Journal of Islamabad Medical & Dental College (JIMDC); 2012(2):93-96 93 Original Article Frequency of Enteric Gram Negative Rods Isolated from Various Clinical Samples Shamim Mumtaz*, Salma Zafar** and Mumtaz Ahmad *** * Professor of Pathology, Islamabd Medical & Dental College, Islamabad ** Assistant Professor Pathology, Islamabad Medical and Dental College, Islamabad *** Associate Professor, Department of Urology, Rawalpindi Medical College, Rawalpindi, (Bahria University, Islamabad) Abstract Objective: To determine the frequency of gram negative rods in both nosocomial and community acquired infections in our setting. Material and Methods: This descriptive study was conducted at the Microbiology Department of Fauji Foundation Hospital, Rawalpindi over a period of two years (April 2004 to March 2006). All pus, urine, blood and HVS received during the study period were included. Samples without properly filled forms, dried swabs and improperly transported samples were excluded. Results: Two thousand and ninety one Enteric Gram Negative Rods (EGNR) were isolated over a period of two years. They were most commonly found in urine samples (40.7%), followed by pus (37.1%), HVS (12.0%), sputum (6.12%) and blood (3.92%) samples. The most common EGNR isolated was Escherichia coli (49.7%) followed by Klebsiella pneumoniae (23.4%), Pseudomonas aeruginosa (19.5%) and Proteus species (1.81%). The most commonly isolated Enteric Gram negative rod from urine samples (n 852) was E. coli (622) followed by Klebsiella pneumoniae (189), Providentia (18) and Enterobacter species (13). The most commonly isolated Gram negative rod from pus samples (n 852) was Pseudomonas aeruginosa (290) followed by E. coli (227), Klebsiella pneumoniae (18) and Proteus species. The most commonly isolated EGNR from HVS samples was E. coli (130) followed by Klebsiella pneumoniae (68) and Pseudomonas aeruginosa (51). The most common EGNR found in sputum (n 128) was Klebsiella pneumoniae (56) followed by Pseudomonas aeruginosa (40) and E. coli (30). In blood samples the most common organism isolated was E. coli, followed by Pseudomonas and Klebsiella. Key Words: Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa Introduction The human life has always been in danger from diseases caused by microorganisms. The history still mourns the death toll of epidemics of influenza, plague & malaria which occurred during the 19th century. Gram negative bacilli (GNB) are a common cause of sepsis, pneumonia, urinary tract infections, and post surgical infections in patients in acute care hospitals.1 During 1970s prevalence of nosocomial infections at specific sites has varied from survey to survey. Gram negative bacilli and Staphylococcus aureus were most frequently isolated from patients with hospital-acquired infection.2 During 1980s there has been a major shift in the etiology of hospital-acquired infections leading to an increase in the laboratory isolation of Coagulase-Negative Staphylococci, Candida, Staph aureus, Enterococci, Pseudomonas aeruginosa and Enterobacter spp.3 Etiologic shifts in nosocomial infections and development of antimicrobial resistance among these pathogens, particularly those isolated from intensive care units are alarming.4 Taken as a whole, the shifts are away from more easily treated pathogens towards more resistant pathogens with fewer options left for therapy.5 Keeping in view the importance of EGNR (especially E. coli, Klebsiella, Pseudomonas) as major pathogens, and the emerging resistance against the commonly used antibiotics, the present study was designed to determine the frequency of gram negative rods in our setting. Material and Methods This study was carried out in the department of microbiology, Fauji Foundation Hospital Rawalpindi, over a period of two years (April 2004 to March 2006). The clinical samples including pus, high vaginal samples, urine, blood and sputum, received from various indoor and outdoor departments were inoculated on the special and enriched media depending on the type of the sample and were identified by the help of standard procedures. The aerobically incubated organisms were identified with the help of colonial morphology, gram staining and biochemical analysis like TSI, indole, urease, citrate and API depending upon the availability.6 Results Two thousand, ninety one EGNR were isolated over a period of two years. The samples received during the study period were: urine samples 852 (40.7 %), followed by pus samples Journal of Islamabad Medical & Dental College (JIMDC); 2012(2):93-96 94 Table 1; Distribution of Enteric Gram Negative Rods in Various Samples Organisms Urine Pus HVS Sputum Blood Total No-% (No) (No) (No) (No) (No) % Escherichia coli 622-73.0 227-29.1 130-51.5 30--23.4% 30-36.5 % 1039 49.7 Klebsiella pneumoniae 189-22.0 171--2.31% 68-26.9% 56--43.7% 6--7.31% 490 23.4 Pseudomonas aeruginosa - 290-37.3% 51-20.2% 40-- 31.2% 26-31.7 % 407 19.5 Proteus species - 33-3.87 - 1 4 38 1.81 Providencia species 18--2.1 11 - - - 29 1.38 Enterobacter species 13--1.52 12 - - 4 29 1.38 Acinetobacter species - 12 - - 1 13 0.62 Citrobacter species 6 2 1 - - 9 0.43 Salmonella species 6 - - 2 8 0.38 Morganella species 7 1 - - 8 0.38 Salmonella typhi - - - - 6 6 0.28 Hafnia species 3 1 - - 4 0.19 Serratia species 1 2 - 1 - 4 0.19 Aeromonas species - 1 - - 3 4 0.19 Yersinia atypical - 1 - - - 1 0.05 Fusobacteria species - 1 - - - 1 0.05 Xanthomonas species - 1 - - - 1 0.05 TOTAL 852 777 252 128 82 2091 Fig 1; Frequency of EGNR in various samples 777 (37.1%) 128 (6.12%) 82 (3.92%) 2091 852 (40.7%) 0 500 1000 1500 2000 2500 Urine Pus HVS Sputum Bloo Total 252 (12.0%) Journal of Islamabad Medical & Dental College (JIMDC); 2012(2):93-96 95 777 (37.1%), HVS 252 (12.0%), sputum 128 (6.12%) and blood 82 (3.92%) (Figure 1). Most common EGNR isolated were Escherichia coli 1039 (49.7%) followed by Klebsiella pneumoniae 490 (23.4%), Pseudomonas aeruginosa 407 (19.5%), Proteus species 38 (1.81%). The rare occurring organisms were Hafnia species, Serratia species, Aeromonas species 04 (0.19%) each followed by Yersini, Fusobacteria, Xanthomonas species 01 (0.05%) each. The most commonly isolated Enteric Gram negative rod from urine sample was E. coli 622 (73.0%) followed by Klebsiella pneumonia 189 (22.0%), Providentia 18 (2.1%) and Enterobacter species 13 (1.52%). The most commonly isolated Gram negative rod from pus sample was Pseudomonas aeruginosa 290 (37.3%) followed by E. coli (227-29.1%), Klebsiella pneumoniae 171 (2.31%) and Proteus species (33-3.87%). Among HVS sample the most frequent EGNR was E. coli 130 (51.5%) followed by Klebsiella pneumoniae 68 (26.9%), Pseudomonas aeruginosa 51 (20.2%). The most frequently found EGNR found in sputum was Klebsiella pneumoniae 56 (43.7%) followed by Pseudomonas aeruginosa 40 (31.2%) and E. coli 30 (23.4%). In blood samples E. coli was the most commonly isolated pathogen 30 (36.5 %) followed by Pseudomonas aeruginosa 26 (31.7 %) and Klebsiella 6 (7.31%) (Table1). Discussion Two thousand, ninety one Enteric Gram Negative Rods (EGNR) were isolated over a period of two years. EGNR were most common pathogens isolated from urine samples (40.7 %), followed by pus samples (37.1%), HVS (12.0%), sputum (6.12%) and blood samples (3.92%). In a previous study conducted on different bacterial isolates, the frequency rates of causative bacteria in various types of infectious diseases were mentioned. Significant urine culture isolates were E. coli and other enterobacteriaceae in uncomplicated UTI, and Pseudomonas spp. in complicated UTI with a urinary catheter. In respiratory tract infections (RTIs), P. aeruginosa, were among common causative organisms. In common with hospital-acquired pneumonia, P. aeruginosa, and enterobacteriaceae were the frequent microorganisms isolated.7 These results correlate to our study which has shown enteric gram-negative and related rods to be the most common isolates from different clinical samples. Most common EGNR isolated were Escherichia coli (49.7%) followed by Klebsiella pneumoniae (23.4%), Pseudomonas aeruginosa (19.5%), and Proteus species (1.81%). This is comparable to previous studies which have shown E. coli (61%) to be the most common pathogen followed by K. pneumoniae (22%), P. aeruginosa (4.0%), A. baumannii (3.0%) and Citrobacter spp (2.0%). 8-10 The most commonly isolated Enteric Gram negative rod from urine sample is E. coli (73.0%) followed by Klebsiella pneumoniae (22.0%), Providentia (2.1%) and enterobacter species (1.52%). Of the 920 tested sample, 100 samples showed growth of pathogens among which the most prevalent were E. coli (61%) followed by Klebsiella spp (22%).10 In a study conducted in Iran, isolated bacteria in urine were as follows : Escherichia coli 72.9%; Klebsiella pneumoniae 24.5%; Citrobacter spp 1%; Proteus mirabilis 0.6% and Pseudomonas spp 1%. 11 The second most common sample from which Gram- Negative Rods were obtained in this study were pus samples (37.1%) followed by High vaginal swabs (12%), sputum samples (6.12%) and blood samples (3.92%). The most commonly isolated Gram negative rod from pus sample is Pseudomonas aeruginosa (37.3%) followed by E. coli (29.1%), Klebsiella pneumoniae (2.31%) and Proteus species (3.87%). The results in previous studies showed E coli (25-31%), K pneumoniae (9.5-10%) & Pseudomonas (8.6-38).12-15 In another study frequencies of pathogens in primary surgical infections in an order of decreasing frequency were Escherichia coli, Pseudomonas aeruginosa, Acinetobacter, Klebsiella pneumoniae and others.15 The most commonly isolated Gram-negative rod from HVS samples is E. coli (51.5%) followed by Kleb. pneumoniae (26.9%) and Pseudomonas aeruginosa (20.2%), comparable to a previous study where E. coli was 21%.16 Chow et al and Izhar et al also made similar observations, as E coli were the commonest organisms (25&45%), followed by K pneumoniae (18 & 25%), P aeruginosa (22 & 28.7%), Acinetobacter spp (7% each) and Enterobacter spp (7 & 11%).17,18 Pseudomonas aeruginosa (30.3%) was the most frequent isolate among Gram-negative organisms, followed by E coli (18.6%), K pneumoniae (16.9%), Acinetobacter baumannii (8.8%) and Enterobacter cloacae (7.1%).19 Majority of the above mentioned organisms are normal flora of female genital tract and may not be implicated with disease production. It seems unlikely that the gynecological infections are caused by the same mechanism throughout the world. The members of normal flora may themselves produce disease under certain circumstances if introduced into foreign locations in large numbers and if predisposing factors are present. The most common EGNR found in sputum was Klebsiella pneumoniae (43.7% ) followed by Pseudomonas aerugonisa ( 31.2% ) and E. coli ( 23.4% ) comparable to a study by Jawad et al 2011 where most prevalent gram negative rods causing respiratory tract infections were Klebsiella pneumoniae (21.4%) & P. aeruginosa (15.3%).20 Whereas the prevalent pathogens in other studies were H influenzae (73%), Moraxella catarrhalis (12%),and H parainfluenzae (5%), Klebsiella spp (7.7%), and P. aeruginosa (2.9%).21,22 Blood stream infections (BSI) revealed E coli (36.5 %) and Pseudomonas aeruginosa (31.7 %) followed by Klebsiella pneumoniae (7.31%). The results were comparable to other studies whereas E. coli (17.2-37%), Klebsiella spp (6.3- 9.6%), and Pseudomonas aeruginosa (6.5%) were the predominant organisms.23 During 1997-2002, a total of 81,213 BSI pathogens from North America, Latin America, and Europe were tested for antimicrobial susceptibility. E. coli, were the most common BSI pathogens in all three regions each year.6 In other studies E coli (8.1-21%), Pseudomonas spp (6.7-17%), Acinetobacter spp (5-10%), Journal of Islamabad Medical & Dental College (JIMDC); 2012(2):93-96 96 Salmonella spp (3.8%) and miscellaneous group (9.2%) were the most common pathogens.24,25 In a study by Mamishi et al, (2005) Klebsiella spp (31%) were most predominant, followed by Escherichia coli (21%) and Pseudomonas aeruginosa (17%). Decousser et al, (2003) established Escherichia coli, as one of the major bacterial isolates in BSI.26,27 In a study by Mehta et al, (2005), among the 567 qualifying samples, Pseudomonas aeruginosa (19.75%), Escherichia coli (15.17%), Klebsiella pneumoniae (14.99%), and Salmonella enterica serovar typhi (12.87%) were the most frequently isolated Gram-negative bacteria.28 Conclusion The sample showing gram-negative rods as a major pathogens were urine samples followed by pus samples. Escherichia coli is the most common gram-negative rod isolated followed by Klebsiella pneumoniae and Pseudomonas aeruginosa. References 1. Yan JJ, Ko WC, Tsai SH, Wu HM, Wu JJ. Outbreak of infection with multidrug-resistant Kleb. pneumoniae carrying bla (IMP-8) in a university medical center in Taiwan. J Clin Microbiol 2001; 39(12):4433-9. 2. John E. McGowan Jr. and Maxwell Finland Infection and Antibiotic Usage at Boston City Hospital: Changes in Prevalence during the Decade 1964–1973 Journal of Infectious Diseases;129(4):421-428. 3. Banerjee SN, Emori TG, Culver DH, Gyanes RP, Jarvis WR and Horan T, Edward JR et al. Secular trends in nosocomial primary bloodstream infections in United States. Am J Med 1991; 16(91): 86-89. 4. Hsueh PR, Chen ML, Sun CC, Chen WH, Pan HJ, Yang LS, Chang SC, Ho SW, Lee CY, Hsieh WC, Luh KT. Antimicrobial drug resistance in pathogens causing nosocomial infections at a university hospital in Taiwan, 1981-1999. Emerg Infect Dis 2002; 8(1):63-8. 5. Schaberg DR, Culver DH, Gaynes RP. Major trends in the microbial etiology of nosocomial infections. Am J Med 1991; 91(3): 72-5. 6. Colles JG and Miles RS. Tests for identification of bacteria. In: Practical Medical Microbiology. eds. Collee JG, Deguid JP, Fraser AG and Marmion BP.13th edition Churchill Livingstone Edinburgh1989, pp141-15. 7. Igari J Current state of causative bacteria in infections diseases and trends in resistance to antimicrobial agents in Nihon Rinsho 1994; 52(2):297-302. 8. Khan MT, Shah SH. Experience with Gram- negative bacilli isolated from 400 cases of Urinary tract infection (UTI) in Abbottabad. J Ayub Med Col 2000; 12(4): 21-3. 9. Kumamoto Y, Tsukamoto T, Hirose T, Yokoo A, Fujime M, Fujita K, Shigeta S, et al. Comparative studies on activities of antimicrobial agents against causative organisms isolated from patients with urinary tract infections; Susceptibility distribution. Jpn J Antibiot.1999; 52(2):93-129. 10. Akram M, Shahid M , Khan A. Etiology and antibiotic resistance patterns of community-acquired urinary tract infections in J N M C Hospital Aligarh, India Ann Clin Microbiol Antimicrob 2007; 6: 4. 11. Irajian G, JazayeriMoghadas A. Frequency of extended- spectrum beta lactamase positive and multidrug resistance pattern in Gram-negative urinary isolates, Semnan, Iran. Jundishapur J Microbiol 2010; 3(3): 107-13. 12. Arshad M, Khan NU, Ali N, Afridi NM. Sensitivity and spectrum of bacterial isolates in infectious otitis externa. J Coll Physicians Surg Pak 2004; 14(3): 146-149. 13. Mumtaz S, Akhtar N, Hayat A. Antibiogram of aerobic pyogenic isolates from wounds and abscesses of patients at Rawalpindi. Pakistan J Med Res 2002; 41(1): 16-18. 14. Fazlul MKK, Zaini MZ, Rashid MA, Nazmul MHM. Antibiotic susceptibility profiles of clinical isolates of Pseudomonas aeruginosa from Selayang Hospital, Malaysia Biomedical Research Biomedical Research 2011;22(3):263- 266. 15. Arya M, Arya PK, Biswas D, Prasad R. Antimicrobial susceptibility pattern of bacterial isolates from post- operative wound infections. Indian J Pathol Microbiol 2005;48(2):266-9. 16. Khan I & Khan UA. A hospital based study of frequency of aerobic pathogens in vaginal infections. J Rawal Med Coll 2004; 29(1):22-25. 17. Chow JW, Satishchandran V, Snyder TA, Harvey CM, Friedland IR, Dinubile MJ. In vitro susceptibilities of aerobic and facultative Gram-negative bacilli isolated from patients with intra-abdominal infections world wide: Study for Monitoring Antimicrobial Resistance Trends (SMART). Surg Infect 2005;6(4):439-48. 18. Izhar M, Khan S, Naqvi A. Anti-Microbial Resistance among Gram-negative Bacteria prevalent in Intensive Care Units. Pakistan J Surg 2001; 17(3):23-6. 19. Kiffer C, Hsiung A, Oplustil C, Sampaio J, Sakagami E, Turner P, Mendes C. MYSTIC Brazil Group. Advisory Group on Antimicrobials and Clinical Microbiology, Fleury Institute, Sao Paulo, Brazil. Antimicrobial susceptibility of Gram-negative bacteria in Brazilian hospitals: the MYSTIC Program Brazil. Braz J Infect Dis 2003; 9(3):216-24. 20. Ahmed J, Jan AH, Nawaz G and Khan M. Epidemiology and antibiotic susceptibility of bacterial isolates from Northern Pakistan African Journal of Microbiology Research 2011; 5(28):4949-4955. 21. Butt T, Rafi N, Ahmed S, Ahmed RN, Salman M, Mirza SH. Community-acquired bacterial pneumonias in Rawalpindi. Pakistan J Pathol 2005; 16(1):14-6. 22. Qureshi AH, Qamar RH, Hannan A. The Prevalence of Bacterial Pathogens in Throat Infections and their Susceptibility Pattern. Pak Armed Forces Med J. 1997;47(1):34-6. 23. Aftab R and Iqbal I. Bacteriological agents of neonatal sepsis in NICU at Nishter hospital Multan. J Coll Physicians Surg Pak 2006; 16(3): 216-19. 24. Asrat D and Amanuel YW. Prevalence and antibiotic susceptibility pattern of bacterial isolates from blood culture in Tikur Anbassa Hospital, Addis Ababa, Ethiopia. Ethiop Med J 2001; 39(2):97-104. 25. Butt T, Afzal RK, Ahmad RN, Salman M, Mahmood A, Anwar M. Bloodstream infections in febrile neutropenic patients: bacterial spectrum and antimicrobial susceptibility pattern. J Ayub Med Coll 2004;16(1):18-22 26. Mamishi S, Pourakbari B, Ashtiani MH, Hashemi FB. Frequency of isolation and antimicrobial susceptibility of bacteria isolated from bloodstream infections at Children's Medical Center Tehran Iran 1996-2000. Int J Antimicrob Agents 2005; 26(5):373-9 27. Decousser JW, Pina P, Picot F, Delalande C, Pangon B, Courvalin P, Allouch P. The ColBVH Study Group; Frequency of isolation and antimicrobial susceptibility of bacterial pathogens isolated from patients with bloodstream infections: a French prospective national survey. J AntimicrobChemother 2003; 51(5):1213-22 28. Mehta M, Dutta P, Gupta V. Antimicrobial susceptibility pattern of blood isolates from a teaching hospital in north India. Jpn J Infect Dis 2005; 58(3):174-6 [