By the present study, it has been revealed that 37.7%, 34.8%, 26.3% resistance against The appropriate antibiotic impregnated discs S. aureus, K. pneumoniae, E. coli against were placed on the agar surface with sterile cefepime, while cephalexin has possessed forceps. Each disc was pressed down gently 77.4%, 89.6%, and 91.7% resistant to S. with the forcep to assure good contact with aureus, K. pneumoniae, E. coli. Cephalothin- agar surface. The disc should be distributed class disc to predict cephalexin susceptibility such that each is at least 24 mm from center discs was emphasized. Respective first and to center of its nearer neighbor and 12 mm fourth generation cephalosporin have from the edge of plate. contained 30 µg content.20 The outcome susceptibility of clinical isolates has been shown in table 2 and fig. 1.

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developed β-lactamase which aid in the DISCUSSION: resistance against bactericidal activity of The main aim of study is to find the difference cephalosporins. Some novel broad spectrum between resistances of microbes among 1st cephalosporins have been soon introduced in generation cephalosporin (cephalexin) and 4th market like ceftaroline23 and ceftobiprole24 for generation cephalosporin (cefepime). It has the prevention and cure of virulent resistant been known mainly resistant virulent bacteria. They also have an edge over pathogens like MRSA and Extended-Spectrum penicillin, as 10% population is sensitive to Beta-lactamase (ESBL) producing bacteria like cephalosporins in 100% penicillin Escherichia coli and Klebsiella pneumoniae. It hypersensitive persons. has been proven that the resistance against Cephalexin, first generation oral cephalosporin β-lactam has been increased. has been marketed by Eli Lilly in 1967. It has been widely prescribed in otitis media, streptococcal pharyngitis, bone and joint infections, pneumonia, cellulitis, and UTIs. The gram-negative bacilli, Escherichia coli, Klebsiella pneumoniae have been less susceptible to cephalexin than the gram-positive cocci, the resistance of E. coli (91.7%) and K. pneumoniae (89.6%) has been shown disappointing position.25 As, it has been found by present study cephaexin has been resistant by methacillin-sensitive S. aureus (MSSA) around 78%. The present study has been supported by Anupurba et al.26 In 1981, Bayston and Swinden25 reported 56% resistant of methacillin-resistant S. aureues (MRSA) against cephalexin. In 2008,

was the most popular cephalosporin In vitro study of antibacterial action has been performed frequently, as consequences of the antibiotic in the United States. The increased sensitivity can be used to conclude how a in resistant of microbes against cephalexin 21 has been developed due to irrational use. drug would act inside the body. By the Cefipime, a fourth generation cephalosporin prolong use of antibiotics, resistance of pathogenic bacteria against antibiotics has developed in 1994. It has been usually been a main concerned. Resistance of prescribed in moderate- severe nosocomial pneumonia, infections antibiotics is also on priority not only for caused by multi-resistant microorganisms health-associated professionals but also a key problem for pharmaceutical industry. The (e.g. P. aeruginosa; MRSA) illogical prescription of antibiotics has and empirical treatment of febrile neutropenia.27 The present study increased the chances of resistance. 28 Moreover, a huge investment of time and has been supported by Nasiri et al. The money of pharma-industries has been lost due outcome of resistance of S. aureus was 37.7% 4,22 in present study. In Taiwan, Liao et al. have to irrational use of antibiotics. reported 33% resistance of E. coli against Cephalosporins are one of the most commonly 29 used belonging to β-lactam class of cephalexin. 89.6% resistance of K. antibiotics. Till now established four pneumoniae against cefepime has been found by our study. Winokur and co-workers have generation of cephalosporins are used by also been reported the resistance of K. physicians and marketed by pharmaceutical 30 industries. Due to its better tolerability, pneumoniae against cefepime. As it has been observed, the resistance of durability, and excellent pharmacokinetic microbes even against 4th generation profile, it is one of the most prescribed antibiotics. Several pathogens have been cephalosporin (cefipime) has been increased

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ARSALAN A., BAQUIR SHAYAM S., etal. FIRST GENERATION AND FOURTH GENERATION with the span of time due to irrational use. Karachi. Annals. Food Sci. Technol. 2013; The treatment of infectious disease is one of 14(2): 318-26. the most troubled issue for health concerned 4. Arsalan A, Naqvi SBS, Sabah A, Bano R, professionals and main cause of morbidity and Ali SI. Resistance pattern of clinical mortality. In underdeveloped countries there is lacking of the usage of antibiotics isolates involved in surgical site guidelines. infections. Pak. J. Pharm. Sci. 2014; 27(1): 97-102. CONCLUSION: 5. Craig WA (1998). The present study has suggested the health Pharmacokinetic/pharmacodynamic associated professional to follow the parameters: rationale for antibacterial guidelines. Moreover, there must a national dosing of mice and men. Clin. Infect. Dis. guideline for health associated professionals to obstruct the illogical use of antibiotics. The 1998; 26: 1-12. antibiotics should be only sale on 6. Sae-Tia L and Chongsomchai C. prescriptions. The culture sensitivity should be Appropriateness of anti-biotic prophylaxis promoted to reduce the use of newly broad in gynecologic surgery at Srina-garind spectrum antibiotics. The pharma-industries Hospital. J. Med. Assoc. Thai. 2006; 89: should be promoted their antibiotics in a 2010-4. specific indication to increase the life of 7. Sadique I, Abid S, Aleem S, Anwar S, susceptibility of their antibiotics. It has been suggested that local bodies should prepared Hafeez M, Pasha MI and Butt F. Single guidelines for initial treatment. Dose Prophylaxis in Obstetrics and Due to financial crisis in underdeveloped Gynaecological Surgeries. Annals, 2009; countries, there is also a deficiency of local 15(4): 176-9. surveillance program. Due to implacable 8. Knox K, Lawsony W, Deanyz B and disobedience of the frequent use of antibiotics Holmesx A. Multidisciplinary antimicrobial in underdeveloped countries, the world renowned regulatory bodies like WHO, NICE, management and the role of the CDC, and several infection control society infectious diseases pharmacist: A UK were steadily recommended the day to day perspective. J. Hosp. Infect. 2003; 53: inspection plan to control the misuse of 85-90. antibiotics. The regulatory bodies also force 9. Fantin B, Farinotti R, Thabaut A, Carbon physician to strictly fellow the guidelines of C. Conditions for the emergence of empiric therapy to avoid the resistance and to resistance to and in tend and treat infections. experimental endocarditis due to REFERENCES: Pseudomonas aeruginosa. J. Antimicrob. 1. Arsalan A, Anwar Z, Ahmad I, Saba A, Chemother. 1994; 33: 563-9. Naqvi SBS. Microbes in pediatric infant 10. Mouton JW, den Hollander JG. Killing of formula. Annals. Food Sci. Technol. 2013; Pseudomonas aeruginosa during 14(1): 90-9. continuous and intermittent infusion of 2. Arsalan A, Anwar Z, Ahmad I, Shad Z, ceftazidime in an in vitro pharmacokinetic Ahmed S. Cronobacter sakazakii: An model. Antimicrob Ag. Chemother. 1994; emerging contaminant in peadiatric infant 38: 931-6. milk formula. Int. Res. J. Pharm. 2013; 11. Davies J, Wendon J, Wyncoll D, Wade J. 4(4): 17-22. Antibiotic infusions reduce the incidence 3. Arsalan A, Naqvi SBS, Ali SI, Anwar Z. of resistant organisms in liver intensive Contamination of microorganisms in care [abstract]. Intensive Care Med. pediatric infant formula marketed in 2000; 26: s269.

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pneumoniae in two regional hospitals in Taiwan. J. Microbiol. Immunol. Infect. Submitted for publication: 02-02-2016 2006; 39(1): 59-66. 30. Winokur PL, Canton R, Casellas JM, Accepted for publication: 20-05-2016 Legakis N. Variations in the prevalence of strains expressing an extended-spectrum β-lactamase phenotype and characterization of isolates from Europe, SR AUTHOR NAME CONTRIBUTION the Americas, and the Western Pacific # Region. Clin. Infect. Dis. 2001; 32(2): 1 Dr. Adeel Arsalan Manuscript Writing S94-103. 2 Dr. Syed Baquir Review of Shayam Naqvi Literature 3 Dr. Rehan Pasha Review of Literature 4 Dr. Osama Proof Reading Shakeel

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