.jpg)
Fig 1: Colonies
of Pseudomonas aeruginosa on Nutrient agar plate
.jpg)
Fig 2: Colonies of Pseudomonas
aeruginosa on Cetrimide agar plate
Identification
of Pseudomonas aeruginosa: The
isolates were identified on the basis of cultural, morphological, and
biochemical characteristics as per Bergey’s Manual of
Systemic Bacteriology [11].
Cultural
characteristics: Plates were observed for production of large, opaque,
irregular colonies with the distinctive, small, rough, strongly cohesive
colony.
Morphological
characteristics: A suspected colony was picked from the plate and smear
preparation was made on clean glass slide and Gram staining was performed and observed
under 100 X objectives.
Biochemical
characteristics: Various biochemical tests were performed for the
identification of P. aeruginosa. Following biochemical were been
undertaken-
Catalase Test: Catalase test was performed by adding 3% hydrogen peroxide (H2O2) solution to trypticase soy agar slant culture. Release of free oxygen gas (O2↑) bubbles indicated positive catalase test. Alternatively the test was performed as slide test in which the inoculum from a plate culture was picked and placed on a clean glass slide. One drop of hydrogen peroxide was added and appearance of bubbles indicated positive catalase test. The appearance of bubble occurred due to breakdown of hydrogen peroxide to water and oxygen by catalase enzyme present in some microorganisms which help them in their survival.
Oxidase
Test: The oxidase test (also known as the cytocrome oxidase test) was used
to look for oxidase enzymes produce by certain
bacteria. Oxidase catalyses electron transport
between substrate acting as electron donors in the bacterium and N,N,N',N'-tetramethyl-p-phenylenediamine
(TMPD)- a redox dye present as hydrochloride. The dye
was reduced to deep-violet blue color in the presence of oxidase
enzymes. The test was performed by wetting strips of filter paper with a fresh
1% N,N,N',N'-tetra methyl-p-phenylenediamine (TMPD)
dye solution. The colony was picked & tested with a loop and rubbed on to
moistened strip. Change in color to deep-violet was observed which show the
positive test for oxidase.
Nitrate Reduction Test: Nitrate reduction medium was inoculated with the organism
and incubated aerobically at 370C for 24 to 48 hrs. After
incubation, α-napthylamine and sulfanilic acid were added. These two compounds react with
nitrite and turn red in color. In few tubes nitrate was further reduced to
ammonia or nitrogen gas. To distinguish between these two reactions, zinc dust
was added. Zinc reduces nitrate to nitrite. The tubes were turn red because
α-napthylamine and sulfanilic
acid are already present in the tube. The test organisms were able to reduce
nitrate. Bright red color after the addition of α-napthylamine
and sufanilic acid and no color change upon the
addition of zinc was recorded as positive nitrate reduction test.
Carbohydrate Fermentation: In this test, a test tube with an inverted Durham’s tube containing a basal medium and the particular sugar (1% conc.) along with a suitable indicator (Bromocresol purple) was used. The medium was inoculated with the help of a loop containing the test organism and then incubated aerobically at 370C for 24 to 48 hrs. Change in color of the medium from purple to yellow indicates acid production and the fermentation of the particular sugar. The presence of air-bubble in the Durham’s tube indicates gas production. The organism was tested for glucose, lactose, sucrose, maltose, and mannitol fermentation. Pseudomonas aeruginosa showed acid production with glucose and mannitol sugars.
Indole hydrolysis: In this test tryptone broth was used containing large amount of tryptophan. In presence of tryptophanase enzyme tryptophan was hydrolyze into an indole and pyruvic acid. The isolated organism was inoculated in tryptone broth. All the inoculated and uninoculated (control) tubes were incubated at 37±0.20C for 48 hrs. After incubation, 5 drops of Kovac’s reagent was added, a red layer at the top of broth indicates positive test where as no change in color indicated negative test.
Methyl red test: The Methyl Red test involves adding the pH indicator methyl red to MR-VP broth. If the organism uses the mixed acid fermentation pathway and produces stable acidic end products, the acids will overcome the buffers in the medium and produce an acidic environment in the medium. The isolated microorganisms were inoculated in Methyl Red-Voges Proskauer broth. All the inoculated and uninoculated (control) tubes were incubated at 37±0.20C for 48 hrs. After incubation, 5-6 drops of methyl red reagent was added. Red color of medium indicates positive test, while no color change in the medium indicated negative result.
Voges- Proskauer test: This test was performed to determine the capability of microorganism to produce non-acidic end products such as ethanol and acetoin (acetyl methyl carbinol) from the organic acid. The isolated microorganisms were inoculated in Methyl Red-Voges Proskauer broth. All the inoculated and uninoculated (control) tubes were incubated at 37±0.20C for 48 hrs. After incubation, 12 drops of freshly prepared VP-reagent I (naphthol solution), 2-3 drops of VP-reagent II (40% KOH) was added in all the inoculated and uninoculated tubes. Development of crimson to pink (red) color indicated positive test where as no change in color indicated negative test.
Citrate utilization: Citrate test was performed to determine the ability of microorganisms to utilize citrate as carbon source. The utilization of citrate depends on the presence of an enzyme citrase that breakdown citrate to oxaloacetic acid and acetic acid. The isolated organism were inoculated in Simmon’s Citrate Agar slant and incubated at 37±0.20C for 48 hrs. After incubation, tubes were examined for change in coloration of slant from green to blue indicating positive test for citrate utilization. If color did not change, it indicated negative test for citrate utilization.
Motility test: This test was done to check the
motility of the bacterium. Tube containing motility agar was stab inoculated.
Positive test is indicated by the growth around the stab line that has radiated
outwards in all directions while no growth around the stab line indicates
negative test.
Urease test: Urea is a major organic waste product of protein digestion. This test was performed to determine the ability of microorganisms to produce enzyme urease. The urease is hydrolytic which attacks the carbon and nitrogen bond amide compounds (e.g. urea) with the liberation of ammonia. Urease test was performed by growing the organism on urea agar medium containing the pH indicator phenol red (pH 6.8). During incubation, microorganism possessing urease reduces ammonia that raises the pH of the medium. As the pH become higher, the phenol red was changed from a yellow color (pH 6.8) to a red or deep pink color, which indicated positive test where as no change in color indicated negative test.
Antibiotic
susceptibility pattern of Pseudomonas aeruginosa: Antibiotic
susceptibility pattern of P. aeruginosa
isolates was studied using Disc Diffusion method described by Bauer et al. [12].
In this test the isolates obtained were swab inoculated on Mueller Hinton agar
plate. Under sterile conditions, antibiotic
discs were placed on the surface of the inoculated plate. The plates were
incubated at 370C, for 24 hrs, observed for the zone of inhibition
and compared with CLSI (Clinical and Laboratory Standards Institute) scale [13]. For conducting the antibiotic susceptibility test the following
antibiotics discs purchased from Hi-media
(Mumbai) were used: Ceftazidime (Ca), Amikacin (Ak), Imipenem (I), Ciprofloxacin (Cf),
Tetracycline (T), Gentamicin (G), Norfloxacin
(Nx), Penicillin (P), Chlorampenicol
(C), Ofloxacin (Of).
Effect of
antiseptic on Pseudomonas aeruginosa:
Antiseptic test was done by using filter paper disk method. In this test the
isolates obtained were swab inoculated on Nutrient agar plate. Sterile disk was prepared by dipping in alcohol
with the help of the forceps, in front of the flame and was then dipped half
way in to a beaker containing the chemical agents (Betadine
and Dettol). Chemical agents were diluted up to 10-4.
Antiseptic discs were prepared by soaking them in dilutions from 10-1
to 10-4. Under sterile condition impregnated antiseptic disc was
placed on the surface of the inoculated plate. The plates were inoculated
at 370C for 24 hrs and observed for the zone of inhibition (mm) [14].
Statistical
Analysis: The data obtained during the course of
investigation was statistically analyzed by applying c2-test at 5%
probability level as well as Z-test and t-test was interpreted accordingly (Panse and Sukhatme [15].
Results and Discussion: In the present study, of the hundred
clinical samples collected from different patients a high prevalence rate of
bacterial pathogen (51%) was observed. Screening of the clinical samples for
incidence of P. aeruginosa showed the
percentage occurrence to correspond to 53% (Table 1, Fig. 3). P. aeruginosa is reportedly the most
frequently isolated non-fermentative gram negative bacterium that has emerged
as a major nosocomial pathogen. With respect to the observations made in the
present study most literature report a similar prevalence rate of P. aeruginosa ranging from 44.7% -
91.67% [16-18].
Certain reports, have however documented a lower incidence of the pathogen in
the range 13%- 31.52% [19-21].
Incidence of P. aeruginosa is
affected by geographical factors, duration of investigation, culture technique
and geographical variations.
.jpg)
The different samples screened for P. aeruginosa included urine, blood,
wound, pus and sputum (20 each). Distribution pattern showed highest isolation
rate from wound (33.33%) followed by sputum (22.22%), pus (18.52%) and urine
(14.81%). The least number of isolates were obtained from blood samples
(11.11%). On analyzing the data, the incidence of bacterial pathogen with
respect to samples type were found to be statistically non-significant (Table 2, Fig 4). Similar studies have
reported maximum isolation of P.
aeruginosa from wound and pus varying in the range 27.81% - 33.30% [9,22].
Table
1: Incidence of Pseudomonas aeruginosa
in clinical samples
|
Total Samples |
Positive samples for
bacterial isolates (%) |
Pseudomonas
aeruginosa (%) |
|
100 |
51(51) |
27(53) |
Table
2: Distribution of P. aeruginosa in
different sample sites
|
Total Isolates
|
Samples sites |
||||
|
27 |
Urine (%) |
Blood (%) |
Wound (%) |
Pus (%) |
Sputum (%) |
|
4 (14.81%) |
3 (11.11%) |
9 (33.33%) |
5 (18.52%) |
6 (22.22%) |
|
c2
=
3.93, NS
NS =
Non-significant, c2 (5%) = 9.49
.jpg)
Fig 4: Distribution of P. aeruginosa with respect to clinical
samples
The clinical
isolates of P. aeruginosa were
subjected to antibiotic sensitivity test. Results revealed an increasing trend
towards development of antibiotic resistance. The resistance rates corresponded
to 100% for Penicillin, Chloramphenicol and
Tetracycline, 92% for Ceftazidime, 85% for Amikacin, Ciprofloxacin [23], Gentamicin, 81% for Ofloxacin, 74% for Norfloxacin,
and 56% for Imipenem. On analyzing the data,
antibiotic sensitivity pattern (Table 3) against P. aeruginosa with respect to antibiotics were found to be
statistically non-significant (Table 4, Fig. 5-6). Several studies have documented a similar pattern of resistance
among P. aeruginosa isolates.
Resistances have been recorded against Penicillin (80.4%), Amikacin
(81%-92.68 %), Tetracycline and Gentamycin (71.6%), Norfloxacin (25.5%), Ciprofloxacin (75.8%-79%), Chloramphenicol (100%), Ceftazidime
(80%) and Ofloxacin (81.6%). [17,20,24] Imipenem
was the only drug in the present investigation towards, which P. aeruginosa was shown least
resistivity. However, resistance rate up to 56% was observed. The resistance
rate to imipenem has been previously shown to be
increasing (24%-60%) [4,25]. The susceptibility pattern of P. aeruginosa has been reported to be
influenced by collateral damage from previous exposure to antibiotics. Further
reduced permeability i.e. down
regulation of porin channels in outer lipopolysaccharide membrane effectively reduces entry of carbapenems and is sufficient for acquisition of
resistance.
Table 3: Antibiotic susceptibility
pattern of P. aeruginosa isolates
|
S. No. |
Antibiotic Disc |
Conc. (µg) |
P.
aeruginosa |
|
1 |
Ceftazidime
(Ca) |
30 |
S |
|
2 |
Amikacin
(Ak) |
30 |
S |
|
3 |
Imipenem
(I) |
10 |
S |
|
4 |
Ciprofloxacin
(Cf) |
30 |
S |
|
5 |
Tetracycline
(T) |
10 |
R |
|
6 |
Gentamicin
(G) |
10 |
S |
|
7 |
Norfloxacin
(Nx) |
30 |
S |
|
8 |
Penicillin
(P) |
30 |
R |
|
9 |
Chlorampenicol
(C) |
30 |
R |
|
10 |
Ofloxacin
(Of) |
5 |
S |
S=Sensitive, R=Resistant
Table 4: Percentage resistance of Clinical isolates
of P. aeruginosa against
antibiotics
|
Total
no. of samples |
Pa isolates |
Percentage
resistance against Antibiotics (%) |
|||||||||
|
100 |
27 |
Ca |
Ak |
I |
Cf |
T |
G |
Nx |
P |
C |
Of |
|
92 |
85 |
56 |
85 |
100 |
85 |
74 |
100 |
100 |
81 |
||
c2= 5.40, Ca = Ceftazidime, G
= Gentamicin
NS =
Non-significant, c2
(5%) = 9.49 Ak = Amikacin, Nx= Norfloxacin
Pa= Pseudomonas aeruginosa I
= Imipenem, P = Penicillin Cf = Ciprofloxacin, C = Chlorampenicol
T = Tetracycline, Of=
Ofloxacin
.jpg)
Fig
5:
Percentage resistance of clinical isolates of P. aeruginosa against antibiotics
.jpg)
Fig 6: Antibiotic susceptibility pattern
of P. aeruginosa
Antiseptics are
used extensively in hospitals and healthcare settings for a variety of topical
applications. In particular they are an essential part of infection control
practices and aid in the prevention of nosocomial infections. The activity of
two commonly used antiseptics (Betadine & Dettol) was evaluated against Pseudomonas aeruginosa. As per the results obtained both antiseptics
were effective against P. aeruginosa.
Dettol exhibited slightly greater inhibition (in
terms of zone diameter) as compared to Betadine
(Table 5, Fig 7). Both antiseptics were
effective at dilutions up to 100 fold. However, no activity was observed at
1000 fold dilution. Thus Dettol and Betadine showed MIC at 100 fold dilution for P. aeruginosa. There was no activity
against the pathogen on subsequent (Betadine & Dettol). Similar to the present study other reports have
documented 100 fold dilutions of antiseptics as Betadine,
Lysol, Dettol, and Savlon
to have an inhibitory effect towards Pseudomonas
aeruginosa [14,26]. Over dilution i.e. dilution of 1000
fold and above has been also previously shown to have very negligible or no
inhibitory effect against the pathogen [27-28].
The effectiveness of antiseptics in controlling
infection is often compromised by the fact that these agents get contaminated
during the preparation process. Further, resistance due to incorrect product
use and ineffective infection control practices cannot be underestimated.
|
S.
No. |
Antiseptics |
Mean
value of zone of inhibition (mm) |
|||
|
10-1 |
10-2 |
10-3 |
10-4 |
||
|
1 |
Dettol |
7.6 |
5.7 |
0 |
0 |
|
2 |
Betadine |
5.6 |
4.3 |
0 |
0 |
Table 5: Antimicrobial
activity of antiseptics (Betadine & Dettol) P. aeruginosa
.jpg)
Fig 7: Antimicrobial activity of antiseptics (Betadine and Dettol) on P. aeruginosa
The study
population was divided into three categories based on their age: 0-30 yrs,
31-60 yrs and 61-90 yrs. Results revealed higher occurrence of Pseudomonas aeruginosa (50%) among
patients of the age group 31-60 yrs followed by age 0-30 yrs (28.33%) and least
in the age 60-90 (21.66%) (Table 6, Fig 8). The data however lost significance
when analyzed statistically. Similar data have been cited in other literature
where greater isolation was obtained in patients of the age groups 0-29 yrs
(15%) and 21- 40 yrs (43.2%) [19,21-22].
In contrast most studies document the old age
group (< 50 yrs) to be predisposed to Pseudomonas
aeruginosa infection [29-31]. Since the majority of patients were in
the intermediate age, hence the greater isolation rates. Infection is
influenced by general health of a patients and risk increases with the exposure
to the pathogens.
Table 6: Age as a risk factor causing infection due
to P. aeruginosa in among patients
|
S. No |
Age group (year) |
Total patients |
Occurrence of P. aeruginosa |
|
|
Total no. of +ve
cases |
Total no. of -ve
cases |
|||
|
1 |
0-30 |
29 |
17(28.33%) |
12(30%) |
|
2 |
31-60 |
51 |
30(50%) |
21(51.22%) |
|
3 |
61-90 |
20 |
13(21.67%) |
7(17.50%) |
t- test = 0.84,
NS
NS =
Non-significant, ttable=12.6
.jpg)
Fig
8: Age as a risk factor causing infection due to P. aeruginosa among
patients
Occurrence of Pseudomonas aeruginosa on the basis of
gender is shown in Table 7 and Fig 9. The results showed higher occurrence in
males (59.26%) and females patients (40.74%). The difference was however found
to be non-significant. The present findings were supported by other studies
where male patients showed higher incidence [21-22,31]. Maintenance of hygienic standards contributes to
acquisition of pathogen by the individual.
Table 7: Gender as a risk factor causing infection
due to P. aeruginosa in among patients
|
S. No |
Gender |
Total patients |
Occurrence of P. Aeruginosa |
|
|
Total no. of +ve cases |
Total no. of –ve cases |
|||
|
1 |
Male |
63
|
16(59.26%) |
47(64.38%) |
|
2 |
Female |
37
|
11(40.74%) |
26(35.62%) |
Z-test = 0.29, NS
NS =
Non-significant, c2 (5%) =1.96
.jpg)
Fig 9: Gender as
a risk factor causing infection due to
P. aeruginosa among patients
Duration of
disease in patient was another factor analyzed for incidence of Pseudomonas aeruginosa infection. Two
categories were defined on the basis of disease duration: 1-10 days and 11-20
days. The occurrence of Pseudomonas
aeruginosa was higher in duration of disease for 11-20 days (81.82%) as
compared to 1-10 days (18.18%). The data was found to be significant when
analyzed statistically (Table 8, Fig 10). Duration of disease over 5 days has
been identified previously as a major risk factor predisposing a patient to
acquire infection due to Pseudomonas
aeruginosa [31]. Duration of disease disposes a patient to acquire
nosocomial infection due to immune-compromised state of the individual.
|
S. No |
Duration of disease (days) |
Total patients |
Occurrence of P. aeruginosa |
|
|
Total no. of +ve
cases |
Total no. of -ve
cases |
|||
|
1 |
1-10 |
42 |
8(18.18%) |
34(60.71%) |
|
2 |
11-20 |
58 |
36(81.82%) |
22(39.29%) |
Table 8: Duration of disease as a risk factor
causing infection due to Pseudomonas aeruginosa among patients
c2 = 18.3, S = Significant, c2 (5%) =3.84
.jpg)
Fig 10: Duration of disease as a risk factor causing
infection due to Pseudomonas
aeruginosa among patients
On the basis of
duration of hospital stay 3 groups were categorized: 1-10 days, 11-20 days and
21-30 days. Highest incidence was observed in 21-30 days duration (53.33%)
followed by 11-20 days (33.33%) and least in 1-10 days (13.33%). On analyzing
the data, the incidence of P. aeruginosa infection
on the basis of hospitalization duration were found to be statistically
significant (Table 9, Fig 11). Similar results were reported by Oguntibeju and Rau [19] and Talon et al. [32], who identified
duration of hospital stay as a major risk factor. Length of hospitalization
affects colonization due to exposure of the individual to various pathogens harbored
in the environment and by constant handling by health care workers.
Table 9:
Hospitalization duration as a risk factor causing infection due to P.
aeruginosa in among patients
c2= 16.89, S=
Significant, c2 (5%) =5.99
.jpg)
Fig 11: Incidence of P. aeruginosa infection on the basis of
Hospitalization duration
CONCLUSIONS- P. aeruginosa is a major cause of nosocomial infection.
Despite advances in sanitation facilities and the introduction of wide variety
of antimicrobial agents with antipseudomonal
activities of P. aeruginosa continue
to be cause of life-threatening infections. With growing concerns about the development of biocidal resistance and cross-resistance with antibiotics,
clinical isolates should be under continual surveillance and other possible
mechanisms of resistance should be investigated. Also, antiseptic and
disinfectant products can varies significantly in their activity despite
containing similar levels of biocides, which underlies the need for close
inspection of efficacy claims. It will also make for more efficient use of antipseudomonal agents clinically with the potential for
design of newer, more effective compounds and products.
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