Chapter 4

Results and discussion

A total of 103 bacterial isolates were obtained from hospital surfaces, 23 from hospital waste
water and 20 from the patient sample of hospital and they were coded as:

Table 4.1: Code name of bacterial isolates from patients,hospital environment,hospital waste
water during 2nd sampling first sampling

Isolates from surface samples of Kamla Nehru hospital Isolates from water sample of Isolates from
Kamla Nehru hospital patient samples
of Kamla
Untreated Treated Nehru hospital
water water

Bronchoscopy unit ICU Emergency Urology Pharmacy KEMSI1 BMSO2 23PSU

KBR B1 KICUF1 KEMB KDIB1 KDSI1 KEMSI3 KMSO2 EM 24

KBRB1 KICUF2 KEMB3 KDIB2 KDSI2 BMSI2 KMSO1 KPI1

KBRB2 KICUF3 KEMB4 KDIB2 KDSI3 BMSI1 BMSO1 KICUPU10

KBRB3 KICUF3 Cre KEMW1 KDIB3 KMSI4 BMSO4 EMPU1

f

KBRB3’ KICUF4 KEMW4 KDIW1 KEMSI1 BMSO3 KPE2

KBR1 KICUW2 KEMW4 KDIW2 BMSI3 KPE3

KBR1’ KICUW4 KEM W KDI W BMSI1 KPE4

cre g

KBR2 KICUWM2 KEMST KDIF1 KPE5

KBR2 KICUWM3 KEMST2 KDIFM2 KPE6

KBR KICUSBM1 KEMF2 KDIFM3 KPE7

KBR Y C KICUFM1 KEMF3 KDIFM4 KPS8

KBRY KICU Cre G KEMFg KDIFM4 KPS9

KBRD1 KICU 3 PIN KEMF Cre KDIC1 KPE10

G
KBRD2 KDIC2 KPS11

KBRT1 KDIT1 KPE12

KBRT2 KPE13

ICU1

1
Table 4.2:Code name of bacterial isolates from patients,hospital environment,hospital waste
water during 2nd sampling

Isolates from surface samples of a tertiary care hospital Isolates from water sample of
a tertiary care hospital
Bronchoscopy ICU Emergency Urology Pharmacy
unit Untreated Treated water
water
SKBR2 SKICUF1 SKEMB SKDIB2 SKDSI1 KMSI11s BMSO2s
SKBR SKICUF2 SKEMB3 SKDIB3 SKDSI2 KMSI13s KMSO2s
SKBR Y C SKICUF3 SKEMB4 SKDIW1 SKDSI3 BMSI2s KMSSO1s
SKBRY SKICUF3 SKEMW1 SKDIW2 SKDSIT1 KMSI4s BMSO1s
Cre f
SKBRD1 SKICUF4 SKEMST2 SKDIFM4 SKDSP1 BMSI1s BMSO4s
SKBRD2 SKICUSBM1 SKEMF2 SKDIFM4 SKDSP2 KMSI5s BMSO3s
SKBRT1 SKICUFM1 SKEMF3 SKDIC1 SKDSIT1 BMSI3s BMSO1s
SKBRT2 SKICU Cre SKEMFg SKDIC2 SKDSIT2 BMSI1s BMSO12s
G
SKBR1 SKICU 3 PIN

Where K represents Kamla Nehru hospital, BR for bronchoscopy unit, EM for emergency, ICU
for ICU ward and DI for dialysis unit of Kamla Nehru hospital KMSO represent an outlet point
of waste water of Kamla Nehru Hospital, KMSI represent an outlet point of waste water of
Kamla Nehru hospital.

Table 4.3: Morphological character wise frequency of bacterial isolates from surfaces,
patients and waste water of a tertiary care hospital:

Hospital Patients Waste water

Morphological Features surfaces
property
Colony surface Glistering 8 4 10
Smooth 35 16 4
Gummy 60 0 9
Wrinkled 0 0 0
Colony margin Entire 85 11 19
Undulate 18 6 4
Lobate 0 0 0
Filamentous 0 0 0
Colony elevation Flat 0 0 0
Raised 18 6 4

2
 Convex 85 14 19
Umbonate 0 0 0
Cell shape Cocci 17 7 0
Bacilli 86 13 23
Cell arrangement Discrete 86 13 23
Staphylococcus 2 1 0
Streptococcus 15 6 0

Bacterial assessment of different sample of hospital environment have performed by

characterizing them on the basis of morphological and biochemical characteristics. Total 146
bacterial colonies were selected and sub cultured for further biochemical characterization and
antibiotic susceptibility testing. Morphological characterization of 103 isolates were performed
by tacking the consideration of their colony color, colony appearance, colony surface, elevation
and edge, gram staining, cell shape and cell arrangement.

During 1st sampling (in 2015) a total 60 bacterial isolates were screened from surfaces and 13
bacterial isolates were screened from waste water associated with hospital. After the six month
variation during the 2nd sapling (in 2016), 43 bacterial isolates were screened from hospital
surfaces and 10 bacterial isolates were screened from waste water of hospital.

Bacterial isolates formed gummy, convex, circular, entire colony were dominant (Table
2).Another criteria used for characterization of bacteria is gram staining and their cell shape and
cell arrangement, total 86 Bacilli and 17 cocci were isolated from surfaces and 13 bacilli and 7
cocci were isolated from a patient sample and 23 bacilli were obtained from waste water. Rod
shaped bacteria were observed in dominant among the bacteria isolated from all the samples of
hospital.

Table 4.4: Biochemical characteristics wide frequency of different bacterial isolates of the
Hospital:

Type of Bacterial isolates Bacterial isolates Bacterial isolates

biochemical test from surface of KN from Patients of KN from waste water of
KN
+ve -ve +ve -ve +ve -ve
Citrate 85 18 20 0 19 4
MR reaction 16 87 11 9 0 23
VP production 35 68 11 9 4 19
Indole production 0 103 0 20 0 23
amylase 8 95 4 16 10 13
Nitrate reduction 93 10 16 4 13 10
Gelatin hydrolysis 77 26 16 4 9 14

3
Urease 25 78 15 5 10 13
Catalase activity 43 60 11 9 10 13

Biochemical characterization was carried out in 146 isolates comprised of 103 surfaces isolates,
23 waste water isolates and 20 isolates belonging to the patient sample of hospital.

The result indicates that maximum of bacteria from hospital surface showed positive results for
nitrate reduction test followed by 85 isolates were citrate positive and 77 isolates were gelatinase
positive. From the patient sample 20 isolates were citrate positive and 16 isolates were nitrate
and gelatinase positive. And bacterial isolates from waste water sample had the highest
frequency of citrate test followed by a nitrate reduction test.

Table 4.5: Frequency of bacterial isolates identified at the genus level, of hospital surfaces,
waste water and patients in hospital

S.No. Bacterial Flora Hospital surfaces Waste water of Patients in

hospital hospital
1 Klebsiella sp 58 8 0
2 Pseudomonas sp 17 4 4
3 Enterococcus sp 15 0 7
4 Escherichia sp 8 10 4
5 Staphyllococcus sp 2 0 1
6 Enterobacter sp 2 1 1
7 Alcaligenes sp 1 0 3

4
 A B C

D E

Fig 4.1: result of biochemical characterization of (A) Pseudomonas sp (B) E.coli ,

(C)Klebsiella sp (D) Enterococcus sp and (E) Staphyllococcus sp

Plate4. 1:KDIF1 isolate on Nutrient agar , MacConkey agar and Hichrome Flexi plate indicate that the isolate may be belong
to Klebsiella sp.

Plate4. 2:KICUWM3 isolate on Nutrient agar , EMB agar and Hichrome Flexi plate(purple colonies) indicate that the isolate
may be belong to E.coli.

Plate 4.3 3:KBRW1 isolate on Pseudomonas isolation agar and Hichrome Flexi plate(cream colonies) indicate that
the isolate may be belong to Pseudomonas sp.

5
Hospitals and clinics are major reservoirs for large numbers of pathogenic bacteria comprised of
resident and community introduced strains (Periasamy et al.,2013). Inanimate surfaces in the
hospital are rapidly contaminated by microorganisms either due to direct contact with the
patient shedding of bacteria, or indirectly due to high-frequency interactions between
healthworkers’ hands and high-touch surfaces (e.g., monitors, ventilator buttons, bedrails), in the
patient zone (Sax et al.,2005).

Biochemical characterization is widely used in identification of bacteria flora the group of

bacteria was identified up to genus level were as Klebsiella spp, Pseudomonas spp,
Enterococcus spp, Escherichia and Staphyllococcus spp.

From hospital surface samples, Klebsiella spp were the most predominant spp(60%)isolated from
different surfaces of the hospital followed by Pseudomonas (27.7%), Enterococcus(25%), Ecoli
(5%)and S.aureus(3.33%). This is in accordance with other studies in India(Bhattacharyya et
al.,2015)and Nigeria(Yusuf et al.,2017), where The frequency of the bacteria varied but the
major varieties of bacteria identified were Klebsiella spp. And Pseudomonas Spp. Similar
results were reported by Garcia-Cruz et al. (2012) where Klebsiella is the most common agent
isolated from indoor surfaces of hospital of Mexico with the prevalence of 50.4% followed by
Pseudomonas (32.1%), E. coli (9.17%). Bakkali et al.( 2015) reported , the highest incidence
was related to Pseudomonas(23.12%), Klebsiella (16.76%) and E. coli (6.94%),study on hospital
surfaces sample of Central university hospital Ibn Sina.

From the waste water sample of hospital the most frequently identified bacteria were E. coli
(43.47%) followed by Klebsiella sp. (39.13%) Pseudomonas spp. Similar results were observed
in the study of Siddiqui et al.(2015),in which E. Cole (30.7%) where the frequently isolated
bacteria followed by Klebsiella spp (25%) and Enterobacter spp 8 (7.7%). Resende et
al.(2009) conducted study on waste water of 10 hospital in Brazil, and found that the Klebsiella
spp (45.5%) were most commonly isolated followed by E.coli(36.4 %) and Pseudomonas spp
(13.6%). Moges et al.(2014) reported Klebsiella (26.6%.2), Pseudomonas (16.8%), E. coli
(11.5%) , Citrobacter spp (11.5%) as the most common Gram-negative bacilli in hospital waste
water of Ethopia. E.coli is a member of fecal coliform bacteria and are commonly found in the
intestines of animals and humans. Presence these enteric bacteria in the hospital waste water
underscores the point that there is a high level of fecal contamination either directly from sewage
or poor hygiene practice(Svanstrom ,2014).

Of the 140 participant growth was obtained from 20(14.25%) samples. Among 20 bacterial
isolates the main isolated organisms from urine samples were Pseudomonas spp (45 %) followed
by E.fecalis (30%) and E .coli (20%). This result is inconsistent from report in Uganda that
Pseudomonas spp have been the frequently isolated from the urine (Najjuka et al.,2016).Study in

6
the Babylon in Hilla Teaching Hospital reported E. coli (20%) , K. pneumoniae (11.6%) and
Pseudomonas spp (3.3%).
Table 4.6: Statistical analysis of antibiotic susceptibility test of sixteen antibiotics against
bacterial isolates of surface, waste water and a patient’s sample of hospital

S.No Name of drugs Surface isolates Water isolates Patients isolates

1 FAR 0.000807 6.74129E-36 3.52882E-13
2 CFS 0.999 0.8875 1.64154E-16
3 CTR 0.225 0.0125 1.29512E-15
4 CLR 0.07410 0.000129 3.08863E-15
5 CTX 0.005 0.0348 4.05321E-25
6 PTZ 0.999 0.99725 0.99384
7 AMC 1.43895E-12 1.10625E-20 1.798812-29
8 GEN 0.8261 0.6628 3.55645E-18
9 AK 0.057 0.8985 8.0548E-19
10 NX 0.9848 2.18521E-05 8.4086E-36
11 LZ 1.320813E-33 1.05261E-31 4.73826E-54
12 CD 1.55599E-19 6.89349E-06 5.79115E-39
13 CIP 0.9560 1.7719E-06 8.38835E-37
14 LE 0.9883 0.5828 1.235235-08
15 OF 0.8693 1.48728E-07 2.89412E-40
16 DO 0.0732 4.56854E-08 5.77465E-31

Sensitivity of all bacterial isolates against 16 antimicrobial agents was evaluated by disc
diffusion method. Mean values were calculated and chi square test was performed to evolutes the
efficiency of different drugs against bacterial isolates from surfaces, waste water and patients
hospital (Table 4). Pipracillin, ciproflox, cefoperazone+salbactum, and levoflox were found to
be effective (93.3, 93.3 86.6 and 81.6%) against bacterial isolates from hospital surfaces.
Pipracillin, gentamycin, norflox, ciproflox and oflox were found to be effective (100%) against
bacterial isolates from hospital waste water samples. Piracillin,cefetrioxane and faropenem were
found to be effective against the patients bacterial isolates and ciproflox,norflox,oflox ,levoflox
were found to be less effective for patients bacterial isolates the reason behind the resistance for
these cephalosporin is that the group cephalosporin was largely prescribed and is used in
antimicrobial therapy used to treat the patients of urinary tract infection from which these
bacterial isolates were belonging.

Antibiotic susceptibility test of bacterial isolates from surface samples of Hospital:

7
The results (fig 1,table 6) showed widespread resistance (2.3– 94.3 %) of the surface isolates of
Kamla Nehru hospital to all the antibiotics, except pipracillin/tazobactum, ofloxacin,
gentamicin(5.66,7.54.9.4 %). The result also showed that isolates surfaces of kamla Nehru
hospital isolate KICUFM3 and KICUF4 were resistant to 7 drug and isolate KEM cre
g,KDIF1,KEMW1,KICUF4 were found to be resistant to 6 drug and KEM floor G were resistant
to 5 drug.

Table 4.7: Frequency of sensitive, resistance, bacterial isolates of different surfaces of the
Kamla Nehru hospital
Antib Pseudomonas spp Enterococci spp E.coli n=3 Alcaligenes fecalis Klebsiella spp n=36
iotics n=10 n=9 n=1
tested Tot Total Resis Tot Total Resis Tot Total Resis Tot Total Resis Tot Total Resis
al resist tance al resist tance al resist tance al resist tance al resist tance
sesi ance perce sesi ance perce sesi ance perce sesi ance perce sesi ance perce
tive ntage tive ntage tive ntage tive ntage tive ntage
PIZ 8 2 20 9 0 11.1 3 0 0 1 0 0 35 1 2.7
AMC 7 7 70 7 2 55.5 1 2 66.6 0 1 100 15 21 61.1
GEN 7 3 30 8 1 22.2 2 1 33.3 1 0 0 26 10 27.7
CLR 5 5 50 7 2 44.4 1 2 66.6 1 0 0 15 19 47.2
CTX 6 4 40 9 0 22.2 3 0 0 1 0 0 16 10 27.7
FAR 4 6 60 7 2 33.3 1 2 100 0 1 100 14 22 55.5
AK 7 3 30 9 0 11.1 3 0 0 0 1 100 27 9 13.8
NX 8 2 20 9 0 0 3 0 0 1 0 0 26 10 11.1
LZ 0 10 100 7 2 66.6 1 2 66.6 0 1 100 4 31 86.1
CIP 10 0 0 9 0 11.1 3 0 0 1 0 0 31 5 13.8
CD 4 6 60 7 2 66.6 1 2 66.6 0 1 1`00 29 7 19.4
CTR 8 2 20 8 1 22.2 2 1 33.3 1 0 0 27 9 25.4
DO 3 7 70 6 3 66.6 0 3 100 0 1 100 16 20 58.3
LE 5 5 50 9 0 111.1 3 0 0 1 0 0 31 5 13.8
CFS 6 4 40 9 0 11.1 3 0 0 1 0 0 33 3 8.3
OF 5 5 50 9 0 22.2 3 0 0 1 0 0 30 6 16.4

Klebsiella spp. was most frequently isolated from surface samples of hospital, shown a high level
of resistance for lenozolid (86.11%) followed by amoxicillin/clavulanate(55.5 %), Doxycycllin
(55.5%), Faropenem (58.33 %),this increase resistance is may be due to the production of
extended spectrum betalactamase ,enzyme that cause resistant to penicillin such as ampicillin
and amoxicillin(Hsu et al.,2007).

Pseudomonas spp. Showed high level of resistance towards linazolid(100%),

amoxicillin/clavulanate(70 %),Doxycycline (70 %) both of the antibiotics belong to the beta
lactum group of antibiotics and the resistance may be due to the presence of β-lactamse enzymes
which destroy β- lactam ring of β-lactam antibiotics, thereby rendering the drug useless. Whereas
ciprofloxacin were found to be effective against the Pseudomonas and had shown (0 %)
resistance.

8
 Klebsiella spp. Pseudomonas sp. E.coli Enterococcus Fecalis

100
80
60
40
20
0

Fig 4.2:: Percentage resistance of bacterial isolates from a surface sample different department of
hospital to different antibiotics. Shows a high level of resistance were observed against linazolid
by all the isolates which may be due to the presence of an intrinsic resistance mechanism in gram
negative isolates towards linazolid,where as cefaperazone and ciproflox were found to be
effective against bacterial isolates.

9
Increased incidence of doxycycline (100 %) resistance was observed in E.coli isolates followed
by amoxicillin,faropenem and clindamycin (66 %) where as norfloxacin, levofloxacin,
ofloxacin, ciprofloxacin,amikacin, pipracillin, cefaperazone/salbactum, cefotaxime were found to
be effective against E.coli isolate. A similar result was observed in a study conducted in Tehran,
where 100 % of resistance towards amoxicillin +clavulinic acid, and 66.6% of resistance towards
penem group of antibiotics were observed (Tajeddien et al.,2016).
Enterococci spp. Showed high level of resistance towards linazolid, clindamycin and
doxycycline (66.6%) followed by amoxicillin/clavulanate(55.5%). This finding is in agreeing
with the previous study ,where 65 % of Enterococcus isolates showed resistance towards
doxycycline and 60 to 100 % resistance were found against linazolid(Bhattacharyya et al.,2015).

Antibiotic susceptibility test of bacterial isolates from treated and untreated water sample
of Kamla Nehru Hospital:

Results show that out of 23 isolates of water sample of hospital, resistance was found to be
(0.076-61.13%) and isolate BMSO2 and KMSI1 were resistant to 7 drugs.

Table 4.8: Frequency of sensitive, resistance bacterial isolates of waste water associated
with Kamla Nehru hospital
Antibiotics Pseudomonas spp n=4 E.coli Klebsiella spp n=9
tested n=10
Total Total Resistance Total Total Resistance Total Total Resistance
sesitive resistance percentage sesitive resistance percentage sesitive resistance percentage
PIZ 4 0 0 10 0 0 9 0 0
AMC 1 3 100 5 5 50 1 7 66.6
GEN 4 0 0 10 0 0 9 0 0
CLR 4 0 0 7 3 33.3 7 2 25
CTX 4 0 0 5 5 50 9 0 0
FAR 4 0 0 4 6 66.6 6 3 40
AK 1 3 100 10 0 0 9 0 0
NX 4 0 0 8 2 16.6 9 0 0
LZ 4 0 0 5 5 50 6 3 40
CIP 2 2 50 6 4 33.2 7 2 25
CD 4 0 0 8 2 16.6 5 4 40
CTR 4 0 0 9 1 16.6 10 0 0
DO 4 0 0 8 2 16.6 10 0 0
LE 4 0 0 10 0 0 10 0 0
CFS 4 0 0 10 0 0 10 0 0
OF 4 0 0 10 0 0 10 0 0

10
 E.coli Pseudomonas spp. Klebsiella spp

100
80
60
40
20
0

Fig 4.3:: Percentage resistance of bacterial isolates from waste water of a tertiary care hospital
with different antibiotics. Figure depicts a high level of resistance against amoxicillin and
faropenem were found to be less effective. Both of these antibiotics belong to the beta lactam
group of antibiotics and this increase resistance is may be due to the production of extended
spectrum betalactumase ,enzyme that cause resistance.

11
High level of resistance found against the faropenem(66.6 %) followed by linazolid, pipracillin
and amoxicillin/clavulanate(50 %),where as no resistance were shown by the E.coli. isolates
towards the amikacin ,oflox,levoflox. Pseudomonas isolates showed resistance to
amoxicillin/clavulanate, faropenem, linazolidclindamicin (50%). High level of resistance was
found against the amoxicillin/clavulanate and faropenem (100 %) followed by linazolid(50%).

Results showed that 23 isolates of water sample were found to be resistant (4.3-65.21%). The
resistance pattern of Klebsiella isolates showed 50 % resistance to linazolid, 66.6 % resistance to
amoxicillin + clavulinic acid. 55.5% resistance rate for amoxicillin +clavulinic acid, and
linazolid was exhibited by E. coli isolates, whereas low level of resistance were found against
amikacin, gentamycin, levoflox, of lox and cefaperazonen. A high resistance rate 87.5% and 55.5
% to faropenem was observed among Pseudomonas and E. coli isolates, low level of resistance
were found against pipracillin+tazobactum, norflox and cifatrioxane. Among 23 bacterial
isolates most striking drug resistant isolate which was resistant to 7 antibiotic tested was
Klebsiella spp, followed by E.coli isolates to 6 antibiotics, Pseudomonas spp to 5 antibiotics.
Among all isolates 1 isolate was susceptible to all antibiotics tested, 3(13.03) were resistant to
only one antibiotic, 8 (34.7) were resistant to 2 antibiotics,5(21.7) for 3 antibiotics,2(8.6) for 4
antibiotics, and 4(17.3) were resistant for 5 or more antibiotics. The overall prevalence of
multiple drug resistance(resistant to 3 or more drug) in this study was 10(43.4).
This part of result indicates that a moderate level of antibiotic resistance was observed in this
study. Overall a high level of resistance was observed against the amoxicillin. The resistance rate
in E.coli, Pseudomonas and Klebsiella isolates for amoxicillin was found 55.5%,75% and 66.6%
respectively. This finding is consistent with the report in Bangladesh that the, resistant rate for
E.coli and Klebsiella isolates were found 99% and 65%(Siddiqui et al.,2015). In our study
resistance rate for faropenem(belong to the penem group of antibiotics) in E.coli, Pseudomonas
and Klebsiella spp were found to be 55.5%,87.1% and 33.5% respectively. This was different
from the other study done in Brazil where low level of resistance were observed by bacterial
isolates for another antibiotics of penem group(Resende et al.,2009).Amoxycillin and faropenem
antibiotics are belong to the beta lactam group of antibiotics and this increase resistance is may
be due to the production of extended spectrum betalactumase ,enzyme that cause resistant to
penicillin such as ampicillin and amoxiillin(Brisse et al.,2005;Hsu et al.,2007),whereas levoflox
,oflox,cefaperazone and gentamycin were found active against the bacterial isolates of waste
water sample. It can be assumed that isolated bacteria in this study have been exposed to
antibiotic residues. As a result mechanism of resistance to antibiotics may be developed in these
isolates .

12
Antibiotic susceptibility test of bacterial isolates from patient’s sample of Hospital:

Table 4.9: Frequency of sensitive, resistance bacterial isolates from patient’s sample
Antibioti Pseudomonas spp n=6 E.coli Enterococci spp n=9 Alcaligenes fecalis
cs tested n=3
Total Total Resistan Total Total Resistan Total Total Resistan Total Total Resistan
sensiti resistan ce sensiti resistan ce sensiti resistan ce sensiti resistan ce
ve ce percenta ve ce percenta ve ce percenta ve ce percenta
ge ge ge ge
PIZ 4 4(66.6) 1 2 66.6 8 1 1.1 0 1 100
AMC 2 2(33.3) 0 3 100 6 2 22.2 0 1 100
GEN 3 3(33.3) 0 3 100 5 3 33.3 1 0 0
CLR 8(88.8) 0 3 100 0 8 88.8 0 1 100
CTX 4(44.4) 0 3 100 4 4 44.4 0 1 100
FAR 2(22.2) 0 3 100 6 2 22.2 0 1 100
AK 4(44.4) 1 2 66.6 4 4 44.4 1 0 0
NX 2(22.2) 0 3 100 4 4 44.4 0 1 100
LZ 4(44.4) 0 3 100 3 5 55.5 0 1 100
CIP 4(44.4) 0 3 100 4 4 44.4 0 1 100
CD 5(55.5) 1 2 66.6 4 4 44.4 0 1 100
CTR 4(44.4) 0 2 66.6 3 5 55.5 0 1 100
DO 4(44.4) 0 3 100 3 5 55.5 1 0 0
LE 5(55.5) 0 3 100 2 6 66.6 0 1 100
CFS 6(66.6) 1 2 66.6 4 4 44.4 0 1 100
OF 4(44.4) 0 3 100 4 4 44.4 0 1 100

High level of resistance (Fig 3, table 8) found against the faropenem(66.6 %) followed by
linazolid,pipracillin and amoxicillin/clavulanate(50 %),where as no resistance were shown by the
E.coli. isolates towards the amikacin ,oflox,levoflox. Pseudomonas isolates showed resistance to
amoxicillin/clavulanate,faropenem, linazolidclindamicin(50%).High level of resistance were
found against the amoxicillin/clavulanate and faropenem(100 %) followed by linazolid(50%).

High level of resistance had shown by the Pseudomonas isolates from patients towards the most
of the drug, that is gentamicin, norflox, clarithromicin, cefotexime(80%) followed by
cefaperazone and levofloxacin (60%),faropenem pipracillin(40%), cefitrioxane and doxycycline
(20%) found to be effective.

E.coli isolates were shown high level of resistance towards 11 of the drug used in the study ,and
shown( 66.6%) of resistance towards amikacin ,clindamicin, doxycycline, cefaperazone,
pipracillin.

13
 Pseudomonas E.coli E.fecalis

100
80
60
40
20
0

Fig 4.4: Percentage resistance of bacterial isolates from patients of hospital to different antibiotics.
Figure depicts a level of resistance were found against the ciproflox, norflox, levoflox, oflox( belong
to the group of fluroquinalone which are commonly used in the treatment of UTI ),this is may be
due the over expression of some efflux pump or presence of the altered drug target
target.

14
Virulence testing of Bacterial isolates: Hospital environment provide a reservoir for
disseminating the bacterial pathogen among patients. these bacteria regulates their pathogenicity
through a series of virulence factors, such as hemolysin production, capsule formation.The
degree of virulence is related directly to the ability of the organism to cause disease. Examples
are toxins, surface coats that inhibit phagocytosis, and surface receptors that bind to host cells.
bacterial pathogens have evolved specific virulence factors that allow them to multiply in their
host or vector without being killed or expelled by the host's defenses. Many virulence factors are
produced only by specific virulent strains of a microorganism. For example extracellular
enzymes hemolysin (extotoxin) that lyse red blood cells in the blood agar (hemolysis). Different
types of hemolysis are produced in Sheep blood agar by bacteria are:

alpha hemolysis (α-hemolysis): is present, the agar under the colony is dark and greenish.

Beta hemolysis (β-hemolysis):sometimes called complete hemolysis, is a complete lysis of red

cells in the media around and under the colonies: the area appears lightened (yellow) and
transparent

gamma hemolysis (γ-hemolysis ):If an organism does not induce hemolysis, the agar under and
around the colony is unchanged, and the organism is called non-hemolytic.

Isolates were screened for the presence of hemolysin were among 20 isolates of patients
17,in water isolates 4, and in surface isolates 37 were found to have hemolysin.

Primary screening of bacterial isolates was made based on the result obtained from disc diffusion
method, 9 bacterial isolates which showed resistance to > 5 antibiotics were selected for further
MIC test and molecular identification. Among 9 selected isolates(table 4.10,Plate 4.1), 4 isolates
belonging to the surface bacterial samples, 3 from patients and 2 isolates were belonging to the
waste water samples of hospital.

15
Table 4.10: Selected bacterial isolates which showed resistance phenotype

Name of Resistance to Sensitive to Source of isolation

isolate
KICUcreg AMC,GEN,CLR,CTX,FAR,LZ,CD, PTZ,AK,NX,CIP,CTR,LE, Surface of hospital
DO CFS,OF
KDIF1 AMC,CLR,FAR,LZ,CD,DO PTZ,GEN,CTX,AK,NX, Surface of hospital
CIP,CTR,DO,LE,CFS,OF
KEMW4 AMC,CLR,CTX,FAR,AK,LZ,CD, PTZ,GEN,FAR,AK,NX, Surface of hospital
CTR,DO CIP,LE,CFS,OF
KBRB1 AMC,CLR,CTX,FAR,AK,LZ,CD, PTZ,GEN,FAR,AK,NX, Surface of hospital
CTR,DO CIP,LE,CFS,OF
2U AMC,GEN,CLR,CTX,AK,NX,LZ, FAR,CFS Patients of hospital
CIP,CD,CTR ,DO,LE,OF
28U AMC,GEN,CLR,FAR,AK,LZ,CIP, CTX,FAR,NX Patients of hospital
CD,CTR,DO,LE,CFS,OF
EM24 AMC,CLR,FAR,LZ,CIP,CD,FAR, GEN,AK,DO Patients of hospital
CTX CTR,LE,CFS,OF
KMSI1 AMC,FAR,LZ,CD,OF GEN,PIZ,CTX,AK,NX, Waste water of
CLR,CTR,DO,LE,CFS,OF hospital
BMSO3 AMC,GEN,CLR,FAR,LZ,CIP,CD PIZ,CTX,AK,NX,CTR, Waste water of
DO,LE,CFS,OF hospital

16
KDIF1 KMSI1 2U EM24 28U

KICUcrg KBRB1 KEMW4

BMSO3

Plate 4.4: Photograph of antibiotic susceptibility results of resistance bacterial isolates

17
 Determination of minimum inhibitory concentration:

As shown in Table 10, 10 bacterial

isolates(EM24,2U,28U,BMSO3,KMSI1,KICUcrG,KBRB1,KEMW4,KDIF1) which were
resistant to more than 5 antibiotics were selected for determination of MIC which ranges from
0.064 to 1200 µg ml-1.

A low MIC value for three bacterial isolates which were belonging to the surface bacterial
isolates, group as Enterobacter sp and Escherichia sp were observed below the range of 3 µg m
l-1for
oflox,ciproflox,levoflox,norflox,amoxicillin,faropenem,amikacin,gentamycin,doxycycline,piprac
illin and azithronam which were found to be below the level of resistance mentioned by
CLSI(2012).

Bacterial isolate 47KDIFI belong to the surface isolates as Alcaligenes faecalis showed a
moderate level of resistance for oflox and levoflox that is 3 µg ml-1 and 250,350 µg ml-1 for
clindamycin and linazolid. Bacterial isolates belong to the patient samples showed a high level of
resistance against the fluroqinalone including oflox,ciproflox,norflox, levoflox(ranged between
125 to 1200 µg ml-1), 200 to 1000 µg ml-1 for cefaperazone,32 to 48 µg ml-1 for amoxicillin and
4to 800 µg ml-1for amikacin.

BMSO3 isolate belonged to the waste water of hospital showed resistance against
oflox,ciproflox,norflox, levoflox(ranged between 300 to 800 µg ml-1), and 128 µg ml-1 for
amikacin and 2 and 400 µg ml-1for doxycycline and clindamycin. Whereas isolate KMSI1
showed a low level of resistance to all these drugs ranged between 1.4 to 500 µg ml-1.

Table 4.11: Minimum inhibitory concentration(µg ml-1)of bacterial isolates

Name AK AMC CEFA CIP CLD DOX FAR GEN LE LZ NOR OF P

of T
isolate Z
EMPU 3.66±0.5 32±0 866.6±11 400±346 1333.3±2 0.5±0 5.6±0.5 2.6±0.5 516.6±10 933.3±2 550±50 200±50 0.46±0.0
1 77 5.4 .4 30.9 4 30.9 5
EM24 466.6±3 47.6±0.5 533.3±23 933.3±2 366.6±57. 64.3±1.5 1.8±0.2 1.5±0.0 416.6±28. 1066.6± 550±50 208.3± 1.0±0.05
05.5 0.9 30.9 7 5 8 461.8 87.7
2U 127.3±1. 47.3±1.1 266.6±11 550±217 233.3±57. 97.6±0.5 1.7±0.4 3±0 416.6±28. 400±0 666.6±2 200±50 1.46±0.0
15 5 5.4 .9 7 6 8 30.9 5
28U 266.6±1 47.3±1.1 266.6±11 333.3±1 433.3±57. 64.3±1.5 2±0 24.3±0. 466.6±11 433.3±5 550±217 166.6± 1.43±0.0
15.4 5 5.4 15.4 7 5 5.4 7.7 .9 57.7 5
BMSO 127±1.7 0.75±0 200±0 533.3±2 400±0 1.8±0.28 0.46±0. 0.46±0. 383.3±28. 1066.6± 666.6±2 193.3± 0.25±0.0
3 30. 05 05 8 230.9 30.9 110.1 05
KMSI1 0.83±0.2 22.6±2.3 133.3±57. 0.018±5. 233.3±57. 1.5±0.05 2.8±0.2 0.74±0. 383.3±76. 933.3±2 533.3±5 1.4±0.1 2±0
8 7 77E-4 7 005 3 30.9 7.7 1
KICUcr 1.43±0.1 0.56±0.0 266.6±11 0.15±0.0 200±0 1.4±0.05 3±0 0.37±0. 0.06±0.00 800±0 0.06±0.0 0.24±0. 1.46±0.0
G 1 5 5.47 05 01 1 01 005 5
KBRB1 0.75±0.0 31.3±1.1 266.6±11 0.15±0.0 133.3±57. 1.5±0 0.13±0. 0.75±0. 0.063±0.0 800±0 0.06±0.0 0.24±0. 1.46±0.0
05 5 5.4 1 7 05 01 01 01 005 5
KEMW 1.46±0.0 0.5±0 266.6±11 0.63±0.0 366.6±57. 8±0 2.6±0.5 0.74±0. 0.5±0.02 1200±0 0.06±0.0 0.063± 0.24±0.0
4 5 5.4 1 7 005 01 0.001 05
KDIF1 3±0 0.75±0.0 333.3±23 0.74±0.0 266.6±57. 246.6±5. 6±0 4±0 1.2±0.05 933.3±2 3±0 2.8±0.2 0.18±0.0
05 0.9 05 7 7 30.9 6 05

18
 OF CIP NOR LEVO AK AMC

Plate 4.5: Microphotograph of result MIC of 2Uisolate showing MIC value of above the range of disc
content 250 µg ml-1 i.e. 150,800,400,400,250,128 µg ml-1 for Oflox, ciproflox, norflox levoflox,amikacin
and amoxycillin respectively.

OF CIP NOR LEVO AK AMC

Plate 4.6: Microphotograph of result MIC of 28Uisolate showing MIC value of above the range of disc
content 250 µg ml-1 i.e. 200,200,400,400,200,48 µg ml-1 for Oflox, ciproflox, norflox levoflox,amikacin
and amoxycillin respectively.

OF CIP NOR LEVO AMC

Plate 4.7: Microphotograph of result MIC of 47 KDIF1isolate showing MIC value of above the range of
disc content i.e. 250 µg ml-1

OF CIP NOR LEVO

19
Plate 4.8: Microphotograph of result MIC of BMSO3isolate showing MIC value of above the range of
disc content of 250 µg ml-1 i.e. 300,800,400,400 µg ml-1 for Oflox, ciproflox, norflox and levoflox
respectively.

MIC value of aminoglycoside ,amikacin was in the range between 0.75 to 800 µg ml-1 that
inhibit protein synthesis. Amikacin resistance with increased MIC value have been reported by
clinical isolates conferring a high level of resistance up to 512 µg ml-1.This increased resistance
is may be due to the presence of aminogloside modifying enzymes(Kim et al.,2008).

Isolates showed extreme resistance to quinalones including Ciproflox, norflox, oflox and
levoflox, 60 % of isolates showed MIC value in between the range of 100-800 µg ml-1. Similar
results were obtained from other study where a high level of resistance was obtained for these
broad spectrum fluroquinalones which ranges from 200-300 µg ml-1 (Mandal et al.,2012).
Fluroquinalones inhibit the bacterial DNA gyrase or topoisomerase IV. Cause of the resistance to
these antibiotics may develop via mutation in bacterial chromosomal genes encoding DNA
gyrase or topoisomerase 1V or by active transport of drug out of the cell(Hooper et al.,2001).

Cefoperazone and amoxicillin have increased MIC to high level as compared to data(ranged
between 0.016- 250 µg ml-1) observed by Soebandrio et al.,(2014) against the pathogenic
bacterial isolates. Both of these drugs belong to the Beta lactam antibiotics group of which
involve in the inhibition of the synthesis of bacterial peptidoglycan cell wall(Tipper,1985).
Resistance to the β-lactam mediated by the action of β- lactamases, These enzymes destroy the
amide bond of the β-lactam ring, causes the antimicrobial to be ineffective (Strateva et al.,2009).

Molecular characterization of selected bacterial isolates:

Molecular identification of bacteria based on similarity of conserved 16SrRNA sequencing is

widely used for the characterization of bacteria and to define microbial communities(Schlaberg
et al.,2012).it used as taxonomic tool and recognize as an effective reference method for
bacterial identification(Froderick et al.,2015).the primers 27F and 1492 are widely used for this
purpose(Mohamad et al.,2013).Since the 16s rRNA is the most conserved (least variable) gene in
all cells. The ribosomes act as a work bench of the protein synthesis. Bacteria has the 70s
ribosomes made up of of two subunits smaller 30s subunit and large 50s subunit. Often present
in dissociated form, bind only during the protein synthesis. The small ribosomal subunit (30s)
contains the 16S rRNA (a most conserved gene in all). The large ribosomal subunit comprised of
two rRNA (the 5S and 23S rRNAs)(Janda et al.,2007). Gene for these three rRNA are co-
transcribed by Oberon, which may be present as a single or multiple copies in the genome of
bacteria. 16S rRNA contains around 1500 nucleotides and 23S rRNA contains around 2900
nucleotides. While 5Sr RNA comprised of 120 nucleotides and is too small for reliable
phylogenetic inference, 23S and 16S are large enough to to these functions(Reller et al.,2007).

20
For the present study 9 potential isolates(4 from hospital surfaces,3 from patients and 2 from
waste water of hospital) were assumed to be potential resistant isolates identified by 16SrRNA
sequencing.

Sequence details of 16SrRNA gene specific amplicons:

Isolate- 2U(Alcaligenes faecalis)
ATTTCCTGGCTCTGGTTACGACTTCACCCCAGTCATGAATCCCACCGTGGTAAGCGCCCTCCTTACGGTTAGGCTACCTACTTCTGG
TGAAACCCACTCCCATGGTGTGACGGGCGGTGTGTACAAGACCCGGGAACGTATTCACCGCGACATTCTGATCCGCGATTACTAG
CGATTCCGACTTCACGCAGTCGAGTTGCAGACTGCGATCCGGACTACGATCGGGTTTCTGAGATTGGCTCCCCCTCGCGGGTGGGC
GAACCCTTTGTTCCCGGAACATTGTATAAGACGTGTGAAAGCCCTACCCCATAAGGGCCACTGGAGGACTTGGACGTCATTCCCC
CACCTTCCTCCGGGTTTGTCACCGGGCAGTCTCCATAAGAGTGCTTCTTGCGGTAGCAAATAATGACAAAGGGTTGCGCTTCGGTG
GCGGGAATTAACCCAACATCTCACGACACGAGCTGACGACAGCCATGCAGCACCCGTGTTTCCGGTTCTTCTGGCGAGCACGGCC
AAATCTTCTTCGGCTTTCCAGACATGTCAAGGGTAGGTAAGGTTTTTCGCGTTGCATCGAATTAATCCACATCATCCACCGCTTGT
GCGGGTCCCCGTCAATTCCTTTGAGTCTTAATCTTGCGACCGTACTCCCCAGGCGGTCAACTTCACGCGTTAGCTGCGCTACTAAG
GCCTAACGGCCCCAACAGCTAGTTGACATCGTTTAGGGCGTGGACTACCAGGGTATCTAATCCTGTTTGCTCCCCACGCTTTCGTG
TCTGAGCGTCAGTATTATCCCAGGGGGCTGCCTTCGCCATCGGTATTCCTCCACATATCTACGCATTTCACTGCTACACGTGGAATT
CTACCCCCCTCTGACATACTCTAGCTCGGCAGTTAAAAATGCAGTTCCAAGGTTGAGCCCTGGGATTTCACATCTTTCTTTCCGAA
CCGCCTACACACGCTTTACGCCCAGTAATTCCGATTAACGCTTGCACCCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGG
TGCTTATTCTGCAGATACCGTCAGCAGCATCCCGTATTAGGGGAAGCCTTTTTCTTCTCTGCCAAAAGTACTTTACAACCCGAAAG
GCCTTCATCAAAACACGCGGGAAGGCTGGAAACAGGGTTTCCCCCATTGTCCAAAATTCCCCAATGCTGCCCTCCCCGTAAGGAA
TCTGGGGCCGGGTTCTCAGTCCCAGTGGTGGCTGGGCGTCCTCTTCAAAACAACCTAAGGAACCTTGCCCTTGGGGAACCTTTTAC
CCCACCAAAAAAGCTAATCCCAAATCTGGGCCGCCCCCAAAAGTGAAAAGGTCTTGCGAAAACCCCCCCTTTCCCCCGTAGGGCG
TATGCGGTATTAGCCACTCTTTCGAGTAGTTATCCCCCGCTACTGGGCACGTTCCGATATATTACTCACCCGTCCGCCACTCGCCAC
CAAGAGAGCAAGCTCTCTCGTGCTGCCGTTCGACTTGCATGGTAAAGCTCCCGAGCCCCTTTT
Isolate- 28U(Alcaligenes faecalis)

ACCCCCCGGCTCCTTGGTTCGACTTCACCCCAGTCATGAATCCCACCGTGGTAAGCGCCCTCCTTACGGTTAGGCTACCTACTTCT
GGTGAAACCCACTCCCATGGTGTGACGGGCGGTGTGTACAAGACCCGGGAACGTATTCACCGCGACATTCTGATCCGCGATTACA
GCGATTCCGACTTCACGCAGTCGAGTTGCAGACTGCGATCCGGACTACGATCGGGTTTCTGAGATTGGCTCCCCCTCGCGGGTTGG
CGACCCTCTGTCCCGACCATTGTATGACGTGTGAAGCCCTACCCATAAGGGCCATGAGGAATTTGGACGTCTATTCCCCCACCTTC
CTTCCGGGTTTGTCAACGGCCAGTCTTCATTTAGAGGGCTTCTTGCGGTAGCAAATAAATGAACAAGGGGTTGCGCTCGGTTGCGG
GAATTAAACCCAAAATTCTCACGGACACGAGCTTGACGACAGCCCATGCAAGCACCTTGTGTTTCCGGTTTCTCTTGCGAGCACG
GGCCAAATCTCTTTCGGCTTTCCAGACATGTCCAAGGGGTAGGTAAGGTTTTTTCGCGTTGCATCGGAATTAATCCACATCATCCA
CCGCTTGTGCGGGTCCCCCGTCAATTCCTTTGAGTTTTAATCTTGCGACCGTACTCCCCAGGCGGTCAACTTCACGCGTTAGCTGCG
CTACTAAGGCCTAACGGCCCCAACAGCTAGTTGACATCGTTTAGGGCGTGGACTACCAGGGTATCTAATCCTGTTTGCTCCCCACG
CTTTCGTGTCTGAGCGTCAGTATTATCCCAGGGGGCTGCCTTCGCCATCGGTATTCCTCCACATATCTACGCATTTCACTGCTACAC
GTGGAATTCTACCCCCCTCTGACATACTCTAGCTCGGCAGTTAAAAATGCAGTTCCAAGGTTGAGCCCTGGGATTTCACATCTTTC
TTTCCGAACCGCCTACACACGCTTTACGCCCAGTAATTCCGATTAACGCTTGCACCCTACGTATTACCGCGGCTGCTGGCACGTAA
TTAGCCGGTGCTTATTCTGCAGATACCGTCAGCAGTATCCCGTATTAGGGGATACCTTTTTCTTCTCTGCCAAAAGTACTTTACAAC
CCGAAAGGCCTTCATCAAAACACGCGGGATGGCTGGATCAGGGTTTCCCCCATTGTCCAAAATTCCCCACTGGCTGCCCTCCCGTA
AGAATCTGGGGCCGTGTCTCCAATCCCAGTGGTGGCTGGCCGTCCCCTTCAAAACAACCACGAAAACCTTGCCCTTTGGAGAGCC
TTTACCCCACCAACTAGCTAATCCGATATCGGCCGCTCCAATAGTGAGAGGTCTTGCGATCCCCCCCTTTCCCCCGTAGGGCGTAT
GCGGTATTAGCCACTCTTTCGAGTAGTTATCCCCCGCTACTGGGCACGTTCCGATATATTACTCACCCGTCCGCCACTCGCCGCCA
AGAGAGCAAGCTCTCTCGCGCTGCCGTTCGACTTGCATGGAAAGCTCCCGAGGCGCAAAACC

Isolate-EM24(Alcaligenes faecalis)

21
GGCCAGGCCCTTGGTTACGACTTCACCCCAGTCATGAATCCCACCGTGGTAAGCGCCCTCCTTACGGTTAGGCTACCTACTTCTGG
TGAAACCCACTCCCATGGTGTGACGGGCGGTGTGTACAAGACCCGGGAACGTATTCACCGCGACATTCTGATCCGCGATTACTAG
CGATTCCGACTTCACGCAGTCGAGTTGCAGACTGCGATCCGGACTACGATCGGGTTTCTGAGATTGGCTCCCCCTCGCGGGTTGGC
GACCCTCTGTCCCGACCATTGTATGACGTGTGAAGCCCTACCCATAAGGGCCATGAGGACTTGACGTCATCCCCACCTTCCTCCGG
TTTGTCACCGGCAGTCTCATTAGAGTGCTCTTGCGTAGCAACTAATGACAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCT
CACGACACGAGCTGACGACAGCCATGCAGCACCTGTGTTCCGGTTCTCTTGCGAGCACGGCCAAATCTCTTCGGCTTTCCAGACAT
GTCAAGGGTAGGTAAGGTTTTTCGCGTTGCATCGAATTAATCCACATCATCCACCGCTTGTGCGGGTCCCCGTCAATTCCTTTGAG
TTTTAATCTTGCGACCGTACTCCCCAGGCGGTCAACTTCACGCGTTAGCTGCGCTACTAAGGCCTAACGGCCCCAACAGCTAGTTG
ACATCGTTTAGGGCGTGGACTACCAGGGTATCTAATCCTGTTTGCTCCCCACGCTTTCGTGTCTGAGCGTCAGTATTATCCCAGGG
GGCTGCCTTCGCCATCGGTATTCCTCCACATATCTACGCATTTCACTGCTACACGTGGAATTCTACCCCCCTCTGACATACTCTAGC
TCGGCAGTTAAAAATGCAGTTCCAAGGTTGAGCCCTGGGATTTCACATCTTTCTTTCCGAACCGCCTACACACGCTTTACGCCCAG
TAATTCCGATTAACGCTTGCACCCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGGTGCTTATTCTGCAGATACCGTCAGC
AGTATCCCGTATTAGGGGATACCTTTTCTTCTCTGCCAAAAGTACTTTACAACCCGAAGGCCTTCATCATACACGCGGGATGGCTG
GATCAGGGTTTCCCCCATTGTCCAAAATTCCCCACTGCTGCCTCCCGAAGAAGTCTGGGCCGGGTCTCAGTCCCAGGGTGGCTGGT
CGTCCTCCCCTCAAAACAACTACGGAACGTTCCCTTGGTGAGACCTTTACCCCACCAAAAAGCTAATCCAAAATCGCCCGCCCCA
ATAAGGGAGAAGGTCTTGCGAAACCCCCCTTTCCCCCATAGGGGCATAGGCGGTAAAAACCACTCTTTAGAGAAGTTATCCCCCC
GCCACGGGGCACGCCCCAATAAATTACGCACCCGTCCGCCACTCGCCGCCAAGAGAGCAAGCTCTCTCGCGCTGCCGTTCGACTT
GCATGTTAAAGCTCCCGAAGCCCC

Isolate-47KDIF1(Alcaligenes faecalis)

GGGGCGGCTCCTGGTTCGACTTCACCCCAGTCATGAATCCCACCGTGGTAAGCGCCCTCCTTACGGTTAGGCTACCTACTTCTGGT
GAAACCCACTCCCATGGTGTGACGGGCGGTGTGTACAAGACCCGGGAACGTATTCACCGCGACATTCTGATCCGCGATTACTAGC
GATTCCGACTTCACGCAGTCGAGTTGCAGACTGCGATCCGGACTACGATCGGGTTTCTGAGATTGGCTCCCCCTCGCGGGTTGGCG
ACCCTCTGTCCCGACCATTGTATGACGTGTGAAGCCCTACCCATAAGGGCCATGAGGACTTGACGTCATCCCCACCTTCCTCCGGT
TTGTCACCGGCAGTCTCATTAGAGTGCTCTTGCGTAGCAACTAATGACAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTC
ACGACACGAGCTGACGACAGCCATGCAGCACCTGTGTTCCGGTTCTCTTGCGAGCACGGCCAAATCTCTTCGGCTTTCCAGACATG
TCAAGGGTAGGTAAGGTTTTTCGCGTTGCATCGAATTAATCCACATCATCCACCGCTTGTGCGGGTTCCCCGTCAATTCCTCTGAG
TTTTAATCTTGCGACCGTACTCCCCAGGCGGTCAACTTCACGCGTTAGCTGCGCTAATAAGGCCTAACGGCCCCAACAGCTAGTTG
ACATCGTTTAGGGCGTGGACTACCAGGGTATCTAATCCTGTTTGCTCCCCACGCTTTCGTGTCTGAGCGTCAGTATTATCCCAGGG
GGCTGCCTTCGCCATCGGTATTCCTCCACATATCTACGCATTTCACTGCTACACGTGGAATTCTACCCCCCTCTGACATACTCTAGC
TCGGCAGTTAAAAATGCAGTTCCAAGGTTGAGCCCTGGGATTTCACATCTTTCTTTCCGAACCGCCTACACACGCTTTACGCCCAG
TAATTCCGATTAACGCTTGCACCCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGGTGCTTATTCTGCAGATACCGTCAGC
AGTATCCCGTATTAGGGGATACCTTTTCTTCTCTGCCAAAAGTACTTTACAACCCGAAGGCCTTCATCAAACACGCGGGAAGGCTG
GAACCAGGGTTTCCCCCATTGTCCAAAATTCCCCACTGCTGCCTCCCCTAAGAATCGGGGCCCGTGTTCCCAGTCCCAGGGTGGGC
TGGTCCTCCCCTCAAAACAACTAAGGAATCGTTCCCTTGGTGGAGGCCTTTACCCCACCAAAAAACTTAACCCAATATCGGCCCCT
CCAAATAGTGAGAGGGTCTTGCGAACCCCCCCTTTTCCCCCGGAGGGCCGAACGCGGAATAACCCACTCTTTCAAGAAATTATCC
CCCCGCTACTGGGGGGGCACGTCCCAAAATATTAAAACTCACCGGCGTTCCGCCACTCGCCGCCAAGAGAGCAAGCTCTCTCGCG
CTGCCGTTCGACTTGCATGTAAAGCTCCCGATGCCCC

Isolate-KICUcrG(Enterobacter bugandensis)

GGGGCTGGTTCCTTGTTCGACTTCACCCCAGTCATGAATCACAAAGTGGTAAGCGCCCTCCCGAAGGTTAAGCTACCTACTTCTTT
TGCAACCCACTCCCATGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGTGGCATTCTGATCCACGATTACTAG
CGATTCCGACTTCATGGAGTCGAGTTGCAGACTCCAATCCGGACTACGACGCACTTTATGAGGTCCGCTTGCTCTCGCGAGGTCGC
TTCTCTTTGTATGCGCCATTGTAGCACGTGTGTAGCCCTACTCGTAAGGGCCATGATGACTTGACGTCATCCCCACCTTCCTCCAGT
TTATCACTGGCAGTCTCCTTTGAGTTCCCGGCCTAACCGCTGGCAACAAAGGATAAGGGTTGCGCTCGTTGCGGGACTTAACCCAA
CATTTCACAACACGAGCTGACGACAGCCATGCAGCACCTGTTCTCAGAGTTCCCGAAGGCACCAAAGCATCTCTGCTAAGTTCTCT

22
GGATGTCAAGAGTAGGTAAGGTTCTTCGCGTTGCATAGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCATT
TGAGTTTTAACCTTGCGGCCGTACTCCCCAGGCGGTCGACTTAACGCGTTAGCTCCGGAAGCCACGCCTCAAGGGCACAACCTCC
AAGTCGACATCGTTTACGGCGTGGACTACCAGGGTATCTAATCCTGTGTGCTCCACACGCTTTCGCACCTGAGCGTCAGTCTTTGT
CCAGGGGGCCGCCTTCGCCACCGGTATTCCTCCAGATCTCTACGCATTTCACCGCTACACCTGGAATTCTACCCCCCTCTACAAGA
CTCTAGCCTGCCAGTTTCGAATGCAGTTCCCAGGTTGAGCCCGGGGATTTCACATCCGACTTGACAGACCGCCTGCGTGCGCTTTA
CGCCCAGTAATTCCGATTAACGCTTGCACCCTCCGTATTACCGCGGCTGCTGGCACGGAGTTAGCCGGTGCTTCTTCTGCGGGTAA
CGTCAATCGACAAGGTTATTAACCTTAACGCCTTCCTCCCCGCTGAAAGTACTTTACAAACCCGAAAGGCCTTCTTCATACACGCG
GCATGGCTGCATCAGGCTTGCCCCCATTGTGCAAAATTCCCCACTGGCTGCCTCCCGTAAGAATCCGGGACCGGGTCTCAGTTCCA
GGGGGGGCTGGGTCATCCTCCCAAACAAACTAGGGAACGCCCCCCTAGGGGAACCCTTACCCCACCCCAAAAACTAAATCCCATC
TTGGGCACAATCTGAAGGCAAAAAGCCCCAAAAGGCCCCCCCTCTTTGGTCTTGCCAACCTTATGCGGGAAAAGAAACCGCTTCC
CAAAAATTATCCCCCTCCATCAGGCAGTTTCCCAGACATTACTCACCCGTCCGCCACTCGTCACCCGAGAGCAAGCTCTCTGTGCT
ACCGTTCGACTTGCATGGTAGGCCCCCCCCGCGCC

Isolate-KBRB1(Eschrichia coli )

GGCGGGGTCTTGGTTCGACTTCACCCCAGTCATGAATCACAAAGTGGTAAGCGCCCTCCCGAAGGTTAAGCTACCTACTTCTTTTG
CAACCCACTCCCATGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGTGGCATTCTGATCCACGATTACTAGCG
ATTCCGACTTCATGGAGGCGAGTGGCAGACTCCAATCCGGACTACGAGCCCACTTTATGAGGTCCGCTTGCTCTCGCGAGGTCGCT
TCTCTTTGTATGCGGCCATTGTAGCACGTGGGTAGCCCCTGGTCGTAAGGGCCATGATGACTTGACGTCATCCCCCACCTTCCTCC
AGTTTATCACTGGCAGTCTCCTTTGAGTTCCCGGCCGGACCGCTGGCAACAAAGGATAAGGGTTGCGCTCGTTGCGGGACTTAACC
CAACATTTCACAACACGAGCTGACGACAGCCATGCAGCACCTGTCTCACAGTTCCCGAAGGCACAATTCCATCTCTGGAAAAATT
TCTGTGGATGTCAAGACCAGGTAAGGTTCTTCGCGTTGCATCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAAT
TCATTTGAGTTTTAACCTTGCGGCCGTACTCCCCAGGCGGTCGACTTAACGCGTTAGCTCCGGAAGCCACGCCTCAAGGGCACAAC
CTCCAAGTCGACATCGTTTACGGCGTGGACTACCAGGGTATCTAATCCTGTTTGCTCCCCACGCTTTCGCACCTGAGCGTCAGTCT
TCGTCCAGGGGGCCGCCTTCGCCACCGGTATTCCTCCAGATCTCTACGCATTTCACCGCTACACCTGGAATTCTACCCCCCTCTAC
GAGACTCAAGCTTGCCAGTATCAGATGCAGTTCCCAGGTTGAGCCCGGGGATTTCACATCTGACTTAACAAACCGCCTGCGTGCG
CTTTACGCCCAGTAATTCCGATTAACGCTTGCACCCTCCGTATTACCGCGGCTGCTGGCACGGAGTTAGCCGGTGCTTCTTCTGCG
GGTAACGTCAATGAGCAAAGGTATTAACTTTACTCCCTTCCTCCCCGCCTGAAAGTACTTTACAACCCGAAAGGCCTTCTTCATAC
ACGCGGCAGGGCTGCATCAGGCTTGGCCCCCATTGGGCAATATTCCCCAATGGCTGCCTCCCGTAAGAATCCGGGACCGGGTCCC
AATTTCCAGGGGGGCTGGGCCATCCCCTCAAGACAAGCTTAGGAAACGTCCCCCTTGGGGAACCCTTACCCCACCCAAAAAGCTT
AATCCCATCTGGGCACATTCCAAAGGCAAGAAGGCCCAAAGGGCCCCCCCCTTTTGGTCTTGGCCACCTTATGCGGGAAAAAACC
ACCCTTTTCCAAAAATTTACCCCCCCCCATCAAGGCAGTTTTCCCCAAACATTACTTCACCCGTCCGCCACTCGTCAGCAAAGAAG
CAAGCTTCTTCCTGTTACCGTTCGACTTGCATGGTAGGCCTGCCCCCCCCACC

Isolate-KEMW4(Enterobacter bugandensis)

GGGGCTGGTTCCTTGGTTCGACTTCACCCCAGTCATGAATCACAAAGTGGTAAGCGCCCTCCCGAAGGTTAAGCTACCTACTTCTT
TTGCAACCCACTCCCATGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGTGGCATTCTGATCCACGATTACTA
GCGATTCCGACTTCATGGAGTCGAGTTGCAGACTCCAATCCGGACTACGACGCACTTTATGAGGTCCGCTTGCTCTCGCGAGGTCG
CTTCTCTTTGTATGCGCCATTGTAGCACGTGTGTAGCCCCTACTCGGAAAGGCCACGGAGGACTTGACGTCATTCCCCACCTTCCT
CCAGGTTATTCACTGGCAGTCTCCTTTGAGTTCCCGGCCTAACCGCTGGCAACAAAAGGATAAGGGTTGCGCTCGTTGCGGGAATT
AACCCAACATTTCACAACACGAGCTGACGACAGCCATGCAGCACCTGTCTCAGAGTTCCCGAAGGCACCAAAGCATCTCTGCAAA
GTTCTCTGGATGTCAAGAGTAGGTAAGGTTCTTCGCGTTGCATCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCA
ATTCATTTGAGTTTTAACCTTGCGGCCGTACTCCCCAGGCGGTCGACTTAACGCGTTAGCTCCGGAAGCCACGCCTCAAGGGCACA
ACCTCCAAGTCGACATCGTTTACGGCGTGGACTACCAGGGTATCTAATCCTGTTTGCTCCACACGCTTTCGCACCTGAGCGTCAGT
CTTTGTCCAGGGGGCCGCCTTCGCCACCGGTATTCCTCCAGATCTCTACGCATTTCACCGCTACACCTGGAATTCTACCCCCCTCTA
CAAGACTCTAGCCTGCCAGTTTCGAATGCAGTTCCCAGGTTGAGCCCGGGGATTTCACATCCGACTTGACAGACCGCCTGCGTGC
GCTTTACGCCCAGTAATTCCGATTAACGCTTGCACCCTCCGTATTACCGCGGCTGCTGGCACGGAGTTAGCCGGTGCTTCTTCTGC

23
GGGTAACGTCAATCGACAAGGTTATTAACCTTAACGCCTTCCTCCCCGCTGAAAGTACTTTACAACCCGGAAGGCCTTCTTCATAC
ACGCGGCATGGCTGCATCAGGCTTGCCCCCCATTGGGCAATATTCCCCACTGGCTGCCCCCCGTAGGGAATCTGGAACCGGGTCT
CCAATTTCCAGTGGGGCCTGGGCATCCCCTTCAAACCACCTAGGGAACCTCCCCCAAGGTGAGCCCTTACCCCACCCCAAAAAAT
TAATCCCATCTGGGGGCACATTCTGAAGGCAAAAAGCCCCAAAGGCCCCCCCCTTTTGGCCTTGCCACCCTTAGGCGGGATTAAA
CAACCCGTTTTCCAAGTAGTTATCCCCCTCCATCAGGCAGTTTCCCAGACATTACTCACCCGTCCGCCACTCGTCACCCGAGAGCA
AGCTCTCTGTGCTACCGTTCGACTTGCATGGTAGGCCGCCCCCCCCC

Isolate-BMSO3(Klebsiella aerogenes)

AAACGGGCCGGTTCCTTGGTTACGACTTCACCCCAGTCATGAATCACAAAGTGGTAAGCGCCCTCCCGAAGGTTAAGCTACCTAC
TTCTTTTGCAACCCACTCCCATGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGTGGCATTCTGATCCACGATT
ACTAGCGATTCCGACTTCATGGAGTCGAGTTGCAGACTCCAATCCGGACTACGACGCACTTTATGAGGTCCGCTTGCTCTCGCGAG
GTCGCTTCTCTTTGTATGCGCCATTGTAGCACGTGTGTAGCCCTACTCGTAAGGGCCATGATGACTTGACGTCATCCCCACCTTCCC
CAGTTTATCACTGGCAGTCTCCTTTGAGTTCCCGGCCTAACCGCTGGCAACAAAGGATAAGGGTTGCGCTCGTTGCGGGACTTAAC
CAACATTTCACAACACGAGCTGACGACAGCCATGCAGCACCTGTCTCAGAGTTCCCGAAGGCACCAAAGCATCTCTGCTAAGTTC
TCTGGATGTCAAGAGTAGGTAAGGTTCTTCGCGTTGCATCGAATTAAACCACATGCTCCCACCGCTTGTGCGGGCCCCCGTCAATT
CATTTGAGTTTTAACCTTGCGGCCGTACTCCCCAGGCGGTCGACTTAACGCGTTAGCCCGGAAGCCACGCCTCAAGGGCACAACC
TCCAAGTCGACATCGTTTACGGCGTGGACTACCAGGGTATCTAATCCTGTTTGCTCCCCACGCTTTCGCACCTGAGCGTCAGTCTTT
GTCCAGGGGGCCGCCTTCGCCACCGGTATTCCTCCAGATCTCTACGCATTTCACCGCTACACCTGGAATTCTACCCCCCTCTACAA
GACTCTAGCCTGCCAGTTTCGAATGCAGTTCCCAGGTTGAGCCCGGGGATTTCACATCCGACTTGACAGACCGCCTGCGTGCGCTT
TACGCCCAGTAATTCCGATTAACGCTTGCACCCTCCGTATTACCGCGGCTGCTGGCACGGAATTAGCCGGTGCTTCTTCTGCGGGT
AACGTCAATCGACAAAGGTTAATAAACCTTATCGCCTTCCCTCCCCGCTGAAAGTACTTTAACAACCCGAAAGGCCTTTCTTCAAA
AACGCGGGCATGGCTGGCATCAAGCTTGCGCCCCATTGGGGCAAAAATTTCCCCACATGGCTGCCTCCCCGTAAGGAAAGTCGGG
GACCGGGTCTTCAATTTCCAGGTGGGGGCCTGGGCATCCCCCCCCCAAAACCACCTAAGGGAATCGTCCCCCCAAGGGGGAAGCC
CTTATCCCCACCCCAACAAAATATATTCCCATCTTTGGGCACATCCGATGGCAAGAGGCCCGAAGGTCCCCCTCTTTGGTCTTGCG
ACGTTATGCGGTATTAGCTACCGTTTCCAGTAGTTATCCCCCTCCATCAGGCAGTTTCCCAGACATTACTCACCCGTCCGCCACTCG
TCAGCAAAGCAGCAAGCTGCTTCCTGTTACCGTTCGACTTGCATGGTAGGCTCCCCCCCACCCATTGGTA

Isolate-KMSI1(Eschrichia coli)

GGGGCTGGTTCCTTGTTCGACTTCACCCCAGTCATGAATCACAAAGTGGTAAGCGCCCTCCCGAAGGTTAAGCTACCTACTTCTTT
TGCAACCCACTCCCATGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGTGGCATTCTGATCCACGATTACTAG
CGATTCCGACTTCATGGAGTCGAGTTGCAGACTCCAATCCGGACTACGACGCACTTTATGAGGTCCGCTTGCTCTCGCGAGGTCGC
TTCTCTTTGTATGCGCCATTGTAGCACGTGTGTAGCCCTGGTCGTAAGGGCCATGATGACTTGACGTCATCCCCACCTTCCTCCAGT
TTATCACTGGCAGTCTCCTTTGAGTTCCCGGCCGGACCGCTGGCAACAAAGGATAAGGGTTGCGCTCGTTGCGGGACTTAACCCA
ACATTCTCACAACACGGAGCTGACGAAAGCCATGCAGCACCTGTCTCACGGTTCCCGAAGGCACATTATCATCTATGAAAAATTC
CGTGGATGTCAAGACCAGGTAAGGTTCTTCGCGTTGCATCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTC
ATTTGAGTTTTAACCTTGCGGCCGTACTCCCCAGGCGGTCGACTTAACGCGTTAGCTCCGGAAGCCACGCCTCAAGGGCACAACCT
CCAAGTCGACATCGTTTACGGCGTGGACTACCAGGGTATCTAATCCTGTTTGCTCCCCACGCTTTCGCACCTGAGCGTCAGTCTTC
GTCCAGGGGGCCGCCTTCGCCACCGGTATTCCTCCAGATCTCTACGCATTTCACCGCTACACCTGGAATTCTACCCCCCTCTACGA
GACTCAAGCTTGCCAGTATCAGATGCAGTTCCCAGGTTGAGCCCGGGGATTTCACATCTGACTTAACAAACCGCCTGCGTGCGCTT
TACGCCCAGTAATTCCGATTAACGCTTGCACCCTCCGTATTACCGCGGCTGCTGGCACGGAGTTAGCCGGTGCTTCTTCTGCGGGT
AACGTCAATGAACAAAGGTATTAACTTTACTCCCTTCCTCCCCGCTGAAAGTACTTTACAACCCGAAGGCCTTCTTCATACACGCC
GGCATGGCTGCATCAGGCTTGCGCCCATTGTGCAAAATTCCCCAATGCTGCCCTCCCGTAGGAATTCGGGACCGGGTCCCAATTCC
AGGGGGGCGGGGCCACCCCCCCAGAACCACCTAGGGATCGTCCCCCCTAGGGGAACCCTTACCCCACCCAAAAAAAAAAACCCC
ATCTGGGGGCACATCCAAAGGCGAAAAGGCCCAAAAGGCCCCCCCCTTTGGTCTTGCGACCCTTATGCGGGAATAAAAACCCTTT
TCCAAAAAATTACCCCCCCCCATCAGGCAGGTTTTCCCAAAAATTAAACTCGAGGGGCCCGATCCGCCACTCGTCAGCGAATGCA
GCAAGCTGCTTCCTGTTACCGTTCGACTTGCATGGTAGGCCTGCCCCCTCCCC

24
 Table 4.12: BLAST homology search results of 16 SrRNA sequences of potential resistance bacterial
isolates:

S.No. Sourse Code Interpreted Query identity E Reference Accession

bacteria coverage value sequence
ID
1. Hospitalize 2U* Alcaligenes 97 94.18 0 NBRC131 MK595695
d Patients faecalis 11

2. Hospitalize 28U* Alcaligenes 97 95.2 0 NBRC131 MK595711

d Patients
faecalis 11

3. Hospitalize EM24 Alcaligenes 98 96 0 NBRC1311 MK595710

d Patients
faecalis 1

4. Hospital 47KDIF1* Alcaligenes 98 95 0 NBRC131 MK595696

surfaces faecalis 11

5. Hospital KICU crG Enterobacter 98 95.15 0 ATCC3382 MK615943

surfaces
bugandensis 1
6. Hospital KBRB1 Eschrichia sp 98 94 0 NBRC1021 MK616099
surfaces
03
7. Hospital KEMW4 Enterobacter 99 94.95 0 KCTC2190 MK615942
surfaces
sp
8. Hospital BMSO3* Enterobacter 98 95 0 KCTC129 MK616100
waste xiangfangensis 0
water
9. Hospital KMSI1 Eschrichia sp 98 94.15 0 NBRC1022 MK436318
waste water 03

* Resistance phenotype based on screening by continieous subculturing up to 5th generation in

presence of antibiotis(Fluroquinalone,beta lactams and lincosamide).

Surface isolate 47KDIF1, patient isolates 2U,28U,EM24 were identified as Alcaligenes fecalis
with94.18,95.2,96,95% similarity of sequences through BLAST analysis and confirmed with the
aid of biochemical characterization. Alcaligenes fecalis is an opportunistic pathogen responsible
for serious infections (Kavuncuoglu et al., 2010);(Chandhuri, 1967) and related to the
contamination of hospital equipment or fluids, with resulting human colonization or

25
infection(Mandal et al.,2000,Almuzara et al.,2010). Momtaz et al (2018) reported these bacteria
as a causative agent of UTI. Wang et al (2010) reported it as a nosocomial pathogen involve in
various surgical site infections.

Surface isolate KEMW4 were identified as Enterobacter sp and KICUcrG as Enterobacter

bugandensis based on 94 % ,95.18% similarity with the species through BLAST search. The
bacterium is non-motile, rod-shaped, Gram-negative, and becomes an important pathogen
involve in different infection in hospital settings (Woldu et al.,2015). The organism found in
environment, food and and intestine of human and animal.the organism cause opportunistic
infection in immune-compromised patients. Godic et al (2016), reported multiple drug resistant
Enterobacter isolated from surfaces of hospital and was found to be resistant to beta lastam,
fluroquinalone.
Based on 94% similarity score KBRB1(surface isolate) and KMSI1(Waste water isolate) were
identified as Escherichia fergusonii and Escherichia coli.
Screening of bacterial isolates:
Pattern of bacterial resistance to antibiotics:

For further screening of bacterial isolates gradient plate method was performed. In the presence
of a gradient of antibiotics in sub inhibitory concentration only resistant bacteria allow to
survive. In this part of the work we allow bacteria to grow up to the 5th generation in gradient
plate containing sub inhibitory concentrations of different antibiotic. Between 9 primarily
screened bacteria, only 4 resistance, bacterial isolates (KDIF1, KM2U, KM28U, BMSO3) were
showing positive growth on plate containing gradient ranging from 50-200 µg ml-1 after
continuous sub culturing and were selected to further plasmid curing and gene identification
through PCR.

Plasmid curing: Finings from the plasmid curing analysis suggest that after the treatment of
acridine orange, all the isolates were revert back to the sensitive form to ciproflox, levoflox and
oflox and cefaperazone (Table 4.13).
3 strains of Alcaligenes faecalis and 1, Enterobacter sp which showed growth in plate containing
sub inhibitory concentrations of antibiotic(ciproflox,levoflox,oflox, amoxicillin,cefaperazone
and linazolid) up to the fifth generation, present an evidence for the genetic marker either present
on genomic DNA or plasmid DNA. For this purpose sub lethal concentrations of the acridine
orange were determined for the strain of Alcaligenes faecalis (KDIF1,KM2U,KM28U) and 1
Enterobacter sp(BMSO3). The highest concentration at which detectable growth of these
bacteria were observed were defined as sublethal concentration. Growth in the presence of
acridine dyes involves loss of the whole plasmid due to a selective interference of acridine
orange (Salisbury et al.,1972,Mobarhan et al.,2007). As a consequence of the loss of these
plasmids, the cured derivatives became sensitive to antibiotics suggesting that antibiotic
determinants were plasmid-borne. The sub lethal concentration for acridene orange were in the
range of 350 to 400 µg ml-1. Warjri et al.,2015 observed a high level of sub inhibitory
concentration which were in the range of 2 to 2.5 mg ml-1.a significant shift was observed from
resistance to susceptibility(Table 4.13 The plasmid profile studies showed plasmid sizes ranging
from 1-10 kb in the isolated resistant strains (fig 4.5).

26
 1 2 3 4 M

12000 bp 10000 bp

200 bp

Fig 4.5:Plasmid DNA of multidrug resistant bacterial isolates analyzed with 0.8 % agarose gel
electrophoresis stained with ethidium bromide. M is 200bp – 10000bp DNA ladder (molecular maker).
Lane1,2,3,4 indicate.KM2U,KM28U,KDI47,BMSO3

27
 )indicating that most of the isolates had plasmids bearing multiple resistance determinants for
three antibiotic classes includes three antibiotic classes such as quinolone,beta lactams and
cephalosporin on them. For the fluoroquinolones, two isolates KDIF1 and KM2U were cured
and became sensitive to the ciproflox,levoflox and oflox, but the isolates BMSO3 and KM28U
which were sensitive to levoflox remained sensitive. For the amoxycillin , all 4 isolates remained
resistant.for the cefaperazone and linazolid all the isolates become sensitive after the curing
experiment.
Table 4.13:MIC(µg ml-1)value of different antibiotics for selected resistant isolates before and after
curing with acridine orange

Group of Antibiotics Alcaligenes Alcaligenes Enterobacter Alcaligenes

antibiotics faecalis 47 faecalis 2U xiangfangensis faecalis 28U
BMSO3
Before After Before After Before After Before After
curing curing curing curing curing curing curing curing
Fluroquinalone Ciproflox 800 160 60 10 30 25 150 150
Levoflox 100 10 50 10 1.3 1.3 0.064 0.064
Oflox 200 20 200 20 80 50 80 0.5
Beta lactam Amoxycillin 16 16 0.6 0.6 0.5 0.5 32 32
Cefaperazone 600 50 800 5 210 180 350 60
lincosamide Clindamycin 1000 200 800 2 1000 1000 800 100

From selected multidrug resistant strains 3(KM2U,KM28U,KDIF1) contained plasmids of 10000bp. To

understand the nature of antibiotic resistance, a plasmid curing experiment was performed which
revealed it was plasmid mediated. Several studies showed that the multi-drug resistant in clinical
and environmental isolates were plasmid mediated (Huang et al.,2012;).Plasmids are the extra
chromosomal element which are capable of self replication(Mbim et al.,2016).plasmid curing
experiment is used to determine the presence of gene in plasmid(Kamruzzaman et
al.,2017),while the plasmid profile analysis used to determining size and number of plasmids in
bacterial isolates(Pattanail et al.,2013).in our study plasmid curing experiment showed the
marker for the resistance were present on the plasmid, furthermore plasmid analysis showed that
all selected resistant bacteria have the plasmid ranges from 1 kb to 10 kb.

Identification of resistance gene on plasmid: in our study selected bacterial isolates were
showed resistance for 4 different class of antibiotics including Fluroquinalone,beta latams and
lincoasamide,for this reason we selected 6 primer for these 3 class of antibiotics(qep,gyr A,qnr
B,qnrS for Fluroquinalone- ibcr6 for brta lactams and cfr gene for lincosamide and
clindamycine).
Out of 6 selected genes only 3 gene were detected in selected resistant isolates. The primer use
for amplification of qnr B, qep, aac-ibcr gene yielded band of approximately 488,596,482 bp
respectively. No band was observed for cfr gene.

28
Identification of resistance gene through PCR amplification showed that, 3 Alcaligenes
faecalis(47KDIF1,2U,28U) harbored the qep A gene.This gene is responsible for the over
expression of Qep A efflux pump which was firstly discovered in 2007 in two E. coli clinical
isolates from Japan and Belgium(Robicsek et al.,2007;Yamane et al.,2007). The finding of qep
gene in these isolates is consistent with reports of Oteo et al.,(2016) and Amaya et al.,(2017)
who revealed that 30.8% and 26% of Alcaligenes faecalis.qep gene in recent years has been
implicated in plasmid mediated fluroquinalone resistance.3 Alcaligenes
faecalis(47KDIF1,2U,28U) harbored the qnrB genes Qnr protein bind to the topoisomerase,
which physically prevents the intercalation of the antibiotic with the target enzyme(Xiong et
al.,2011).cfr gene was present in Alcaligenes faecalis(47KDIF1,2U) and
Enterobacter(BMSO3).none of the isolate showed the presence of qnr S and aac6ibcr gene.Since
the gene for the resistance are located mainly on plasmids and thus have the potential to spread
their resistance to other environmental bacteria.

29
 1 2 3 4 5 6 7 8 9 10 11 12 13 M

565 bp(qep gene)

3000bp

485 bp(qnr B gene) 500 bp

100 bp

Fig 4.6: PCR amplification of the qnr genes using the primers qnrS,qepA, qnrB (C), M is 100bp – 3000bp
DNA ladder (molecular maker). Lane1,2,3,4,5,6,7,8,9,10,11,12,13indicate.qnrS-2U,28U,47,BM,qep-
2U,28U,47,BM,qnr 2U,28U,47,BM respectively.

M 1 2 3 4 5 6 7 8 9 10 11 12 13 14

3000 bp

482bp(aacibcrgene
500 bp
)
100 bp

Fig 4.7: Fig 6: PCR amplification of the gyr(Lane 2-5), aacib-cr gene (Lane6-9) and cfr gene (10-14) M is
100bp – 3000bp DNA ladder (molecular maker). Lane1,2,3,4,5,6,7,8,9,10,11,12,13indicate.qnrS-
2U,28U,47,BM,qep-2U,28U,47,BM,qnr 2U,28U,47,BM respectively

30
Study of whole cell protein profiles of resistant isolates through SDS-PAGE
SDS-PAGE is currently one of the most commonly used techniques for the characterization and
analysis of proteins and it has been used as a taxonomic tool for identification of various
bacterial species and yielding valuable information on the similarity and dissimilarity amongst
bacterial cultures (Chung, 1987). The polyacrylamide gel electrophoresis (PAGE) of protein
analysis have been used widely in typing of many bacterial strains. Protein patterns offer
considerable potential for typing bacterial strains of clinical interest, especially for species with
other typing methods are not available (Holmes et al., 1991; Malik et al., 2003). In the present
study, protein profiles were very similar and characteristic among the isolates of each group of
microorganisms and several isolates exhibited characteristic proteins that may be useful markers
for epidemiological investigation. SDS-PAGE of total cell protein extracts of 4 tested resistant
isolates treated with different antibiotics(Ciproflox,amoxicillin and cindamycin) produced
characteristic patterns containing about 14 discrete bands with molecular weightsin the range
from 11to 245 KDa estimated by polyacrylamide gel electrophoresis. The patterns among all
tested isolates were nearly the same; however, few differences were observed. In the present
study, three different total cell protein patterns were detected by SDS-PAGE (Figure 4.8 & 4.9).
The first pattern was represented by isolates trested with amoxycillin(KM2U,KM28U,KDIF1)
the second pattern was represented by 3isolates isolates trested with ciproflox
(KM2U,KM28U,KDIF1).and the third pattern was represented by 3 isolates isolates trested with
clindamycin (KM2U,KM28U,KDIF1) .In pattern one 14 bands were observed ranges from 1 1 to
63 kDa.pattern 3 represent 10 bands with molecular weight of 11 to 254 kda,11 band were
observed in pattern for, molecular weight of the band were ranging between 11 kDa to 245 kDa.
The main difference between the five total cell protein patterns were the bands at about 63 and
20 KDa.

31
 1 2 3 4 5 6 7 8 M

245 kDa

30 kDa

25kDa

11kDa

Fig 4.8:SDS page result analysis of selected resistant bacterial isolates M is 11 to 245kDa protein
marker.Lane1,2,3,,4,5,6,7,,8indicate treated strain BMSO3,KM2U,KM28U,KDIF1 with clindamycin,
,5,6,7,,8indicate treated strain BMSO3,KM2U,KM28U,KDIF1 with ciproflox

M S 1 2 3 4

245kDa

35 kDa

25 kDa

11 kDa

Fig 4.9:SDS page result analysis of selected resistant bacterial isolates M is 11 to 245kDa protein
marker.Lane1,2,3,,4 indicate treated strain KM2U,KM28U,KDIF1,BMSO3, with amoxycillin

32
 CONCLUSIONS AND FUTURE PROSPECTIVES
Present work enables us to understand the evolution and status of antibiotic resistance among
different clinical environment evaluation. the study present the phenotypic and genotypic (with
special emphasis on plasmid-bearing resistance) differences among the different species of
bacteria from clinical care settings. The highlights of this study are listed below:

A total of 143 isolates including 103 isolates from hospital surfaces,23 isolates from hospital
waste water and 20 isolates from the hospital patients, were analyzed initially for their resistance
against 16 different antibiotics. The β-lactam
antibiotic ampicillin, combination antibiotic piperacillin/tazobactam, 3rd
generation cephalosporins (CTX and CAZ), and the 1st and 2nd generation
quinolones (NA and CIP) were found to be ineffective for these isolates as almost
all of them were found to be resistant to these antibiotics. A relatively low
resistance was exhibited against AZM, GEN, MRP and C. Among this C was

Resistance to multiple antibiotics was also found to be prominent among the

isolates. Among E.coli, 33 (75%) were found to be MDR while 36 (88%)
Klebsiella isolates were MDR. Five E.coli (11%) and 18 (44%) Klebsiella spp.
were found to be resistant to all the 10 antibiotics (MAR index 1). Plasmids were
obtained from a total of 54 (63.5%) in which 25 (57%) E.coli and 29 (71%)
Klebsiella were included. An inspection of the plasmid bearing and plasmid
negative isolates with the highest MAR index of 1 revealed a significant
association between multiple antibiotic resistance and presence of plasmid in
Klebsiella spp.(P<0.01).
Phenotypic and genotypic variability among the isolates was analysed by
correlating the various antibiotic resistance phenotypes with the plasmid profile
types and also by constructing an RAPD dendrogram. By careful analysis of the
antibiotic resistance phenotypes and plasmid profiles, a total of 16 types of

33
resistance phenotypes and 31 types of plasmid profiles were assigned. A comparison of these
two parameters clearly revealed similarities in the antibiotic
resistance phenotypes of isolates exhibiting similar plasmid profiles. Analysis of
the distribution of isolates in various plasmid profile types also clearly showed
the plasmid variability in Klebsiella spp. wherein only 6 (21%) were included
under shared profile type (a) compared to 13 (52%) E.coli isolates (P<0.05).
Also, in case of E.coli, similarities were also apparent between the sub-types of
shared plasmid profile type (a) which was not observed amongst Klebsiella spp.
Similarly, 9 (36%) E.coli and 13 (45%) Klebsiella spp. were found to exhibit
unique profiles. RAPD analysis also revealed a similar phenomenon in which
most of the clonally related E.coli isolates exhibited similarities in terms of their
area of collection, antibiotic resistant phenotypes and plasmid profile type
whereas these were not evident among Klebsiella spp. From the dendrogram, the
genotypic similarities among E.coli isolates were apparent - 18 different clones
were obtained in dendrogram consisting of 25 isolates. In contrast, the genetic
unrelatedness was obvious amongst Klebsiella, since 27 different clones were
found to exist in the dendrogram comprising of 29 isolates (P<0.05) with low
similarities among isolates belonging to the same cluster.
Further phenotypic and genotypic characterizations involved checking for presence of different
classes of integrons, β- lactamase production and biofilm
formation. Among the three classes of integrons screened by PCR, class 1 was
found to be the predominant one followed by class 2 (P<0.01). Class 3 integron
was found to be totally absent. Interestingly, the sequence results of a class 2
integron showed similarity with mgrB gene disrupted with IS5 like element,
which was an indication of acquired colistin resistance in that isolate. This
unexpected finding is indeed a warning for the emergence of Klebsiella spp.
resistant to the „last resort‟ antibiotics.
Beta-lactamase production was also found to be predominant with ESBL
production being more prominent among E.coli (all the isolates were ESBL
producers) (P<0.01) than Klebsiella spp., which were comparatively higher MBL
producers. blaNDM-1 was amplified from four E.coli and five Klebsiella spp. The phylogenetic
analysis of this particular gene sequence showed close
relationship within the samples under study and between those from other states
and countries - a clear indication of horizontal exchange of the gene under study.
E.coli isolates also exhibited a prominent ability to form biofilms in which most
of them were strong biofilm producers compared to Klebsiella spp. Also, curli
fimbriae, reportedly associated significantly with biofilm formation in E.coli and
Salmonella spp., was found to be amplified from all the E.coli isolates under
study (P<0.01). On the otherhand, presence of type3 fimbriae reported to play
significant role in biofilm formation in Klebsiella spp., was found to exhibit an
inverse relation with the ability of the isolates to form biofilms. Thus this study
concluded that, it is the fimbrial expression rather than the mere presence of the
gene, which play a decisive role in the ability of the isolate to form biofilms.
Plasmid replicon typing performed on selected Klebsiella spp. revealed a
prevalence of IncFIIk followed by IncR replicons and that most of these existed
in a multireplicon state. IncFIIk again was found to be present in all the isolates

34
which carried blaNDM-1 gene. These observations are indicative of a
challenging situation since these plasmids are equipped with the inbuilt ability of their stable
maintenance (addiction systems) throughout the bacterial community
with a capability to spread to a wider host range.
Overall, the present investigation clearly warrants the need for advanced
studies in this area especially with respect to the State of Kerala, where few studies
of this nature have been conducted as evidenced by the sparse reports published till
date. Moreover, a unique situation prevails in this state with a high percentage of its
population seeking employment outside the state and the country, in addition to the
flourishing tourism. This paves the way for high mobility of people, which, in turn
contributes significantly to the spread of microbial pathogens. The antibiotic
resistance of clinical pathogens analysed in this study by various phenotypic and
molecular investigations necessitates further expansion in terms of larger sampling
of pathogens from across the state followed by a critical analysis of multiple loci
involved in microbial resistance and their molecular characterization

35
 1 2 3 4 5 6 7 8 M

245 kDa

30 kDa

25kDa

11kDa

Fig 6:SDS page result analysis o selected resistant bacterial isolates M is 11 to 245kDa protein
marker.Lane1,2,3,,4,5,6,7,,8indicateAMCBM,CIP2U,CIP28U,CIP47,AMCBM,AMC2U,AMC28U,AMC47

M S 1 2 3 4 5 6 7 8

48 kDa

35 kDa

25 kDa

20 kDa

Fig 7:SDS page result analysis o selected resistant bacterial isolates M is 11 to 245kDa protein
marker.Lane1,2,3,,4,5,6,7,,8indicate CEF2U,CEF28U,CEFBM,CEF47,AK2U,AK28U,AK47,AKBM.

36
Diversity indices:

Diversity of bacterial isolates from Kamla nehru hospital:

Hospital surfaces patients Wastewater

Dominance(D) 0.41 0.33 0.30
Simpson(1-D) 0.58 0.66 0.69
Shannon(H) 1.15 1.19 1.275
Evenness(e H/S) 0.63 0.82 0.89
Margalef 0.977 1.00 0.97
Taxa(S) 5 4 3
Indiveduals 60 20 26

37
. Jalalvandi F, Teimouri B, Sohrabi N, et al. Microbial Contamination of Operating Rooms Equipments in
Selec Original Article
Bangladesh J Microbiol, Volume 32, Number 1&2, June-December 2015, pp 21-24

Untreated Liquid Hospital Waste: Potential Source of Multidrug

Resistant
Bacteria
Md. Kamruzzaman Siddiqui, Nazma Khatoon, Pravas Chandra Roy*
Department of Microbiology, Jessore University of Science and Technology, Jessore-7408, Bangladesh ted Hospitals in Kermanshah.
Iran J Infect Dis Trop Med 2013; 17(59): 49-52.

Detection of antimicrobial-resistant gram-

negative bacteria
in hospital effluents and in the sewage treatment
station
of Goiânia, Brazil
Detección de bacterias gram negativas resistentes a antimicrobianos en
efluentes hospitalarios y en la
estación de tratamiento de aguas residuales de Goiânia, Brasil
Detecção de bactérias gram-negativas antimicrobiano-resistentes em
efluentes de hospitais e na estação
tratamento de esgoto de Goiânia, Brasil

Isolation and characterization of multiple

drug
resistance bacterial pathogens from waste
water
in hospital and non-hospital environments,
Northwest Ethiopia
Feleke Moges1*, Mengistu Endris1, Yeshambel Belyhun2,3 and Walelegn Worku4 Moges et al. BMC
Research Notes 2014, 7:215
http://www.biomedcentral.com/1756-0500/7/215

38
16S rRNA Gene Sequencing for Bacterial
Identification in the Diagnostic Laboratory:
Pluses, Perils, and Pitfalls
J. Michael Janda, Sharon L. Abbott
JOURNAL OF CLINICAL MICROBIOLOGY, Sept. 2007, p. 2761–2764
2761 Vol. 45, No. 9 0095-1137/07/$08.000
1137/07/$08.000
doi:10.1128/JCM.01228-07 07 Copyright © 2007, American Society for Microbiology. All Rights Reserved

Detection
etection and Identification of
Microorganisms by Gene Amplification and
Sequencing
L. Barth Reller Melvin P. Weinstein Cathy A. Petti
Clinical Infectious Diseases,, Volume 44, Issue 8, 15 April 2007, Pages 1108
1108–
1114,https://doi.org/10.1086/512818
https://doi.org/10.1086/512818
Published:

15 April 2007

Journal of Babylon University/Pure and Applied Sciences/ No.(3)/ Vol.(22): 2014

1355
Study of some virulence factors of some
urepathogenic bacteria
Jwan Ahmed AL-Hamawandi,
College of Medicine, University of Babylon..

Outbreak of Alcaligenes faecalis pseudobacteraemia in neonatology and

paediatric unitsApril 2010Volume 74, Issue 4, Pages 397–399
M. Almuzaraa, , Correspondence information about the author M. Almuzara Email the author M. Almuzara

M. Matteob

R. Cittadinic

39
, E. Bertonac

R. Armitanob

M. Catalanob

M. del Castilloc

C. Vaya,c

PlumX Metrics

Journal of General Microbiology (I 972), 70,443-452 Printed in Great Britain 443 Two Modes of ‘ Curing’
Transmissible Bacterial Plasmids By VYVYAN SALISBURY, R. W. HEDGES AND NAOMI DATTA Department
of Bacteriology, Royal Postgraduate Medical School, London, W I 2 oHS (Accepted for publication 7
December I 97 I)

2007 Trade Science Inc. - INDIAuseful tool for some genetic analysis and engineering. BioTechnology
KEYWORDS Curing; Acriflavine; Acridine orange; Acridine dyes; Plasmid; Escherichia coli. Abbas Rezaee
1*, Mandana Mobarhan 1 , Mohamad Salam2 1EnvironmentalHealthDept.,Faculty
ofMedicalSciences,Tarbiat Modarres University,Tehran,(IRAN) 2Faculty of
PublicHealth,BagiyatallahMedical Sciences University,Tehran,(IRAN) Fax: +98 21 88013030 E-mail:
abbasrezaee@yahoo.com; rezaee@modares.ac.ir Received: 6 th September, 2007 ; Accepted: 11 st
September, 2007)

Detection and characterization of extended-spectrum β-lactamases

(blaCTX-M-1 and blaSHV) producing Escherichia coli, Salmonella spp. and
Klebsiella pneumoniae isolated from humans in Mizoram
Iadarilin Warjri, T. K. Dutta, H. Lalzampuia and Rajesh Chandra
Department of Veterinary Microbiology, Central Agricultural University, Selesih, Aizawl, Mizoram, India.
Corresponding author: T. K. Dutta, e-mail: tapandutta@rediffmail.com, IW: iadawarjri@gmail.com,
HL: ateahlawndo@gmail.com, RC: rajeshchandra2k@rediffmail.com
Received: 18-12-2014, Revised: 05-04-2015, Accepted: 11-04-2015, Published online: Veterinary World, EISSN:
2231-0916

Available at www.veterinaryworld.org/Vol.8/May-2015/7.pdf

Surveillance of bacterial colonisation on contact surfaces in different medical wards Karmen Godič
Torkar and Sanja Ivić Department for Sanitary Engineering, Faculty of Health Sciences, University of
Ljubljana, Ljubljana, Slovenia [Received in September 2016; Similarity Check in September 2016;
Accepted in May 2017

40