Indian Pediatrics 1999;36: 1029-1032

Prevalence of Anti-microbial Resistance Among Respiratory Isolates of Haemophilus Influenzae

From the Departments of Microbiology and Pediatrics*, University College of Medical Sciences and Guru Teg Bahadur Hospital, Delhi, India.

Reprint requests: Dr. Jyoti Puri, Assistant Professor, Department of Microbiology, G.B. Pant Hospital, New Delhi 110 002, India.
Manuscript received: December 29, 1998;
Initial review completed: February 5, 1999;
Revision accepted: May 21, 1999

Haemophilus influenzae causes a variety of bacterial infections in young children. Non capsulated influenzae are a part of the normal flora in the upper respiratory tract of children(1,2). They can spread contiguously from the upper respiratory tract and cause sinusitis, otitis media and pneumonia(3). Capsulated strains of Pittman type b colonize less frequently but are responsible for a variety of invasive infections like meningitis, septic arthritis and facial cellulitis(2,4).

There is also increasing evidence that non capsulated H. influenzae is an inportant patho-gen of pneumonia and other lower respiratory tract infections in developing countries(3,5-7). Strep. pneumoniae and H. influenzae are the most frequent bacterial agents of acute respiratory infections (ARI) in children in developing countries(8-11).

Life threatening invasive infections more commonly caused by encapsulated type b strains have largely disappeared at least in circumstances and areas in which the protein conjugated type b capsular polysaccharide vaccine has been used(3). Since localized infections such as those caused by non type b H. influenzae isolates are often treated empirically a knowledge of antibiotic resistance determined on the basis of systematic surveill-ance studies is essential. The problem of b-lactamase mediated ampicillin resistance in H. influenzae is becoming more prevalent(3). We therefore conducted this prospective study to define the contemporary levels of resistance to 8 antimicrobial agents for isolates of H. influenzae.

Material and Methods

Between May 1997 to April 1998 naso-pharyngeal swabs were collected from 200 cases of children between 6 months to 10 years with features of upper and lower ARI(2) who were attending the Pediatric OPD or casualty services. Exclusion criteria were administration of an antibiotic in the preceding 72 hours. For the control group, nasopharyngeal swabs were taken from 50 children of the same age group who were attending the Immunization Clinic of the hospital and having no evidence of respiratory infection.

Nasopharyngeal samples were cultured for H. influenzae on selective chocolate agar (i.e., chocolate agar with 300 mg/ml of bacitracin) and sheep blood agar plates. The identification of H. influenzae was based on growth characte-ristics and requirements for growth factors(12). The isolates were tested serologically for the presence of the type b polysaccharide capsule, but not for types a, c, d, e or f. The isolated H. influenzae strains are termed noncapsulated in the present study, although some may be rare capsular non-b types.

Antibiotic susceptibility of H. influenzae confirmed isolates was done by National Committee for Clinical Laboratory Standards technique(13). The antimicrobial sensitivity was tested to the following antibiotics-Ampicillin (10 mg). Amoxycillin - Clavulanate (20/10 mg), Ciprofloxacin (5 mg). b-lactamase detection tests were done by Iodometric and Acidometric methods. b-lactamase detection was also done by Cefinase discs (Nitrocefin) from Becton Dickinson Microbiology Systems. USA.

Results

Twenty eight isolates (i.e., 14%) of H. influenzae were obtained from nasopharyngeal swabs taken from 200 patients with ARI. From the control group, 3 isolates were obtained. Among these, 3 isolates (9.7%) were encapsulated type b strains and 28 (90.3%) were non type b strains. The largest number of isolates (both type b and non type b) were obtained from patients between the ages of 2 months and 2 years (Table I).

Table I^__Strains Isolated From Various Age Groups.

The in-vitro activities of the various antimicrobial agents against the 31 study isolates are shown in Table II. Ampicillin resistance was documented in 32.2% cases. All the ampicillin resistant strains were found to be b lactamase positive when tested by Iodometric, Acidometric and Cefinase tests.

Table II^__ Antimicrobial Susceptibility Pattern of Isolated H. influenzae strains (n=31).

Discussion

In the present study, there was maximum recovery rate of H. influenzae from 6 months- 2 years of age (both type b and non type b). This relationship between age and isolation of H. influenzae was similar to previous reports(2,4). Ampicillin resistance was present in 32.2% of strains compared with 20% (20% b-lactamase positive) in the US study(3) and tetracycline resistance was 3.2% as compared to 0.5% and chloramphericol resistance was not seen as compared to 0.5% in the previous study(3).

The incidence of beta-lactamase producing strains alters our consideration of amoxycillin as the drug of choice for otitis media, sinusitis and mild to moderate pneumonia(13). The impact of nontypable strains is changing with the diminished importance of type b strains in immunized children especially in developed countries. In a recent study of eleven develop-ing nations(14), 6% of strains were found to be resistant to ampicillin. All the isolates in the above study produced b-lactamase which is similar to results of our study. However in contrast, in another study from Bangui(15) only 1.4% of the isolates from outpatients and none from the inpatients was resistant to ampicillin. Strains of H. influenzae responsible for respira-tory tract infections may be subdivided on the basis of b-lactamase production and susceptibility to Ampicillin or Penicillin G. Addition of Clavulanic acid, a b-lactamase inhibitor, to oral amoxycillin protects the drug from inactivation by the enzyme. Although first generation oral cephalosporins (cephalexin) have limited activity against H. influenzae (22.5% resistant in the current study), the activity is enhanced in some third generation cephalosporins like cefotaxime (no resistance in Current study) Chloramphenicol resistant strains have been reported in India(16-18). However, among type b isolates we did not find chloramhenicol resitance in any of our isolates. A sensitivity of 100% was also seen against Ciprofloxacin and Cefotaxime.

In the present study, b lactamase mediated ampicillin resistance was quite high (32.2%). Depending on the geographic region 20 to 30% of strains of H. influenzae produce b lactamase(3,19) and can inactive susceptible penicillins like Ampicillin and Amoxycillin. Addition of clavulanic acid, a b-lactamase inhibitor to oral amoxycillin protects the drug from inactivation by the enzyme. Although first generation oral cephalosporins like Cephalexin have limited activity against H. influenzae, third generation cephalosporins like cefotaxime, have excellent activity against this organism. Other effective drugs include chloramphenicol, macrolides and tetracycline which have vari-able activity(19). All the ampicillin resistant strains are found to be b-lactamase positive because H. influenzae strains that are resistant to Ampicillin by mechanisms other than production of TEM type b-lactamase are extremely uncommon(14).

Since resistance levels vary dramatically with geographic region and there is paucity of data from developing countries like India on the antibiotic susceptibility of non typable H. influenzae, more surveillance studies are needed.

References

1. Faden H, Duffy L, Williams A, Pediatrics TW, Krystofik DA, Wolf J. Epidemiology of nasopharyngeal colonization with non typeable Haemophilus influenzae in the first 2 years of life. J Infect Dis 1995; 172: 132-135.

2. Korppi M, Katila ML, Jaaskelainen J. Leinonen M. Role of noncapsulated Haemophilus influenzae as a respiratory pathogen in children. Acta Paediatr 1992; 81: 989-992.

3. Doern GV, Brueggemann AB, Pierce G, Holley HP, Rauch A. Antibiotic resistance among clinical isolates of Haemophilus influenzae in the United States in 1994 and 1995 and detection of beta-lactamase - positive strains resistant to amoxycillin - clavulanate: Results of a national multicentre surveillance study. Antimicrob Agents Chemother, 1997; 41: 292-297.

4. Howard AJ, Dunkin KT, Millar GW. Nasopharyngeal carriage and antibiotic resistance of Haemophilus infuenzae in healthy children. Epidem Inf 1988; 100: 193-203.

5. Montgomery JM, Lehman D, Smith T. Bacterial colonization of the upper respiratory tract and its association with acue lower respiratory tract infections in highland children of Papua New Guinea. Rev Infect Dis 1990; 1 2: S1006-S1016.

6. Munson RS, Kabeer Jr. MH, Lenoir AA, Granoff DM. Epidemiology and prospects for prevention of disease due to Haemophilus influenzae in developing countries. Rev Infect Dis 1989; 11: S588-S597.

7. Shann F, Gratten M, Germer S, Linnemann V, Hazlett D, Payne R. Etiology of pneumonia in children in Goroka Hospital, Papua New Guinea. Lancet 1984; 2: 537-541.

8. Selwyn BJ. The epidemiology of acute respiratory tract infection in young children: Comparison of findings from several develop-ing countries. Rev. Infect Dis 1990; 12: S870-S888.

9. Shann F. Etiology of severe pneumonia in children in developing countries. Pediatr Infect J 1986; 5: 247-252.

10. Pio A. Acute respiratory infections in children in developing countries: An international point of view. Pediatr Infect Dis 1986; 5: 179-183.

11. A programme for controlling acute respiratory infections in children: Memorandum from a WHO meeting. Bull WHO 1984; 62: 47-58.

12. Murray PR, Baron EJ, Pfaller MA, Tenover FC, Yoken RH. Manual of Clinical Microbiology, 6th edn. Washington DC, ASM Press, 1995; pp556-565.

13. Doern GV, Jones RN. Antimicrobial susceptibility testing of Hemophilus influenzae, Branhamella catarrhalis and Neisseria

gonorrhoeae. Antimicrob Agents Chemother 1988; 32: 1747-1753.

14. Doern GV. In vitro susceptibility testing of Haemophilus influenzae: Review of New National Committee for Clinical Laboratory Standard Recommendations. J Clin Microbiol 1992; 30: 3035-3038.

15. MMWR. Antibiotic resistance among nasopharyngeal isolates of Streptococcus pneumoniae and Haemophilus influenzae - Bangui 1995. JAMA 1997; 277: 621.

16. Singh R, Thomas S, Kirubabaean C, Lallitha MK, Raghupathy. Occurrence of multiple antimicrobial resistance among Haemophilus influenzae type b causing meningitis. Indian J Med Res 1992; 95: 230-233.

17. Amarnath SK, Kanungo R, Rao RS. Srinivasan S. Multiple resistant H. influenzae type b causing meningitis. Indian J Med Res 1992; 45: 187-189.

18. Ayyagari A, Sharma P, Chakraborti A. Isolation and detection of H. influenzae from patients of respiratory tract infections and their anitbiotic susceptibility patterns in Chandigarh. Indian J Chest Dis All Sci 1984; 7: 230-234.

19. Klein JO. Haemophilus influenzae: Its role in pediatric infections. Pediatr Infect Dis J 1997; 16 (Suppl): S3-S4.