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IDENTIFICATION AND CHARACTERIZATION OF BACTERIA ISOLATED FROM

RESPIRATORY TRACT OF MAN
Article · July 2010

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Int. J. BioRes. 1(6): 27-30 June, 2010 Islam et al.

Full Length Research Paper

IDENTIFICATION AND CHARACTERIZATION OF BACTERIA ISOLATED FROM

RESPIRATORY TRACT OF MAN
1 1,2 1 1 1 1
M Islam , *M M Rahman , M S R Khan , J Akhter , M T Hossain and M P Siddique
1
Department of Microbiology and Hygiene, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh
2
Dept. of Medical Microbiology & Immunology, Faculty of Medicine, UKM, Cheras-56000, Kuala
Lumpur, Malaysia *Correspondence: mmr@ppukm.ukm.my; mostabau@yahoo.com Accepted on
27 June 2010

ABSTRACT
The study was designed to characterize the bacteria isolated from the respiratory tract of man considering their health status.
A total of 60 throat swab samples were aseptically collected comprising 30 from apparently healthy man and 30 from sick
man. The samples were cultured on nutrient agar and blood agar media for isolation of bacteria and identified according to
their morphology, staining, motility, cultural and biochemical properties. The isolated bacteria were also subjected to
characterize their pathogenicity and antibiotic sensitivity. The bacterial flora present in the throat swab of man with their
percentage of distribution were Staphylococcus spp. (39.44%) of which Coagulase positive Staphylococcus (21.13%) and
Coagulase negative Staphylococcus (18.31%), Klebsiella spp. (19.72%), Pseudomonas spp. (15.49%), Proteus spp.
(4.23%), E. coli (9.86%) and Bacillus spp. (11.27%). Among the isolates Staphylococcus, Klebsiella and Pseudomonas
were the predominant species. Percentages of identified bacteria were higher in sick man (53.52%) than apparently healthy
one (46.48%). All coagulase positive Staphylococcus, Klebsiella spp. and Pseudomonas spp. isolated from sick man were
found to be pathogenic. The isolated bacteria were resistant to amoxicillin and ampicillin but sensitive to ciprofloxacin and
norfloxacin. Isolated Pseudomonas spp. showed multidrugs resistant properties.
Key words: Characterization, respiratory tract, man, bacteria, pathogenicity, antibiotic sensitivity
INTRODUCTRION

Nasopharynx is not only the primary settlement of opportunistic pathogen but also the chief carrier of common respiratory
pathogens (Belliveau, 1973). More than 200 species of bacteria colonize on upper respiratory tract (Nadel et al., 1999) of
which Staphylococcus aureus, Streptococcus pneumoniae, Neisseria meningitides, Klebsiella pneumoniae, Pseudomonas
aeruginosa and Haemophilus influenzae were potential pathogens (Todar, 2008).
These bacteria are responsible for various respiratory illness of human being. Treatment of the illness may not be judicious if
proper identification of the causal agent is not performed perfectly. Moreover, multidrugs resistant strains are being developed
due to indiscriminate use of antibiotics irrespective of the identification of causal agents. Therefore, the present research was
undertaken to isolate and identify the bacteria from respiratory tract of apparently healthy and sick man and to study their
pathogenicity and antibiotic sensitivity.
MATERIALS AND METHODS
This study was conducted in the Department of Microbiology and Hygiene, Bangladesh Agricultural University (BAU),
Mymensingh during the period of January to June 2009.
Sample collection
A total of 60 throat swab samples were aseptically collected in which 30 from apparently healthy man and 30 from sick man
with the help of sterile cotton buds. Immediately after collection the samples were inoculated into nutrient broth. These were
then transferred to the laboratory of Microbiology & Hygiene department, BAU, Mymensingh-2202, Bangladesh.
Isolation and Characterization of bacteria
The collected samples were processed as per the procedure of Cheesbrough, (2006). For isolation and characterization of
bacterial flora, the procedure suggested by Ryan and Ray, (2004) were followed throughout the experiment. Briefly, the
samples were then inoculated into Blood agar (BA) media and incubated at 37°C for 24 hours. Characteristic colonies from
the plates were isolated and then sub cultured to obtain pure culture. The isolated organisms were identified based on colonial
morphology, microscopic study and biochemical tests according to standard laboratory methods. Stock culture was maintained
in both Agar slant and 20% sterile buffered glycerin.
Pathogenicity tests
The isolated Staphylococcus, Klebsiella and Pseudomonas organisms were subjected to pathogenicity test in 2 months old
mice by intra-peritoneal inoculation. The virulence of Staphylococcus isolates were determined according to the method
described by Torres and Stanislawa, (1970); virulence of Klebsiella spp. was determined following the method of Yu et al.,
2009; virulence of Pseudomonas spp. was determined by the method suggested by Daniel et al. 1992.
27
Int. J. BioRes. 1(6): 27-30 June, 2010 Islam et al.

Antibiotic sensitivity tests

Antibiotic sensitivity tests were performed using disc diffusion test of the method described by Kirby-Bauer (Bauer et al.,
1966). Commercially available antimicrobial discs (Mast Group Ltd., Merseyside, Uk) were used for the determination of the
drug sensitivity. The concentration of antimicrobial agent per disc was: Gentamycin (10 µg), Pefloxacin (5 µg), Enrofloxacin
(5 µg), Norfloxacin (10 µg), Ciprofloxacin (5 µg), Furazolidone (50 µg), Amoxicillin (25 µg) and Ampicillin (10 µg). For this
purpose, 01-02 ml of freshly growing broth culture were poured on NA and spread uniformly. Antibiotic discs were placed
apart onto the surface of the inoculated plates aseptically with the help of a sterile forceps and incubated at 37°C for 24 hours.
After incubation, the plates were examined and the diameters of the zone of inhibition were measured and were interpreted
with the standard diameters of NCCLS, (1999) and recorded as sensitive (S), intermediate (I) and resistant (R).
RESULTS AND DISCUSSION
Bacterial flora isolated from throat swab of human being
The bacteria isolated and identified from the throat swabs of apparently healthy and sick man are presented in Table-1. It
reveals from the table that 15 were coagulase positive Staphylococcus sp. (21.13%), 13 were Coagulase negative
Staphylococcus sp. (18.31%), 14 were Klebsiella spp. (19.72%), 11 were Pseudomonas spp. (15.49%), 3 were Proteus spp.
(4.23%), 7 were Escherichia coli (9.86%) and 8 were Bacillus spp. (11.27%). Among the 71 isolates, 33 (46.48%) were found
from apparently healthy and 38 isolates (53.52%) from sick man. Similar studies were conducted by Kabra et al. (2004),
Dedeic et al. (2007) and Berkovitch (2009). Kabra et al. (2004) isolated Staphylococcus aureus and Pseudomonas aeruginosa
and Dedeic et al. (2007) isolated Klebsiella pneumoniae, Pseudomonas spp., Acinetobacter baumannii and Serratia
marcescens. Berkovitch (2009) also isolated Staphylococcus aureus from throat swab of man. Research conducted by Todar
(2008) was showed the predominant normal bacterial flora of respiratory tract of human included Staphylococcus epidermidis,
Staphylococcus aureus, Streptococcus spp., Klebsiella spp., Pseudomonas spp., Proteus spp. and E. coli.
Table 1. Bacteria isolated from throat swab of man based on health status
Name of isolates Apparently healthy Sick Total
Frequency of isolate Percent Frequency of isolate Percent Number Percent
Coagulase positive Staphylococcus 03 4.23 12 16.90 15 21.13
Coagulase negative Staphylococcus 10 14.08 03 4.23 13 18.31
Klebsiella spp. 05 7.04 09 12.68 14 19.72
Pseudomonas spp. 04 5.63 07 9.86 11 15.49
Proteus spp. 03 4.23 00 0.00 03 4.23
Escherichia coli 04 5.63 03 4.23 07 9.86
Bacillus spp. 04 5.63 04 5.63 08 11.27
Total 33 46.48 38 53.52 71 100
Pathogenicity tests
The inoculated mice died within 48 hours of inoculation, revealed symptom of labor pneumonea including coughing, sneezing
and respiratory distress before death. The other mice did not show any sings of illness and finally survived. No symptoms or
death was observed in any of the control group of animal. The animals those died revealed the hemorrhages and congestion of
lung after post mortem examination. The organisms were re-isolated from the lung specimens. The results of pathogenicity
tests are presented in Table2. Torres and Stanislawa (1970) determined the pathogenicity of Staphylococcus aureus in mice by
injecting through the intra-cerebral and intra-peritoneal routes, Domenico et al. (1982) determined the virulence of K.
pneumoniae by injecting intra-peritoneally into mice and similarly Daniel et al. (1992) performed pathogenicity test of
Pseudomonas spp. through intranasal and intra-peritoneal route. All the authors’ findings are in agreements of the present
4
study. Helen et al., (2007) also reported that intratracheal inoculation with 7x 10 CFU of a strain of Klebsiella pneumoniae
induced 100% mortality in C57BL/6J mice within 5 days post infection.
Table 2. Results of Pathogenicity tests (Route of inoculation= Intraperitoneal, No. of mice in all cases= 3)
Name of bacteria Sample Sources Dosage of inoculum *Number of death Pathogenicity
within 48 hours
Coagulase positive Staphylococcus Apparently Healthy 0.5 ml inoculum 0/3 Non pathogenic
6
Sick man [3.7 x 10 CFU 3/3 Pathogenic
Coagulase negative Staphylococcus Apparently Healthy (Colony forming 0/3 Non pathogenic
Sick man unit)) 0/3 Non pathogenic
Klebsiella spp. Apparently Healthy 0.5 ml inoculum 0/3 Non pathogenic
Sick man 5 3/3 Pathogenic
(5 x 10 CFU)
Pseudomonas spp. Apparently Healthy 0.5 ml inoculum 0/3 Non pathogenic
Sick man 7 3/3 Pathogenic
(2 x 10 CFU)
Nutrient broth (Control) 0.5 ml 0/3 Non pathogenic
28
Int. J. BioRes. 1(6): 27-30 June, 2010 Islam et al.

Antibiotic sensitivity tests

All the bacterial isolates were 100% sensitive to Norfloxacin and Ciprofloxacin and were 100% resistant to Amoxicillin and
Ampicillin (Table 3) . Enrofloxacin was found effective against Coagulase positive Staphylococcus (CPS) and Klebsiella but
not effective against Pseudomonas. Klebsiella was found sensitive to Pefloxacin where as CPS was intermediately sensitive
and Pseudomonas was 100% resistant. Wayne (2003) isolated Coagulase-positive and negative Staphylococcus strains, and
tested by disc-diffusion, for sensitivity to the following antimicrobials: oxacillin (10 µg), cephazolin (30 µg), ciprofloxacin (5
µg), clavulanic acid + amoxicillin (30 µg), vancomycin (30 µg), azitromycin (15 µg), clindamycin (2 µg), amoxicillin (10 µg),
enrofloxacin (5 µg) and ampicillin (10 µg) and found variable sensitivity pattern of the isolates. Cheesbrough (2006) showed
Klebsiella often produce beta-lactamases and were resistant to ampicillin and some Klebsiella strains showed multiple drug
resistance. Ndip et al. (2005) conducted antimicrobial susceptibility of Pseudomonas aeruginosa by the disc diffusion assay.
The resistance pattern of cefotaxime, gentamicin and tetracycline was the most common (21.6%) amongst the isolates and
there was a significant difference in the susceptibility of isolates to ciprofloxacin (98%), amikacin (90.2%) and netilmicin
(80.4%) compared with other drugs used in that study. Michelim et al., 2005 also found multidrug resistant Staphylococcus
strains.
Table 3: Results of antibiotic sensitivity tests
Name of Sensitivity Name of isolates
antibiotics and Resistant patterns Coagulase positive Staphylococcus Klebsiella spp. Pseudomonas spp.
Number Percent Number Percent Number Percent
Pefloxacin Sensitive 2 33.33 6 100 0 00
(PEF) Intermediate 4 66.67 0 00 0 00
Resistant 0 00 0 00 6 100
Amoxicillin Sensitive 0 00 0 00 0 00
(AML) Sensitive 0 00 0 00 0 00
Resistant 6 100 6 100 6 100
Ampicillin Sensitive 0 00 0 00 0 00
(AMP) Sensitive 0 00 0 00 0 00
Resistant 6 100 6 100 6 100
Furazolidone Sensitive 0 00 0 00 0 00
(FR) Sensitive 3 50 0 00 0 00
Resistant 3 50 6 100 6 100
Gentamycin Sensitive 2 33.33 2 33.33 0 00
(CN) Sensitive 4 66.67 4 66.67 0 00
Resistant 0 00 0 00 6 100
Norfloxacin S Sensitive 6 100 6 100 6 100
(NOR) Sensitive 0 00 0 00 0 00
Resistant 0 00 0 00 0 00
Ciprofloxacin Sensitive 6 100 6 100 6 100
(CIP) Sensitive 0 00 0 00 0 00
Resistant 0 00 0 00 0 00
Enrofloxacin Sensitive 6 100 6 100 0 00
(ENR) Sensitive 0 00 0 00 3 50
Resistant 0 00 0 00 3 50
CONCLUSION
It may be concluded that Staphylococcus spp. , Klebsiella spp. and Pseudomonas spp. were predominant of the bacteria
isolated from throat swab of man. Percentages of identified bacteria were higher in sick man than apparently healthy one.
Coagulase positive Staphylococcus (CPS), Klebsiella spp. and Pseudomonas spp. isolated from sick were pathogenic.
Amoxicillin and ampicillin were not recommended antibiotics to use against Staphylococcus, Klebsiella and Pseudomonas
infection because of their resistance. Ciprofloxacin and norfloxacin were found to be effective antibiotics to treat
Staphylococcal, Klebsiella and Pseudomonas infections. Isolated Pseudomonas spp. showed multidrug resistant properties.
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