1. SCREENING, CHARACTERIZATION AND RESISTOGRAM
STUDIES OF PATHOGENIC MICRO-ORGANISMS
FROM DIFFERENT LABORATORY
SPECIMENS
PRESENTED BY
DHANJI P. RAJANI
GUIDE
DR. Y. A. SHELAT
Ex - Head, Research Guide & P. G Associate Professor of Microbiology,
Ex. Co-ordinator: P.G.D.M.L.T Microbiology Department,
Sir P.P Institute of Science,
Bhavnagar .

2. INTRODUCTION
Microbes may be the most significant life forms sharing this planet with humans because
of their pervasive presence and their utilization of any available food source, including
humans whose defenses may be breached.
Infectious diseases are still major cause of morbidity and mortality worldwide.
According to WHO’s report of more than 17 million out of 52 million deaths were due to
infectious diseases.
On the contrary 30 new infectious diseases were recognized in the world during last 20
years, (WHO. 2010).
Yet the high death toll from these infectious diseases is only a part of story. Ingoing ill-
health is one of the main reasons why poor stay poor.
There are diverse group of organisms causing various infections of human anatomical
systems like central nervous system, respiratory system, gastro intestinal system,
reproductive system, urinary system and even skin infections.

3. Numerous clinical specimens like urine, blood, sputum etc. containing enormous amount of
infectious microbial flora, which are causative agents of dangerous communicable diseases.
These samples are tested in microbiological laboratories, to present data which indeed is
helpful in the prevention, diagnosis and treatment of human diseases. Particularly for
nosocomial infections these type of testing is useful in cases of antibiotic treatment failure
against resistant microorganism.
Resistance to antimicrobial agents has been recognized since the dawn of antibiotic era. Paul
Ehrlich, the father of modern chemotherapy, observed that, during treatment of trypanosome
infection, organism sometimes emerged were resistant to agents being used.
Ehrlich observed that resistance, once acquired, was stably inherited and in 1908 proposed
that resistance was due to “reduced avidity of chemoreceptor so that they are no longer able to
take up” the drug (Ehrlich et al. 1909).
Earlier, resistance was categorized as either natural or acquired. For example, natural
resistance to gentian violet was a property of Gram negative as compared to Gram positive
organisms

4. The natural resistance of Gram negative agents was attributed to an outer membrane barrier.
(Nikaido 1996). Acquired resistance properly involved reduced susceptibility of an organism
that was previously more sensitive to drug.
 Plasmids carry genes for resistance to many other antimicrobial agents. Some genes codes
for enzymes that modify or inactivate the agents, others for enzymes that alter drug targets in
the cell or provide alternate biosynthetic pathways. Genes for antibiotic efflux
(chloramphenicol, tetracycline) were also found to be plasmid determined.
Antibiotics resistant mechanism.

5.  Antibiotic resistance is an inevitable consequence of antibiotic use. Surveys have shown
that as much as 50% of all antimicrobial use is inappropriate (Livermore , 2009).
Determining the drug resistant in terms of quantity and quality is a real need of village,
city or district.
In short, our country needs to
(i) Spread knowledge regarding antibiotics from authoritative sources rather than
commercial pamphlets.
(ii) Standardize antibiotic resistance testing to ensure comparability.
(iii) Have mandatory institutional mechanisms to regular antibiotic prescription and control
drug resistance.
(iv) Have a national policy for treating community infections.
(v) Establish a National Institute to study antibiotic resistance in nation as a whole.

6. AIMS & OBJECTIVES
To find out the prevalence of pathogens in various clinical samples.
To study the resistogram of pathogenic micro organisms.
To study the prevalence of infection caused by pathogenic bacteria including
Mycobacteria.
To study the identification and cultivation of pathogens
To study the drug resistance pattern of routinely isolated organism from laboratory
specimen.
To study incidence of fungus infections on the basis of fungal isolates from various
clinical specimens.

7. METHODOLOGY
 Present study was carried out between August 2007 to August 2009
We had analyzed totally 6372 different samples such as urine, blood, pus, sputum, body
fluids, stool, semen and swabs from wound and throat.
All clinical samples used for the study were collected as per WHO guideline and as per
mentioned by the standard book (Isenberg, 2nd edition. 2007.)
Before screening for their morphological characteristics the samples urine, stool, sputum,
pus, body fluids and semen were analyzed for their physical characteristics. The screening of
samples was carried out by Gram’s staining technique reported their characteristics.
Sample Collection Tray & Container

8. All samples were streaked on differential media and moderately selective or highly
selective media i.e. Mac-conkey’s agar, sheep blood agar, Cysteine Lactose Electrolyte-
Deficient Agar, brain heart infusion, Thioglycollate broths , chocolate agar, Xylose lysine
deoxycholate agar, tellurite agar, selenite F broth , alkaline peptone water, TCBS agar.
 Then incubate at 37 C for 24 to 48 hrs aerobically.
 The isolates collected from various selective and differential medium were further
characterized. Isolates were identified by morphological and biochemical characteristics
as per standard guidelines.
 Antibiotic Susceptibility test:
 Antibiotic susceptibility tests were carried out by Kirbey-Bauer disk diffusion
technique.
The plates were then incubated at 370C for 24 hours. After 24 hour’s incubation,
each plate was examined and the diameters of the zone of inhibition were noted using
a zone reader scale for antimicrobial disc and result interpreted as per CLSI
guidelines.

9. MRSA detection:
 A direct colony suspension of each S. aureus isolate was prepared to a 0.5 McFarland
standard and plated on Mueller-Hinton agar containing 2-4% NaCl.
 An oxacillin (1 µg) and 5µg methicillin discs were placed on the surface and incubated
at 35°C for 24 hours.
Zone of inhibition were measured following incubation.
ESBL DETECTION:
 All the gram negative lactose fermentor isolates under study were also tested for their
extended spectrum β-Lactamase production.
 Combination Disk diffusion method was used to confirm ESBL production by gram
negative lactose fermentor isolates.
 Plates are inoculated as per the standard disc diffusion method as recommended in CLSI
guidelines).
Zone of inhibition were measured following overnight incubation aerobically at 37°C.
The test organism was regarded as an ESBL producer if the zone of inhibition around the
combination disc is at least 5mm larger than that of the cephalosporin alone, or if the zone
diameter is expanded by 50% in the presence of the clavulanic acid regardless of zone
diameters.

10. Isolation and cultivation of fungi:
 Samples suspected to be having fungal pathogen were collect in sterile containers.
 They were inoculated into the Sabouraud dextrose agar.
 The inoculated plates were sealed with gas permeable tape and incubated at 22 0 c for
up to 15 days.
 Primary plates were read daily for the first week and every other day for the second
week. When growth appears, differentiation between yeast and filamentous forms was done
by microscopic examination. (Isenberg. 2007).
Isolation and cultivation of Mycobacterium tuberculosis:
 We have analyzed sputum, pus, urine and some fluid samples suspected to be collected
from patients suffering from tuberculosis.
 The smears prepared from untreated samples were subjected to ZNCF stain and
results were noted as per RNTCP gradation.
 The sputum and pus samples were subjected to pretreatment for digestion and
decontamination by Petroff’s method (Paramshivam CN, 1998), and cultured on the L.J
medium. (Khatri GR et al., 2002).
 They were incubated at 370 c for up to 8 week. Cultures were examined starting from
1st week up to 8th week.
 Isolated colonies were studied in detail for their morphological, cultural and
biochemical characteristics. All positive cultures were further tested for drug susceptibility
towards first line drugs (rifampicin, isoniazid, streptomycin, ethambutol,) by 1% proportion
method. (SOP for Mycobacteriology laboratory, 2007).

11. Sr.no Sample No. of Samples
1 Urine 2452
2 Blood 2276
3 Pus 1033
4 Wound swab 57
5 Body fluid 203
6 Sputum 141
7 Stool 91
8 Abscess 71
9 Throat swab 37
10 Semen 11
Total 6372
Distributions of number of sample
Sr.n
o
Sample Male Female Total
1 Urine 1466 986 2452
2 Blood 1024 1252 2276
3 Pus 658 375 1033
4 Body fluid 118 85 203
5 Sputum 90 51 141
6 Stool 54 37 91
7 Abscess 39 32 71
8 Throat swab 22 15 37
9 Wound Swab 21 36 57
10 Semen 11 0 11
Distribution based on Gender wise and sample type
0
10
20
30
40
50
60
70
80
90
100
% male
%female
39%
36%
16%
1%
3% 2%
1%
1%
1%
0%
Urine Blood Pus Swab
Body fluid Sputum Stool Abscess

12. Sr.
no.
Sample Pure Mix No growth
1 Urine 1437 29 986
2 Blood 574 0 1702
3 Body fluid 25 3 175
4 Semen 7 0 4
Screening of pathogen isolated from sterile clinical samples
0
20
40
60
80
100
Urine Blood Fluid Semen
% pure
%Mix
% no growth
Sr.no. Sample Pure Mix No growth
1 Pus 663 10 364
2 Sputum 96 38 7
3 Stool 84 5 2
4 Abscess 17 0 54
5 Throat swab 31 6 0
6 Wound Swab 30 0 23
Screening of pathogen isolated from non sterile clinical samples
0
10
20
30
40
50
60
70
80
90
100
% pure
%Mix
% no growth

13. Sr.
no
Sample Gram-ve
Gram
+ve
Acid fast
bacilli
Fung
al*
Total
1 Urine 1282 124 0 60 1466
2 Blood 347 173 0 54 574
3 Pus 260 285 7 14 562
4 Body fluid 23 27 4 1 52
5 Sputum 50 67 25 24 148
6 Stool 83 0 0 6 89
7 Abscess 5 12 0 0 17
8 Throat swab 5 31 0 1 37
9 Swab 13 11 0 3 27
10 Semen 1 6 0 0 7
Morphological characteristic of bacterial isolates from diverse clinical samples
(*) Gram resistant yeast culture or fungal spore.
0% 50% 100%
Urine
Pus
Sputum
Abscess
Swab
Gram-ve
Gram +ve
Acid fast
bacilli
Sr.no Organism Urine
1 Escerichia coli 945
2 Klebsiella spp. 229
3
Pseudomonas
aeruginosa
90
4 Proteus spp. 18
5 Group D Enterococci 69
6 Staphylococcus aureus 53
7 Streptococci spp. 2
8 Candida spp. 60
Distribution of pathogens isolated from urine samples
0
10
20
30
40
50
60
70

14. Sr.no Organism Blood
1 Escherichia coli 191
2 Klebsiella spp. 116
3 Pseudomonas aeruginosa 24
4 Salmonella typhi 2
5 Acinetobacter spp. 14
6 Staphylococcus aureus 143
7 Coagulase negative staphylococci 23
8
Methicillin resistant staphylococcus
aureus 7
9 Candida spp. 54
Distribution of pathogens isolated from blood samples
Distribution of pathogens isolated from body fluid samples
Sr.no Organism Fluid
1 Escherichia coli 10
2 Pseudomonas aeruginosa 9
3 Klebsiella spp. 4
4 Staphylococcus aureus 4
5 Candida spp. 1
36%
32%
14%
14%
4%
Escherichia coli
Pseudomonas
aeruginosa
Klebsiella spp.
Staphylococcus
aureus
Candida spp.

15. Distribution of pathogens isolated from sputum samples
Sr. no Organism Sputum
1 Escherichia coli 24
2 Pseudomonas aeruginosa 15
3 Klebsiella spp. 11
4 Streptococci spp. 61
5 Staphylococcus aureus 6
6 Candida spp. 23
7 Aspergillus spp. 1
17%
11%
8%
43%
4%
16%
1%
Escherichia coli
Pseudomonas
aeruginosa
Klebsiella spp.
Strptococci spp.
Staphylococcus
aureus
Distribution of pathogens isolated from stool samples
Sr.no Organism Stool
1 Escherichia coli 55
2 Klebsiella spp. 26
3 Salmonella typhi 2
4 Candida spp. 6
62%
29%
2%
7%
Escherichia coli
Klebsiella spp.
Salmonella typhi
Candida spp.

16. Distribution of pathogens isolated from abscess samples
Sr.no Organism Abscess
1 Escherichia coli 1
2 Klebsiella spp. 2
3 Pseudomonas aeruginosa 1
4 Acinetobacter spp, 1
5 Streptococci spp. 1
6 Coagulase negative staphylococci 1
7 Staphylococcus aureus 10
6%
11%
6%
59%
6%
6%
6%
Escherichia coli
Klebsiella spp.
Pseudomonas
aeruginosa
Staphylococcus
aureus
Streptococci spp.
Distribution of pathogens isolated from throat swab samples
Sr.no Organism Throat swab
1 Pseudomonas aeruginosa 4
2 Klebsiella spp. 1
3 Streptococci spp. 30
4 Staphylococcus aureus 1
5 Candida spp. 1
11% 2%
81%
3% 3%
Pseudomonas aeruginosa
Klebsiella spp.
Streptococci spp.
Staphyloccous aureus
Candida spp.

17. Distribution of pathogens isolated from semen samples
Sr. no Organisms Semen
1 Escherichia coli 1
2 Staphylococcus aureus 6
Distribution of pathogens isolated from pus samples
Sr. no Organisms Pus
1 Escherichia coli 105
2 Pseudomonas aeruginosa 102
3 Klebsiella spp. 51
4 Proteus mirabilis 1
5 Proteus vulgaris 1
6 Streptococci spp. 16
7 Staphylococcus aureus 251
8 Coagulase negative staphylococci 13
9
Methicillin resistant staphylococcus
aureus 5
10 Candida spp. 14 0 10 20 30 40 50
Escherichia coli
Psedomonas aeruginosa
Klebsiella spp.
Proteus mirabillis
Proteus vulgaris
Streptococci spp.
Staphylococcus aures
Coagulase negative…
Methicillin resistant…
Candida spp

18. Distribution of pathogens isolated from wound and other swab samples
Sr. no Organisms Swab
1 Escherichia coli 9
2 Klebsiella spp. 2
3 Pseudomonas aeruginosa 2
4 Staphylococcus aureus 8
5 Streptococci spp. 2
6 Methicillin resistant staphylococcus aureus 1
7 Candida spp. 3
33%
8%
7%
30%
7%
4%
11%
Escherichia coli
Klebsiella spp.
Pseudomonas
aeruginosa
Staphyloccous
aureus

19. Results of the ESBL detection from all isolated microorganisms
Name of
Antibiotic
Symbol Positive Negative Positive (%) Negative (%)
Ceftazidime/C
lavulanic acid CAC
558 1269 30.54 69.46
Cephotaxime/
Clavulanic
acid CEC
608 1219 33.27 66.73
Results of the MRSA detection from all Staphylococcus aureus isolated organisms
Name of Antibiotic Symbol Resistant Sensitive Resistant (%) Sensitive (%)
Methicillin M 13 528 2.4 97.6

20. Antibiogram/resistogram study of Escherichia coli isolated from all clinical samples
0
20
40
60
80
100
120
% S
% R

21. 0
20
40
60
80
100
120
% S
% R
Antibiogram/resistogram study of Pseudomonas aeruginosa isolated from all
clinical samples

22. Antibiogram/resistogram study of Klebsiella spp. isolated from all clinical samples
0
20
40
60
80
100
120
% S
% R

23. 0
10
20
30
40
50
60
70
80
90
100
% S
% R
Antibiogram/resistogram study of Salmonella typhi isolated from all clinical
samples

24. Antibiogram/ resistogram study of Proteus spp. isolated from all clinical samples
0
10
20
30
40
50
60
70
80
90
100
% S
% R

25. 0
10
20
30
40
50
60
70
80
90
100
% S
% R
Antibiogram/ resistogram study of Acinetobacter spp. isolated from all clinical
samples

26. Antibiogram/resistogram study of Staphylococcus aureus isolated from all clinical
samples
0
20
40
60
80
100
120
% S
% R

27. 0
20
40
60
80
100
% S
% R
Antibiogram/resistogram study of Streptococci spp. isolated from all clinical
samples

28. Antibiogram/resistogram study of Coagulase negative staphylococci isolated from
all clinical samples
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
% S
% R

29. 0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
% S
% R
Antibiogram/resistogram study of Methicillin resistant staphylococcus aureus
isolated from all clinical samples

30. Antibiogram/resistogram study of Group D Enterococci isolated from all clinical
samples
0
20
40
60
80
100
120
% S
% R

31. Sr. no Sample Yeast Mould
01 Sputum 20 3
02 Pus 29 1
03 Urine 60 0
04 Body fluid 1 0
05 Blood 54 0
06 Stool 6 0
07 Throat Swab 1 0
Distribution of fungal pathogen isolated from Clinical samples

32. Distribution of Mycobacteria tuberculosis isolation from clinical samples
Sr.no Sample No. of sample
01 Sputum 43
02 Pus 30
03 Urine 09
04 Body fluid 08 0.00%
10.00%
20.00%
30.00%
40.00%
50.00%
60.00%
SPUTUM PUS URINE FLUID
PERCENTAGE [%]
Acid fast bacilli gradation wise distribution as per Revised National
Tuberculosis Control Programme
Sr. no Gradation No. of samples
01 1+ 15
02 2+ 15
03 3+ 6
05 Total 36
0.00% 10.00% 20.00% 30.00% 40.00% 50.00%
1+
2+
3+
SCANTY
PERCENTAGE (%)

33. Distribution of Mycobacterium spp. based on biochemical test
Organism No. of culture Biochemical test
PNB CATALASE NIACIN
Mycobacterium
tuberculosis
32 - - +
Mycobacteria
other than
tuberculosis
01 + + -
M.tuberculosis
MOTT
Sensitivity pattern of Mycobacterium tuberculosis
Sr.n
o
Group No. of
strain
01 Multi Drug Resistant 13
02 Sensitive to all Drugs 02
03 Mono Drug & Other than
multi drug resistant
18
0 5 10 15 20
MDR
SENSITIVE TO ALL DRUGS
MONO DRUG & OTHER THAN
MDR
NO.OF STRAIN

34. Multi Drug Resistant patterns (13 strains)
Drugs No. of strains
HR 4
HSR 3
HER 2
HESR 4
TOTAL 13 0.00%
5.00%
10.00%
15.00%
20.00%
25.00%
30.00%
35.00%
HR HSR HER HESR
PERCENTAGE(%)

35. CONCLUSION
 We can conclude that various gram negative organisms causing urinary tract infection,
respiratory tract infection, genital tract infection, blood stream infection, gastro intestinal
infection, pyogenic infection and cerebrospinal and other effusion were reported.
We found staphylococcus and streptococci spp. as gram positive isolates causing various
infections in our body. Our result shows that the gram negative bacteria were highest among all
bacterial pathogens where as the fungal pathogens were meager in number.
In case of uropathogens majority of them were gram negative bacteria and Escherichia coli
found to be prevalent. Samples collected in respiratory tract infection like sputum shows majority
of gram positive organisms i.e. Streptococci spp.
In the present study the pathogens isolated from blood causing bacteremia were several bacteria
as well as fungi .The prevalence found among them was of Escherichia coli but several fungal
pathogens like Candida spp. was also reported. Stool samples collected in gastro intestinal
infection shows the dominating organism was Escherichia coli.

36. In our study we found Escherichia coli and Pseudomonas aeruginosa were highest in number
from body fluid samples. The only prevalent organism found in case of abscess and semen was
gram positive isolate i.e. Staphylococcus aureus but Streptococci spp. was found more in number
in case of throat swab samples.
In pyogenic infection gram positive dominating isolates was Staphylococcus aureus, while in
case of wound and other swab gram negative bacteria Escherichia coli was prevalent.
Finally we can conclude that Escherichia coli were found to be the most prevalent isolates
among various clinical specimens.
The main objective of present study was to find out resistogram among the microbial pathogen.
We found Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus Klebsiella and
Streptococci spp. showing highest resistance among all clinical samples from their resistogram
study.
 While testing the antibiotic susceptibility for various drugs recommended by Clinical
Laboratory Standard Institute (CLSI), ampicillin was found to be resistant for majority of gram
negative organisms.

37. In our study among gram negative isolates we found Escherichia coli was highest resistant for
ampicillin, amoxycillin+clavunic acid, co-trimoxazole and tetracycline group, whereas
Pseudomonas aeruginosa was resistant for 1st generation of cephalosporin and 2nd generation of
cephalosporin , co trimoxazole and tetracycline group.
Klebsiella spp. was more resistant to penicillin group, 1st generation of cephalosporin,
amoxycillin+clavunic and co-trimoxazole, while in case of Salmonella typhi only one drug was
resistant i.e. ampicillin.
We have observed that for Proteus spp. more resistant drugs were ampicillin, co-trimoxazole
and tetracycline group. Acinetobacter isolates were more resistant for ampicillin, tetracycline
group, amoxycillin+clavunic acid, aztreonam and Cefazolin.
From gram positive pathogens Staphylococcus aureus was highest resistant for penicillin G,
ampicillin, ciprofloxacin and Azithromycin, while Streptococci spp. was resistant to only one
antibiotic i.e. ciprofloxacin.
 Methicillin resistant Staphylococcus aureus shows more resistant to cephalosporin,
tetracycline, Azithromycin group and ciprofloxacin.

38. In case of Group D Enterococci ciprofloxacin, Ofloxacin, Lomefloxacin, clindamycin and
Azithromycin group were highest resistant.
In our study we found ESBL positive isolates were 32.% when tested with CE/CEC and
CA/CAC combination. We have observed low rate of ESBL production by Enteropathogens.
We also concluded that among Staphylococcus aureus majority of them were methicillin sensitive
i.e. 97.6%, only 2.5% of them were methicillin resistant.
In our study we found fungal pathogen from various clinical samples and majority was
Candida spp.
The mould infections were found only in case of sputum samples (13.04% ) and pus (3.33%)
where as the infection with yeast was found to be nearly 100% in all other samples like urine,
body fluid, stool, blood and throat swabs.
The Mycobacterium infection was found to be highest in case of sputum samples suggesting the
prevalence of pulmonary tuberculosis.

39. REFERENCES