Respiratory Bacterial Flora from Healthy as well as Respiratory Symptoms Subjects
Respiratory system is the primary settlement place of opportunistic organisms and considered as chief carrier of common respiratory pathogens. The aim of the study was to know the opportunistic organisms present in the healthy subjects as well as subjects that were suffering from respiratory symptoms. The organisms were identified as per standard bacteriological protocol and pathogenicity tests of the identified organisms were performed in mouse model. Antibiotic sensitivity of the identified organisms was performed. The bacterial flora present in the throat swab of apparently healthy as well as subjects suffering from respiratory symptoms 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 respiratory symptoms exhibiting individuals (53.52%) than apparently healthy individuals (46.48%). All coagulase positive Staphylococcus, Klebsiella spp. and Pseudomonas spp. isolated from respiratory symptoms subjects 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. The study provided information about the pathogenic organisms present respiratory systems of apparently healthy as well as subjects suffering from respiratory symptoms. The pathogenic natures of the isolated organisms were determined to make aware of scientists as well as clinicians. Antibiotics sensitivity assays would provide information to the clinicians for the selection of appropriate antibiotics to treat their patients.
Received: April 14, 2011;
Accepted: May 20, 2011;
Published: July 13, 2011
Respiratory system is the important avenue of bacterial infection. All potential
respiratory pathogens primarily adhere to the respiratory mucosa and become
infective under favorable circumstances. In this regard, viruses play a vital
role to perpetuate such infections (Adderson, 2006).
Therefore, respiratory tract is considered as primary settlement site for opportunistic
organisms as well as chief carrier of common respiratory pathogens (Belliveau,
1973). It is well known that in immunocompromised hosts, microorganisms
present in oral cavity and cause opportunistic infections and systemic diseases
such as bacterial endocarditis, aspiration pneumonia and osteomyelitis (Philip
et al., 2009). Among the bacteria, Escherichia coli, Klebsilla
pneumoniae, Haemophilus influenza, Streptococcus pneumoniae,
Staphylococcus aureus, Pseudomonas aeruginosa and Proteus vulgaris
are the common bacteria of nosocomial infections for respiratory tract infections
(Sittiwet et al., 2009).
In this regard, Das et al. (2010) pointed out
that the presence of respiratory pathogen such as Klebsiella might be attributed
to the bacterial aerosols generated due to sneezing and coughing in public places.
It proves that healthy carrier may transmit the infection to susceptible individuals.
It is known that 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, 2011).
Recent study suggests that bacteria found in the throat, as well as in the mouth,
can be drawn into the lower respiratory tract. This can cause infections or
worsen existing lung conditions. People with respiratory diseases, such as chronic
obstructive pulmonary disease, typically suffer from reduced protective systems,
making it difficult to eliminate bacteria from the lungs (AAP,
2011) Treatment of the illness may not be judicious if proper identification
of the causal agents 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 subjects suffering from respiratory symptoms and to study their
pathogenicity and antibiotic sensitivity. This would provide us information
of the types of bacteria present in respiratory systems during healthy life
as well as at stress condition with respiratory symptoms. Moreover, pathogenicity
of isolated organisms as well as their antibiotic sensitivity pattern would
provide treatment guidelines to the clinicians.
MATERIALS AND METHODS
This study was conducted in the Department of Microbiology and Hygiene, Bangladesh Agricultural University (BAU), Mymensingh during the period of January 2009 to December 2009.
Samples: A total of 60 throat swab samples were aseptically collected in which 30 from apparently healthy adult subjects and 30 from respiratory symptoms adult subjects with the help of sterile cotton buds. Immediately after collection, the samples were inoculated into nutrient broth. These were then transferred to the laboratory, Department of Microbiology and Hygiene BAU, Mymensingh-2202, Bangladesh.
Isolation and characterization of bacteria: The collected samples were
processed as per the procedure of Cheesbrough (2006).
Briefly the samples were then inoculated into Blood Agar (BA) media and incubated
at 37°C for 24 h. 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 of Cheesbrough
Pathogenicity tests: The isolated Staphylococcus, Klebsiella and Pseudomonas organisms were subjected to pathogenicity test in 2 months old mice by intra-peritoneal inoculation to observe their pathogenicity.
Antibiotic sensitivity tests: Antibiotic sensitivity tests were performed
using disc diffusion test of the method described by CLSI
RESULTS AND DISCUSSION
Bacterial from healthy subjects as well as subjects suffering from respiratory
symptoms: The bacteria isolated and identified from the throat swabs of
apparently healthy and subjects suffering from respiratory symptoms have been
presented in Table 1. It reveals from the Table
1 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 identified from apparently
healthy subjects and 38 isolates (53.52%) from respiratory symptoms exhibiting
subjects. Similar studies were conducted by Kabra et
al. (2004), Dedeic et al. (2007) and
Berkovitch et al. (2002). 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 et al.
(2002) also isolated Staphylococcus aureus from throat swab of man.
Study conducted by Todar (2011) 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.
Pathogenicity tests: Some of the inoculated mice died within 48 h of
inoculation, revealed symptom of labor pneumonia 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 have been presented
in Table 2. 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. Findings of almost all authors
described above are in agreements of the present study.
|| Bacteria isolated from throat swab of apparently healthy
and respiratory symptomatic showing subjects
|| Results of pathogenicity tests performed in mouse
|AH: Apparently healthy man, S: Sick man, I/P: Intraperitoneal,
CFU: Colony forming units, P: Pathogenic, NP: Non pathogenic
|| Results of antibiotic sensitivity tests
|PEF: Pefloxacin, AML: Amoxicillin, AMP: Ampicillin, FR: Furazolidone,
GN: Gentamycin, NOR: Norfloxacin, CIP: Ciprofloxacin, ENR: Enrofloxacin,
R: Resistant, I: Intermediate, S: Sensitive
Results of antibiotic sensitivity tests: The results of antibiotic sensitivity
were categorized into Resistant (R), Intermediate (I) and Sensitive (S) based
on diameter of zone of inhibition. The results in Table 3
shows that Coagulase positive Staphylococcus was sensitive from 33.33
to 66.67% to Pefloxacin, 100% resistant to Amoxicillin and Ampicillin, 50% resistant
to Furazolidone and 66.67% sensitive to Gentamycin. But then, this organism
was found to be 100% sensitive to Norfloxacin, Ciprofloxacin and Enrofloxacin.
Klebsiella spp. were 100% sensitive to Pefloxacin and 100% resistant
to Amoxicillin, Ampicillin and Furazolidone. In Gentamycin these species were
found to be 66.67% sensitive, however, in case of Norfloxacin, Ciprofloxacin
and Enrofloxacin these were proved 100% sensitive. In Pseudomonas spp.
antibiotics Pefloxacin, Amoxicillin Ampicillin, Furazolidone and Gentamycin
were 100% resistant. On the other hand, Norfloxacin, Ciprofloxacin were 100%
sensitive, however, Norfloxacin was only 50% sensitive. 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). 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. A study was conducted by Gamal
et al. (2010) and reported that quinolones, cefipime and linezolide
were the most efficient antibiotics in treatment of lower bacterial respiratory
tract infections in Egypt. The predominant isolates in that case were Heamophilus
Influnza (32%), Streptococcus pneumoniae (30%), Moraxella catarrhalis
(14%), Klebsiella pneumoniae (10%) and Chlamydia pneumoniae (7%).
The difference of identified bacteria with the present study might be due to
isolation was done here from the clinical cases.
Similarly, Parvez et al. (2004) isolated and
studied bacterial pathogens: Staphylococcus aureus, Enterobacter aerogenes
and Escherichia coli from clinical specimens of hospitalized patients
against three antibiotics viz., erythromycin, tetracycline and penicillin G
and reported that all the isolates were sensitive to tetracycline and erythromycin.
But then, Sittiwet and Puangpronpitag (2009) reported
that S. aureus and E. coli causing serious nosocomial infection
and indiscriminate use of antibiotics showed multi-drug resistant since the
bacteria have ability to change the susceptible gene and become resistant to
antibiotics. The study highlighted judicious use of antibiotics with proper
sensitivity tests following the correct regiment.
It was observed from the present study that Staphylococcus spp.,
Klebsiella spp. and Pseudomonas spp. were predominant of the bacteria
isolated from throat swab of subjects. Percentages of identified bacteria were
higher in subjects suffering from respiratory symptoms than apparently healthy
subjects. Coagulase Positive Staphylococcus (CPS), Klebsiella spp.
and Pseudomonas spp. isolated from respiratory symptoms subjects
were found to be 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 multidrugs resistant
properties. The study provided information about the harboring of potential
pathogenic organisms in respiratory systems of apparently healthy subjects and
subjects suffering from respiratory symptoms. The pathogenic natures of the
isolated organisms were studied that had been evidenced by mouse pathogencity
tests. In addition, antibiotics sensitivity guidelines were studied so that
the clinicians could treat the patients with appropriate antibiotics.
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