12 chapter 4 - shodhgangashodhganga.inflibnet.ac.in/bitstream/10603/78949/12/12_chapter 4.p… ·...

“Characterization of uropathogenic strains of Escherichia coli”

Observations and Results

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CHAPTER 4CHAPTER 4CHAPTER 4CHAPTER 4

OBSERVATIONS AND RESOBSERVATIONS AND RESOBSERVATIONS AND RESOBSERVATIONS AND RESUUUULTSLTSLTSLTS



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4.1 Clinical Data:4.1 Clinical Data:4.1 Clinical Data:4.1 Clinical Data:

All the clinical samples were collected from five hospitals from

different areas in Thane district during all the seasons in one year.

Total numbers of urine samples collected were 305 from patients

showing signs and symptoms of urinary tract infection (Table 11).

Out of which 250 (81.96 %) were from female patients. From these

investigations it was observed that in urinary tract infections, the

number of patients was higher in females compared to males (81.96

% vs 18.04 %).

Similarly, 66 stool samples were collected from diarrheal cases.

Higher incidence of diarrhea was recorded in males than females

(51.52 % vs 48.48 %)

TABLE TABLE TABLE TABLE ---- 11: Data of collected samples 11: Data of collected samples 11: Data of collected samples 11: Data of collected samples ––––

Sr. Sr. Sr. Sr.

No.No.No.No.

SourceSourceSourceSource Total no. of Total no. of Total no. of Total no. of

samples collectedsamples collectedsamples collectedsamples collected

Total No. isolated &Total No. isolated &Total No. isolated &Total No. isolated &

ididididentified as entified as entified as entified as E. coliE. coliE. coliE. coli

1. Urinary

infection

305 289

2. Diarrheal

infection

66 50

As far as seasonal effect is concerned, in urinary tract infection,

higher incidence was recorded during the rainy season i.e. months of

July to October, with a peak in July and August (high humidity period)

dropping till the winter month of December (Figure 9).

Also, female cases were observed to be maximum during this period.

The diarrheal incidence is higher during the rainy months of the year

(July - October), may be due to polluted water supply and drops in



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the ensuing months gradually but has a sudden peak of diarrhea in

months of April – May (Figure 10).

The higher prevalence of the disease in this season was recorded in

males than females here.

FIGURE FIGURE FIGURE FIGURE ---- 9: Incidenc9: Incidenc9: Incidenc9: Incidenceof eof eof eof E. coliE. coliE. coliE. coli in UTI cases during various seasons.in UTI cases during various seasons.in UTI cases during various seasons.in UTI cases during various seasons.



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FIGURE FIGURE FIGURE FIGURE ----10: Incidence of 10: Incidence of 10: Incidence of 10: Incidence of E. coliE. coliE. coliE. coli in diarrhea cases during various in diarrhea cases during various in diarrhea cases during various in diarrhea cases during various

seasons.seasons.seasons.seasons.

The urine and stool samples from patients of both sexes were

microbiologically tested for isolation of E. coli which was further

identified biochemically and serologically.



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4.2 Results4.2 Results4.2 Results4.2 Results:

4.2.1 Laboratory isolation and identification of 4.2.1 Laboratory isolation and identification of 4.2.1 Laboratory isolation and identification of 4.2.1 Laboratory isolation and identification of E. coliE. coliE. coliE. coli from from from from

urinary and diarrhea cases:urinary and diarrhea cases:urinary and diarrhea cases:urinary and diarrhea cases:

From the 305 UTI cases tested 289 were isolated as E.coli, out of

which 237 (82.1%) were from female patients and 52 (17.9%) were

males (Table 12i & Figure 11).

Out of 66 diarrhea cases, 50 were isolated as E.coli, out of which

27 (54%) were from male patients and 23 (46%) were from females

(Table 12ii & Figure 12).

TABLE TABLE TABLE TABLE ----12: Percentage of positive cases gender wise and season 12: Percentage of positive cases gender wise and season 12: Percentage of positive cases gender wise and season 12: Percentage of positive cases gender wise and season

wise.wise.wise.wise.

i) i) i) i) E. coliE. coliE. coliE. coli isolated from cases of Urinary Tract Infection isolated from cases of Urinary Tract Infection isolated from cases of Urinary Tract Infection isolated from cases of Urinary Tract Infection --------

Sr. Sr. Sr. Sr.

No.No.No.No.

SEASONSEASONSEASONSEASON GENDERGENDERGENDERGENDER

Male FemaleMale FemaleMale FemaleMale Female

NO. OF NO. OF NO. OF NO. OF

ISOLATESISOLATESISOLATESISOLATES

PERCENTPERCENTPERCENTPERCENT

1. Winter

(December to

March)

14 42 56 19.4

2. Summer

(April to June)

10 29 39 13.5

3. Rainy

(July to

October)

28 166 194 67.1

4. TOTAL 52 237

289

5. PERCENT 17.9 % 82.1%

100



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FIGUREFIGUREFIGUREFIGURE----11: Incidence of 11: Incidence of 11: Incidence of 11: Incidence of E. coliE. coliE. coliE. coli in UTI cain UTI cain UTI cain UTI cases gender wise and season ses gender wise and season ses gender wise and season ses gender wise and season




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TABLE TABLE TABLE TABLE ----12: Percentage of positive cases gender wise and season 12: Percentage of positive cases gender wise and season 12: Percentage of positive cases gender wise and season 12: Percentage of positive cases gender wise and season


ii) ii) ii) ii) E. coliE. coliE. coliE. coli isolated from diarrhea cases isolated from diarrhea cases isolated from diarrhea cases isolated from diarrhea cases ----

Sr. Sr. Sr. Sr.

No.No.No.No.

SEASONSEASONSEASONSEASON GENDERGENDERGENDERGENDER

Male FemaleMale FemaleMale FemaleMale Female

NO. OF NO. OF NO. OF NO. OF

ISOLATESISOLATESISOLATESISOLATES

PERCENTPERCENTPERCENTPERCENT

1. Winter

(December to

March)

05 05 10 20

2. Summer

(April to June)

10 08 18 36

3. Rainy

(July to

October)

12 10 22 44

4. TOTAL

27 23 50

5. PERCENT

54 % 46% 100



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FIGUREFIGUREFIGUREFIGURE----12: Incidence of 12: Incidence of 12: Incidence of 12: Incidence of E. coliE. coliE. coliE. coli in diarrhea cases gender wise and in diarrhea cases gender wise and in diarrhea cases gender wise and in diarrhea cases gender wise and

season wiseseason wiseseason wiseseason wise.



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4.2.2 Colony count:4.2.2 Colony count:4.2.2 Colony count:4.2.2 Colony count:

To obtain a urine culture, urine sediment was placed on MacConkeys

and CLED agar dishes, incubated and colony-forming units (cfu)

counted.

In the past, a quantitative urine culture yielding greater than

100,000 colony-forming units (cfu) of bacteria per millimeter of

urine was termed significant bacteriuria.

Even in asymptomatic persons, this value has a high specificity for

true infection. For that reason, it was chosen as the standard.

However, many studies have established that one third of

asymptomatic women have cfu counts below this level. Additionally,

bacterial counts of 100 cfu per mL of urine in symptomatic women

have a high positive predictive value for cystitis.

Some laboratories only report counts greater than 10,000 cfu per

mL, resulting in the under diagnosis of low-coliform-count infections

(1).

Of the 289 urine samples in which E.coli was isolated, 270 had

significant bacteriuria with counts 105 colonies/mL and 19 had

probably significant bacteriuria with counts between 104-105

colonies/mL.



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4.2.34.2.34.2.34.2.3 Cultural and Biochemical Characteristics of Cultural and Biochemical Characteristics of Cultural and Biochemical Characteristics of Cultural and Biochemical Characteristics of E.E.E.E.colicolicolicoli isolated from isolated from isolated from isolated from

Urine and Faeces specimens:Urine and Faeces specimens:Urine and Faeces specimens:Urine and Faeces specimens:

All Escherichia coli related to this study were identified on the basis

of their cultural and biochemical characteristics prior to proceeding

for serotyping.

In general, most isolates, regardless of host source, were able to

ferment lactose. However, 5 of UPEC and 11 from EPEC did not give

a positive reaction on MacConkey agar for lactose fermentation,

while 5 isolates from diarrhael specimens were found to be late

lactose fermenters.

All cultures identified as E.coli have shown lactose fermenting (pink)

colonies on MacConkey’s agar and a greenish metallic sheen on

EMBA. (Table -13)



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TABLE TABLE TABLE TABLE ---- 13: Characteristics observed on selective media. 13: Characteristics observed on selective media. 13: Characteristics observed on selective media. 13: Characteristics observed on selective media.

Sr. Sr. Sr. Sr.

No.No.No.No.

Inoculums Inoculums Inoculums Inoculums

usedusedusedused

Selective Selective Selective Selective

mediamediamediamedia

Colony Colony Colony Colony

characteristicscharacteristicscharacteristicscharacteristics

No.No.No.No. of of of of

isolatesisolatesisolatesisolates

No. No. No. No.

identified identified identified identified

as as as as E.E.E.E.colicolicolicoli

1 Urine MacConkey

agar

Total tested

- 305

Lactose

fermenters

300 289

Non-lactose

fermenters

05

E.M.B. agar

Total tested

- 305

Greenish metallic

Sheen

289 289(96.3%)

Without metallic

sheen

16

2 Stool MacConkey

agar

Total tested

- 66

Lactose

fermenters

50 50

Non-lactose

fermenters

11

Late lactose

fermenters

05

E.M.B. agar

Total tested

- 66

Greenish metallic

Sheen

50 50(75.7%)

Without black

sheen

16

Cultures fermenting lactose and showing metallic sheen on EMBA

were selected for further studies.

On Gram staining they were found to be Gram negative motile rods.

Acidic butt and slant, with H2S production was observed on TSI agar.

Urea and oxidase was found negative. Indole production and Methyl

red test positive while V-P and Citrate utilization was negative.

Glucose, lactose, mannitol and xylose were fermented and sucrose

was found to be variable (Table - 14).



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TABLE TABLE TABLE TABLE ---- 14: Cultural and Biochemical Characteristics of 14: Cultural and Biochemical Characteristics of 14: Cultural and Biochemical Characteristics of 14: Cultural and Biochemical Characteristics of Escherichia Escherichia Escherichia Escherichia

colicolicolicoli observed observed observed observed during the present study.during the present study.during the present study.during the present study.

Sr. Sr. Sr. Sr.

NoNoNoNo

CharacteristicsCharacteristicsCharacteristicsCharacteristics ObservationsObservationsObservationsObservations

1. Morphology Gram negative, rods.

2. Motility Motile

3. Cultural

Characteristics

Lactose fermenting(pink color) colonies on

MacConkey’s agar

Greenish, metallic sheen on EMBA.

Deep yellowish, opaque colonies on CLED.

4. Reaction on

TSI

Slant - Acidic(yellow)

Butt - Acidic(yellow)

Gas - +

H2S - ---

5. Urea ---

6. Oxidase ---

7. IMViC Indole production ---

Methyl red ---

Voges-Proskaur ---

Citrate utilization ---

8. Carbohydrate

fermentation

Glucose (+)

Lactose (+)

Mannitol (+)

Xylose (+)

Sucrose V

Key:

+ = positive

(+) = Positive with gas.

--- = negative.

V = Variable.



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4.3 Results of phenotypic tests:4.3 Results of phenotypic tests:4.3 Results of phenotypic tests:4.3 Results of phenotypic tests:

4.3.1 Hemolysis test:4.3.1 Hemolysis test:4.3.1 Hemolysis test:4.3.1 Hemolysis test:

Organisms from both sources were assessed for their hemolytic

reaction on 5% sheep blood agar plates using standard methods (2)

E.coli is the most common opportunistic pathogen associated with

extraintestinal infections and several studies have shown that β -

hemolytic (Hly) production is a frequent and important virulence

factor of pathogenic strains.

Hemolytic E.coli strains may be virulent because of a toxic effect on

various cells and in addition this effect may be strengthened because

the growth of the bacterium is stimulated by iron made available by

lysis of the erythrocytes (3).

A total of 289 E.coli strains isolated from urinary infection and 50

faecal samples from diarrhea cases were examined for production of

β-hemolysin (Hly) (Table 15).

Among strains causing urinary tract infections, hemolysin production

was detected in 241(90%) and 40(80%) of faecal isolates from

diarrhael illness synthesized hemolysin on blood agar suggesting that

these organisms produce β -hemolysin.

In both cases, hemolysis was shown by more than 70% of isolates

collected from females. As usual this indicated higher incidence of

the urinary tract disease in females than males.



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One strain from stool sample while nine of urinary strains showed

partial hemolysis on blood agar. 18 cultures from UTI and 9 from

stools were non-haemolytic (Figure -13).

TABLE TABLE TABLE TABLE ---- 15: Hemolysis pattern in both clinical groups.15: Hemolysis pattern in both clinical groups.15: Hemolysis pattern in both clinical groups.15: Hemolysis pattern in both clinical groups.

Total no. of isolates tested - UTI = 268 & Diarrhea = 50.

Sr. Sr. Sr. Sr.

No.No.No.No.

SourceSourceSourceSource No. ofNo. ofNo. ofNo. of isolatesisolatesisolatesisolates

With clear With clear With clear With clear

zone ofzone ofzone ofzone of

hemolysis.hemolysis.hemolysis.hemolysis.

No. of No. of No. of No. of

isolates isolates isolates isolates

With partial With partial With partial With partial

hemolysis.hemolysis.hemolysis.hemolysis.

No. of isolates No. of isolates No. of isolates No. of isolates

showing no zone showing no zone showing no zone showing no zone

of hemolysis.of hemolysis.of hemolysis.of hemolysis.

1. UTI 241 (90 %)

09 18

2. Diarrhea 40 (80%)

01 09



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FIGURE FIGURE FIGURE FIGURE ----13: Distribution of types of haemolysis in UTI and di13: Distribution of types of haemolysis in UTI and di13: Distribution of types of haemolysis in UTI and di13: Distribution of types of haemolysis in UTI and diarrhea arrhea arrhea arrhea

cases.cases.cases.cases.

Strains designated as hemolytic were clearly positive on blood agar

plates and, in practically all cases (>90%), strongly positive from

both clinical cases.

4.3.2 Serological test:4.3.2 Serological test:4.3.2 Serological test:4.3.2 Serological test:

Serotyping was carried out at the National Salmonella and

Escherichia Center, Central Research Institute, Kasauli, H.P. based

on 'O' antigen of E.coli.



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Serogroups of E.coli cultures were identified on the basis of somatic

“O” antigen with the help of group-specific antisera, using slide

agglutination technique.

Blanco et al. ( 1990) (4) have described that E.coli isolated from

sepsis belong to a limited number of serogroups, mainly O2, O4, O6,

O7, O8 and O75 and show virulence factors similar to those shown

by strains responsible for urinary tract infections (UTI).

According to WHO proceedings in 1987, the following E.coli O

serogroups should be considered EPEC O serogroups: O26, O55,

O86, O111, O114, O119, O125, O126, O127, O128, O142 and O158.

Many of the UPEC and EPEC isolates used in this study could be

classified using standard antisera. Overall, about 90% of both groups

were typable to serogroups recognized previously as UPEC and

EPEC.

Some isolates in both groups were classified as non-typable, may be

as they were rough strains, showing multiple serogroups, or

autoagglutinated, preventing their assignment to a serogroup.

A total of 268(93.7%) were found to be typable in the Urinary group

and 46(92%) from the diarrheal group. Those that could be classified

into a single serogroup, a high degree of diversity were seen (Table

-16). Both UPEC and EPEC contained isolates identified as O2, O4,

O6, O7, O8, O11, O18, O25, O26, O27, O36, O75, O78, O81, O101,

O112, O117, O125, O126 and O158.



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Among the 268 typable UPEC, 15 different serogroups were found,

with 11 of these unique to UPEC. Serogroups containing the typable

isolates for UPEC were O101> O75> O2> O6>O7> O18> O4> O8>

O27> O81> O112 and for EPEC, they were O25> O78> O158>

O44>O26>O125 (Figure 14 & 15).

Other serogroups found common in both groups were O11, O36,

O117 and O126.

The O101 and O75 serogroup was among the most commonly

occurring serogroups in UPEC, followed by O2 and O6 serogroup.

In the EPEC group, O25 was the serogroup most commonly found

followed by O78.

TABLE TABLE TABLE TABLE –––– 16: Data of Serot16: Data of Serot16: Data of Serot16: Data of Serotyping.yping.yping.yping.

Sr. Sr. Sr. Sr.

NoNoNoNo

HeadsHeadsHeadsHeads SourceSourceSourceSource

UTI UTI UTI UTI DiarrheaDiarrheaDiarrheaDiarrhea

1. Total no. of isolates send for

serotyping

289 50

2. Total no. of strains serotyped 268(92.7 %) 46 (9 %)

3. No. of untypable strains 21 (7.3 %) 04 (8 %)

OUT OF SEROYTPED SOUT OF SEROYTPED SOUT OF SEROYTPED SOUT OF SEROYTPED STRAINSTRAINSTRAINSTRAINS -- 268 46

1. No. of strains with known

pathogenic serogroups

242 (90 %) 42 (91.3 %)

2. No. of strains with common

serogroups

26 (9.7 %) 04 (8.7 %)



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Hemolytic isolates from UTI were most common among serotypes

O75(36/36 Hly+),O4(08/08), O8(08/08), O101(70/79), O2 (30/34),

018(14/16) and 07(19/21), whereas strains of the EPEC serogroups

O25(15/15), O78(12/12), O158(03/06), O44(02/05) and O26 and O125

each 2 strains were hemolytic (Table -17).

FIGURE FIGURE FIGURE FIGURE –––– 14: Distribution of various serotypes in UTI 14: Distribution of various serotypes in UTI 14: Distribution of various serotypes in UTI 14: Distribution of various serotypes in UTI cases.cases.cases.cases.



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FIGURE FIGURE FIGURE FIGURE ----15: Distribution of various serotypes in diarrhea cases.15: Distribution of various serotypes in diarrhea cases.15: Distribution of various serotypes in diarrhea cases.15: Distribution of various serotypes in diarrhea cases.



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TABLE 17TABLE 17TABLE 17TABLE 17: Distribution of : Distribution of : Distribution of : Distribution of E coliE coliE coliE coli serogroups.serogroups.serogroups.serogroups.

Source Source Source Source -------- Urinary Tract Infection (UPEC)Urinary Tract Infection (UPEC)Urinary Tract Infection (UPEC)Urinary Tract Infection (UPEC)

Total No. of isolatesTotal No. of isolatesTotal No. of isolatesTotal No. of isolates---- 289. 289. 289. 289. No. of serotypable No. of serotypable No. of serotypable No. of serotypable strains strains strains strains -------- 268.268.268.268.

Sr. NoSr. NoSr. NoSr. No SerogroupsSerogroupsSerogroupsSerogroups Total No.Total No.Total No.Total No.

PercentPercentPercentPercent No. of No. of No. of No. of

haemolytichaemolytichaemolytichaemolytic

StrainsStrainsStrainsStrains

No. selected No. selected No. selected No. selected

for further for further for further for further

investigationinvestigationinvestigationinvestigation

1111 O101O101O101O101 79797979 32.6432.6432.6432.64 70707070 60606060

2222 O75O75O75O75 36363636 14.8814.8814.8814.88 36363636 35353535

3333 O2O2O2O2 34343434 14.0514.0514.0514.05 30303030 30303030

4444 O6O6O6O6 29292929 11.9811.9811.9811.98 23232323 20202020

5555 O7O7O7O7 21212121 8.688.688.688.68 19191919 15151515

6666 O18O18O18O18 16161616 6.616.616.616.61 14141414 10101010

7777 O4O4O4O4 08080808 3.313.313.313.31 08080808 05050505

8888 O8O8O8O8 08080808 3.313.313.313.31 08080808 05050505

9999 O27O27O27O27 05050505 2.062.062.062.06 01010101 NilNilNilNil



TotalTotalTotalTotal 242242242242 90.390.390.390.3 211211211211 180180180180

Source Source Source Source -------- Diarrhea (EPEC)Diarrhea (EPEC)Diarrhea (EPEC)Diarrhea (EPEC)

Total No. isolates Total No. isolates Total No. isolates Total No. isolates –––– 50. No. 50. No. 50. No. 50. No. of serotypable strainsof serotypable strainsof serotypable strainsof serotypable strains -------- 46.46.46.46.

1 1 1 1 O25O25O25O25 15151515 35.7135.7135.7135.71 15151515 15151515

2222 O78O78O78O78 12121212 28.5828.5828.5828.58 12121212 11111111

3333 O158O158O158O158 06060606 14.5914.5914.5914.59 03030303 03030303

4444 O44O44O44O44 05050505 11.8011.8011.8011.80 02020202 02020202

5555 O26O26O26O26 02020202 4.664.664.664.66 02020202 02020202

6666 O125O125O125O125 02020202 4.664.664.664.66 02020202 02020202

Total Total Total Total 42424242 91.391.391.391.3 36363636 35353535

Source Source Source Source --------Urinary Tract Infection and Diarrheal (Common serogroups)Urinary Tract Infection and Diarrheal (Common serogroups)Urinary Tract Infection and Diarrheal (Common serogroups)Urinary Tract Infection and Diarrheal (Common serogroups)

No. of serotypable straiNo. of serotypable straiNo. of serotypable straiNo. of serotypable strains ns ns ns -------- UTI = 26 + Diarrheal = 04.UTI = 26 + Diarrheal = 04.UTI = 26 + Diarrheal = 04.UTI = 26 + Diarrheal = 04.

UTIUTIUTIUTI DIARDIARDIARDIAR UTIUTIUTIUTI DIARDIARDIARDIAR

1111 O11O11O11O11 10101010 01010101 40.0040.0040.0040.00 05050505 01010101 NilNilNilNil




TotalTotalTotalTotal 26262626 04040404 18181818 04040404

UntypableUntypableUntypableUntypable 21212121 04040404 12121212 NilNilNilNil NilNilNilNil



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4.3.34.3.34.3.34.3.3 Congo red binding test:Congo red binding test:Congo red binding test:Congo red binding test:

The ability to bind the dye Congo red is also recognized as a

phenotype that differentiates between virulent and avirulent colony

types of many bacteria, including Yersinia pestis, Aeromonas

salmoncida, Neisseria meningitides and Vibrio cholerae (5, 6). For

Shigella species, smooth colonies that bind Congo red are virulent,

while smooth colonies that do not bind the dye are avirulent (6, 7).

Although rough colony types of Shigella are avirulent, they are able

to bind Congo red because loss of the hydrophilic O polysaccharide

from the LPS during the smooth to rough transition makes the cell

surface more hydrophobic and increases binding of the dye

molecules to the lipid A core of the LPS (7).CR has been routinely

used to distinguish virulent from avirulent invasive organisms like

Shigella, (7, 8) Yersinia and Aeromonas (9).

In the present study, all the isolates showing hemolysis on blood

agar plates were inoculated on Congo red medium. Out of the 241

urinary strains, 166 (68.8 %) showed red colored colonies in 18 to 24

hours incubation, while 15 ( 6.2%) strains were showing red colonies

after 36 hours and 60 (24 %) did not bind the dye even after 48

hours and were therefore declared negative. It was observed the

99% strains were of the serotypeable groups.

While, from the diarrheal group, out of the 40 hemolytic isolates

inoculated on Congo red agar, 38 (95 %) isolates developed into dark

red colonies on incubation for 18 - 24 hours and among these all the

serotypable strains showed perfect red colonies.



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Hemolytic strains i.e. showing clear zone of haemolysis on Blood

agar, producing perfect red colored colonies on Congo red agar and

which were of known recognized serogroups were selected for

further investigation.

4.3.4 Hemagglutination in presence of D4.3.4 Hemagglutination in presence of D4.3.4 Hemagglutination in presence of D4.3.4 Hemagglutination in presence of D----mannose (MRHA) :mannose (MRHA) :mannose (MRHA) :mannose (MRHA) :

P-fimbriae are rod-like rigid heteropolymeric appendages that

protrude from the cell-surface of E.coli bacteria. The P-fimbriae

are encoded by the pap chromosomal gene cluster. P-fimbriae

mediate the mannose-Resistant hemagglutination (MRHA) of

erythrocytes from individuals with common blood group P1, but fail

to agglutinate erythrocytes of the more uncommon blood group P

(10, 11).

Type 1-fimbriae are widely spread among different E.coli strains

causing urinary tract infections (12, 13). Addition of D-mannose

inhibits this hemagglutination. The Type 1-Fimbriae are encoded by

the chromosomal fim gene cluster.

A total of 149 (82.7 %) among 180 urinary cases and 25 (71.5 %)

among 35 diarrhael showed mannose resistant haemagglutination, i.e.

agglutinated within 2mins, There was no significant difference in

MRHA between UTI and diarrheal cases (Table -18).

Of the 180 urinary isolates, 24(13.3%) exhibited weak (within 5 mins)

hemagglutination and 13(7.5%) did not hemagglutinate (Figure -16).

But, from diarrheal cases, isolates that did not show

haemagglutination were more (7 i.e. 20 %) than the weakly

agglutinating ones (03 i.e. 8.5%) (Figure -17).



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TABLE TABLE TABLE TABLE ---- 18: Mannose Resistance Hema18: Mannose Resistance Hema18: Mannose Resistance Hema18: Mannose Resistance Hemagglutination (MRHA) and gglutination (MRHA) and gglutination (MRHA) and gglutination (MRHA) and

haemolysis production by selected serogroups of E. coli from UTI and haemolysis production by selected serogroups of E. coli from UTI and haemolysis production by selected serogroups of E. coli from UTI and haemolysis production by selected serogroups of E. coli from UTI and

Diarrheal cases.Diarrheal cases.Diarrheal cases.Diarrheal cases.

Sr. Sr. Sr. Sr.

No.No.No.No.

SourceSourceSourceSource SerotypeSerotypeSerotypeSerotype No. of No. of No. of No. of

strains strains strains strains

showing showing showing showing

HemolysisHemolysisHemolysisHemolysis

No. selected No. selected No. selected No. selected

for further for further for further for further


MRHAaMRHAaMRHAaMRHAa

No. of strainsNo. of strainsNo. of strainsNo. of strains showing showing showing showing

agglutination withinagglutination withinagglutination withinagglutination within

2 mins 2 mins 2 mins 2 mins

(+)(+)(+)(+)

5 mins5 mins5 mins5 mins

(weak)(weak)(weak)(weak)

No agglNo agglNo agglNo aggl

((((----))))

1.1.1.1. UTIUTIUTIUTI O101O101O101O101 70707070 60606060 50505050 07070707 03030303

2.2.2.2. UTIUTIUTIUTI O75O75O75O75 36363636 35353535 26262626 08080808 O1O1O1O1

3.3.3.3. UTIUTIUTIUTI O2O2O2O2 30303030 30303030 30303030 ---------------- ----------------


5.5.5.5. UTIUTIUTIUTI O7O7O7O7 19191919 15151515 14141414 ---------------- 01010101


7.7.7.7. UTIUTIUTIUTI O4O4O4O4 08080808 05050505 04040404 01010101 ----------------

8.8.8.8. UTIUTIUTIUTI O8O8O8O8 08080808 O5O5O5O5 03030303 02020202 ----------------

TotalTotalTotalTotal 208208208208 180180180180 149149149149 24242424 13131313

PercentPercentPercentPercent 82.7%82.7%82.7%82.7% 13.3 %13.3 %13.3 %13.3 % 7.2 %7.2 %7.2 %7.2 %

1.1.1.1. DiarrheaDiarrheaDiarrheaDiarrhea O25O25O25O25 15151515 15151515 10101010 ---------------- 05050505

2. 2. 2. 2. DiarrheaDiarrheaDiarrheaDiarrhea O78O78O78O78 12121212 11111111 10101010 01010101 ----------------

3.3.3.3. DiarrheaDiarrheaDiarrheaDiarrhea O158O158O158O158 03030303 03030303 02020202 01010101 ----------------



6.6.6.6. DiarrheaDiarrheaDiarrheaDiarrhea O125O125O125O125 02020202 02020202 01010101 01010101 ----------------

TotalTotalTotalTotal 36363636 35353535 25252525 03030303 07070707

PercentPercentPercentPercent 71.5%71.5%71.5%71.5% 8.5 %8.5 %8.5 %8.5 % 20 %20 %20 %20 %

a Haemagglutination resistance to 0.5%(w/v) of D-Mannose (MRHA)

is noted.



- 147 -

FIGURE FIGURE FIGURE FIGURE ----11116666: Prevalence of isolates in serogroup: Prevalence of isolates in serogroup: Prevalence of isolates in serogroup: Prevalence of isolates in serogroups showing MRHA s showing MRHA s showing MRHA s showing MRHA

from UTI cases.from UTI cases.from UTI cases.from UTI cases.



- 148 -

FIGURE FIGURE FIGURE FIGURE ––––17: Prevalence of isolates in serogroups showing MRHA 17: Prevalence of isolates in serogroups showing MRHA 17: Prevalence of isolates in serogroups showing MRHA 17: Prevalence of isolates in serogroups showing MRHA

from diarrhea cases.from diarrhea cases.from diarrhea cases.from diarrhea cases.



- 149 -

4.3.5 Salt Aggregation Test 4.3.5 Salt Aggregation Test 4.3.5 Salt Aggregation Test 4.3.5 Salt Aggregation Test ---- (SAT): Hydrophobicity assay(SAT): Hydrophobicity assay(SAT): Hydrophobicity assay(SAT): Hydrophobicity assay

A total of 180 strains of E.coli isolated from urinary tract infections

and 36 from diarrhea were compared for relative surface

hydrophobicity in the salt aggregation test (14). The SAT is based

on precipitation of cells by salts, that is, the higher the

hydrophobicity of the proteins, the lower the salt concentration

required aggregating the cells.

It has been shown that bacterial aggregation caused by low salt

concentrations is an index of high surface hydrophobicity (15).

The SAT revealed that 116 (64.4%) UPEC strains aggregated at low

salt concentrations (i.e. 0.5 M to 1.4 M ammonium sulfate). In case of

EPEC, 15 strains (42.8 %), out of 35 tested showed aggregation at

lower concentration of ammonium sulfate (Table – 19).

Bacteria were defined as extremely hydrophobic when showing

autoaggregation in saline or in 0.002 M sodium phosphate buffer (pH

6.6).

Using this definition, it was found that a large number of strains

isolated from diarrhea to be very hydrophobic. The auto aggregating

strains in diarrheal cases were 15(42.8%), out of 35.

While, out off 180 E. coli strains from patients with Urinary tract

infections, 49 strains (27.3%) showed cell clumping in physiological

saline (pH 6.8); and were defined auto aggregating strains.



- 150 -

Therefore, the incidence of autoaggregating strains among strains

isolated from diarrhea cases were more than from urine [ 42.8%(15

out of 35) ] while in case of urinary cases it was only 27.3% ( 49 out

of 180 ).

Therefore, the results indicate high hydrophobicity of UPEC strains

and according to some authors (16); this suggests adhesion capacity

of this bacterium.

Cell surface hydrophobicity as measured by SAT was a stable

property.

Frequency of SAT and autoaggregating in different serogroups from

urinary and diarrheal isolates is shown in Table - 19, Figure -18 &

19.



- 151 -

TABLE TABLE TABLE TABLE –––– 19191919: Frequency of SAT and autoaggregating Frequency of SAT and autoaggregating Frequency of SAT and autoaggregating Frequency of SAT and autoaggregating ----

Sr. Sr. Sr. Sr.

NoNoNoNo

SourceSourceSourceSource

SerotypeSerotypeSerotypeSerotype

Total Total Total Total


strains strains strains strains

testedtestedtestedtested

Salt Aggregation Salt Aggregation Salt Aggregation Salt Aggregation

[ M (NH4)[ M (NH4)[ M (NH4)[ M (NH4)2222SOSOSOSO4444]]]]

No. of strains showing No. of strains showing No. of strains showing No. of strains showing

clumping inclumping inclumping inclumping in

AutoagglutinationAutoagglutinationAutoagglutinationAutoagglutination

In saline(pH 6.8)In saline(pH 6.8)In saline(pH 6.8)In saline(pH 6.8)

0.5 M to 1.4 M 0.5 M to 1.4 M 0.5 M to 1.4 M 0.5 M to 1.4 M 1.6 to 2.4 M1.6 to 2.4 M1.6 to 2.4 M1.6 to 2.4 M

UTIUTIUTIUTI

1.1.1.1. O101O101O101O101 60606060 50505050 02020202 08080808

2.2.2.2. O75O75O75O75 35353535 15151515 05050505 15151515

3.3.3.3. O2O2O2O2 30303030 12121212 04040404 14141414

4.4.4.4. O6O6O6O6 20202020 16161616 ------------ 04040404

5.5.5.5. O7O7O7O7 15151515 09090909 03030303 03030303

6.6.6.6. O18O18O18O18 10101010 05050505 01010101 04040404

7.7.7.7. O4O4O4O4 05050505 04040404 ------------ 01010101

8.8.8.8. O8O8O8O8 O5O5O5O5 05050505 ------------ ------------

180180180180 116(64.4%)116(64.4%)116(64.4%)116(64.4%) 15(8.3%)15(8.3%)15(8.3%)15(8.3%) 49(27.3%)49(27.3%)49(27.3%)49(27.3%)

DiarrheaDiarrheaDiarrheaDiarrhea

1.1.1.1. O25O25O25O25 15151515 06060606 02020202 07070707

2.2.2.2. O78O78O78O78 11111111 06060606 02020202 03030303

3.3.3.3. O158O158O158O158 03030303 02020202 ------------ 01010101

4.4.4.4. O44O44O44O44 02020202 ------------ ------------ 02020202

5.5.5.5. O26O26O26O26 02020202 ------------ 01010101 01010101

6.6.6.6. O125O125O125O125 02020202 01010101 ------------ 01010101

35353535 15(42.8 %)15(42.8 %)15(42.8 %)15(42.8 %) 5(14.4%)5(14.4%)5(14.4%)5(14.4%) 15(42.8%)15(42.8%)15(42.8%)15(42.8%)



- 152 -

FIGURE FIGURE FIGURE FIGURE ––––18: Prevalence of isolates in serogroups showing salt 18: Prevalence of isolates in serogroups showing salt 18: Prevalence of isolates in serogroups showing salt 18: Prevalence of isolates in serogroups showing salt

aggregation in UTI cases.aggregation in UTI cases.aggregation in UTI cases.aggregation in UTI cases.



- 153 -

FIGURE FIGURE FIGURE FIGURE ––––19: Prevalence of isolates in serogroups showing salt 19: Prevalence of isolates in serogroups showing salt 19: Prevalence of isolates in serogroups showing salt 19: Prevalence of isolates in serogroups showing salt

aggregation in diarrhea cases.aggregation in diarrhea cases.aggregation in diarrhea cases.aggregation in diarrhea cases.



- 154 -

4.3.6 Serum Bactericidal test (SBT):4.3.6 Serum Bactericidal test (SBT):4.3.6 Serum Bactericidal test (SBT):4.3.6 Serum Bactericidal test (SBT):

The susceptibility of microorganisms to antimicrobial agents is most

often estimated in the laboratory by measuring the inhibitory activity

of the agent. Tests which measure only inhibition of growth may not

always provide sufficient information to guide the therapy of certain

infections

Moreover, certain antimicrobial agents such as beta-lactam agents,

who are considered to be "bactericidal," can no longer be assumed to

kill all isolates (17 - 20). There is the need, therefore, for additional

laboratory methods that can assess the bactericidal activity of an

antimicrobial agent.

In urinary tract infection, resistance to serum is related to the site of

infection (21) and to the severity of symptoms (22). Investigations

into the basis of serum resistance have until recently focused upon

chromosomally encoded cell surface antigens.

Serum bactericidal test was performed according to Siegfried et al

(1994) (22).

To evaluate the function of serum complement in killing bacteria, the

complement proteins were inactivated by heat treatment at 56oC for

30 mins in a water bath and 20 μl aliquots was used to test the

bactericidal activity.

Total No of isolates tested - UPEC = 180 & EPEC = 35.

In case of UTI isolates, it was found that, E.coli colonies grown on

agar plates at 0 - hour incubation, varied from 36 - 56 x 1010

cfu/mL.



- 155 -

After treating the bacterial suspension with normal serum and

incubating, the number of colonies varied from 3 -7.5 x 1010 cfu/mL,

after 3 hours incubation.

From diarrheal isolates, the number of colonies, at 0-hour incubation

was 45 - 68 x 1010 cfu/mL.

The number of colonies after treatment with serum from diarrheal

isolates, varied from 0.13 to 45 x 1010cfu/mL.

The urinary isolates had lower bactericidal activity in normal serum

than that of diarrhael strains.

The bactericidal activity of inactivated serum from the urinary

isolates varied from 26.8 - 47.4 %. Where as from the diarrheal

isolates varied from 15.6 - 23.8%. The bactericidal activity of

inactivated serum from the urinary isolates was found to be higher

(Table-20).

These experiments show that both types exhibited highly significant

bactericidal activities.



- 156 -

TABLE TABLE TABLE TABLE ---- 20: Growth response20: Growth response20: Growth response20: Growth response1111 of of of of E. coliE. coliE. coliE. coli isolated from UTI and isolated from UTI and isolated from UTI and isolated from UTI and

Diarrheal cases, after incubation, in SBT.Diarrheal cases, after incubation, in SBT.Diarrheal cases, after incubation, in SBT.Diarrheal cases, after incubation, in SBT.

Sr. Sr. Sr. Sr.

No.No.No.No.

AssayAssayAssayAssay After 0 After 0 After 0 After 0 ---- hr hr hr hr

incubation in serumincubation in serumincubation in serumincubation in serum

UPEC UPEC UPEC UPEC EPECEPECEPECEPEC

After 3 After 3 After 3 After 3 ---- hr incubation in hr incubation in hr incubation in hr incubation in

serumserumserumserum

UPEC UPEC UPEC UPEC EPECEPECEPECEPEC

1. Normal

serum

36 - 56 45 - 68 3 - 7.5

(8.3-13.4%)

0.13 - 4.5

(0.28-6.6%)

Bactericidal

activity

86.6-91.7% 90.3-99.7%

2. Heat

inactivated

Serum2

38 - 68 42 - 64 20 - 50

(47.6-73.5%)

32 - 54

(7- 84.4%)

Bactericidal

activity

26.8 - 47.4%

15.6-23.8%

1 Colony forming units log 10 /mL after incubation.

2 Serum heated at 56oC for 30 minutes.

Uropathogenic Escherichia coli growth responses occurred in 70.2

% of urinary isolates in normal serum as compared with 35.6 % of

diarrhael isolates.

4.3.74.3.74.3.74.3.7 Antibiotics Sensitivity Test (AST) : Antibiotics Sensitivity Test (AST) : Antibiotics Sensitivity Test (AST) : Antibiotics Sensitivity Test (AST) :

Despite the widespread availability of antibiotics, UTI remains the

most common bacterial infection in the human population (9).

A high incidence of resistance to an antibiotic within a given

population has long being accepted as evidence of widespread use of

that antibiotic.

The disc diffusion test was done for each isolates and the Mueller

Hinton Agar was used as growth media with 4% sodium chloride.



- 157 -

In this study, the antibiotic sensitivities were determined against

impregnated disks obtained from Hi media. Sensitivity and

resistance pattern to various antibiotics followed in this study was

based on NCCLS protocol.

The definition of sensitivity and resistance to the various antibiotics

based on this protocol are listed in Table -21.

TABLE TABLE TABLE TABLE –––– 21: Standard antibiotic disc sensitivity pattern.21: Standard antibiotic disc sensitivity pattern.21: Standard antibiotic disc sensitivity pattern.21: Standard antibiotic disc sensitivity pattern.

Sr.

no

Sr.

no

Sr.

no

Sr.

no

Antibio

tic

Antibio

tic

Antibio

tic

Antibio

tic

Pro

duct

Pro

duct

Pro

duct

Pro

duct

code

code

code

code

Sym

bol

Sym

bol

Sym

bol

Sym

bol

Dis

cD

isc

Dis

cD

isc

conte

nt

conte

nt

conte

nt

conte

nt

Resis

tant

Resis

tant

Resis

tant

Resis

tant

mm

mm

mm

mm

Inte

rmedia

teIn

term

edia

teIn

term

edia

teIn

term

edia

te

mm

mm

mm

mm

Sensitiv

eSensitiv

eSensitiv

eSensitiv

e

mm

mm

mm

mm

1 Amikacin SD035 Ak 30 µg 14 15-16 17

2 Ampicillin SD

002

A 10 µg 13 14-15 17

3 Cefopime SD213 Cpm 30 µg 14 15-17 19

4 Ciprofloxacin SD060 Cf 5 µg 15 16-20 21

5 Co--Trimoxazole

(Trimethoprim/

Sulphamethoxazole)

SD010

Co

1.25

10

11-15

15

6 Gentamicin SD016 G 10 µg 12 13-14 15

7 Kanamycin SD017 K 30 µg 13 14-17 18

8 Nalidixic acid SD022 Na 30 µg 13 14-18 19

9 Norfloxacin SD057 Nx 10 µg 12 13-16 17

10 Piperacillin SD066 Pc 100

µg

17 18-20 21

11 Streptomycin SD031 S 10 µg 11 11-14 15

Ref: In accordance to Performance Standards for Antimicrobial disk

Susceptibility Tests CLSI (formerly NCCLS).



- 158 -

With regard to the prevalence of antibiotic resistant E.coli from

hospitalized and non-hospitalized UTI patients, 25.5% responded to

one antibiotic, while 68.8% UPEC isolates showed multi drug

resistance (Table - 22).

Resistance to ampicillin and trimethoprim-sulfamethoxazole was

observed in all the urinary serogroups, but not in the

enteropathogenic strain.

Resistance to nalidixic acid, norfloxacin and ciprofloxacin among

E.coli serogroups ranged from 50-60 %.

The common serogroup O75 showed resistance up to 97 % or more

to three or more antibiotics.

All the UPEC strains revealed resistance to at least three of the

antibiotics tested (TABLE - 22 & Figure - 20).



- 159 -

TABLE TABLE TABLE TABLE –––– 22:22:22:22: Multiple Drug Resistance (MDR) Multiple Drug Resistance (MDR) Multiple Drug Resistance (MDR) Multiple Drug Resistance (MDR) PatternsPatternsPatternsPatterns of urinary of urinary of urinary of urinary

E.E.E.E.colicolicolicoli serogroups isolatedserogroups isolatedserogroups isolatedserogroups isolated....

Sr. Sr. Sr. Sr.

No.No.No.No.

SeSeSeSerogrouprogrouprogrouprogroup Resistance pattern to Resistance pattern to Resistance pattern to Resistance pattern to

commonly used commonly used commonly used commonly used

antibioticsantibioticsantibioticsantibiotics


MDR MDR MDR MDR



StudiedStudiedStudiedStudied

%%%%

1 O101 A, Ak, Co, Na, Nx, Cf 34 60 56.7

2 O75 A, Ak, Co, Na, Nx, Cf 34 35 97.0

3 O2 A, Co, Na, Nx, Cf 23 30 76.7

4 O6 A, Co, Na, Nx, Cf 17 20 85.0

5 O7 A, Co, Nx, G 09 15 60.0

6 O18 A, Co, K 05 10 50.0

7 O4 A, Co, Na 01 05 20.0

8 O8 A, Ak, Co, Na, Nx, Cf 01 05 20.0

Isolates showing multi

drug resistance

124 180 68.8

Isolates showing

resistance to single

antibiotic

46 25.5

Isolates showing

sensitivity

10 05.7



- 160 -

FIGURE FIGURE FIGURE FIGURE –––– 20: 20: 20: 20: Antibiotic ResistanceAntibiotic ResistanceAntibiotic ResistanceAntibiotic Resistance Pattern of UPEC serogroupsPattern of UPEC serogroupsPattern of UPEC serogroupsPattern of UPEC serogroups....



- 161 -

In case of EPEC, multiple drug resistance was observed in 23

(65.7%) of isolates an all strains of O158 was found to be resistant to

four antibiotics. (Table - 23 & Figure -21).

TABLE TABLE TABLE TABLE –––– 23: Antibiotic Resistance Pattern of EPEC serogroups:23: Antibiotic Resistance Pattern of EPEC serogroups:23: Antibiotic Resistance Pattern of EPEC serogroups:23: Antibiotic Resistance Pattern of EPEC serogroups:

Sr. Sr. Sr. Sr.

No.No.No.No.

SerogroupSerogroupSerogroupSerogroup Resistance pattern to Resistance pattern to Resistance pattern to Resistance pattern to

commonlycommonlycommonlycommonly

used antibioticsused antibioticsused antibioticsused antibiotics


MDR MDR MDR MDR



StudiedStudiedStudiedStudied

%%%%

1 O25 Ak, Pc, Cf 14 15 93.3

2 O78 A, Na, Cf 04 11 9.09

3 O158 Ak, Pc, Nx, Cf 03 03 100

4 O44 Ak, Ax, Cf 01 02 50.0

5 O26 A, Ax, G 00 02 00.0

6 O125 A, K 00 02 00.0

Isolates showing multi

drug resistance

23 35 65.7

Isolates showing

resistance to single

antibiotic

03 8.6

Isolates showing

sensitivity.

09 25.7



- 162 -

FIGURE FIGURE FIGURE FIGURE ---- 21:21:21:21: Antibiotic ResistanceAntibiotic ResistanceAntibiotic ResistanceAntibiotic Resistance Pattern of EPEC serogroups.Pattern of EPEC serogroups.Pattern of EPEC serogroups.Pattern of EPEC serogroups.



- 163 -

Those serogroups which revealed multiple drug resistance (MDR)

were taken for studying the ESBL activity.

4.3.8 Extended 4.3.8 Extended 4.3.8 Extended 4.3.8 Extended Spectrum for BetaSpectrum for BetaSpectrum for BetaSpectrum for Beta----lactamase activity: ESBLlactamase activity: ESBLlactamase activity: ESBLlactamase activity: ESBL::::

There are not enough data on the prevalence of ESBL producers in

urinary tract infection.

Hence, the present study was undertaken to find out prevalence of

ESBL producers in urinary isolates of E.coli and also a comparison

made with diarrhael isolates.

The treatment of E.coli infections is increasingly become difficult

because of multi-drug resistance exhibited by bacteria

(23).Extended spectrum β-lactamase (ESBL) producing organisms

pose a major problem for clinical therapeutics. The incidence of

ESLB producing strains of E.coli among clinical isolates has been

steadily increasing over the past few years resulting in limitations of

therapeutic options (24).

ESBLs are clinically important because they destroy cephalosporins

given as first-line agents to many severely ill patients and delayed

recognition and inappropriate treatment of severe infections caused

by ESBL producers with cephalosporins has been associated with

increased mortality (25). Many ESBL producers are multi-resistant

to non - β-lactam antibiotics such as quinolones, aminoglycosides

and trimethoprim, narrowing treatment options.



- 164 -

The phenotypic profile for ESBL detection test result is expressed in

Figure -22. Out of 124 urinary isolates exhibiting MDR, 54 isolates

were suspected of ESBL production i.e. Ceftazidime screen positive.

And out of 54 only 15 isolates were confirmed for ESBL production

in DDST.

In case of enteropathogenic isolates, 23 isolates were taken for

testing the ESBL production and only 5 gave ESBL screen test

positive, while in DDST only 2 were confirmed as ESBL producers.

FIGURE FIGURE FIGURE FIGURE –––– 22: The phenotypic profile of ESBL detection 22: The phenotypic profile of ESBL detection 22: The phenotypic profile of ESBL detection 22: The phenotypic profile of ESBL detection teststeststeststests.



- 165 -

The percentage of ESBL producing strains in E.coli from UTI and

Diarrhea was found to be 12.0% (15/124) and 8.6% (2/23)

respectively.

Table - 24 shows the summary of phenotypic traits in the studied

groups of E.coli.

Frequency of virulence-associated phenotypes among E.coli isolates

from patients with urinary tract infections and diarrhea.

TABLETABLETABLETABLE ---- 24: Summary of phenotypic profiles of study isolates from 24: Summary of phenotypic profiles of study isolates from 24: Summary of phenotypic profiles of study isolates from 24: Summary of phenotypic profiles of study isolates from

recognized serogroups recognized serogroups recognized serogroups recognized serogroups

Sr.

Sr.

Sr.

Sr.

No.

No.

No.

No.

Sourc

eSourc

eSourc

eSourc

e

Sero

gro

ups

Sero

gro

ups

Sero

gro

ups

Sero

gro

ups

No.

No.

No.

No.

sero

typed

sero

typed

sero

typed

sero

typed

Haem

oly

sin

Haem

oly

sin

Haem

oly

sin

Haem

oly

sin

pro

ducers

pro

ducers

pro

ducers

pro

ducers

No.

No.

No.

No.

sele

cte

dsele

cte

dsele

cte

dsele

cte

d

for

furt

her

for

furt

her

for

furt

her

for

furt

her

Stu

die

s.

Stu

die

s.

Stu

die

s.

Stu

die

s.

MR

HA

MR

HA

MR

HA

MR

HA

SA

TSA

TSA

TSA

T

MD

RM

DR

MD

RM

DR

UTIUTIUTIUTI

1.1.1.1. O101O101O101O101 79797979 70707070 60606060 50505050 50505050 34343434

2.2.2.2. O75O75O75O75 36363636 36363636 35353535 26262626 15151515 34343434

3.3.3.3. O2O2O2O2 34343434 30303030 30303030 30303030 12121212 23232323

4.4.4.4. O6O6O6O6 29292929 23232323 20202020 14141414 16161616 17171717

5.5.5.5. O7O7O7O7 21212121 19191919 15151515 14141414 09090909 09090909

6.6.6.6. O18O18O18O18 16161616 14141414 10101010 08080808 05050505 05050505

7.7.7.7. O4O4O4O4 08080808 08080808 05050505 04040404 04040404 01010101

8888 O8O8O8O8 08080808 08080808 O5O5O5O5 03030303 05050505 01010101

TOTALTOTALTOTALTOTAL 231231231231 208208208208 180180180180 149149149149 116116116116 124124124124

PERCENTPERCENTPERCENTPERCENT 82.7%82.7%82.7%82.7% 64.4%64.4%64.4%64.4% 68.8%68.8%68.8%68.8%

DiarrhealDiarrhealDiarrhealDiarrheal

1.1.1.1. O25O25O25O25 15151515 15151515 15151515 10101010 06060606 14141414

2.2.2.2. O78O78O78O78 12121212 12121212 11111111 10101010 06060606 04040404

3.3.3.3. O158O158O158O158 06060606 03030303 03030303 02020202 02020202 03030303

4.4.4.4. O44O44O44O44 05050505 02020202 02020202 01010101 ------------ 01010101

5.5.5.5. O26O26O26O26 02020202 02020202 02020202 01010101 ------------ 00000000

6.6.6.6. O125O125O125O125 02020202 02020202 02020202 01010101 01010101 00000000

TOTALTOTALTOTALTOTAL 42424242 36363636 35353535 25252525 15151515 23232323

PERCENTPERCENTPERCENTPERCENT 71.5%71.5%71.5%71.5% 42.8%42.8%42.8%42.8% 65.7%65.7%65.7%65.7%



- 166 -

4.4 Results of Molecular tests:4.4 Results of Molecular tests:4.4 Results of Molecular tests:4.4 Results of Molecular tests:

Whole cell proteins were analyzed by SDS-PAGE and representative

gel profile is shown in Figures U1 – U8 for UPEC strains and E1, E2

for EPEC strains.

Samples were processed in 2 series - A and B.

Series A containing 8 groups of samples processed from UTI and

Series B containing 2 groups of samples processed from diarrhea.

In Statistical analysis, In Statistical analysis, In Statistical analysis, In Statistical analysis, Dice coefficient was used for calculation in

results.

Analysis of whole cell protein of Analysis of whole cell protein of Analysis of whole cell protein of Analysis of whole cell protein of Escherichia coliEscherichia coliEscherichia coliEscherichia coli

Cells were grown in LB broth (pH-7) at 37oC overnight. Protein

profile was prepared and analyzed by SDS-PAGE (12.5% poly-

acrylamide gel) as described in Chapter 3: Materials and Methods.



- 167 -

TABLE TABLE TABLE TABLE ---- 25: Series and groups of Whole Cell Proteins studied.25: Series and groups of Whole Cell Proteins studied.25: Series and groups of Whole Cell Proteins studied.25: Series and groups of Whole Cell Proteins studied.

i) Series A: Urinary pathogenic isolates.i) Series A: Urinary pathogenic isolates.i) Series A: Urinary pathogenic isolates.i) Series A: Urinary pathogenic isolates.

Sr. NSr. NSr. NSr. No.o.o.o. SerogroupSerogroupSerogroupSerogroup Total No. of strains Total No. of strains Total No. of strains Total No. of strains

in each groupin each groupin each groupin each group

S. No. of strainsS. No. of strainsS. No. of strainsS. No. of strains Group No.Group No.Group No.Group No.

1. O101 12 U1 – U12. 1

2. O75 9 U13 – U21. 2

3. O2 8 U22 – U29 3

4. O6 7 U30 - U36 4

5. O7 7 U37 – U43 5

6. O18 6 U44 – U49 6

7. O4 5 U50 – U54 7

8. O8 4 U55 – U58 8

ii)ii)ii)ii) Series B : Diarrheal isolates.Series B : Diarrheal isolates.Series B : Diarrheal isolates.Series B : Diarrheal isolates.

Sr. No.Sr. No.Sr. No.Sr. No. SerogroupSerogroupSerogroupSerogroup Total No. of strains Total No. of strains Total No. of strains Total No. of strains

in each groupin each groupin each groupin each group

S. No. of strainsS. No. of strainsS. No. of strainsS. No. of strains Group No.Group No.Group No.Group No.

1. O25 8 E1 – E 8 1

2. O78 7 E9 – E15 2

Lane M in all the gels contains protein Markers viz

i) BSA - Bovine Serum Albumin - 65 KDal.

ii) EA - Egg Albumin - 45 KDal.

iii) LYZ - Lysozyme - 14.4 KDal.



- 168 -

Gel Profiles of the whole cell proteins:Gel Profiles of the whole cell proteins:Gel Profiles of the whole cell proteins:Gel Profiles of the whole cell proteins: Series A:Series A:Series A:Series A:

Grouped on the basis of serotypes isolated from urinary tract infection Grouped on the basis of serotypes isolated from urinary tract infection Grouped on the basis of serotypes isolated from urinary tract infection Grouped on the basis of serotypes isolated from urinary tract infection

(UPEC).(UPEC).(UPEC).(UPEC).

Group No. 1: Group No. 1: Group No. 1: Group No. 1: Serogroup O101:Serogroup O101:Serogroup O101:Serogroup O101: total isolates selected = 12.total isolates selected = 12.total isolates selected = 12.total isolates selected = 12.

Figure U1: GelFigure U1: GelFigure U1: GelFigure U1: Gel No.1.No.1.No.1.No.1.

KDal M U1 U2 U3 U4 U5 U6 U7 U8 U9 U10 U11 U12

BSA 65

EA 45

LYZ 14.4

Figure U1: UPEC group 1 Figure U1: UPEC group 1 Figure U1: UPEC group 1 Figure U1: UPEC group 1 ––––

Lanes U1 to U12 contains isolates no. UPEC 1, 2, 3, 4, 5, 6, 7, 8, 9,

10, 11 and 12 respectively.

Gel No. 1 shows profile of strains No. 1 to 12, isolated from urinary

infections.

Protein band shown by strains No. U7 ranged from 43 to 75 KDal.

Bands shown by strains No. U1 to U6 are more or less similar to

strains No. U7, but these proteins were not run smoothly for some

unknown reason.

Lane No. U10 and U11 were different from profile of other strains

since a doublet could be seen approximately at 75 & 73 Kdal.

Strain No. U12 showed a greater number of protein bands as

compared to other strains.



- 169 -

Group No. 2 : Serogroup O75 : total isolates selected = 9.Group No. 2 : Serogroup O75 : total isolates selected = 9.Group No. 2 : Serogroup O75 : total isolates selected = 9.Group No. 2 : Serogroup O75 : total isolates selected = 9.

Figure U2: Gel No. 2.Figure U2: Gel No. 2.Figure U2: Gel No. 2.Figure U2: Gel No. 2.

KDal M U13 U14 U15 U16 U17 U18 U19 U20 U21

BSA 65

EA 45

LYZ 14.4

Figure Figure Figure Figure U2:U2:U2:U2: UPEC group 2 UPEC group 2 UPEC group 2 UPEC group 2 ----

Lanes U13 to U21 contains isolates no. UPEC 13, 14, 15, 16, 17, 18,

19, 20 and 21 respectively.

Gel No. 3 shows banding pattern of protein of strains U13 to U 21.

Six protein bands of approximately molecular weight ranging from 36

to 49 KDal were detectable. No significant variation in the protein

pattern was observed in the protein profile.



- 170 -

Group No. Group No. Group No. Group No. 3:3:3:3: Serogroup Serogroup Serogroup Serogroup O2O2O2O2:::: total isolates selected = 8.total isolates selected = 8.total isolates selected = 8.total isolates selected = 8.

Figure U3: Gel No. 3Figure U3: Gel No. 3Figure U3: Gel No. 3Figure U3: Gel No. 3....

U22 U23 U24 U25 U26 U27 U28 U29 M KDal

65 BSA

45 EA

14.4 LYZ

Figure Figure Figure Figure U3:U3:U3:U3: UPEC group 3 UPEC group 3 UPEC group 3 UPEC group 3 ----

Lanes U22 to U29 contains isolates no. UPEC 22, 23, 24, 25, 26, 27,

28 and 29 respectively.

Total proteins of strains U22 to U29 separated on gel No. 3,

demonstrated similarity between strains U22, U23, U24, U26 and

U29, while U25 and U27 were found different from the others.



- 171 -

Group No. Group No. Group No. Group No. 4:4:4:4: Serogroup Serogroup Serogroup Serogroup O6O6O6O6:::: total isolates seltotal isolates seltotal isolates seltotal isolates selected = 7.ected = 7.ected = 7.ected = 7.


U30 U31 U32 U33 U34 U35 U36 M KDal

65 BSA

45 EA

14.4 LYZ


Lanes U30 to U36 contains isolates no. UPEC 30, 31, 32, 33, 34, 35

and 36 respectively.

Strains No. U30, U31, U32, U33, U34 and U36 showed protein bands

of molecular weight 80, 68, 63 and 54 KDal. The only variation was

observed in strain U35 which showed a prominent and distinct band

of approximately 65 KDal.



- 172 -


Figure Figure Figure Figure U5:U5:U5:U5: Gel No. 5.Gel No. 5.Gel No. 5.Gel No. 5.

M U37 U38 U39 U40 U41 U42 U43 M M KDal

65 BSA

45 EA

14.4 LYZ

Figure U5: UPEC group 5 Figure U5: UPEC group 5 Figure U5: UPEC group 5 Figure U5: UPEC group 5 ----

Lanes U37 to U43 contains isolates no. UPEC 37, 38, 39, 40, 41, 42

and 43 respectively.

Gel No. 5 shows the electrophoretically separated total proteins of

strains U37 to U43. Strains U37, U39, U40 and U41 seemed to be

similar strains. Strain U38 showed two doublets of molecular weights

40 and 38 KDal. Strains U43 showed an extra band of approximately

41 Kdal.



- 173 -

GrouGrouGrouGroup No. p No. p No. p No. 6:6:6:6: Serogroup Serogroup Serogroup Serogroup O18O18O18O18:::: total isolates selected = 6.total isolates selected = 6.total isolates selected = 6.total isolates selected = 6.


KDal M U44 U45 U46 U47 U48 U49

BSA 65

EA 45

LYZ 14.4

Figure U6: UPEC Figure U6: UPEC Figure U6: UPEC Figure U6: UPEC group 6 group 6 group 6 group 6 ––––

Lanes U44 to U49 contains isolates no. UPEC 44, 45, 46, 47, 48 and

49 respectively.

Gel No. 6 shows total protein separations of strains U44 to U49.

Strains U46 and U49 showed more or less similar banding patterns

while strain U47 contained somewhat less bands.



- 174 -



U50 U51 U52 U53 U54 M KDal

65 BSA

45 EA

14.4 LYZ


Lanes U50 to U54 contains isolates no. UPEC 50, 51, 52, 53 and 54

respectively.

Gel No. 7 shows total protein of strains U50 to U54. Protein banding

for U50, U52 and U53 were found to be similar. The molecular

weight of these proteins ranged from 35 to 75 KDal.



- 175 -

Group No. 8: Serogroup Group No. 8: Serogroup Group No. 8: Serogroup Group No. 8: Serogroup O8O8O8O8: total isolates selected = 4.: total isolates selected = 4.: total isolates selected = 4.: total isolates selected = 4.


U55 U56 U57 U58 M KDal

65 BSA

45 EA

14.4 LYZ


Lanes U55 to U58 contains isolates no. UPEC 55, 56, 57 and 58

respectively.

Gel No. 8 shows proteins separated from strains U55 to U58. Each

strain showed some differences. More than 7 dense bands could be

identified in addition to some faint bands.



- 176 -

Series Series Series Series B:B:B:B:

Containing 2 groups of samples processed from diarrhea(EPEC).Containing 2 groups of samples processed from diarrhea(EPEC).Containing 2 groups of samples processed from diarrhea(EPEC).Containing 2 groups of samples processed from diarrhea(EPEC).

Group 1: Serogroup Group 1: Serogroup Group 1: Serogroup Group 1: Serogroup O25: total isolates selected = 8O25: total isolates selected = 8O25: total isolates selected = 8O25: total isolates selected = 8

Figure E1: Gel No. 9.Figure E1: Gel No. 9.Figure E1: Gel No. 9.Figure E1: Gel No. 9.

KDal M E1 E2 E3 E4 E5 E6 E7 E8

BSA 65

EA 45

LYZ 14.4

Figure 9: EPEC group 1 Figure 9: EPEC group 1 Figure 9: EPEC group 1 Figure 9: EPEC group 1 ––––

Lanes E1 to E8 contains isolates no. EPEC 1, 2, 3, 4, 5, 6, 7 and 8

respectively.

Gel No. 9 shows protein bands separated from strains isolated from

diarrheal cases E1 to E8.

Strains E1 and E8 seemed to be similar while Strain E2 showed an

extra band at 46.5 KDal.

Strain E4 showed some variation at 50,33,32,30 and 27.5 KDal

respectively.



- 177 -

Group 2: SGroup 2: SGroup 2: SGroup 2: Serogroup O78erogroup O78erogroup O78erogroup O78 : total isolates selected = 7: total isolates selected = 7: total isolates selected = 7: total isolates selected = 7

Figure E2: Figure E2: Figure E2: Figure E2: Gel No. 10.Gel No. 10.Gel No. 10.Gel No. 10.

KDal M E9 E10 E11 E12 E13 E14 E15

BSA 65

EA 45

LYZ 14.4

Figure E2: Figure E2: Figure E2: Figure E2: EPEC group 2 EPEC group 2 EPEC group 2 EPEC group 2 ––––

Lanes E9 to E15 contains isolates no. EPEC 9, 10, 11, 12, 13, 14 and

15 respectively.

Total proteins of strains E9 to E15 separated on gel No. 10.

Strains E11 and E12 showed similar banding except for the presence

of an extra band at 68 KDal.

An extra band of low molecular weight (27.5 KDal) is visible in strain

E14.

Strain E13 showed three mid range bands of molecular weights 42,

39, 36 KDal.



- 178 -

TABLE TABLE TABLE TABLE ----26:26:26:26: Average percent similarity of Whole Cell Protein Average percent similarity of Whole Cell Protein Average percent similarity of Whole Cell Protein Average percent similarity of Whole Cell Protein

Profile of Profile of Profile of Profile of Escherichia coEscherichia coEscherichia coEscherichia colililili isolates from urinary infection.isolates from urinary infection.isolates from urinary infection.isolates from urinary infection.

i) Series i) Series i) Series i) Series A:A:A:A: UPEC:UPEC:UPEC:UPEC: Group 1Group 1Group 1Group 1

Isolate

No. U1 U2 U4 U5 U6 U7 U9 U10 U11 U12

U1 80 60 66.66 59.25 74.28 82.35 80.90 88.88 89.47

U2 62.5 75 61.53 70 66.66 69.29 74.28 64.56

U4 73.33 58.33 76 83.87 71.79 72.72 74.28

U5 66.66 75 83.87 71.09 78.78 80

U6 61.43 64 48.48 59.25 55.17

U7 43.20 78.04 91.72 81.08

U9 70 70 70.58

U10 76.1 81.81

U11 78.94

U12 78.94

Maximum percent similarity = 91.43

Minimum percent similarity = 43.24.



- 179 -

ii) Series ii) Series ii) Series ii) Series A:A:A:A: UPEC:UPEC:UPEC:UPEC: Group 2Group 2Group 2Group 2

Isolate

No. U13 U14 U15 U16 U17 U18 U19 U20 U21

U13 70 64 57.14 65 57.11 71.9 65 62.82

U14 66.6 51.50 66.66 51.65 74.27 72.37 59.0

U15 71.22 72.62 85.71 87.5 78.9 77.9

U16 51.61 61.54 66.55 74.50 54.8

U17 64.5 85.70 72.22 64.5

U18 66.77 77.49 68.9

U19 84.9 73.2

U20 70.76

U21 69.9

Maximum percent similarity = 87.71.




- 180 -

iii) Series iii) Series iii) Series iii) Series A:A:A:A: UPEC:UPEC:UPEC:UPEC: Group 3Group 3Group 3Group 3

Isolate

No. U22 U23 U24 U25 U26 U27 U28 U29

U22 58.05 66.56 60.80 56.90 68,78 51.09 65.54

U23 60 58.07 60.31 65.67 54.90 65.56

U24 57.13 59.11 64 53.84 57.24

U25 56.67 64.1 54.80 58.19

U26 57.7 49 53.59

U27 57 74.06

U28 63.64

U29 63.64


Minimum percent similarity = 49.



- 181 -

iv) iv) iv) iv) Series Series Series Series A:A:A:A: UPEC:UPEC:UPEC:UPEC: Group 4Group 4Group 4Group 4

Isolate

No. U30 U31 U32 U33 U34 U35 U36

U30 92.9 81 91 76.77 68.5 87.77

U31 87.76 83.67 75.67 52 61.63

U32 84.97 82.35 66.55 61.73

U33 80 70 66,55

U34 65 81.49

U35 63.64

U36 63.64





- 182 -

v) Series v) Series v) Series v) Series A:A:A:A: UPEC:UPEC:UPEC:UPEC: Group 5Group 5Group 5Group 5

Isolate

No. U37 U38 U39 U40 U41 U42 U43

U37 53.67 73.54 65.1 53.07 54 68.67

U38 65.45 74.57 64.3 58.43 77.78

U39 66.67 63.12 53 68.7

U40 62.28 52.9 71.19

U41 65 72

U42 69.34

U43 69.3





- 183 -

vi) Series vi) Series vi) Series vi) Series A:A:A:A: UPEC:UPEC:UPEC:UPEC: Group 6Group 6Group 6Group 6

Isolate

No. U44 U45 U46 U47 U48 U49

U44 60.38 53.12 58.84 59.58 68

U45 57.13 54.55 65 54.58

U46 60 55.55 59.57

U47 60 57.89

U48 53.45

U49

Maximum percent similarity = 60.




- 184 -

vii) Series vii) Series vii) Series vii) Series A:A:A:A: UPEC:UPEC:UPEC:UPEC: Group 7Group 7Group 7Group 7

Isolate

No. U50 U51 U52 U53 U54

U50 55.55 70.8 61.54 68

U51 66.55 60 71.1

U52 68.09 65.66

U53 72.9

U54 72.8





- 185 -

viii) Series viii) Series viii) Series viii) Series A:A:A:A: UPEC:UPEC:UPEC:UPEC: Group 8Group 8Group 8Group 8

Isolate

No. U55 U56 U57 U58

U55 61.53 54.05 60

U56 55.55 55.16

U57 59.58 48

U58 48





- 186 -

Average percent similarity of Whole Cell Average percent similarity of Whole Cell Average percent similarity of Whole Cell Average percent similarity of Whole Cell Protein Profile of Protein Profile of Protein Profile of Protein Profile of

Escherichia coliEscherichia coliEscherichia coliEscherichia coli isolates from diarrheal isolates from diarrheal isolates from diarrheal isolates from diarrheal infection:infection:infection:infection:

ix) Series ix) Series ix) Series ix) Series B:B:B:B: EPEC:EPEC:EPEC:EPEC: Group 1Group 1Group 1Group 1

Isolate

No. E1 E2 E3 E4 E5 E6 E7 E8

E1 51.16 57.04 57.57 53.08 49 57.16 55.81

E2 60 60 66.60 60.37 55 54.17

E3 57.18 56.60 53.45 61.43 55.31

E4 75 58.18 57 65

E5 57.79 56.52 66.76

E6 57.66 60.37

E7 58

E8 55

Maximum percent similarity = 75.




- 187 -

x) Series x) Series x) Series x) Series B:B:B:B: EPEC:EPEC:EPEC:EPEC: Group 2.Group 2.Group 2.Group 2.

Isolate

No. E9 E10 E11 E12 E13 E14 E15

E9 57.15 53.65 54.90 55 56 56.43

E10 56.41 53.07 68.43 64.84 71.27

E11 58.33 59.45 59.45 66.66

E12 59.57 46.56 47.82

E13 61.16 68.57

E14 57.13

E15





- 188 -

TABLE TABLE TABLE TABLE –––– 27: Data of collected samples and isolates used for studies.27: Data of collected samples and isolates used for studies.27: Data of collected samples and isolates used for studies.27: Data of collected samples and isolates used for studies.

Sr.Sr.Sr.Sr.

No.No.No.No. SourceSourceSourceSource Urinary infectionUrinary infectionUrinary infectionUrinary infection DiarrheaDiarrheaDiarrheaDiarrhea

1. Total no. of samples Total no. of samples Total no. of samples Total no. of samples

collectedcollectedcollectedcollected 305 66

2.

Total No. isolated & Total No. isolated & Total No. isolated & Total No. isolated &

identifiedidentifiedidentifiedidentified

as as as as E. coliE. coliE. coliE. coli 289 50

3. HemolyticHemolyticHemolyticHemolytic 241 40

3. SerotSerotSerotSerotypable ypable ypable ypable

Of known serotypes Of known serotypes Of known serotypes Of known serotypes

268

242

46

42

4. Congo red binding testCongo red binding testCongo red binding testCongo red binding test

166(+ve) &

15(+ve after some

incubation)

38

Selected number for Selected number for Selected number for Selected number for

further further further further


180 35

5. MRHA +veMRHA +veMRHA +veMRHA +ve 149 25

6. SAT SAT SAT SAT +ve+ve+ve+ve 116 15

7. SBT +veSBT +veSBT +veSBT +ve 127 12

8. MDR MDR MDR MDR +ve+ve+ve+ve 124 23

9. ESBL ESBL ESBL ESBL +ve+ve+ve+ve 15 / 124 02 / 23

10. Analysis of whole cell Analysis of whole cell Analysis of whole cell Analysis of whole cell

proteinsproteinsproteinsproteins 58 15

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