112 J I M D C 2 0 1 8 112 Open Access F u l l L e n g t h A r t i c l e Antibiotic Susceptibility Pattern of Gram Negative Bacilli Isolated from Different Clinical Specimens in a Tertiary Care Hospital Lubna Ghazal 1, Ayaz Hussain Qureshi 2, Naila Iqbal 3 1 Assistant Professor, 2 Professor /Head of Department 3 Registrar (Department of Microbiology, Wah Medical College) A B S T R A C T Objective: To find out the antibiotic susceptibility pattern of gram-negative bacilli isolated from different clinical specimens received in a tertiary care hospital at Wah. Material and Methods: This cross-sectional study was carried out to determine antibiotic susceptibility pattern of gram- negative bacilli, cultured from different clinical specimens received in POF Hospital laboratory at Wah. One hundred and forty-four clinical isolates of gram-negative rods from different clinical specimens from April 2015 to March 2016 were included in the study. All the isolates were processed by standard microbiological methods. The antibiotic susceptibility pattern was carried out by disk diffusion method as recommended by Clinical Laboratory Standard Institute guidelines (CLSI). Results: Out of one hundred and forty-four Gram-negative bacilli, one hundred (69.44%) were from Enterobacteriaceae family and forty-four (30.56%) were from non-Enterobacteriaceae group. The commonest isolated organism was Escherichia coli (47.3%), followed by Pseudomonas aeruginosa (17.36%) and Acinetobacter baumannii (13.19%). These isolates were highly resistant to the most of the commonly prescribed antibiotics. The members of the family Enterobacteriaceae showed better sensitivity for amikacin and cefoperazone-sulbactam. Resistance rate for carbapenems was significantly high for K.pneumoniae and Proteus mirabilis. Among non-Enterobacteriaceae, P.aeruginosa showed better susceptibility for cefoperazone-sulbactam, amikacin, imipenem and meropenem. The multi- drug resistant pattern was observed for Acinetobacter.baumannii. Conclusion: The isolates depict highly resistant patterns to available oral antibiotics as well as commonly prescribed injectable third generation cephalosporins and carbapenems. Establishment and implementation of infection control practices are required to combat this grave situation. Key words: Antibiotics, Antibiotic susceptibility, Enterobacteriaceae, Gram negative bacilli, Non-Enterobacteriaceae. Author`s Contribution 1 Conception, synthesis, planning of research and manuscript writing Interpretation and discussion 2 Data analysis, interpretation and manuscript writing, 3 Active participation in data collection. Address of Correspondence Lubna Ghazal Email: doctor.lubna@yahoo.com Article info. Received: July 28, 2017 Accepted: December 12, 2017 Cite this article. Ghazal L, Qureshi AH, Iqbal N. Antibiotic Susceptibility pattern of Gram Negative Bacilli Isolated from different Clinical Specimens in a Tertiary Care Hospital. JIMDC.2018; 7(2):112-117 Funding Source: Nil Conflict of Interest: Nil I n t r o d u c t i o n Antibiotics have enabled tremendous advances in the discipline of infectious diseases since their emergence in 1930. Unfortunately, the occurrence of resistant bacteria is endangering the efficacy of antibiotics, which have transformed medicine and saved millions of lives.1 The infections caused by multiple-drug resistant (MDR) gram- negative organisms have created entire classes of antibiotics redundant and threatened to bring about the end of the 'antibiotic era'. 2,3 Extended-spectrum beta- lactamases, Metallo-beta-lactamases and Amp-C O R I G I N A L A R T I C L E 113 J I M D C 2 0 1 8 113 mediated beta-lactamases produced by Enterobacteriaceae and other non-lactose fermenters are increasingly implicated in outbreaks through the dissemination of mobile genetic elements rendering emergence of resistant mutants.4 This grave situation demands optimization of therapy primarily because of substantial increases in the frequency with which these organisms affect the health care settings as well as the community acquired infections. The challenge of heightened antimicrobial resistance among gram-negative pathogens has been exacerbated by the stagnation in the development of novel antimicrobials. Studies have provided convincing evidence that effective initial empirical antibiotic therapy, based on ultimate drug susceptibility results, improves survival.2 The constantly evolving antimicrobial resistance patterns render antibiotic susceptibility profile in one region at a specific period, inapplicable to other region or in another period. Thus, antimicrobial susceptibility data from any given regional, national, or international surveillance study cannot reliably predict the drug resistance profiles of pathogens isolated from an individual patient.5 The current study was designed to document the commonly isolated gram-negative bacilli from different clinical specimens and their susceptibility patterns in tertiary health care hospital at Wah. This will be an effort to rationalize the empirical treatment by clinicians resulting in evidence-based practice and better results in terms of early recovery from infections, shorter duration of hospitalization and cost effectiveness. Moreover, this effort will contribute to safeguard the remaining therapeutic options for the clinicians and encourage a focused, concerted effort against key human pathogens. M a t e r i a l a n d M e t h o d s This cross-sectional study was carried out at Microbiology section of Pakistan Ordinance Factories Hospital laboratory from April 2015 to March 2016. One hundred and forty-four clinical specimens from patients either gender, of all ages, yielding growth of Gram-negative bacilli were included in the study and selected by convenient sampling. Duplicate samples of the same patient from the same site were not included.The specimens were inoculated on appropriate culture medium like blood agar, MacConkey agar, chocolate agar (sputum) and cysteine lactose electrolyte deficient agar (urine) and incubated at 35-37˚C under aerobic conditions for 24 hours. After overnight incubation, the agar plates were examined for growth of bacteria and their colonial morphology. Gram-negative rods were identified based on Gram staining, catalase test, oxidase test and motility.6 Microbact Gram-negative 24E identification kits (Oxoid, Basingstoke, UK) were used for confirmation of isolates. Antimicrobial susceptibility tests were performed on the Muller–Hinton agar plates with disk diffusion method, as recommended by clinical laboratory standards institute.7 The bacterial suspensions of isolates equivalent to 0.5 McFarland standard turbidity were applied on Mueller- Hinton agar (Oxoid, Basingstoke, UK). The antimicrobial disks (Oxoid, Basingstoke, UK) were evenly placed on the inoculated plates and included Ampicillin (10 µg), amoxicillin-clavulanate (20/10 µg), trimethoprin- sulfamethoxazole (1.25/ 23.75 µg), ceftriaxone (30 µg), ciprofloxacin (5 µg), gentamicin (10 µg), amikacin (30 µg), cefoperazone-sulbactam (75/ 30 µg), imipenem (10 µg) and meropenem (10 µg). Concurrent quality control testing was performed with Escherichia coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853. After overnight incubation, the diameter of each zone of inhibition around the antimicrobial disk was measured. The susceptibility results were interpreted according to recommendations of CLSI as sensitive, intermediate and resistant.7 The data were entered and analyzed using SPSS version 19. For qualitative variables (Gram- negative bacilli, gender, type of samples and organisms isolated) frequencies and percentages were calculated. Mean ± SD was presented for age. R e s u l t s A total of one hundred and forty-four (144) isolates of Gram-negative bacilli were included in the study. Majority of isolates were yielded from urine (n=83, 57.6%), followed by pus (n=23, 16%) and respiratory specimens (n=17, 11.9%). The other isolates were from blood, high vaginal swabs, catheter tips, ear swabs, tissue and body fluids. The distribution of specimens along with their breakup is presented in table 1. Out of one hundred and forty-four (144) Gram-negative bacilli, one hundred (69.44%) were members of the family Enterobacteriaceae 114 J I M D C 2 0 1 8 114 Figure 1: Frequency of isolated gram-negative bacilli (n=144) and forty-four (30.56%) were from non- Enterobacteriaceae. and forty-four (30.56%) were from non-Enterobacteriaceae. The commonest isolated organism was Escherichia coli (47.3%), followed by Pseudomonas aeruginosa (17.36%) and Acinetobacterbaumannii (13.19%). (Figure-1) Out of one hundred and forty-four isolates, 53.47% were recovered from male patients and 46.53% from female patients. Mean age of the patients was 49.32years+ 23.72 SD. Age distribution of different age groups which yielded Gram-negative isolates is shown in Figure-2. Figure 2: Distribution of different age groups, which yielded gram- negative bacilli Thirty-seven isolates (33.3%) were isolated from outdoor patients, while the remaining (n=107, 66.7%) were from patients admitted in different wards. Their distribution is presented in Figure 3. The resistance frequency of E.coli against ampicillin, amoxicillin-clavulanate and trimethoprim- sulfamethoxazole was 93.3%, 81.3% and 76% respectively. None of the isolates was 100% susceptible to any of the antimicrobials assessed in the study. Table 1: Break up of clinical specimens yielding gram-negative bacilli (n=144) Frequency Percentage Blood 8 5.6 Tracheostomy discharge 8 5.6 Endotracheal tubes 6 4.2 CVP 1 .7 CSF 1 .7 Vitreous tap 1 .7 Bile 1 .7 Tissue 1 .7 Bronchoalveol ar lavage 1 .7 Peritoneal fluid 2 1.4 Urine 83 57.6 High vaginal Swabs 1 .7 Sputum 2 1.4 Catheter tips 4 2.8 Pus 23 16.0 Ear swab 1 .7 115 J I M D C 2 0 1 8 115 Table 2: Antimicrobial resistance pattern in Enterobacteriaceae Escherichia coli (n=75) Klebsiella pneumonia (n=15) Enterobacter Spp (n=08) Proteus mirabilis (n=02) n R% n R% n R% n R% AMP 70 93.3 15 100 7 87.5 2 100 AMC 61 81.3 14 93.3 7 87.5 2 100 AK 14 18.6 7 46.6 3 37.5 2 100 G 32 42.6 8 53.3 3 37.5 1 50 COT 57 76 11 73.3 6 75 2 100 CRO 51 68 11 73.3 4 50 1 50 IMP 17 22.6 8 53.3 1 12.5 1 50 MNP 21 28 8 53.3 2 25 1 50 SCF 10 13.3 5 33.3 2 25 1 50 CIP 51 68 8 53.3 3 37.5 1 50 The antibiotic susceptibility pattern of members of Enterobacteriaceae, including E.coli, K.pneumoniae, Enterobacter sp and Proteus mirabilis has been shown in Table 2. Figure-3: Sites of collection of gram-negative bacilli. The susceptibility pattern of members of non- Enterobacteriaceae including Pseudomonas aeruginosa and Acinetobacter baumannii is displayed in Table 3. Table 3: Antimicrobial resistance pattern in Non- Enterobacteriaceae Pseudomonas Aeruginosa (n=25) Acinetobacter Baumannii (n=19) n R% n R% AMP _ _ 19 100 AMC _ _ 19 100 AK 13 52 15 78.9 G 20 80 17 89.4 COT _ _ 18 94.7 CRO _ _ 17 89.4 CAZ 24 96 _ _ IMP 13 52 18 94.7 MNP 13 52 18 94.7 CIP 18 72 17 89.4 SCF 12 48 12 63.1 DOX _ _ 13 68.4 AMP-Ampicillin, AMC-Amoxicillin-clavulanate, AK- Amikacin, G-Gentamicin, COT-Trimethoprim- sulfamethoxazole , CRO- Ceftriaxone, CAZ- Ceftazidime, IMP- Imipenem , MNP- Meropenem, SCF- Cefoperazone- sulbactam , CIP- Ciprofloxacin ,DOX- Doxycycline, R- Resistant 116 J I M D C 2 0 1 8 116 D i s c u s s i o n The susceptibility pattern of Gram-negative isolates revealed an alarming resistance ratio to commonly used antibiotics. In the present study, E.coli, Klebsiella pneumoniae, Enterobacter spp and Proteus mirabilis are highly resistant to first-line drugs including ampicillin, trimethoprim-sulfamethoxazole and amoxicillin- clavulanate. These findings are in agreement with a study carried out at Fauji Foundation Hospital Rawalpindi by Nabi et al.8 It is worrisome to note the high rates of resistance of members of Enterobacteriaceae to the third generation cephalosporins (ceftriaxone) and also to the commonly used flouroquinolone (ciprofloxacin) in our study. Similar resistance patterns have been reported in other studies from Rawalpindi4 as well as from Iran.9 This situation is much different when compared to the resistance rate prevailing in England as published in English surveillance programme for antimicrobial utilization and resistance (ESPAUR) report, 2014.10 The contrasting results provide an evidence of injudicious and imprudent use of these antibiotics in our setup. The resistance frequency of E.coli against gentamicin (42.67%) and amikacin(18.67%) is comparable to a study conducted on uropathogens by Nabi et al in Dhaka in 2014.11 A low percentage of resistance of E.coli against amikacin indicate that this antibiotic may be a useful therapeutic agent in our setup when considered as empirical choice. Klebsiella pneumoniae is fairly resistant to gentamicin and amikacin. This finding is in concordance with those reported by Bhat et al.12 A high and moderate level of resistance of Proteus mirabilis against amikacin and gentamicin respectively are depicted in our study, a situation different from the one reported by Bahashwan et al.13 A disturbing situation existing in our hospital is 53.3% resistance against imipenem and meropenem conferred by K.pneumoniae, and 50% for both members of carbapenems by P.mirabilis. Similar results have been reported from other parts of the subcontinent.11,13 Poor infection control measures contribute to the development of high-level resistance to these relatively safe and effective antibiotics. Resistance of E.coli and Enterobacter spp against carbapenems are also significantly high when compared to studies conducted in two different institutions at Rawalpindi.4,8 Our study also revealed A.baumannii is significantly resistant (89.4%) to each of ceftriaxone, gentamicin and ciprofloxacin. This bug shows 94.4% resistance to carbapenems and trimethoprim- sulfamethoxazole. The resistance pattern in our settings is in accordance when compared to data by Fayyaz et al16 in Rawalpindi and Sohail et al in Lahore.17 Cefoperazone- sulbactam and doxycycline are relatively effective drugs against A.baumannii (63.1% & 68.4% respectively) in our setting , which is a similar finding as demonstrated by Fayyaz et al.16 Our data suggests 96% resistance of P.aeruginosa against ceftazidime, 72% against ciprofloxacin and 52% against amikacin , imipenem and meropenem respectively. Comparison with ESPAUR report revealed a stupendous difference in susceptibility pattern of P.aeruginosa, indicative of a failure of antibiotic stewardship in our settings.10 Antibiograms in context to local data unveiled an increased resistance ratio to antipseudomonal antibiotics as compared to our clinical settings.4,18 Our study is laboratory-based and has no correlation with the clinical outcomes after antibiotics administration to treat the specific pathogen. Despite this limitation, the study will be helpful for local clinicians to make an appropriate choice of antibiotic for empirical therapy. C o n c l u s i o n E.coli is the most common Gram-negative bacillus, followed by P.aeruginosa and A.baumannii isolated from the culture of clinical specimens in POF Hospital, Wah. The isolates depict highly resistant patterns to available oral antibiotics as well as commonly prescribed injectable third generation cephalosporins and carbapenems. 1. Gram-negative bacilli reveal relatively better susceptibility against Cefoperazone-sulbactam. 2. Antibiotic resistance is a dynamic phenomenon which emphasizes continuous monitoring of infection control practices and regular surveillance of antibiotics susceptibility patterns in our health care setting. 3. Strict implementation of polices for judicious use of antibiotic and efficient infection control practices are strongly recommended. 117 J I M D C 2 0 1 8 117 R e f e r e n c e s 1. Boucher HW, Talbot GH, Bradley JS et al. Bad bugs, no drugs: no ESKAPE! An update from the Infectious Diseases Society of America. Clin. Infect. Dis. 2009;48(1):1–12. 2. Centers for Disease Control and Prevention, Office of Infectious Disease Antibiotic resistance threats in the United States, 2013. 3. Pendleton JN, Gorman SP, Gilmore BF. Clinical Relevance of the ESKAPE Pathogens. Expert Rev Anti Infect Ther. 2013; 11(3): 297-308. 4. Khan IU, Mirza IA, Ikram A, Afzal A, Ali S, Hussain A, et al. Antimicrobial Susceptibility Pattern of Bacteria Isolated from Patients with Urinary Tract Infection. J Coll Physicians and Surg Pakistan. 2014; 24 (11): 840- 44. 5. BalanK1 ,Sujitha K2 , Vijayalakshmi TS. Antibiotic Susceptibility Pattern of Gram Negative Clinical Isolates in a Teaching Tertiary Care Hospital. Sch. J. App. Med. Sci., 2013; 1(2):76-79. 6. Schreckenberger PC, Janda JM, Wong JD, Baron EJ. Algorithms for identification of aerobic gram-negative bacteria. Manual of Clinical Microbiology. 1999; 7:438-52. 7. Murray PR, Baron EJ, Jorgensen JH, Landry ML, Pfaller MA (editors). Manual of Clinical Microbiology. 9thed. Washington, D.C: ASM press 2007. 371-6. 8. CLSI. Performance standards for antimicrobial susceptibility testing; Twenty-fifth informational supplement. CLSI document M100–S25. Wayne, PA: Clinical and Laboratory Standards Institute; 2015. 9. Nabi SG, Zaffar G, Mumtaz S. Antimicrobial Resistance Pattern of Gram Negative Bacilli Isolated from Clinical Samples. JIMDC. 2014; 3(1):25-28. 10. Rabiradi N, Mohammad PM, Lari R, Shojaie A, Bayat R, Alebouyeh M. Antimicrobial susceptibility patterns of the gram-negative bacteria isolated from septicemia in Children's Medical Center, Tehran, Iran. J prev med hyg. 2014; 55(1): 23-26. 11. Public Health England: English surveillance programme antimicrobial utilization and resistance (ESPAUR) report - Publications - GOV.UK. 2014. 12. Nabi SN, Haider KMTS, Rahimgir M, Uddin MN, Shapla NR. Current Trends of Urinary Pathogens and their Antimicrobial Susceptibility Pattern in a Tertiary Care Hospital. JAFMC. 2015;10(2): 71-74. 13. Bhat V, Gupta S, Kelkar R, Biswas S, Khattry N, Moiyadi AA, et al. Bacteriological profile and antibiotic susceptibility patterns of clinical isolates in a tertiary care cancer center. Indian J Med PaediatrOncol. 2016; 37(1): 20-4. 14. Bahashwan SA, El Shafey HM. Antimicrobial resistance patterns of Proteus isolates from clinical specimens. European Scientific Journal. 2013; 9(27): 188-202. 15. Kalam K, Qamar F, Kumar S, et al. Risk factors for carbapenem resistant bacteraemia and mortality due to gram-negative bacteraemia in a developing country. J Pak Med Assoc. 2014;64(5): 530-6. 16. Kim YJ, Kim SI, Hong KW, Kim YR, Park YJ, Kang MW. Risk factors for mortality in patients with carbapenem- resistant Acinetobacterbaumannii bacteremia: impact of appropriate antimicrobial therapy. J Korean Med Sci. 2012; 27(5):471-5. 17. Fayyaz M, Khan IU, Hussain A, Mirza IA, Ali S, Akbar N. Frequency and Antimicrobial Susceptibility Pattern of Acinetobacter Species Isolated from Pus and Pus Swab Specimens. J Coll Physicians Surg Pak. 2015; 25(5): 346- 349. 18. Sohail M, Rashid A, Aslam B, Waseem M, Shahid M, Akram M, et al. Antimicrobial susceptibility of Acinetobacter clinical isolates and emerging antibiogram trends for nosocomial infection management. Rev Soc Bras Med Trop. 2016; 49(3): 300-304.