Antibiotic Susceptibility Profile of E. coli Isolated from urine pus of Leady reading hospital Peshawar Pakistan
, Sarwat Moon2
, Nelli Rasheed3
, Madiha Farooq5
, Asad ullah7
, Muhammad Ubaid8
, Fariha Farooqi9
, Farzana anwar10
1Department of Microbiology and health sciences Abasyn University Peshawar Pakistan [email protected]
2Department of Microbiology university of Karachi, Karachi Pakistan [email protected]
3The school of allied health sciences Lahore Pakistan [email protected]
4Department of Microbiology and health sciences Abasyn University PeshawarPakistan [email protected]
5Department of Microbiology and health sciences Abasyn University Peshawar Pakistan [email protected]
6Department of Biosciences, Capital University of sciences and technology Islamabad, Pakistan.
7Department of Pharmacy University of swabi Pakistan [email protected]
8Department of allied health sciences Radiology Khyber medical university Pakistan [email protected]
9Department of Microbiology and health sciences Abasyn University Peshawar Pakistan [email protected]
10Department of Microbiology, university of Baluchistan, Pakistan [email protected]
11Department of emergency care Gandhara university Peshawar Pakistan [email protected]
12Institute: institute of nursing science Khyber Medical University Peshawar Pakistan.
13Rahman colleges of allied health sciences Peshawar Pakistan [email protected] Corresponding author [email protected]
The bacterial resistance to antibiotics is one of the main problem worldwide. This study investigated the antibiotics sensitivity and resistance pattern of isolated bacteria from UTIs patients, thus big challenge for antibiotics era. Total 30 urine pus samples isolates during 1 month’s period to study and check antibiotic pattern of various organisms.
Organisms were identified as per standard operative procedure and drug sensitivity was done. Commonly founded organism was E.coli but some mixed growth also found such as S.aureus and pseudomonas. It was showing sensitivity towards vancomycin, clindamycin and cefoxitin whereas gram negative organisms were showing sensitivity towards penicillin, gentamycin and eztromycine. The study observes that the prevalence of urine pus is high among females
Principally due to the reason sexually or menstrual cycle or physical factors in females. Among male increase prevalence of pus infection was recorded in young age in 20-35 year group.
The study demonstrate that E.coli remain and causing agent organism which cause uropathogen being responsible for 60% of puss infection both male and female. This is inconsistence with finding of other studies in which E.coli was the most frequently reported isolated from patient with pus culture. Following over study show S.aureus spp 27%, and pseudomonas 27% , and E.coli is 40% are the other common uropathogens. Antimicrobial resistance pattern of several antibiotics against E.coli as below ciprofloxacin is 55%, gentamycin 30%, ampicillin 45%, penicillin 25%, tetracycline 24%, erythromycin 15%. Its showed that ciprofloxacin and ampicillin is the best choice toward E.coli spp. Antimicrobial therapy allow to stop microbes growth and mutate into new forms that help them to survive antibiotic treatment and common pathogens such as E.coli other microbes and their resistance pattern can best idea for clinician to choose appropriate antibiotic for experimental treatment of patients.
Keywords: E.coli, Antibiotic, susceptibility, pus, culture, urine Introduction
E. coli is a gram-negative rod-shaped bacteria which belong the genus Escherichia spp that is commonly found in the esophagus and fundus region of the stomach lower intestine of human . Most E. coli strains are beneficial and help out in digestion and produce metabolites and help in enzymatic activity but some strain of E.coli serotypes can cause serious food poisoning in their hosts . E. coli found 0.1% of gut flora in human intestine, and spread through fecal oral route which cause disease such urine trace infection, digestive problem and some strain typhoid fever. E.coli are able to survive outside the body for short time, E.coli is responsible for causing UTI urine trace infection and other skin infection .
The virulence strain which cause diarrhea worldwide . The E. coli globally, is becoming resistant to antimicrobial agents and has been rising day by day. These strain is particular combination of virulence genes include enterotoxins, endotoxin, capsulated, and lipopolysaccharide. E. coli differentiated by serotyping, based. E.coli cause certain disease Urinary tract infection (UTI) 70%, Septic infections of wound 56% , Diarrhea 89% ,Dysentery70% ,Septicemia 40%
,Pneumonia 15% ,Neonatal meningitis80% ,Abscess in various organs 23% [5,6].
Diagnosis of E.coli Phenotypic/conventional Method, Culturing, E.coli can be detected by its colony morphology, physical appearance (Colour, shape, size, margins etc), Microscopy. Microscopy technique can be applied to visualize the cells morphology. The following techniques can also be utilized to detect the E.coli, Biochemical techniques, Serological techniques, Molecular techniques [7,8].
Antibiotic therapy and resistance
Antibiotics are usually uses to treat bacterial infection. E. coli are showed resistant to many antibiotics that are effective against gram-positive organisms.9 Antibiotics which may be used to treat E. coli infection include tetracycline , clavulanic acid , gentamycin , as well as other generation such penicillin’s, cephalosporin’s, carbapenems, cefixime , trimethoprim-sulfamethoxazole, ciprofloxacin, aminoglycosides and nitrofurantoin. Broad spectrum Cephalosporin, Aminoglycoside and Fluoroquinolones are the drugs of choice against E.coli. Antibiotic resistance is a particular problem in Gram-negative bacilli. Some strains are showing high level resistance to aminoglycosides, β-lactam, and quinolones [9,10,11,12].
Antibiotic resistance is a major problem now a days due to misuses of antibiotics. Research study published in the journal of life Science in August 2018 found that the rate of mutations in E. coli is much higher than other organism
“on the order of 10−5 per genome per generation to generation. Which may have significance for the study and management of bacterial antibiotic resistance [13, 14, 15 ].
E.coli may pass the gene to other species to showed antibiotic resistance this process is called horizontal transfer gene mechanism. E. coli bacteria carry multiple drug resistance genes, and under stress situation transfer those plasmids genes to other species is called conjugation process.
Infections with MDR multi drug resistance E.coli increasing with resistance by extended spectrum b-lactamase (ESBL) production. These isolates from hospitalized patients and as well increased cause of community acquired infection too. The risk factors for infection include UTI, ESBLs are able to hydrolyze of the b-lactam antibiotics, including second and third generation such cephalosporin, gentamycin. In addition showed co-resistance to trimethoprim and aminoglycosides. Generally carbapenems considered the drug of choices for the treatment of ESBL- E.coli infections. Extended Spectrum Beta-Lactamases (ESBLs) are the enzymes that persuade resistance to several beta-lactam antibiotics like monobactam, cephalosporin and penicillin. The beta-lactamases production by the bacteria is the main protection tool of Enterobacteriaceae against β-lactam antibiotics. These microorganisms are proficient of dropping the efficacy of updated extended spectrum cephalosporin excluding carbapenems and cephamycins through the production of plasmid mediated ESBLs [17,18,19].
Material and methods
This study was carried out in Microbiology Research Laboratory, Abasyn University Peshawar.
Collection of samples
In the proposed study, about 100 samples collected from indoor and outdoor patients and medical equipment from Lady Reading Hospital, Peshawar by using sterile swab, and further processing will be conducted in microbiology research laboratory of Abasyn University Peshawar.
Isolation of E.coli
Samples from the clinical patients and medical equipment (BP apparatus, catheters and respiratory equipment, etc) will be inoculated on nutrient agar, Blood agar and MacConkey agar. On nutrient agar E.coli appear as pink colour..
MacConkey agar was used as a selective media for the growth of gram negative bacteria and E.coli was identified as lactose fermenter on MacConkey. Samples was streaked on media and the plates was at incubated at 37°C for 24hours.
Identification and characterization of E.coli isolates
The following tests and procedures were carried out for the identification and characterization of E.coli in collected samples.
Morphological and Cultural Characterization
Cultural characteristic is useful for identification of target microbes. Nutrient agar was used for the growth and colonies selected on the bases of size, shape and pigmentation.
Microscopy and Gram staining
Through Gram staining method the bacterial species identified and classified into gram positive and gram negative.
Clinical samples will be biochemically characterize according to Bergey’s Manual of Determinative Bacteriology (9th edition) and for characterization different biochemical tests were be performed (22,).
For characterization and identification of E.coli following tests were performed:
⮚ Triple Sugar Iron test (TSI): glucose and lactose fermentation.
⮚ Indole test
⮚ Citrate Utilization test
⮚ Catalase test
⮚ Oxidase test
⮚ Urease test
Synergy disc diffusion method
Synergy Disc Diffusion method will be performed for the detection of ESBL producers. The antibiotic discs of Ceftriaxone (CRO), Cefotaxime (CTX), Ceftazidime (CAZ) and (AZM) will placed at 25 to 30 mm from Clavulanic acid-Amoxicillin. Clavulanic acid + Amoxicillin (AMC =20+10g) placed in the center of inoculated Muller Hinton agar (MHA) plates according to the recommendation of Clinical and Laboratory Standards Institute (CLSI). After overnight incubation, zones of inhibition around the Aztreonam and third-generation Cephalosporin discs will be observed. If the zones of inhibition around the 3G Cephalosporin disc and Aztreonam will close or extended sides to AMC, the isolate bacterial specie will be ESBL producer .
Antibiotics sensitivity testing
Disk diffusion method used to determine the susceptibility of the E.coli isolates to antimicrobial agents according to Clinical and Laboratory Standards Institute (CLSI, 2006; 2007) guidelines. The antibiotic disc to be used placed on the media plates for 24hours at 37 °C. Zones of inhibition measured for each antibiotic .
Following antibiotics were used.
1. Ttracyline 2. Erthromycine 3. Gentmycine 4. Ciprofloxacin 5. Ampecillin 6. Pincilline
Clinical specimens and processing for bacterial isolation
In the current research study among total 30 samples 24 samples were positive for bacterial growth while 6 samples had no growth on culture plates were collected from pathology section and surgical B ward Lady Reading hospital Peshawar Pakistan. Total 24 samples were positive on culture plates for bacterial growth including Gram negative and Gram positive bacteria. Identified bacterial species were Staphylococcus aureus, P. aeruginosa, E. coli, and. These clinical samples through pure culture techniques including various types of media i.e Nutrient agar (N.A). Blood agar, Mannitol salt agar (MSA), Eosin Methylene blue (EMB), and MacConkey agar. While characterization of these collected isolates were screened out by performing microscopy and different biochemical tests, which showed in percentage in graph. In gram staining Staphylococcus aureus, was Gram positive and remaining isolates were Gram negative. It was observed that E.coli showed gram negative and rod shape bacilli and acidic with no colour change in TSI test but showed negative response toward oxidase, urease, citrate and coagulase test. E.coli also showed indole positive response.
Table. 1. Isolated bacterial species from the deep wounds surgical infection.
S.NO Identified species No. of isolated species (n)
1 Staphylococcus aureus 6 27 %
2 Pseudomonas aeruginosa 7 27 %
4 Escherichia coli 11 46 %
Identification of collected bacterial isolate
Table 2. Detailed description of microscopy and biochemical test response of test bacterial organisms.
Bacterial species identified
Gra m stai nin g
Biochemical tests La
De xtr ose
Su cro se
S Tes t
In dol e tes t
Cit rat e tes t
Ur eas e tes t
Ca tal ase test
E. coli - AG AG - - + - - + A/NC
Pseudomonas sp., - - - + + + +
S. aureus + - - - - + + - + N/A
Key: + = Positive reaction, - = Negative reaction, d = Variable reaction, Cat = Catalase, Oxi = Oxidase, Cou = Cougulase, Cit = Citrate utilization, Ind = Indole production, Urea = Urease production, TSI = Triple sugar iron KK
=no fermenter, K = Red, A = Yellow, K = Alkaline, A= Acidic, NA= Not applicable Multi drug resistant bacterial isolates
Total 24 multi drug resistant bacterial species were screened out and total 6 Antibiotic were used against E.coli. The multi-drug resistant bacteria E. coli isolate 1 to 7 revealed resistance to Penicillin, Tetracycline, and Ampicillin, Erythromycin and Gentamycin and ciproflxine. Multi drug resistant E. coli isolate 1 to 7 showed 18, 16, 14, 15, 18, 17, 14mm inhibition zone respectively when Ciprofloxacin was used.
Sample P TET AMP CIP E GN
E. coli 001 R 12mm R 17mm R 12mm
E .coli 002 R 8mm R 14mm 8mm 18mm
E. coli 003 9mm R 10mm 13mm R R
Staphylococcus aureus Pseudomonas aeruginosa Escherichia coli
E.coli 005 8mm R 9mm 18mm R 9mm
E. coli 006 11mm R 9mm 17mm R R
E. coli 007 R 9mm R 14mm 16mm R
Key: R= Resistant, P= Penicillin, TET=Tetracycline, AMP= Ampicillin, CIP= Ciprofloxacin, E=
Erythromycin and GN= Gentamycin.
(c) Fig 3 E.coli drug resistivity and culture shown in figure
0 2 4 6 8 10 12 14 16 18 20
E. coli 001
E .coli 002
E. coli 003
E. coli 004
E. coli 006
E. coli 007
Antibiotic-resistant is a global issue worldwide, but under developing countries are more in threat because of less hygienic conditions and poor clinical infrastructure. In the present study, urine pus infection was among the highest reported clinical diagnosis. In bacterial pathogen, E. coli was reported in the maximum number of studies. UTI are the great issue needs hospitalization among patients. It is also usually the great cause of non-traumatic lower extremity amputations. The Physicians play an important role in the safety measurement, early investigations and management of UTI problems. Management however involves an extensive knowledge of the major risk factors. In the present research study bacterial species were isolated from pus sample. Which were S. aureus, P.aeruginosa, E.
coli species from urine pus samples The multi drug resistant bacteria E.coli isolate 1 to 8 revealed resistance to different sort of antibiotics like Penicillin, Tetracycline, Ampicillin, Erythromycin and Gentamycin. Multi drug resistant bacteria E.coli isolate 2, 3, 6, 7 and 8 showed to Ciprofloxacin while isolate 1 and 5 showed susceptibility towards Ciprofloxacin making inhibition zone like 16 and 15mm respectively. Ciprofloxacin is least potent antibiotics towards E.coli. This result which ciprofloxacin was most effective antibiotic. Penicillin, Tetracycline, Ampicillin and Erythromycin were the most ineffective antibiotics against all isolated E.coli spp Gentamycin, Erythromycin and antibiotics were ineffective against E. coli in our study. Ciprofloxacin and ampicillin is the most effective antibiotics against all type of E.coli
The present study is design to identify resistance antibiotics against E.coli and determine prevalence of in different clinical samples.
1. Collect urine samples from male & female patients having urine pus problem.
2. Identify bacterial pathogens by growth on selective media and different biochemical tests.
3. Check the antibiotic susceptibility of ESBL& MBL production of the isolates towards commercially available antibiotic.
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