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Non-fermenting Gram Negative Bacilli Associated withAcute Respiratory Infections in Children in Madras by C. N. Paramasivan, K. Sivadasan, Manjula Datta, R. S. Vallishayee and R. Prabhakar Tuberculosis Research Centre, Indian Council of Medical Research, Madras 600031, India Non-fermenting Gram negative bacilli (NFGNB) were isolated as the most predominant organism from
children suffering from acute respiratory illness (ARI) and accounted for 40 out of 151 children reported
in the study. Five of the NFGNB were isolated in pure culture and all of them were from children having
lower respiratory infection. The isolation of NFGNB was higher when the duration of illness exceeded 7
days (44 per cent) as compared to 22 per cent with less than 7 days duration (P= 0.03). Detailed identifi-
cation studies on 61 isolates of NFGNB showed that 56 (92 per cent) of them belonged to Pseudomonas

pseudoalkaligenes alkaligenes group. Four strains were identified as Ps. pseudomallei group and one as
Ps. stutzeri. The majority of the NFGNB isolates showed multidrug resistance.
Introduction
blood agar, and Mac Conkey agar (MA) plates. Thenon-lactose fermenting colonies on MA were further Till recently non-fermenting Gram negative bacilli tested for fermentation of mannitol, lactose, sucrose, (NFGNB) other than Pseudomonas aeruginosa have and glucose in peptone water base. Those isolates been considered as either saprophytes or commensals.
which failed to ferment any of the sugars were The increasing frequency of isolation of these regarded as NFGNB and taken for further identifica- NFGNB1-4 in different clinical conditions has led to tion. Only those clinical specimens which yielded a re-evaluation of their role as probable human path- moderate (21-79 colonies) or heavy ( > 80 colonies) ogens, and necessitated their identification and classi- growth of NFGNB were subjected for detailed identi- fication. The following is a report on the incidence, probable significance, and sensitivities to antibioticsof the NFGNB isolated from children suffering fromacute respiratory infections (ARI) in Madras.
IdentificationThe NFGNB were identified by Gram stain, motility,growth at 4°C and 42°C pigment production, penicil- Materials and Methods
lin susceptibility and other biochemical reac- tions. 1, 2, 4-6 The biochemical reactions employed Throat swabs, laryngeal swabs, nasal swabs, and were hydrogen sulphide and indole production, nasal secretions were obtained from children below 6 production of acid from 10 per cent solutions of glu- years of age suffering from ARI and attending the cose, lactose, and mannitol in ammonium salt solu- outpatient department of the Institute of Child tions; production of urease, arginine decarboxylase, Health and Hospital for Children, Egmore, Madras.
gelatinase, phenylalanine deaminase, catalase, and From October 1985 to March 1986, 151 children with oxidase; utilization of citrate and malonate; oxi- dation/fermentation (O/F) of glucose, lactose, malt-ose, and xylose; nitrate reduction, nitrite reduction The specimens were inoculated on to chocolate agar, Acknowledgements
In vitro antibiotic susceptibility test was done on The authors are grateful to the Director, Institute of Child Muller-Hinton agar by the disc diffusion method.’ Health and Hospital for Children, Egmore, Madras, for pro- The drugs used were penicillin (10 units), strepto- viding facilities and help for conducting this study. The mycin (10 µg), erythromycin (15 µg), chloramphenicol authors also thank Mr P. G. Gopi for statistical assistance, (30 µg), tetracycline (30 µg), cephalosporin (30 µg), Mr G. K. Loganathan for technical assistance and Mr B.
kanamycin (30 µg), ampicillin (10 µg), gentamicin (10 µg), colistin (10 µg), and cotrimoxazole (25 µg).
Correspondence: Dr C. N. Paramasivan.
All the drugs tested in this study were obtained from tract. Five patients yielded NFGNB in pure culture Isolation of NFGNB according to clinical diagnosis and all of them were suffering from LRI. On fiveoccasions NFGNB were found associated with other bacteria incriminated in ARI. In two patients it was found mixed with Klebsiella pneumoniae, in another two with Haemophilus influenzae, and in one with βhaemolytic streptococci and H. influenzae. Of the 77 NFGNB isolated, complete characteriza- tion was done for 61 isolates. All these were oxidase positive, ruling out the genus Acinetobacter. Since all these were resistant to penicillin, they may not belong to the genus Moraxella either. As all of them were non-pigment producers, pigment-producing organ- isms such as Chromobacterium violaceum, Pseudo-monas aeruginosa, Ps. fluorescens and Ps. putida were also ruled out. Based on the biochemical tests men-tioned earlier, 56 (92 per cent) strains of NFGNB Figures in parenthesis indicate pure growth.
belonged to Pseudomonas pseudoalkaligenes alkali- genes group. Four strains were identified as Pseudo- monas pseudomallei group and one strain as Ps.
stutzeri.
The pattern of sensitivity to antibiotics in these NFGNB isolates is presented in Table 3. All of them I s o l a t i o n o f N F G N B a c c o r d i n g t o were resistant to penicillin and cephalosporin. A majority of them showed resistance to streptomycin,erythromycin, and ampicillin followed by gentamicin.
Approximately 60 per cent of them were sensitive to chloramphenicol and tetracycline and colistin. Of all the drugs tested, cotrimoxazole was found to be mosteffective, 90 per cent of the isolates being sensitive fol- lowed by Kanamycin (77 per cent). On the whole, the NFGNB isolates showed lowest resistance to colistin(2 per cent), when the moderately sensitive strains (35 per cent) were considered as sensitive along with thehighly sensitive strains (63 per cent).
Discussion
In all, 77 strains of NFGNB were isolated from 40 (26per cent) of the 151 children included in the study.
In the present study, out of 151 children with ARI, 40 The number of NFGNB isolated according to the (26 per cent) yielded NFGNB. This is a significant clinical diagnosis and duration of illness are shown in finding considering the fact that other commonly Tables 1 and 2, respectively. The isolation rates of incriminated respiratory pathogens like Haemophilus NFGNB in upper respiratory tract infection (URI) influenzae and β -haemolytic streptococci were isolated and lower respiratory tract infection (LRI) were simi- from only 13 and 11 per cent of the children, respect- lar, being 29 and 24 per cent, respectively. Also, it is ively. NFGNB as pure culture was obtained from five seen from Table 2 that the isolation was maximum children, all with LRI. Only on five occasions when the duration of illness exceeded 7 days. The age NFGNB were found mixed with other potential path- adjusted isolation rate was 44 per cent in children like Klebsiella pneumoniae, Haemophilus with a duration of illness of more than 7 days as com- influenzae, and β -haemolytic streptococci. pared to 22 per cent in children with a duration of ill- A large number of species of NFGNB are known to ness of 1-7 days (P=0.03). In this study, the other exist and many of them have been isolated from clin- organisms isolated according to the order of fre- ical material3 In the study reported here, the great quency were Haemophilus influenzae (13 per cent), β− majority (92 per cent) of the NFGNB isolates haemolytic streptococci (11 per cent), Klebsiella pneu- belonged to Ps. pseudoalkaligenes alkaligenes groups.
moniae (7 per cent), Pneumococcus (3 per cent), and Four strains were identified as Ps. pseudomallei coagulase positive Staphylococcus aureus (1 per cent).
group. As this is the first report from India describing In most of the patients, the NFGNB were isolated the isolation of NFGNB from children with ARI, along with other commensal flora of the respiratory comparison of the results from the present study with Antibiotic susceptibility pattern of 60 isolates of NFGNB Ery. Chl. Tet. Cep. Kan. Amp. Gen. Col. Cot.
that from other Indian reports is not possible. How- References
ever, Pedersen8 in his review on NFGNB associated Pickett MJ, Pedersen MM. Non-fermenting bacilli asso- with man has mentioned that nearly 60 per cent of the ciated with man II. Detection and identification. Am J isolates of Ps. pseudoalkaligenes obtained from USA were from sputum. Ps. pseudamallei group has 2. Bhujwala RA. Laboratory manual on non-fermentative already been described as a possible respiratory path- gram negative bacilli (Non-fermenters). A guide to iden- ogen in a recent review by Levison and Kaye.9 tification. New Delhi: Indian Association of Medical As regards the aetiopathogenic role of NGFNB in ARI, it is at present of uncertain significance. So far it Misra B, Bhujwala RA, Shrinivas. Non-fermenters in has been believed that the NFGNB in general are human infection. Ind J Med Res 1986; 83: 561-6.
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ever, recently there have been reports about their emergence as secondary or opportunistic pathogens 5. Parker MT. Pseudomonas. In Topley and Wilsons Prin- in human disease process. 3, 10 When the NFGNB are ciples of bacteriology, virology and immunity. In Wilson present in large numbers, it is quite likely that their G, Miles A, and Parker MT (eds), 7th edn., Vol. 2.
endotoxin content itself may have some deleterious effect on the host.10 Even the established respiratory 6. Collins CH, Lyne PM. Microbiological methods, 5th pathogens like Haemophilus influenzae, β -haemolytic streptococci, pneumonococcus, and some members of 7. Bauer AW, Kirby WMM, Sherries JC, Turk M. Anti- enterobacteriaceae, reside in the respiratory tract of biotic susceptibility testing by a standardised single discmethod. Am J Clin Pathol 1966; 45: 493-6.
healthy individuals as commensals on occasions. The 8. Pedersen MM, Marso E, Pickett MJ. Non-fermentative exact role of these potential pathogens in the primary bacilli associated with man. III Pathogenicity and anti- aetiology of ARI is not clear. Viruses probably act as biotic susceptibility. Am J Clin Pathol 1970; 54: 178-92.
initiators of respiratory infections and predispose the 9. Levison ME, Kaye D. Pneumonia caused by gram- bacteria to invade and establish themselves.11-12 The negative bacilli: an overview. Rev Infect Dis 1985; precise mechanism is still uncertain, but, transient immunosuppression caused by viruses,13 destruction 10. Cruickshank R, Duguid JP, Manion BP, Swain RHA.
of the integrity of the respiratory epithelium,14 and Medical microbiology, 12th edn, Vol. II. New York: increased adherence of bacteria to the cells infected 11. Sellers TF, Schulman J, Bouvier C, Mccune R, Kil- with viruses15 have been postulated. In this context, bourne ED. The influence of influenza virus infection on the isolation of NFGNB in an appreciable proportion exogenous staphylococcal and endogenous murine bac- of children with ARI gains importance.
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The pattern of sensitivity to antibiotics show that a very high proportion of the strains were sensitive to 12. Jakab CJ, Dick EC. Synergistic effect in viral-bacterial colistin (98 per cent), cotrimoxazole (90 per cent), and infection. Combined infection of the murine respiratory kanamycin (77 per cent). Approximately 60 per cent tract with sendai virus and Pasteurella pneumotropica. of them were sensitive to chloramphenicol and tetra- cyclin. Majority of the strains showed multidrug 13. Woodruff JE, Woodruff JJ. Viral immunology and immunopathology. London: Academic Press, 1975.
resistance. This multidrug resistance will be of im- 14. Mims CA, White DO. Viral pathogenesis and immuno- portance should these organisms be shown to have logy. London: Blackwell Scientific Publication, 1984.
any role in the aetiopathogenesis of ARI in children.
15. Sanford BA, Skelokov A, Ramsay MA. Bacterial adher- More studies pertaining to the biological properties of ence to virus infected cells: a cell culture model of bacter- NFGNB are necessary to elucidate this.
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