Original Article. Speciation of oxidase positive non fermentative gram negative bacilli

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1 Original Article Speciation of oxidase positive non fermentative gram negative bacilli Usha Kalawat, Sumathi I, Jaya Prada, Satish Kumar Reddy, Abhijit Chaudhury, KK Sharma Department of Microbiology, Sri Venkateswara Institute of Medical Sciences, Tirupati, India Abstract Background: Due to lack of simple and economic methods for speciation of oxidase positive non fermentative gram negative bacilli (NFGNB) and the belief that most of them are saprophytes, the oxidase positive isolates are reported as Pseudomonas aeruginosa without further testing for identification. Any commensal or saprophyte can cause infections when a suitable opportunity arises. This is also true for oxidase positive NFGNBs which are widely being reported from different clinical settings among immunocompromised as well as immunocompetent patients. There are contrasting differences in the antibiotic sensitivities of these organisms making identification and sensitivity testing a necessity. Objectives: This study was conducted to speciate various oxidase positive NFGNBs from different clinical samples and to study their antibiotic sensitivity profile. Materials and Methods: Oxidase positive clinical isolates from various samples were characterized by biochemical analysis using a battery of tests. Antibiotic sensitivity was done according to National Committee on Clinical Laboratory Standards (NCCLS) guidelines for antibiotic susceptibility testing for P. aeruginosa. Results: We isolated 78 different types of oxidase positive species from different clinical samples. The various samples included urine, sputum, Bronchoalveolar lavage, Cerebrospinal fluid (BAL, CSF), blood, pus, catheter tips, and body fluid. P. aeruginosa was the commonest isolate comprising 25 (32%), followed by Moraxella catarrhalis (11.5%), Pseudomonas sp. CDC group 17 (8.9%), and Moraxella osloensis and CDC Vb 3 (each 5.1%) among others. Conclusions: Identification and antibiotic sensitivity testing of oxidase positive NFGNBs are necessary for apt therapy as they are widely prevalent in the environment and can cause a wide spectrum of infections. Key Words: Non fermentative gram negative bacilli, oxidase positive organism, oxidase positive gram negative bacilli INTRODUCTION Oxidase positive non fermentative gram negative bacteria (NFGNB) are generally thought to be harmless environmental saprophytes and only Pseudomonas aeruginosa is clinically thought to be important. [1] In recent years, the number of diagnoses of infections caused by non fermentative bacteria has increased, largely because of a heightened awareness of the possible roles of these bacteria as pathogens rather than as contaminants. [2,3] In most of the laboratories, all the oxidase positive NFGNB are reported as P. aeruginosa without further identification. Most of the other isolates we find as case reports Quick Response Code: Access this article online Website: DOI: / only, due to some incidental identification. But there are contrasting differences between the sensitivity patterns to different antibiotics as reported by some studies. [4] With the exception of P. aeruginosa, the Kirby Bauer method has not been standardized for these organisms. This study was taken up to speciate oxidase positive NFGNB by standard biochemical methods and to perform their antibiotic susceptibility testing. MATERIALS AND METHODS Oxidase positive organisms from different clinical samples were processed for identification using a battery of biochemical tests and to study their antibiotic sensitivity pattern by disk diffusion method. [1] Methods employed for identification included: Gram staining, hanging drop preparation, catalase test, pigmentation on blood agar plate, oxidase test by Kovac s wet filter paper method, indole test, triple sugar iron agar, 10% lactose, lysine decarboxylase test, arginine dihydrolase test, nitrate reduction, Hugh Leifson s O F test, urease test, phenylalanine Address for correspondence: Dr. Usha Kalawat, Department of Microbiology, Sri Venkateswara Institute of Medical Sciences, Tirupati , Andhra Pradesh, India. E mail: ukalawat@yahoo.com 22 Scholars' Research Journal / Jan-Dec 2012 / Vol 2 Issue 1 and 2

2 [Downloaded free from on Monday, June 23, 2014, IP: ] Click here to download free Android application for this journa Kalawat, et al.: Oxidasepositive NFGNB deaminase test, esculin hydrolysis, gelatin hydrolysis, and starch hydrolysis [Figure 1]. Polymyxin B (300 units) and penicillin (1.2 µg disks) were also used for identification purpose [Figure 2 and 3]. Antibiotic sensitivity was carried out as per guidelines for P. aeruginosa. All antibiotic disks and media used were obtained from Himedia laboratories PVT. LTD., Mumbai, India. CDC group II c, and Brevundimonas vesicularis contributed one (1.2%) each to the total of NFGNB isolates [Table 2]. RESULTS Total number of oxidasepositive NFGNBs isolated was 78 from 72 different clinical samples. The various infections from which these organisms were isolated are depicted in Figure 4. Most commonly, the organisms were isolated from respiratory tract infection (RTI) both from outpatients and inpatients followed by urinary tract infection (UTI), wound infection, postoperative central nervous system infection, septicemia/bacteremia in decreasing frequency. Among the RTI, three were cancer patients and two were organophosphorus poisoning cases. Six soft tissue infections were in diabetic patients and four in nondiabetic ulcers which were mainly posttrauma infections. Most of the isolates from wound infections were P. aeruginosa. Figure 1: Starch hydrolysis by oxidasepositive NFGNB spp Of the 72 clinical samples, 46 (63.88%) were males and 26 were females (36.11%). The age and sex distribution of samples are presented in Table 1. The youngest patient was 1yearold female child and the oldest patient was 87yearold male. P. aeruginosa was the commonest isolate comprising 25 (32%), followed by Moraxella catarrhalis (11.5%), Pseudomonas sp. CDC group 17 (8.9%), and Moraxella osloensis and CDC Vb3 each comprised four (5.1%). Delftia acidovorans, Achromobacter piechaudii, Pseudomonas mendocina (Vb2), and Flavobacterium comprised three (3.8%) each. Gilardi rod group 1, Ralstonia picketti, Achromobacter xylosoxidans, CDC EO2, and Alcaligenes faecalis contributed two (2.5%) each. Bordetella hinzii, Alcaligenes denitrificans, Figure 2: Flavobacterium spp. showing resistance to polymyxin B and penicillin Table 1: Age and sex distributions of various clinical samples (n=72) Age (years) Males no. (%) Females no. (%) Total no. (%) Total 2 (100) 6 (50) 1 (33.3) 7 (63.6) 10 (55.5) 16 (84.2) 2 (100) 2 (100) 46 (63.88) 3 (100) 6 (50) 2 (66.6) 4 (36.3) 8 (44.4) 3 (15.7) 26 (36.11) 3 (4.1) 2 (2.7) 12 (16.6) 3 (4.16) 11 (15.2) 18 (25) 19 (26.3) 2 (2.7) 2 (2.7) 72 (100) Scholars' Research Journal / Jan-Dec 2012 / Vol 2 Issue 1 and 2 Figure 3: Pseudomonas spp. resistant to penicillin and sensitive to polymyxin B 23

3 Figure 4: Distribution of various sites infection from which isolates were cultured Table 2: The different oxidase positive NFGNB species isolated and their percentage distribution (n=78) Species isolated No. of organisms Percentage Pseudomonas aeruginosa M. catarrhalis Pseudomonas CDC group I M. osloensis CDC Vb D. acidovorans A. Piechaudii P. mendocina (Vb 2) Flavobacterium Gilardi rod group Ralstonia picketti A. xylosoxidans Alcaligenes faecalis CDC EO Shewanella putrefaciens Bordetella hinzii P. pseudoalcaligenes A. denitrificans CDC group II c B. vesicularis Total There were single isolates of B. hinzii, P. pseudoalcaligenes, A. denitrificans, CDC group II c, and B. vesicularis. B. hinzii from sputum, P. pseudoalcaligenes and A. denitrificans from CSF, CDC group II c from urine, and B. vesicularis from blood (n=78), NFGNB: Nonfermentative gram-negative bacilli Imipenem was the most effective antibiotic and 87% of NFGNB were sensitive to it. Sensitivity to cefoperazone/sulbactum and piperacillin/tazobactun was observed among 79.4% and 75.6% of the isolates, respectively. Among aminoglycosides, amikacin was the most effective antibiotic in vitro. Tobramycin was the least effective antibiotic. Sensitivity to cephalosporins ranged from 44% (ceftazidime) to 76% (cefotaxime) [Tables 3 and 4]. Four patients expired. Among these three patients had RTI, and one had UTI due to flavobacterium spp which was resistant to all the antibiotics tested. Three of these patients had organophosphorus/supervasmol poisoning and one was a Chronic obstructive pulmonary disease (COPD) patient. DISCUSSION The oxidase positive NFGNB are nowadays gaining importance as nosocomial pathogens and infections with them are increasingly being reported from hospitalized patients due to heightened awareness. Although no studies have been carried out exclusively on the prevalence of oxidase positive NFGNB and our approach to be the first study of its kind, there are several case reports of infections due to different oxidase positive NFGNB. This study includes all the oxidase positive NFGNB from all clinical samples irrespective of outpatient or inpatient. But isolates were more from inpatients (41) as our hospital is a tertiary care hospital and most of the patients are referred from other hospitals for management of complications or maintenance on life supporting equipment. NFGNB are known to cause infection in extremes 24 Scholars' Research Journal / Jan-Dec 2012 / Vol 2 Issue 1 and 2

4 Table 3: Antibiotic sensitivity pattern of oxidase positive NFGNB to β lactam group of antibiotics and their inhibitor combination (n=78) Isolate CE CA CFS CAR PT MR I AZ Total P. aeruginosa 17 (68) 10 (40) 22 (88) 17 (68) 23 (92) 16 (64) 22 (88) 13 (52) 25 M. catarrhalis 9 (100) 0 9 (100) 9 (100) 0 9 (100) 9 (100) 9 (100) 9 (100) P. CDC group I 4 (57.1) 4 (57.1) 4 (57.1) 4 (57.1) 5 (71.4) 4 (57.1) 7 (100) 4 (57.1) 7 M. osloensis 3 (75) 0 3 (75) 3 (75) 4 (100) 3 (75) 3 (75) 1 (25) 4 CDC Vb 3 3 (75) 3 (75) 4 (100) 3 (75) 4 (100) 2 (50) 4 (100) 3 (75) 4 D. acidovorans 3 (100) 3 (100) 3 (100) 3 (100) 2 (66.6) 2 (66.6) 1 (33.3) 3 (100) 3 A. piechaudii 3 (100) 3 (100) 3 (100) 3 (100) 3 (100) 3 (100) 2 (66.6) 3 (100) 3 P. mendocina (Vb 2) 2 (66.6) 0 3 (100) 2 (66.6) 3 (100) 2 3 (100) 0 3 Flavobacterium 2 (66.6) 2 (66.6) 2 (66.6) 2 (66.6) 2 (66.6) 2 (66.6) 3 (100) 2 (66.6) 3 Gilardi rod group 1 1 (50) 0 1 (50) 1 (50) 1 (50) 1 (50) R. picketti 1 (50) 1 (50) 2 1 (50) (50) 2 A. xylosoxidans A. faecalis 2 1 (50) (50) 2 2 CDC EO 2 1 (50) 1 (50) 1 (50) (50) 1 (50) 2 S. putrefaciens 2 1 (50) (50) 2 The single isolate of CDC group II c was sensitive to all the antibiotics tested but single isolates of Bordetella hinzii, B. vesicularis were sensitive to all except aztreonam. The single isolate of P. pseudoalcaligenes was resistant to all β lactam antibiotics except imipenem, A. denitrificans was resistant to β lactam antibiotics Table 4: The antibiotic sensitivity pattern of different oxidase positive NFGNB to antibiotics other than β lactam group of antibiotics and their inhibitor combinations (n=78) Isolate AK G NT TB CF Total P. aeruginosa 20 (80) 15 (60) 13 (52) 12 (48) 19 (76) 25 M. catarrhalis (100) 9 P. CDC group I 5 (71.4) 4 (57.1) 5 (71.4) 3 (42.8) 3 (42.8) 7 M. osloensis 4 (100) 4 (100) (75) 4 CDC Vb 3 3 (75) 2 (50) 3 (75) 2 (50) 4 (100) 4 D. acidovorans 2 (66.6) 2 (66.6) 2 (66.6) 2 (66.6) 3 (100) 3 A. Piechaudii 2 (66.6) 3 (100) 3 (100) 2 (66.6) 2 (66.6) 3 P. mendocina (Vb 2) 3 (100) 2 (66.6) 1 (33.3) 1 (33.3) 1 (33.3) 3 Flavobacterium 3 (100) 3 (100) 2 (66.6) 2 (66.6) 3 (100) 3 Gilardi rod group 1 2 (100) 1 (50) 1 (50) 1 (50) 1 (50) 2 Ralstonia picketti 2 (100) 2 (100) 1 (50) 0 2 (100) 2 A. xylosoxidans (50) 2 A. faecalis 2 (100) 1 (50) 0 2 (100) 2 (100) 2 CDC EO 2 1 (50) 2 (100) 2 (100) 0 2 (100) 2 S. putrefaciens 2 (100) 2 (100) 2 (100) 2 (100) 2 (100) 2 The single isolates of Bordetella hinzii, B. vesicularis, and CDC group II c were sensitive to all the antibiotics tested. The single isolate of P. pseudoalcaligenes was sensitive to amikacin and gentamicin and A. denitrificans was sensitive to only amikacin, Abbreviations for antibiotics used CE: Cefotaxime, CA: Ceftazidime, CFS: Cefoperazone/sulbactum, CAR: Carbenicillin, PT: Piperacillin/tazobactum, MR: Meropenem, I: Imepenem, AZ: Aztreonam, AK: Amikaci, G: Gentamicin, NT: Netilmicin, TB: Tobramycin, CF: Ceprofloxacin of age which was also observed in our study; 25.3% of isolates were from patients in the age group of years and 4% from patients in their first decade of life. Predominance of infection in the extreme age groups could be due to immature and weakening immune system, respectively. Majority of strains were isolated from urine (24.12%). Urine forms the major chunk of samples in our laboratory; this explains the maximum number of isolates from urine in our study. Oxidase positive NFGNB have been reported from different kinds of samples from various types of infections from patients in different clinical settings from India and rest of the world. There are few case reports of infections due to rare oxidase positive NFGNB from India also. A case series of infections due to Shewanella spp. was published from our center. Soft tissue infections due to Shewanella spp. infections also have been reported from Delhi and Manipal, India. [5 7] A study emphasizing the clinical significance of M. catarrhalis and dire consequences of ignoring these as normal flora isolates from RTIs was published from Delhi, India. [8] Other case reports from India Scholars' Research Journal / Jan-Dec 2012 / Vol 2 Issue 1 and 2 25

5 by oxidase positive isolates include bacteremia in a female infant by B. vesicularis and meningitis due to Flavobacterium meningosepticum in a new born. An article reported that complicated nosocomial UTIs can be caused by non fermenters, Alcaligenes, Flavobacter CDC group II, and Agrobacter Weeksiella. [9 11] Studies from around the world have beyond doubt established the pathogenic potential of M. catarrhalis once considered a doubtful pathogen when isolated from sputum. In our study, M. catarrhalis was isolated in pure culture from nine cases of respiratory infections. Studies have emphasized that pure isolation was associated with pathogenic potential of these bacteria and that the confusion of M. catarrhalis with Neisseria spp. was probably responsible for the original misconception that M. catarrhalis is predominantly a commensal organism. [12,13] M. osloensis infections have been reported by several authors from different clinical specimens. [14] In our study, it was isolated from septicemia, post surgery central nervous system infections, and pleural effusion cases. D. acidovorans was isolated from wound infections and RTIs. Previously, it has been reported from cases of bacteremia in different kinds of patients. [15,16] Different oxidase positive organisms designated as CDC groups were isolated from patients suffering from UTI, RTI, and wound infections. Potvliege et al. reported a case of Vb 3 septicemia in a patient with multiple myeloma. CDC EO 2 was studied by Maryam et al. from different clinical samples. It has been recommended that CDC group EO 2 be re classified as P. immobilis based on transformation studies. [17 19] Pseudomonas CDC group 1 mimics Pseudomonas alcaligenes in biochemical tests which can lead to its wrong identification, therefore leading to less reporting and doubtful human pathogen. [1] P. mendocina was isolated from cases of cases of UTI and RTI. Palleroni and others in 1970 isolated it from soil and water samples in the province of Mendoza, Argentina and has been reported from cases of endocarditis and bacteremia. [1,20,21] R. pickettii, B. hinzii, Achromobacter, and Alcaligenes were isolated were from RTI, UTI, and septicemia. In literature, several reports are available describing them as pathogens in several types of infections. [22 26] In this study, Flavobacterium spp. were isolated from UTI and wound infections. [27] It is reported from sepsis in burn patient. No studies are available on the antibiotic sensitivity testing of oxidase positive NFGNB except for P. aeruginosa. In our study, P. aeruginosa showed high resistance to cephalosporins but susceptibility to carbapenems and β lactam/β lactam inhibitors combinations was appreciable and corroborates to the published data. [28] R. picketti has been shown to develop resistance to ciprofloxacin, trimethoprim sulfamethoxazole, piperacillin tazobactam, imipenem cilastatin, and ceftazidime. [22] Of the four patients expired, three had RTI due to P. aeruginosa which were sensitive strains and one had UTI due to flavobacterium spp. which was resistant to all the antibiotics tested. P. aeruginosa were sensitive strains even then patients expired. Fatal outcome in patients having infection with sensitive strains of P. aeruginosa could be due to the general critical conditions of these patients alone. M. catarhalis has been reported to show highest sensitivity to cefotaxime followed by tetracycline, ciprofloxacin, erythromycin, amikacin, gentamicin, and cefazolin. [8] In our study, the isolates were sensitive to all the cephalosporins and resistant to all the aminoglycosides tested. The findings of our study suggest that the oxidase positive NFGNB other than P. aeruginosa are also common pathogens and many of them can be easily isolated and identified by means of conventional methods. Most of the NFGNBs are capable of causing a variety of infections in community as well as hospital settings and there is sufficient evidence to establish them as important pathogens in the form of case reports. The paucity of data is due to lack of prospective studies owing to lack of simple and economic methods to speciate them. Whatever tests available are costly and laborious. Another reason for lack of reporting is the belief that they are non pathogenic or saprophytic in nature which leads to discarding of these isolates in laboratories which carry out the identification tests. With the longer survival of the immunocompromised patients due to advancement of medical techniques and facilities in the present era, the conversion of commensals or saprophytes to opportunistic pathogens cannot be ruled out. Therefore, considering them as contaminants can be hazardous. Identification is also important because the antibiotic sensitivity pattern differs tremendously. CONCLUSION Oxidase positive NFGNB are a group of opportunist bacteria that can cause a vast range of infections in hospitals as well as community settings. Identification of the oxidase positive non fermenters will help to establish their role in infections and also to confirm 26 Scholars' Research Journal / Jan-Dec 2012 / Vol 2 Issue 1 and 2

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Afr J Microbiol 2011;5: How to cite this article: Kalawat U, Sumathi I, Prada J, Reddy SK, Chaudhury A, Sharma KK. Speciation of oxidase-positive nonfermentative gram-negative bacilli. Scho Res J 2012;2:22-7. Source of Support: Nil, Conflict of Interest: None declared. Scholars' Research Journal / Jan-Dec 2012 / Vol 2 Issue 1 and 2 27