International J. of Healthcare and Biomedical Research, Volume: 06, Issue: 03, April 2018, 23-29

Original research article Prevalence of anaerobes in upper respiratory tract infection

1Dr. Sonal Jindal, 2Miss Anju Rani, 3Dr. Sonpal Jindal

1MBBS, MD, Assistant Professor 2M.Sc., Tutor 3MBBS, MD, Assistant Professor Department of Microbiology , KD Medical College, Mathura-281406, Uttar Pradesh Address for Correspondence: Dr. Sonal Jindal , Assistant Professor , KD Medical College, Mathura-281406.

ABSTRACT Introduction: Anaerobic bacteria are a common cause of URI infections, some of which can be serious and life-threatening. Because anaerobes are the predominant normal flora of the skin and mucous membranes, they are common cause of infections of endogenous origin. The management of anaerobic infection is often difficult because of slow growth of the anaerobic organisms, their polymicrobial nature and their increasing antimicrobial resistance. It is a global problem accounting for over 50 million deaths of each year and occurs in both community and health care settings. Aim and Objectives: To find out the prevalence of anaerobes in various upper respiratory tract infections like chronic recurrent tonsillitis, otitis, pharyngitis, , etc. Material and Methods: This study was conducted in Department of Microbiology, Teerthankar Mahaveer Medical College & Research Center, Moradabad (U.P) India from February 2014 - January 2015. One hundred samples were collected for the study of isolation of anaerobic bacteria from patients with upper respiratory tract infection. Results: facultative anaerobes isolated were Haemophilus influenza (10%), Moraxella cattarhalis (5.71%), Staphylococcus aureus (10%), GABHS (17.85%), Klebsilla sp, (2.86%). Obligate anaerobic bacteria isolated were Fusobacterium sp. (28.57%), Bacteroids sp. (11.43%), Peptostreptoccus sp. (4.29%), sp 1.43 %,) Propionibacterium sp (4.29%) . Mostly observed facultative anaerobe was GABHS and strict anaerobe was Fusobacterium spp. (28.57%). Conclusion: Upper respiratory infections were usually either polymicrobial or mixed anaerobic-aerobic infections. This study indicates the frequency and prevalence of anaerobic bacteria involved in URI. Recovery from an anaerobic infection depends on adequate and rapid management. The main principles of managing anaerobic infections are neutralizing the toxins, preventing their local proliferation and spread to healthy tissues. The best way to prevent the spread of infection is to practice good personal hygiene and regular hand washing. Keywords: Upper Respiratory Tract, Anaerobes

INTRODUCTION Upper respiratory tract infection (URTI) causes one-half of all symptomatic illness in the community, exacting huge tolls that can be measured as morbidity, direct health care costs, and overuse of antibiotics leading to the emergence of drug-resistant bacteria. It is a global problem accounting for over 50 million deaths of each year and occurs in both community and health care settings. The nose, mouth, and pharynx are exposed to circulating viruses and are normally colonized by large numbers of bacteria including potential pathogens such as S. aureus , S. pneumoniae , H. influenzae , and group A streptococci. 1 Anaerobic bacteria are a common cause of URTI, some of which can be serious and life-threatening. Because some anaerobes are the normal flora of the skin and mucous membranes, they are also the common cause of infections of endogenous origin. 2 The

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management of anaerobic infection is often difficult because of the slow growth of anaerobic organism, their polymicrobial nature and their increasing antimicrobial resistance.3 Respiratory infections spread through air or by direct contact. This could be by inhalation of small infectious nuclei containing the pathogenic organisms, by sharing cups and other eating materials with infected persons; by kissing, by direct inhalation of the pathogens released by infected persons during sneezing, coughing or talking. 4In the mouth, anaerobes and aerobes are present in approximately equal numbers. The current use of more defined methods of collection, cultivation and identification of anaerobic bacteria will undoubtedly show them to be even more numerous than our present knowledge indicates. 5 Aspiration and lung are frequently caused by non-spore forming anaerobes such as sp , Fusobacterium sp, anaerobic cocci and Eubacterium . Spirochetes also inhabit the mouth and are found in oral and pulmonary disease but their significance is unknown. 6 Acute respiratory infections accounts for 20-40% of outpatient and 12-35% of inpatient attendance in a general hospital. Symptoms range from the common cold—typically a mild, self-limited, catarrhal syndrome of the nasopharynx—to life-threatening. 7, 8 Nasopharyngitis, pharyngitis, tonsillitis and constitute 87.5% of the total episodes of respiratory infections. 9Polymicrobial infections may occur due to both aerobic/facultative anaerobes and obligate anaerobes. Group A beta hemolytic Streptococci (GABHS), Anaerobic Gram-negative rods ( Bacteroides, Prevotella, Porphyromonas ), Anaerobic Gram-positive cocci ( spp.), Anaerobic Gram-positive spore-forming( spp.) and non-spore forming bacilli ( , Eubacterium, Lactobacillus and Bifidobacterium spp.); and Anaerobic Gram-negative cocci (Veillonella spp.) comprises mixed URTI.10 ,11 In day to day practice, samples for URTI are processed only aerobically and patients continue to have problems even after antimicrobial therapy. 12 This facilitates the importance of anaerobes in URTI and need of routine processing of samples for anaerobic identification and culture. The present study was conducted in a tertiary care hospital with an objective of focusing the importance of isolation of anaerobes in various URTI. MATERIAL AND METHODS This study was conducted in Department of Microbiology, Teerthankar Mahaveer Medical College & Research Center, Moradabad (U.P) India from February 2014 - January 2015. One hundred samples of both nasopharygeal and throat swabs were collected from the patients suffering from various URTI, with consent for the study. Infections like Common Cold, Laryngotracheitis, , Epidural or subdural , were excluded. Dental infections were also excluded. Nasopharyngeal and throat Swabs were collected and immediately sent to the laboratory for analysis and processing, following standard guidelines. The samples were inoculated in transport media Robertson’s Cooked Meat Broth in case of delay of inoculation of samples for anaerobic cultures, to prevent the exposure of oxygen to strict anaerobes. Samples were inoculated in Anaerobic brain heart infusion Agar (HIMEDIA M211) supplemented with vitamin K and 5-10% sheep blood, placed in McIntosh - Fildes' anaerobic jar with anaerobic gas packs (HIMEDIA REF LE002A). Incubation was done at 37° C for 5-7 days. Maintenance of anaerobiosis inside jar was monitored with help of biological indicator ( Pseudomonas aeruginosa ATCC 27583) and chemical indicator (HIMEDIA anaero indicator tablet). From Robertson's Cooked Meat Broth, subcultures were done. (Figure 1)

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The plates were observed regularly for bacterial growth. All isolates were presumptively identified by conventional methods. Bacterial isolates were identified on basis of gram staining, cultural characteristics like haemolysis, pigmentation, Brick red fluorescence under UV light and biochemical reactions as per standard protocol.13-16 RESULTS Out of 100 samples, 70 samples show bacterial growth. Facultative anaerobes isolated were Haemophilus influenza (10%), Moraxella cattarhalis (5.71%), Staphylococcus aureus (10%), GABHS (17.85%), Klebsilla sp. (2.86%). Facultative anaerobic gram positive cocci GABHS was isolated in maximum number (17.85%). Obligate anaerobes isolated were Fusobacterium (28.57%), Bacteroids (11.43%), Peptostreptoccus (4.29%), Prevotella (1.43 %,) Propianobacterium (4.29%). Strict anaerobic gram negative bacilli Fusobacterium was isolated in maximum number (28.57) (Table 1). (Figure 2a, 2b, 3) Highest percentages of pathogens were isolated in patients of Age group of 20-40 years. A total of 100 individuals (62 males and 38 female) were observed. The prevalence in males was approximately double of that of females. DISCUSSION The people have different culture, custom, and life style and most of them are dependent upon natural medicine. Due to poor economic condition, they are not able to avail the facility of modern medicinal system. This is probably the first study in this group of people to understand prevalent organism for URI. In this study, we found that percentage of upper respiratory tract infection with significant bacteria was 70%. A total of 100 patients were screened for having upper respiratory tract infections. In our study, obligate anaerobes isolated were Fusobacterium 28.57%, Peptostreptoccus 4.29% Prevotella 1.43%, Propionibacterium 4.29% . Among these, highest percentage was of Fusobacterium 28.57%. Facultative anaerobes isolated were Haemophilus influenza (10%), Moraxella cattarhalis (5.71%), Staphylococcus aureus 10%, GABHS 17.85%, klebsiella pneumoniae 2.86%. Our findings are in accordance with the study by El-Mahmood et al., 2010 in Yola, Okesola and Oni, 2009; Nwanze et al., 2007 in Ibadan and Lagos. 18-20 In terms of frequency of occurrence, Fusobacterium was the commonest bacteria (47.44%) in URI pa- tients, but with a different rate obtained from other populations in U.S.A. study (75.5% - 87.0%), where the number are 68.69% and 83.0%. However, it should be pointed out that other investigators have observed Fusobacterium (28.57 %) as the common bacterium in significant bacteria .Our results were in accordance with the study conducted in other countries such as Cameroun (Ndip et al., 2008), South Africa (Liebowitz et al., 2003).21,22 The occurrence of bacterial pathogens varies with age, age group ranging from 20-40 years reported the highest number. The least age group in terms of occurrence, were within the ranges of between 61-70 years in which out of total patients examined, only 2 % reported the occurrence. This might be probably due to the fact that, most of the people in these age groups in this community are more exposed to agents responsible for causing respiratory tract infections than the elderly ones. Sex-related occurrence of pathogens reveals that male subjects reported higher number of pathogens compared to females. This is due to more prevalent associated risk factors (e.g. smoking and chronic alcoholism) of respiratory infections in males than females. In our study same males are more susceptible to URI than females.

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These results contradicts the data obtained by El- Mahmood et al., 2010, in which in a similar study, out of 232 total isolates, 114 (49.1%) were from males while 118 (50.9%) from females. The aim of this study is to strengthen the surveys of the cases and the causes of the respiratory tract infection. It also strengthens the importance of cultural morphology in early identification of anaerobes, which is helpful for early treatment. CONCLUSION Anaerobic bacteria were predominant components of normal oral cavity, upper respiratory tract. Upper respiratory infections were usually either polymicrobial or mixed anaerobic-aerobic infections. The incidence, attack rates and related socio-economic factors give special epidemiologic importance to these infections. Recovery from an anaerobic infection depends on adequate and rapid management. The main principles of managing anaerobic infections are neutralizing the toxins, preventing their local proliferation and spread to healthy tissues. The best way to prevent the spread of infection is to practice good personal hygiene and regular hand washing.

REFERENCES 1. Dr Charles Bryan Emeritus Professor University of South Carolina School of Medicine 2005, 85: 576- 81 2. Hentges DJ. The anaerobic microflora of the human body. Clin Infect Dis 1993; 164:175–80. 3. Jousimies-Somer HR, Summanen P, Baron EJ, Citron DM, Wexler HM, Finegold SM. Wadsworth- KTL anaerobic bacteriology manual. 6th ed. Belmont, CA: Star Publishing, 2002. 4. Jafari NJ, Ranjbar R, Haghi-Ashtiani MT, Abedini M, Izadi M. The study of prevalence and antimicrobial susceptibility of tracheal bacterial strains isolated from paediatric patients. Pak J Biol Sci 2009; 12:455-8. 5. Balows A, DeHaan RM, Dowell VR, Guze LB (eds): Anaerobic Bacteria. Charles C Thomas, Springfield, IL, 1974. 6. Finegold SM, George WL (eds): Anaerobic Infections in Humans. Academic Press, San Diego, 1989. 7. Morris JG. The physiology of obligate anaerobiosis. Adv Microb Physiol 1975; 12:169–246. 8. Sutter VL, Citron DM, Edelstein MAC, Finegold SM. Wadsworth Anaerobic Bacteriology Manual, 4th Ed. Star Publishing, Belmont, CA, 1985. 9. Aldridge KE, Ashcraft D, Cambre K, Pierson CL, Jenkins SG, Rosenblatt JE. Multicenter survey of the changing in vitro antimicrobial susceptibilities of clinical isolates of group, Prevotella, Fusobacterium, Porphyromonas, and Peptostreptococcus species. Antimicrob Agents Chemother 2001; 45:1238-43. 10. Jousimies-Somer HR, Summanen P, Baron EJ, Citron DM, Wexler HM, Finegold SM. Wadsworth- KTL anaerobic bacteriology manual. 6th ed. Belmont, CA: Star Publishing, 2002. 11. Nagy E. Anaerobic infections: update on treatment considerations. Drugs 2010; 70:841-58. 12. Brook I. Anaerobic Infections Diagnosis and Management. A Textbook. Informa Healthcare USA, Inc. New York. 2007.

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13. Collee JG, Marr W, Watt B, Miles RS. Culture of Bacteria and Tests for Identification of Bacteria. In: Collee JG, Fraser AG, Marmion BP, Simmons A, eds. Mackie and McCartney’s Practical Medical Microbiology, 14 th edition. New York: Elsevier Churchill Livingstone. 2012.p. 113-49. 14. Betty AF, Daniel FS, Alice SW. Overview and General Considerations, Laboratory Considerations. In: Bailey & Scott's Diagnostic Microbiology, 12th edition; 2007.p.216-53. 15. Jousimies-Somer HR, Summanen P, Baron EJ, Citron DM, Wexler HM, Finegold SM. Wadsworth- KTL anaerobic bacteriology manual, 6th edition, Belmont, CA, Star Publishing; 2002.p.49-70. 16. Mahon CR, Lehman DC, Manuselis G, Engelkirk GP, Engelkirk DJ. Anaerobes of Clinical Importance. In: Textbook of Diagnostic Microbiology, 3rd edn.; 2007.p.587-639. 17. Betty AF, Daniel FS Alice SW. Overview and general considerations of Anaerobic of Bacteriology. In: Bailey & Scott's Diagnostic Microbiology, 12th edition; 2007.p.455-77. 18. El-Mahmood AM, Isa H, Mohammed A, Tirmidhi AB. Antimicrobial susceptibility of some respiratory tract pathogens to commonly used antibiotics at the Specialist Hospital, Yola, Adamawa State, Nigeria. J Clin Med Res 2010; 2:135-42. 19. Okesola AO, Oni AA. Antimicrobial resistance Among Common Bacterial Pathogens in South Western Nigeria. Am-Eurosian. J Environ Sci 2009; 5:327-30. 20. Nwanze P, Nwaru LM, Oranusi S, Dimkpa U, Okwu MU, Babatunde BB, et al. Urinary tract infection in Okada village: Prevalence and antimicrobial susceptibility pattern. Sci Res Essays 2007; 2:112-6. 21. Ndip RN, Ntiege EA, Ndip LM, Nkwelang G, Akoachere TK. Antimicrobial resistance of bacterial agents of the upper respiratory tract of school children in Buea, Cameroon. Health Popul Nutr 2008; 26:397-404. 22. Liebowitz D, Slabbert M, Huisamen A. National Surveillance programme on susceptibility patterns of respiratory pathogens in South Africa: moxifloxacin compared with eight other antimicrobial agents. J Clin Pathol 2003; 56:344-7.

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Figure 1: Mc-Intosh Filde’s jar with gas pack and inoculated anaerobic BHI Blood Agar plates for the growth of anaerobic bacteria.

TABLE 1: Number and P ercentage of isolated both facultative and strict anaerobic bacteria

Type and name of Bacteria Out of 70 positive Percentage of Percentage sample isolates Out of 70 isolates Total no. of isolates

Gram-positive 28 40 %

Staphylococcus aureus 07 25 % 10% GABHS 15 44.64 % 17.85 % Propionibacterium 03 10.72 % 4.29%

Peptostreptococcus 03 10.72 % 4.29%

Total 100 % 40 % Gram-negative 42 60 %

Hemophilus influenza 07 16.67 % 10% Moraxella catarahhalis 04 9.52% 5.71% Klebsiella pneumoniae 02 4.77% 2.86% Bacteroides 08 19.05% 11.43% Prevotella 01 2.38% 1.43% Fusobacterium 20 47.61% 28.57%

Total 100% 60 %

Figure 2a) Growth of anaerobic Gram negative bacilli Fusobacterium species on anaerobic BHI . Arrow shows characteristic “Fried egg colonies”.

Figure 2 b) Gram stain morphology ‰ gram negative slender bacilli with tapering ends.

Fig. 2a Fig. 2b

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Figure 3: Growth of anaerobic Gram negative bacilli Bacteroides species on anaerobic BHI . Black Arrow shows pale yellow colonies of >1mm diameter. Yellow arrowarrow showsshows the characteristic “pitting of agar.”

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