UJMR, Volume 6 Number 1, June, 2021, pp 38 - 46 ISSN: 2616 - 0668

https://doi.org/10.47430/ujmr.2161.005

Received: 07th Feb, 2021 Accepted: 13th May, 2021

Antibiotic Sensitivity Pattern and Plasmid Profile of Bacteria Isolated from Diabetic
Ulcers in Mbano Metropolis, Imo State, Southeastern Nigeria

Nwankwo, E. O., Nwagbara, E. E., Onusiriuka, K. N.

Department of Microbiology, College of Natural Sciences, Michael Okpara University of Agriculture,
Umudike, Abia State
Corresponding author Email: [email protected]
Abstract
The study was undertaken to evaluate the bacteriology and antibiogram of isolates from diabetic
patients with chronic foot ulcers in Nigeria. A total of 150 pus samples were collected and
processed according to standard aerobic and anaerobic microbiological methods. Antibiogram
was done using Kirby-Bauer method. Biofilm tests, ESBL & AmpC production was conducted using
Congo red agar, Double disc synergy test and Cefoxitin disc test respectively. Total number of
isolates obtained was 210. The Plasmid profiles of some of the Multi-Drug Resistance (MDR)
isolates were carried out using the alkaline lysis method for plasmid extraction and
electrophoresis on agarose gel with standard markers. The most frequently isolated aerobic
organism in the study was Escherichia coli (32.1%) while the least occurring was Enterobacter
spp (1.57%). For the anaerobes, Peptostreptococcus spp (40%) was the highest isolated bacterium.
Percentage of Extended Spectrum -lactamase ( ESBL) producers among E. coli isolates was
44%. Percentages of biofilm formation potential among the isolates were: E. coli (36.8%), S.
aureus (23.1%) and Proteus vulgaris (4.2%). Escherichia coli and S. aureus showed considerable
levels of resistance to some common antibiotics. No methicilin resistant S. aureus was
encountered. AmpC producers encountered were Klebsiella pneumonia (10%) and E. coli (8.1%).
Post-curring antibiogram tests revealed that nine isolates carried plasmids, suggesting that the
mode of resistance may be plasmid mediated.
Keywords: Diabetic ulcers, Bacteria, Antibiogram, Plasmid profile.

INTRODUCTION organisms multiply in the wound which may

Diabetic ulcer is a serious clinico-pathologic lead inexorably to tissue damage. Utilizing a
outcome of diabetes with recent studies murine chronic wound model, some researchers
revealing that life time risk of developing a found that DNA protected P. aeruginosa in the
foot ulcer in diabetic patients could be as wounds of insulin-treated diabetic mice from
high as 25% which leads to proximate and non- antibiotic treatment (Watters et al., 2014). Due
traumatic causes of leg amputation (Lipsky, to common or recurrent infections, diabetic
2004; Ahmed et al., 2006). Presently there is patients have more antibiotic treatments
increasing empirical evidence suggesting that compared with other subjects which can
diabetes is a risk factor for antibiotic-resistant increase the antibiotic resistance rates in the
Streptococcus pneumonia, Methicillin bacteria (Watters et al., 2014). In a study in
resistant Staphylococcus aureus (MRSA) with Malaysia, S. aureus was found to be
basal sensitivity to vancomycin and predominant followed by Klebsiella pneumonia
vancomycin-resistant enterococci as well as and Pseudominas aeruginosa (Mohanasoundram,
ESBL producing Gram negative bacteria and 2012). The increased relationship of multi-
carbapenem-resistant Pseudomonas spp drug resistant (MDR) pathogens with diabetic
(Lyndmila et al., 2013). ulcers, further compounds the problem faced
The prevalence of foot infections in persons by physicians or surgeons in treating diabetic
with diabetes ranges from a lifetime risk of ulcers without resorting to amputation (Yoga et
up to 25% in persons with the disease, to 4% al., 2006). Infection with MDR pathogens
yearly in patients treated in a diabetic foot incurs a lot of expenses and leads to delayed
center (Singh et al., 2005). In diabetic foot hospital stay, and sometimes increases the
ulcers, various organisms inhabit the wound chances of mortality in diabetic patients.
and in most patients one or more species of

38
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 UJMR, Volume 6 Number 1, June, 2021, pp 38 - 46 ISSN: 2616 - 0668

Careful selection of antibiotics based on the incubated at 37°C for 24hrs (Turgeon, 2012).
susceptibility pattern of the isolates from the Isolation of anaerobic bacteria
lesions is most reliable for the proper The second swab sample was inoculated in
management of these infections. neomycin sulphate blood agar and cooked meat
Reported cases of diabetic infections which broth immediately. The blood agar plate was
investigated the prevalence of microbes and incubated anaerobically for 48hrs at 370C in an
their associated multi-drug resistance have anaerobic jar with Gaspak Oxoid BROO38B (Gas
been published in developed countries. Generating Kit). Saccharolytic reaction is
Contrastingly, the bacteriology of diabetic shown by reddening of the meat with a
ulcers in Nigeria has not been studied rancid smell due to carbohydrate
extensively especially in the south-eastern decomposition while proteolytic reaction is
part of the country. This study was designed shown by blackening of the meat with a
to identify the common bacterial agents very unpleasant smell due to protein
found in foot ulcers o f d i a b e t i c p a t i e n t s decomposition.
in Imo State and t o d e t e r m i n e their in Identification of Organisms
vitro susceptibility to routinely used The isolates were identified by standard
antibiotics. techniques on the basis of their cultural
morphology, motility, Gram staining reaction
MATERIALS AND METHODS and biochemical properties according to CLSI
Study Location (2015).
The study was conducted in Ihitte Uboma Antibiotic Sensitivity test
town in Imo State. Samples were collected Antimicrobial susceptibility testing was
from Madonna Austrian Hospital Ihitte which performed using the disk diffusion method
serves as a referral hospital in the locality. according to Clinical Laboratory Standard
Institute (2015) on Mueller Hinton agar (Hardy
Study Design Diagnostics, USA). Mueller Hinton culture
This is a cross sectional surveillance study. plates were inoculated using a sterile cotton
wool swab d i p p e d into an overnight growth
Study Population and study sample suspension of the test organism
The study population comprised of all diabetic prepared to the density of 0.5 McFarland
patients that reported to Madonna Austrian standard. The antibiotics tested were
Hospital Ihitte between June, 2018 and cotrimoxazole (25µg), levofloxacin (20µg),
January, 2020. A total of 150 diabetic patients streptomycin (30µg), ciprofloxacin (10µg),
w i t h d i a b e t i c f o o t u l c e r s were enrolled amoxicillin (10µg), amoxicillin/clavulanate
for the study, out of which 65 patients were on (30µg), gentamicin (10µg), perfloxacin (10µg),
admission while 85 were out-patients. Some of ofloxacin (10µg), ceftriaxone (30µg),
the patients were referrals from other ceftazidime (30µg), erythromycin (30µg). After
healthcare centres in the neighbouring towns. overnight incubation, examination of the
The collect foot ulcer samples were processed control and test plates were carried out to
at the Microbiology laboratory of Michael ensure the growth is semi- confluent. Using a
Okpara University, Umudike and Oevent ruler on the underside of the plate of each zone
Research Laboratory Uzuakoli Road Umuahia of inhibition was measured in mm. Escherichia
respectively. coli ATCC 25922 was used as the control strain
for this study. This organism was obtained from
Sample collection from the diabetic foot Aminu Kano Teaching Hospital (AKTH), Kano.
ulcers patients ESBL Screening and confirmation
The surface of the wound was cleaned with The isolates were tested against third
physiological saline to avoid skin generation cephalosporins (Cefpodoxime,
contaminants. With the aid of two sterile cefotaxime, and ceftrixone) using the WHO
swab sticks, pus samples were collected from modified Kirby Bauer diffusion method (2003).
the foot ulcers of each diabetic patient. The Zone diameters were interpreted using the
sample collection was carried out before wound revised Clinical Laboratory Standard Institute
dressing. document (2015). Isolates with reduced
susceptibility to cefpodoxime (<17mm),
Isolation of aerobic bacteria cefotaxime (<27mm) and ceftriaxone (<25mm)
The swab was inoculated on to blood agar, were considered to be possible ESBL producers.
MacConkey agar and Mannitol Salt agar and

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 UJMR, Volume 6 Number 1, June, 2021, pp 38 - 46 ISSN: 2616 - 0668

Phenotypic Confirmation Test was carried out Plasmid Extraction

using Double Disc Synergy test. Disc containing Plasmid extraction was done following the
the standard 10µg of cefpodoxime and the 30 g method described by Birnboim, et al., (1979).
of ceftazidime/ ceftriaxone was placed 15mm This analysis was carried out using the alkaline
apart (edge to edge) with amoxicillin-clauvlanic lysis method and gel electrophoresis.
acid disc containing 10µg of the latter Plasmid Curing
compound mounted exactly at their center. Ten millilitres of each bacterial culture
After 16-20 hours of incubation at 35°C any inoculated into peptone water and incubated
enhancement of the zone of inhibition for 24hrs was introduced into a set of 20
between a beta-lactam disk and that test tubes, respectively. Ethidium bromide in
containing the -lactamase inhibitor is various concentrations of 0, 20, 50, 100, 150,
200, 250, 300, 350, 400, 450, 500, 550,
indicative of the presence of an ESBL (Jacoby
600,650, 700, 750 and 800 µl/ml were then
et al., 2004).
introduced accordingly into the test tubes and
Screening for AmpC -lactamase incubated at 37ºC for 24hrs to determine the
Screening for AmpC β-lactamase production sub-lethal concentrations of ethidium bromide.
was done by placing a cefoxitin disk (30µg) After 24hrs of incubation, 1ml aliquot from
on Mueller-Hinton agar. Isolates showing an each test tube was inoculated onto nutrient
inhibition zone diameter of < 18mm were agar plates and incubated, after which colonies
considered positive on the screening. were selected and inoculated onto freshly
Amp C Detection test prepared Muller Hinton agar plates. Then,
A 0.5 McFarland suspension of multidrug antibiotic discs of prior resistance were
resistant bacteria was inoculated on the surface aseptically introduced into the plates,
of a MHA plate. A 30µg cefoxitin disc (Oxoid, ensuring that the discs made appropriate
England) was placed on the inoculated surface contact with the surface of the agar. These
of the agar. A sterile plain disc inoculated with were incubated for 24 hrs at 37ºC after which
several colonies of the test organism was plates were examined for cured colonies
placed beside the cefoxitin disc almost (Raghada et al., 2013).
touching it, with the inoculated disk face in
contact with the agar surface. The plates were RESULTS
incubated at 370C for 24 hours. After Table 1 shows the age and sex
incubation, the plates were examined for either distribution of Diabetic ulcer patients. O f
an indentation or a flattening of the zone of t h e 150 patients enrolled for this study, 90
inhibition, indicating enzymatic inactivation of (60%) were male and 60 (40%) were female.
cefoxitin (negative result) (CLSI, 2015). Their ages ranged from 41 to >90 and the
Test for Methicillin Resistant Staphylococcus. highest number of patients were found in the
Susceptibility to cefoxitin was determined by age group of 61-70 years (28%), followed by
the Disk Diffusion method on Mueller–Hinton those the aged of 81-90 years (20.6% ) then the
agar plates using a bacterial suspension with age groups 41-50 (9.3% ) and >90 (9.3%).
the turbidity adjusted to a 0.5 McFarland Table 2 shows the distribution of aerobic and
standard. Plates were incubated at 35°C for 24 an aero bic isolates. A total 210 isolates were
hrs. Results were interpreted according to CLSI obtained from this study out of which 137
(2013) guidelines. The interpretive criteria for (65.5%) were Gram negative, 53 (25.5%) were
cefoxitin were: S. aureus, sensitive 22mm, Gram positive and 20 (9.5%) were anaerobes.
resistant 21 ug/ml; Coagulase Negative The most frequently isolated organism in this
Staphylococcus (CoNS), sensitive 25mm, study was Escherichia coli (32.1%), and the
least was Enterobacter spp (1.57%) for the
resistant 24mm.
aerobes. The anaerobes, Peptococcus spp
Test for Biofilm Formation Potential
(15%), Peptostreptococcus spp (40%),
This was done by the Congo Red Agar Method.
Bacteroides spp (30%) and Fusobacterium spp
Blood agar base supplemented with sucrose
(15%).
and Congo red was used. Plates were
Table 3 shows the d is t r i bu t io n of
inoculated with the tested isolates and
o rg a n is ms w ith b io f il m f o rm a t io n ,
incubated aerobically for 24hrs at 37°C.
betalactamase pr o du c in g p o t e n t ia l s and
Positive result was indicated by black colonies
AmpC p r o du c e rs . The percentage of biofilm
with a dry crystalline consistency. Non slime
forming organisms is as follows: E coli (36.8), S
producers usually remained pink (Mathur et al.,
aureus (23.1%), Klebsiella spp (12.6%), P.
2006).
aeruginosa (8.4%), P. mirabilis, COANS (7.3%)
and P. vulgaris at (4.2%). It shows the
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percentage of Extended Spectrum Beta- Coagulase negative Staphylococcus spp (COANS)

lactamse (ESBL) as follows: E coli (44%), were sensitive to Ofloxacin at the rate of 63%
Klebsiella spp and P. mirabilis (17%) each, P. and 100% respectively. Enterococcus spp was
aeruginosa (15%) and P. vulgaris (4%). Also, 100% sensitive to Streptomycin while COANS
Table 3 the AmpC producers encountered as showed 100% sensitivity to Ofloxacin, Amoxil
Klebsiella spp (28.5%) and E. coli (71.4%). and Streptomycin (Table 4). Citrobacter spp
Table 4 shows the antibiogram for Gram was 100% sensitive to Perfloxacin,
negative and Gram positive isolates. E coli Ciprofloxacin, Ofloxacin and Gentamicin while
recorded the highest sensitivity rate with Enterobacter spp was 100% sensitive to
Ciprofloxacin (57.3%) and Gentamicin (40.9%) Ofloxacin and Pefloxacin (Table 4).
(Table 4). Similarly, Klebsiella spp recorded Table 5 shows the distribution of plasmids and
higher sensitivity rate of 70% to t he ir m o l e c u l a r w e ig h t. It a lso re ve a l s
Ciprofloxacin and 55% to Perfloxacin and the resistance pattern of isolates before and
Ofloxacin (Table 4). The antibiogram for the after curing.
Gram-positive organisms indicated that
organisms like Staphylococcus aureus and

Table 1: Age and Sex Distribution of Diabetic Ulcer Patients

Age (Years) No. examined Male Female Percentage (%)
41-50 14 7 7 9.3
51-60 21 11 10 14
61-70 42 32 10 28
71-80 28 18 10 18
81-90 31 16 15 20.6
>90 14 6 8 9.3
Total 150 90 60

Table 2: Distribution of aerobic and anaerobic Isolates

Aerobic Isolates
Organisms Number of Occurence Percentage (%)
Klebsiella spp 20 10.5
E coli 61 32.1
P. mirabilis 20 10.5
Enterobacter spp 3 1.57
COANS 10 5.2
S. aureus 30 15.7
P. aeruginosa 19 10
E. faecalis 8 4.2
Strept spp 5 2.6
Citrobacter spp 3 1.57
Total 190

Anaerobic Organisms
Peptococcus spp 3 15
Peptostreptococcus spp 8 40
Bacteroides spp 6 30
Fusobacterium spp 3 15
Total 20
CONS = Coagulase Negative Staphylococci

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Table 3: Distribution of Organisms with Biofilm Formation, Beta-lactamase producers and AmpC producers Number Positive (Percentage)
Organisms Number Biofilm forming Beta-lactamase AmpC
Isolated potential producers producers
Klebsiella spp 20 12 (12.6%) 8 (17%) 2 (28.5%)
E. coli 61 35 (36.8%) 20 (44%) 5 (71.4%)
P.vulgaris. 11 4 (4.2%) 2 (4%) Nil
P. mirabilis 20 7 (7.3%) 8 (17%) Nil
Enterobacter spp. 3 Nil Nil Nil
CoNS 10 7 (7.3%) Nil Nil
S.aureus 30 22 (23.1%) Nil Nil
P. aeruginosa. 19 8 (8.4%) 7 (15%) Nil
E. faecalis 8 Nil Nil Nil
Streptococcus spp. 5 Nil Nil Nil
Citrobacter spp. 3 Nil Nil Nil

Table 4: Antibiogram result of the bacterial isolates

Organism No. No. sensitive
Gram tested PEF CPX CRO S CAZ AUG SXT OFX GN PN
Negative
E coli 61 13 (21.3) 35 (57.3) 10(16.3) 9 (14.7) 3 (4.9) 2 (3.2) 3 (4.9) 20(32.7) 25(40.9) -
Klebsiella
20 11 (55) 14 (70) 5(25) 1(5) 0 (0) 2 (10) 0 (0) 11(55) 7(35) 6(30)
spp
P.
19 6 (31) 9 (47) 0(0) - 3 (15.7) 1 (5.2) 1 (5.2) 11(57) 8(42)
aeruginosa
P. vulgaris 11 7 (63) 3 (27.2) 1(9) 2 (18) 1(9) 1 (9) 0 (0) 9(81) 2(18) 3(27)
P.mirabilis 20 12 (60) 7 (35) 3 (15) 3 (15) 3 (15) 2 (10) 1 (5) 16(80) 4(20) 7(35)
Citrobacter
spp. 3
3(100) 3(100) 0(0) - 0 (0) - 0 (0) 3(100) 3(100) -
Enterobacter
3 3(100) 2(66.7) 1(33.3) 1(33.3) - 2 (66.7) 0 (0) 3(100) 2(66.7) -
spp.

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Table 4 continue

No. No. sensitive

Gram positive
tested PEF LEV CPX OFX ERY GN APX AUG AM S R
S. aureus 30 10 18 (60) 15 (50) 19 15 (50) 21(70) 0(0) 9 (30) 0 (0) 5 (16)
5(16)
(33.3) (63)
Streptococcus 5 0 (0) 0 (0) 1 (20) 3 (60) 3 (60) 1(20) 1(20) 0(0) 1(20) 0 (0)
2(40)
spp
CoaNS 10 0 (0) 0 (0) 7 (70) 10 9 (90) 5 (50) 0(0) 0(0) 10 10
8(80)
(100) (100) (100)
Enterococcus 8 0 (0) 0 (0) 1 (12.5) 3 1 (12.5) 1 (12.5) 1(12.5) 1(12.5) 0 (0) 8
1(12.5)
spp (37.5) (100)

KEY: PEF= Perfloxacin, CPX=Ciprofloxacin, CRO = Ceftriaxone, S = Streptomycin, CAZ = Ceftazidime, AUG=Amoxicillin-clavulanate,
OFX=Ofloxacin, LEV=Levofloxacin, ERY= Erythromycin, APX= Ampicillin, AM=Amoxicillin, SXT= Septrin, GN=Gentamicin, PN=Ampicillin,
CoNS= Coagulase Negative Staphylococci, Strept. Spp = Streptococcus spp, AM= Amoxicillin, R=Rifampicin, APX= Ampiclox

Table 5: Distribution of Plasmids among Resistant Isolates, Cured Plasmids and Antibiotic Resistance
Organisms Base pair (bp) Resistance before curing Resistance after curing
E1 PEF,CPX,CAZ,CRO,S,GN
E2 CPX,S,OFX,CRO,CAZ,AU
E3 23130 OFX,CPX,CAZ,CRO,PEF,GN OFX, CPX, PEF
E4 23130 OFX,CAZ,CRO,CPX,AU,S CRO,OFX,CAZ
E5 23130 CAZ,CRO,OFX,GN,AU,PEF CRO,AU,OFX,
E6 23130 CRO,CAZ,OFX,GN,AU,PEF CAZ,CRO,PEF,OFX
E7 23130 CPX,S,CRO,CAZ,PEF,GN CRO,CAZ
E8 23130 CRO,CAZ,CPX,PEF,AU,S S, CPX, PEF
E9 23130 OFX,CPX,CRO,CAZ,PEF,S OFX,CPX,CRO,
E10 23130 CAZ,CRO,OFX,PEF,AU,S CAZ, PEF, S
E11 23130 PEF,AU,CPX,CAZ,CRO,GN CRO,OFX,S
E12 CRO,CAZ,AU,PEF,CPX,S PEF,CRO, CPX
E13 CPX,PN,OFX,CRO,CAZ,
E14 CRO.CPX,PN,GN,PEF,S
E15 CAZ,CRO,OFX,CPX,AU,S.

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Fig 1: Plasmid profile of resistant isolates from the study.

profile of resistant isolates from the study.

DISCUSSION (NFGNB).
The prevalence of diabetic ulcers among male The Gram-negative organisms isolated from this
subjects was found to be 60% and 40% in females. study were sensitive to perfloxacin ciprofloxacin,
This may be due to higher level of outdoor ofloxacin and gentamycin. The Gram-positive
activity among males compared to females. This organisms were susceptible to ofloxacin,
corresponds w i t h t h e f i n d i n g s o f s o m e erythromycin and gentamicin. This is consistent
r e s e a r c h e r s as t h e y i n d i c a t e d t h e with the findings in Kumasi, Ghana where most of
preponderance of diabetes among males the Gram-negative organisms were sensitive to
compared to females (Lilian et al., 2015; ciprofloxacin and gentamicin (Brenyah et al.,
Mohanasoundram, 2012; Nwachukwu et al., 2014). Similarly, Gram positive organisms like S.
2009). Majority of t h e patients were in the age aureus and CONS were observed to be highly
group of 61-70, suggesting that age c o u l d b e sensitive to ofloxacin. This sensitivity pattern
a r i s k factor as t h e elderly are naturally agrees with the findings of Lilian et al. (2015).
predisposed to infections. This agrees with a Five out of seven Gram negative organisms
report in South India by Viswanathan et al. isolated from this study were observed to be ESBL
(2002). producers. Among the Gram negative, E. coli
The study findings i n d i c a t e d t h a t Gram ( a t 44%) had the highest number of ESBL
negative microbes were t h e predominant producers while, Proteus vulgaris had the least
pathogens isolated with 65% Gram negative, 25% at4%. This is in accordance with the study of
Gram positive and 9.5% anaerobes. A similar Bansal et al. (2008) and Shashikala et al. (2016)
result was documented in Malaysia revealing who reported a high level of ESBL production
more Gram-negative bacteria (52%) than Gram among Ecoli isolates. However, this contradicts
positive bacteria ( 45%) (Raja, 2007). In addition, with the work of Samir et al. (2009) w h o
this agrees with the report of Lilian et al. (2015) reported Proteus spp as having the highest ESBL
but contradicts with that of Mohanasoundram producing potential from their study. This
(2012) whose study showed S. aureus as the most variance could be associated with the difference
prevalent followed by E coli, Klebsiella spp, in sample size as Samir et al. (2009) used 75
Pseudomonas aeruginosa, Enterococcus faecalis, patients for their study. The increasing
a n d Non Fermenting Gram Negative Bacteria prevalence of ESBL producing organisms is

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disturbing because of the problems associated showed that nine out of fifteen samples
with antibiotic prescription. contained plasmids. Hence, the resistance is
Recent studies have shown that biofilm plasmid mediated because upon re-exposure of
associated microorganisms can be up to 1000 the isolates to antibiotics after plasmid curing,
times more resistant to antibiotics than free they were observed to be sensitive.
floating planktonic bacteria. In the present CONCLUSION
study, seven organisms were biofilm formers. E. This study established the presence of ESBL and
coli had the highest biofilm forming potential AMPC production among biofilm forming bacterial
followed by S. aureus, Klebsiella spp, P. isolates obtained from samples of diabetic
aeruginosa, P. mirabilis, CONS and P. vulgaris. patients with foot ulcers. This is of great public
This r e s u l t a g r e e s w i t h t h a t o f Gordon health concern as this could contribute to the
et al. (2008) on the biofilm forming nature of resistant of these bacterial pathogens to
Staphylococci. This result disagrees with the antibiotic like carbapenems.
study of Asima et al. (2015) who reported S
.aureus as having the highest potential for RECOMMENDATIONS
biofilm formation followed b y P. aeruginosa, Regular studies of the antibiotic susceptibility
Citrobacter spp and Ecoli. The unusual number of pattern of diabetic ulcer isolates commonly
biofilm forming E.coli may be as a result of the observed in Mbano will guide clinical judgment
high prevalence rate of E. coli in this study. and sustain veritable antibiotic prescriptions.
This study reports a high level of AmpC Discoveries from such surveys will illuminate
production in K. pneumonia and Ecoli. This is at the current knowledge on multi drug resistant
variance with the report from researches at isolates and proffer solutions about the right
Kolkata which recorded a high level of AmpC choice of antibiotics. The detection of ESBL
production in P. aeruginosa (Subha et al., 2003; producers, AmpC producers, Biofilm forming
Suranjana et al., 2005). Agarose gel organisms and plasmids are vital.
electrophoresis of plasmid DNA in this study
It is recommended that ESBL producers be studies are encouraged to better understand
subjected to further molecular procedures to the dynamics of multidrug resistance.
define their various types and transformation

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