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Research Article - (2014) Volume 5, Issue 1

Isolation and Identification of non-plasmid Multidrug Resistant E.coli from Poultry Wastes in Chittagong Region, Bangladesh

Muhammad Shahjalal Khan1, Naznin Akhtar2, Muhammad Ehteshamul Haque1, Abanti Barua1, Tasneem Chowdhury1, Romel Mullick1 and Abu Sayeed Mohammad Mahmud3*
1Department of Microbiology, University of Chittagong, Chittagong, Bangladesh
2Tissue Banking and Biomaterial Research Unit, Atomic Energy Research Establishment, Dhaka, Bangladesh
3Industrial Microbiology Research Division, Bangladesh Council of Scientific and Industrial Research, Chittagong, Bangladesh
*Corresponding Author: Abu Sayeed Mohammad Mahmud, Industrial Microbiology Research Division, Bangladesh Council of Scientific and Industrial Research, Chittagong, Bangladesh, Tel: 01746700196 Email:

Abstract

In two branches of poultry culture; small local ones and big industrial ones, tetracycline is a common antibiotic, which has been taken as a standard antibiotic in this study. 20 isolates were taken from big poultry farms like Agha Ltd and Denm Poultry. 10 isolates were taken from small local poultry farms like Rahat Poultry and Star Poultry. After collection of samples, total numbers of bacteria with and without tetracycline were counted. In both cases numerous bacterial growths were observed. The normal dose of tetracycline is 30 μg/ml which failed extremely to regulate high bacterial growth. Two dilutions (10-3 and 10-4) of sample 1, 2, 3 and 4 were taken and allowed to grow at different concentrations of tetracycline like 30,60 and 100 μg/ml, where bacterial growth was observed. High concentration of antibiotics for example, above 100 μg/ml may be harmful to humans and animals. After performing sensitivity test against other commonly used antibiotics in poultry, it was found that isolated tetracycline-resistant E. coli were 100% resistant to penicillin and erythromycin,100 sensitive to imipenem, 93.34% resistant to tetracycline, 23.03% resistant to gentamycin and 53.33% resistant to chloramphenicol. These indicated the multidrug resistant property of isolates. Subsequent agarose gel electrophoresis showed no plasmid DNA band in the gel indicating non-existence of any bacterial plasmid and also proved that the observed resistance was chromosomal gene-mediated or at least not plasmid mediated.

Keywords: E. coli; Non-plasmid; Multi-drugs resistant; Poultry wastes

Introduction

The hope ushered by the discovery of antimicrobials has been tainted by the emergence of bacterial strains which are able to resist this therapeutics. Due to the use and misuse of antimicrobials in the last few decades, today’s clinically important bacteria are not only single drug resistant but also multiple antibiotics resistant. These multidrug resistant bacteria are increasing public health hazard all over the world [1]. Antimicrobial susceptible bacteria are substantially less responsible for causing infections compared to the antimicrobial-resistant bacteria which actually cause infections leading to higher rates of morbidity and mortality [2]. The reason behind this high rate is that, these antimicrobial-resistant microorganisms are resistant to conventional treatment and can cause serious infection resulting in prolonged illness and greater death risk. Annually, about 440,000 new cases of Multidrugresistant Tuberculosis (MDR-TB) are reported, causing no less than 150,000 deaths. In most malaria-endemic countries, widespread resistance to earlier generation antimalarial medicines, such as, chloroquine and sulfadoxine-pyrimethamine is seen [3]. Over the past decade, intercontinental spread of methicillin resistant Staphylococcus aureus [4] and penicillin resistant Streptococcus pneumonia [5], has progressed and has given rise to concerns about increasing resistance of Salmonella typhi [6]. It has proved the parochial approach to be a failure. Most antibiotic use is in two areas: in humans in the community, and in animals for growth promotion and prophylaxis. 20-50% human uses of antibiotics are unnecessary and 40-80% agricultural uses of antibiotics are highly questionable [7]. In the Southern Netherlands, almost 80 percent of raw chicken supplied by the grocery stores was found to be containing multidrug-resistant bacteria. When these germs were compared with the speciments collected from hospital patients, researchers found that, the predominant resistant genes were identical [8]. Antimicrobial resistance has been recognized by the World Health Organization (WHO) as a global problem that calls for global response. Keeping the problem in view, WHO issued the global principles for the containment of antimicrobial resistance in animal intended for food. After some recommended interventions, the WHO global strategy for the containment of antimicrobial resistance will hopefully enable local authorities to reduce the spread of resistance and slow down its emergence in diverse setting [9,10]. These guidelines recommend prudent use of antimicrobials and the establishment of surveillance programmes for antimicrobial consumption and resistance and further research as well.

Collection of sample

Samples were collected from four poultry farms

1) Agha Poultry Ltd, Roufabad, Hathajari, Chittagong

2) DENM Poultry Farm, North Fatehabad, Chittagong

3) Star Poultry, University of Chittagong campus area

4) Rahat Poultry, Mogoltuli, Chittagong

Sample-1 (Agha Poultry) and Sample-2 (DENM Poultry) are big commercial poultry farms. Sample-3 (Star Poultry) and Sample-4 (Rahat Poultry) are small local poultry farms. Samples collected from each of these poultries were- a) raw feces from the inside of the farms, b) feces from the open fields beside the farms which were thrown away as waste products.

Transportation of the sample

After collection the samples were placed in a sterile ice-bag containing ice and were transported to the laboratory of Department of Microbiology, University of Chittagong.

Processing of samples

Samples were allowed to reach room temperature and then 10 gm of fresh fecal sample was mixed with 90ml of sterile normal saline and shook to form homogenous mixture. All samples were mixed by vigorous shaking.

Bacteriological count

All the bacteriological enumerations were carried out by pour plate method. In this case total number of bacteria and total number of resistant bacteria were counted [11].

Total Viable Count (TVC) with and without antibiotic

1 ml of from 10-2, 10-3, 10-4 and 10-5 dilutions were poured into different sterile Petri plates. The Nutrient agar media (temperature 45°C) were poured into each petri-plate. After solidification, the plates were incubated at 37°C for 24 hours at inverted position. After 24 hours, plates with 30-300 bacterial colonies were counted.

There is a difference between total viable count without antibiotic and total viable count with antibiotic. In case of total viable count with antibiotic, antibiotics (30 μg/ml tetracycline) were mixed to the sterilized media (temperature 45°C) and were shaken well before plating.

Transferring single colonies from NA plates to EMB agar media

Single colonies were picked up randomly by sterile tooth picks from plates (with different concentration of Tetracycline). The colonies were then streaked on individual EMB agar containing 30 μg/ml tetracycline. The EMB plates were incubated at 37° C for 24 hours.

Transferring to broth culture

After incubation, presence of growth with green metallic sheen was observed on the EMB plates. One loopful from such growths was transferred randomly to 3 ml of nutrient broth (in 10ml screw cap tubes) containing 30 μg/ml tetracycline samples. 30 such growths (10 from sample-1, 10 from sample-2, 5 from sample-3 and 5 from sample-4) were transferred patching from all of the samples. The 30 culture tubes were then allowed for incubation at 37°C for 24 hours with loose capping and vigorous shaking of over 250 rpm.

Identification of the Isolated E. coli

Microscopic examination of morphology bacteria

The size, shape, arrangements and Gram reactions of the 24 hour bacterial cultures were observed in a microscopical field [12].

Conventional biochemical test for the identification of E. coli

Conventional Biochemical tests were carried out for the identification of E. coli. The tests are- Indole test, Methyl-red test, Voges-proskauer test, Citrate test and Motility test. Tetracycline (30 μg/ ml) was present in all biochemical tests.

Antimicrobial susceptibility of the microorganisms to antibiotics

The standard disc diffusion method also known as Kirby Bauer method [13] was used for the in vitro determination of the sensitivity to the antimicrobial agents.

Antibiotic disc used

Antibiotics were chosen so that some of them were used during sample collection (e.g. tetracycline), some of them were continuously used in the poultry in addition to the running antibiotics, some of them were moderately or rarely used in poultry farms, some of them were not used (e.g. Imipenem and Gentamycin) (Table 1).

Antibiotics name Symbol Concentration of antibiotics applied  
Tetracycline T 30 µg
Gentamycin G 10 µg
Imipenem I` 10 µg
Chloramphenicol C 30 µg
Penicillin P 10 µg
Erythromycin E 15 µg

Table 1: Six antibiotics that were tested against the E. coli isolates using standard disc.

Plate preparation

A cotton swab was dipped in the suspension prepared in compliance with McFarland solution, excess fluid was removed by pressing and rotating the cotton bar inside the wall of the tube just above the fluid level. Then the swab was streaked over the surface of the Muller-Hinton agar medium to obtain uniform inoculums and some plates were also prepared by pour plate method.

Preparation and application of the disc to the plates

The discs were then placed on the surface of the seeded plates at appropriate spatial arrangement by using a sterile forceps. Then the plates were inoculated at 37°C for 24 hours and observed for the clear zone of inhibition.

Observation of clear zone of inhibition

After incubation the zones of complete inhibition were measured by using MD8 Scan Zone Reader.

Plasmid isolation

Plasmid extraction procedure was carried out following the protocol developed by ICDDRB. The extracted plasmid was then isolated using a horizontal 1% Agarose Gel Electrophoresis technique.

Preparation of the sample

The pure cultures were transferred to 10 ml screw cap tubes containing 3 ml Luria Bertani (LB) broth with 30 μg/ml tetracycline. The broths were then incubated at 37°C with loose capping and vigorous shaking (200 rpm) for overnight. Then inoculums were transferred to another 3 ml LB broth at a 1:200 ml rate containing same concentration of tetracycline and incubated for 4-6 hours at 37°C with loose capping and vigorous shaking (200 rpm). After sufficient growth with slight turbidity the incubation stopped and the cells were prepared for extraction.

Plasmid extraction

1.0ml of overnight culture was taken in an Eppendorf ’s tube (1.5ml) and cells were collected by centrifugation for 7 minutes at 12,000 rpm. The supernatant was discarded and the pellet was thoroughly suspended in 100 μl of solution I and the solution was kept at room temperature (32°C) for 10 min.

Then 200 μl of solution II (lysis solution) was added and mixed gently by inverting the tube for a few times. After that 150 μl of ice-cold solution III (neutralizing solution) was added and mixed vigorously by vortexing for a few seconds. The tubes were kept on ice for 5 minutes. The mixture was then centrifuged at 12,000 rpm for 15 minutes to pellet the chromosomal DNA. The clear supernatant (approximately 400 μl) was taken to fresh Eppendorf ’s tubes. Then two volumes of cold 95% ethanol (800 μl) were added in each tube and vortexes for a few seconds to mix well. It was then kept in room temperature for about 20 minutes for DNA precipitation. The precipitated DNA was collected by centrifugation for 15 minutes at 12,000 rpm. The supernatant was discarded and the pellet was dried in a drier at 45°C for 20 minutes. Finally the dried DNA was dissolved in 30 μl TE buffer and kept at 4°C.

Separation of plasmid DNA by agarose gel electrophoresis

Plasmid DNA was separated by horizontal electrophoresis in 1% agarose slab gels in a Tris-Acetate EDTA (TAE) buffer at room temperature at 80 volt (50 mA) for 3 h. briefly, 30 μl of plasmid DNA solution was mixed with 3 μl of tracking dye (Appendix) and was loaded into the individual well of the gel. The gel (5mm thick) was stained with 0.5 μg/ml of ethydium bromide for 15 min at room temperature and then distained with distilled water for 10 min.

Results

Bacterial enumerations

Total count of bacteria with and without antibiotics (tetracycline): Total number of bacteria (without antibiotics) in the samples collected from Agha Ltd, Denm poultry (big commercial poutries) and Rahat poultry, Star poultry (small local poultries) were counted and the results were given in Table 2 and presented in the Figure 1. The numbers of total bacteria differ from sample to sample. Total average count of the fecal wastes collected from a small local poultry -Rahat poultry showed highest number of bacteria 34510000/ml (sample-4). The second highest count (31140000/ml) was also from a small local poultry -Star poultry (sample-3). Total average count of sample- 1(big commercial poultry-Agha Ltd.) and sample-2 (big commercial poultry- Demn poultry) were 11276667/ml and 15970000/ml respectively. The highest count (from small local poutry Rahat poultry) was 3.07 times greater than that of lowest count (from a big commercial poultry- Agha Poultry). In total bacterial count with antibiotics (tetracycline) of same sample (sample-4, Star poultry, small local poultry) showed highest bacterial count (3980000) and sample-1 (Agha, Big commercial poultry farm) exhibited the lowest bacterial count (8000/ml). The highest one was 497.5 times greater than lowest one. It is important to note that the amount of tetracycline resistant bacteria in local poultries (sample-1 and 2) is much higher than that of sample 3 and 4 (Figure 2 and Tables 3-5).

Sample Dilution No.of  Colony No.of microrganisms/ ml Average CFU/ml
Sample – 1 AGHA 10-2 10-4 10-6 Too Numerous 83 33 × 830000 33000000   11276666.67
Sample – 2 DEMN 10-2 10-4 10-6 Too Numerous 91 47 × 910000 47000000   15970000
Sample – 3 STAR 10-2 10-4 10-6 Too Numerous 142 92 × 1420000 92000000   31140000
Sample – 4 RAHAT 10-2 10-4 10-6 Too Numerous 153 102 × 1530000 102000000   34510000

Table 2: Total Count of Bacteria without Antibiotics (tetracycline).

Sample Dilution No. of  Colony No.of microorganisms/ ml Average CFU/ml
Sample – 1 10-2 10-4 10-6 100 23 0 10000 230000 ×   80000
Sample – 2 10-2 10-4 10-6 120 33 0 12000 330000 ×   114000
Sample – 3 10-2 10-4 10-6 Too Numerous 83 7 × 830000 7000000   2610000
Sample – 4 10-2 10-4 10-6 Too Numerous 94 11 × 940000 11000000   3980000

Table 3: Total Count of Bacteria with Antibiotics (tetracycline).

Sample Dilution Concentration of tetracycline (m/ml) No. of Colony No. of Bacteria/ml
1 10-3 10-3 10-3 30 60 100 53 24 13 53×10-3 24×10-3 13×10-3
2 10-3 10-3 10-3 30 60 100 77 39 19 77×10-3 39×10-3 19×10-3
3 10-3 10-3 10-3 30 60 100 103 61 27 103×10-3 61×10-3 27×10-3
4 10-3 10-3 10-3 30 60 100 185 73 53 185×10-3 73×10-3 53×10-3

Table 4: Bacterial count (dilution 10-4) with different concentration of tetracycline.

Sample Dilution Concentration of tetracycline (m/ml) No. of Colony No. of Bacteria/ml
1 10-4 10-4 10-4 30 60 100 33 11 3 33×10-4 11×10-4 3×10-4
2 10-4 10-4 10-4 30 60 100 53 24 6 53×10-4 24×10-4 6×10-4
3 10-4 10-4 10-4 30 60 100 91 33 11 91×10-4 33×10-4 11×10-4
4 10-4 10-4 10-4 30 60 100 102 43 13 102×10-4 43×10-4 13×10-4

Table 5: Bacterial count (dilution 10-4) with different concentration of tetracycline.

bacteriology-parasitology-total-viable-count

Figure 1: Result of total viable count of four types of samples collected from poulties.

bacteriology-parasitology-Total-resistant-bacterial

Figure 2: Total resistant bacterial count in the samples 1,2,3,4.

Isolation and identification of tetracycline resistant E. coli

A total of 30 individual colonies of E. coli were isolated and were characterized according to the biochemical properties. Following figures show the characteristic metallic sheen on EMB agar plate of the isolates and the biochemical properties (Figures 3-7).

bacteriology-parasitology-Tetracycline-Resistant

Figure 3: Tetracycline (30 μl/ml) Resistant E. coli on EMB.

bacteriology-parasitology-Citrate-Test

Figure 4: Citrate Test.

bacteriology-parasitology-VP-Test

Figure 5: VP Test.

bacteriology-parasitology-MR-Test

Figure 6: MR Test.

bacteriology-parasitology-Motility-Test

Figure 7: Motility Test.

Antimicrobial Susceptibility

Antimicrobial susceptibility patterns of the isolates

Six antibiotics were tested against the E. coli isolates using standard disc.

1. Tetracycline (T,30 μg)

2. Gentamycin (G,10 μg)

3. Imipenem (I,10 μg)

4. Chloramphenicol (C,30 μg)

5. Penicillin (P,10 μg)

6. Erythromycin (E,15 μg)

After performing sensitivity test it was found that isolated tetracycline-resistant E. coli were 100% resistant to penicillin and erythromycin, 100% sensitive to imipenem, 93.34% resistant to tetracycline, 23.03% resistant to gentamycin and 53.33% resistant to chloramphenicol (Figure 8 and Tables 6-8).

Antimicrobial agents (µg) Diffusion zone breakpoints (mm)
Aminoglycosides
Gentamycin ≤ 12
Cephalosporins
Penicillin ≤ 13
Imipenem ≤ 13
Phenicols
Chloramphenicol ≤ 12
Macrolides  
Erythromycin ≤ 14
Tetracycline
Tetracycline ≤ 14

Table 6: Standard range of antimicrobial susceptibility.

Antimicrobial agents Resistant (R) isolates (%) Intermediate (I) isolates (%) Sensitive (S) isolates (%)
Gentamycin 7 (23.3%) 0 23 (76.7%)
Penicillin 30 (100%) 0 0
Imipenem 0 0 100 (100%)
Chloramphenicol 16 (53.33%) 0 14 (46.67%)
Tetracycline 28 (93.24%) 1 (3.34%) 1 (3.34%)
Erythromycin 30 (100%) 0 0

Table 7: Susceptibilities of 30 isolates from sample 1, 2, 3 and 4 to different antibiotics.

  Antibiotics Concentration (µg/ml) Zone of inhibition (mm) Remarks     Antibiotics Concentration (µg/ml) Zone of inhibition (mm) Remarks
JE1 Penicillin 10µg 2 R JE16 Penicillin 10µg 0 R
Gentamycin 10 µg 14 R Gentamycin 10 µg 8 R
Erythromycin 15 µg 0 R Erythromycin 15 µg 2 R
Tetracycline 30 µg 0 R Tetracycline 30 µg 16 I
Chloramphenicol 30 µg 0 R Chloramphenicol 30 µg 0 R
Imipenem 10 µg 29 S Imipenem 10 µg 22 S
JE2 Penicillin 10µg 0 R JE17  Penicillin 10µg 0 R
Gentamycin 10 µg 18 R Gentamycin 10 µg 24 S
Erythromycin 15 µg 9 R Erythromycin 15 µg 0 R
Tetracycline 30 µg 10 R Tetracycline 30 µg 0 R
Chloramphenicol 30 µg 20 S Chloramphenicol 30 µg 3 R
Imipenem 10 µg 41 S Imipenem 10 µg 36 S
JE3 Penicillin 10µg 0 R JE18  Penicillin 10µg 0 R
Gentamycin 10 µg 15 R Gentamycin 10 µg 27 S
Erythromycin 15 µg 6 R Erythromycin 15 µg 6 R
Tetracycline 30 µg 11 R Tetracycline 30 µg 11 R
Chloramphenicol 30 µg 0 R Chloramphenicol 30 µg 9 R
Imipenem 10 µg 32 S Imipenem 10 µg 29 S
JE4 Penicillin 10µg 0 R JE19  Penicillin 10µg 0 R
Gentamycin 10 µg 4 R Gentamycin 10 µg 19 S
Erythromycin 15 µg 0 R Erythromycin 15 µg 3 R
Tetracycline 30 µg 10 R Tetracycline 30 µg 9 R
Chloramphenicol 30 µg 9 R Chloramphenicol 30 µg 0 R
Imipenem 10 µg 28 S Imipenem 10 µg 27 S
JE5 Penicillin 10µg 5 R JE20  Penicillin 10µg 5 R
Gentamycin 10 µg 9 R Gentamycin 10 µg 20 S
Erythromycin 15 µg 0 R Erythromycin 15 µg 0 R
Tetracycline 30 µg 11 R Tetracycline 30 µg 13 R
Chloramphenicol 30 µg 0 R Chloramphenicol 30 µg 19 S
Imipenem 10 µg 33 S Imipenem 10 µg 41 S
JE6 Penicillin 10µg 0 R JE21  Penicillin 10µg 0 R
Gentamycin 10 µg 19 S Gentamycin 10 µg 16 S
Erythromycin 15 µg 5 R Erythromycin 15 µg 9 R
Tetracycline 30 µg 9 R Tetracycline 30 µg 6 R
Chloramphenicol 30 µg 19 S Chloramphenicol 30 µg 0 R
Imipenem 10 µg 37 S Imipenem 10 µg 24 S
JE& Penicillin 10µg 4 R JE22  Penicillin 10µg 0 R
Gentamycin 10 µg 23 S Gentamycin 10 µg 24 S
Erythromycin 15 µg 0 R Erythromycin 15 µg 3 R
Tetracycline 30 µg 11 R Tetracycline 30 µg 22 S
Chloramphenicol 30 µg 18 S Chloramphenicol 30 µg 19 S
Imipenem 10 µg 42 S Imipenem 10 µg 24 S
JE8 Penicillin 10µg 9 R JE23  Penicillin 10µg 0 R
Gentamycin 10 µg 16 S Gentamycin 10 µg 23 S
Erythromycin 15 µg 0 R Erythromycin 15 µg 9 R
Tetracycline 30 µg 13 R Tetracycline 30 µg 0 R
Chloramphenicol 30 µg 11 R Chloramphenicol 30 µg 9 R
Imipenem 10 µg 31 S Imipenem 10 µg 26 S
JE9 Penicillin 10µg 0 R JE24  Penicillin 10µg 0 R
Gentamycin 10 µg 16 S Gentamycin 10 µg 24 S
Erythromycin 15 µg 2 R Erythromycin 15 µg 11 R
Tetracycline 30 µg 2 R Tetracycline 30 µg 0 R
Chloramphenicol 30 µg 20 S Chloramphenicol 30 µg 25 S
Imipenem 10 µg 26 S Imipenem 10 µg 22 S
JE10 Penicillin 10µg 0 R JE25  Penicillin 10µg 0 R
Gentamycin 10 µg 20 S Gentamycin 10 µg 19 S
Erythromycin 15 µg 7 R Erythromycin 15 µg 8 R
Tetracycline 30 µg 11 R Tetracycline 30 µg 0 R
Chloramphenicol 30 µg 19 S Chloramphenicol 30 µg 22 S
Imipenem 10 µg 31 S Imipenem 10 µg 27 S
JE11 Penicillin 10µg 0 R JE26  Penicillin 10µg 0 R
Gentamycin 10 µg 21 S Gentamycin 10 µg 17 S
Erythromycin 15 µg 0 R Erythromycin 15 µg 8 R
Tetracycline 30 µg 11 R Tetracycline 30 µg 0 R
Chloramphenicol 30 µg 0 R Chloramphenicol 30 µg 25 S
Imipenem 10 µg 34 S Imipenem 10 µg 23 S
JE12 Penicillin 10µg 0 R JE27  Penicillin 10µg 0 R
Gentamycin 10 µg 26 S Gentamycin 10 µg 21 S
Erythromycin 15 µg 4 R Erythromycin 15 µg 0 R
Tetracycline 30 µg 11 R Tetracycline 30 µg 0 R
Chloramphenicol 30 µg 25 S Chloramphenicol 30 µg 0 R
Imipenem 10 µg 44 S Imipenem 10 µg 26 S
JE13 Penicillin 10µg 0 R JE28  Penicillin 10µg 0 R
Gentamycin 10 µg 22 S Gentamycin 10 µg 24 S
Erythromycin 15 µg 3 R Erythromycin 15 µg 0 R
Tetracycline 30 µg 15 R Tetracycline 30 µg 0 R
Chloramphenicol 30 µg 0 R Chloramphenicol 30 µg 0 R
Imipenem 10 µg 37 S Imipenem 10 µg 34 S
JE14 Penicillin 10µg 0 R JE29  Penicillin 10µg 0 R
Gentamycin 10 µg 22 S Gentamycin 10 µg 24 S
Erythromycin 15 µg 2 R Erythromycin 15 µg 0 R
Tetracycline 30 µg 9 R Tetracycline 30 µg 0 R
Chloramphenicol 30 µg 19 S Chloramphenicol 30 µg 4 S
Imipenem 10 µg 36 S Imipenem 10 µg 21 S
JE15 Penicillin 10µg 0 R JE30  Penicillin 10µg 0 R
Gentamycin 10 µg 8 R Gentamycin 10 µg 17 S
Erythromycin 15 µg 8 R Erythromycin 15 µg 0 R
Tetracycline 30 µg 15 R Tetracycline 30 µg 11 R
Chloramphenicol 30 µg 8 R Chloramphenicol 30 µg 22 S
Imipenem 10 µg 33 S Imipenem 10 µg 24 S

Table 8: Antimicrobial susceptibility of all of the poultry isolates showing different zone of inhibition (mm).

bacteriology-parasitology-Antibiotic-sensitivity-Test

Figure 8: Antibiotic sensitivity test.

Total 30 isolates were subjected to plasmid DNA extraction and they were analyzed in 1% Agarose. The results are negative and no band was found (Figure 9).

Discussion

Random use of antibiotics without medical indication in Poultry and adult dairy cows are a common phenomenon these days. This contributes to the increase of antimicrobial resistance and indirectly exposes human beings to these pathogens [14]. In this study, poultry, a popular and widespread business was selected to observe its contribution to the development of multi-drug resistant E. coli. We have divided Poultry two branches-small local culture and big industrial culture. Various types of antibiotics are being used in these poultry industry. The most common types of antibiotic that is used in poultry are tetracycline-which was used as standard antibiotic in this study. Other most common type of antibiotics like penicillin, imepenem, chloramphenicol, erythromycin and gentamycin were used to observe multi-drug resistance. 20 isolates were taken from big poultry farms like Agha Ltd and Denm Poultry. 10 isolates were taken from small local poultry farms like Rahat Poultry and Star Poultry. After collection of sample, total number of bacteria with and without antibiotics was counted. In both cases numerous bacterial growths were observed. The normal dose of tetracycline is 30 μg/ml which failed extremely to regulate high bacterial growth. The samples labeled with number 1, 2, 3, and 4 were allowed to grow at different concentrations of tetracycline (30, 60 and 100 μg /ml) where bacterial growth was observed. After performing sensitivity test against other commonly used antibiotics in poultry, it was found that isolated tetracycline-resistant E. coli were 100% resistant to penicillin and erythromycin, 100% sensitive to imipenem, 93.34% resistant to tetracycline, 23.03% resistant to gentamycin and 53.33% resistant to chloramphenicol. These indicated the multidrug resistant property of isolates. A statistically significant [12] Increase in antibiotic resistance was observed among outpatient and inpatient isolates of E. coli. Subsequent Agarose Gel Electrophoresis showed no plasmid-DNA band in the gel indicating non-existence of any bacterial plasmid proving that observed resistance was chromosomal genemediated or at least not plasmid mediated. Observation of the multidrug resistance character of poultry fecal isolates is a terrible warning to natural environment [15,16]. The poultry feces used by farmers as manure can poison the crop. Poultry feces is also used as a common feed for fish, so these fish containing multi-drug resistant culture of bacteria like E. coli can be deadly for humans and animals, that is, for any fish eaters. Antibiotics resistance in bacteria associate with food animals and the use of antibiotics for agricultural purposes, particularly for growth enhancement, contributed to the increased prevalence of antibiotic-resistant bacteria. Our finding proposed that proper antibiotics should be used at proper doses to avoid the development of multi-drug resistant bacteria. To perform these, skilled workers with sound knowledge of antibiotics are essential. For personal-small poultry farm, the related individuals should take training on the use antibiotics. The waste of poultry should be disposed off properly to avoid the spread of multi-drug resistant bacteria in the environment.

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Citation: Khan MS, Akhtar N, Haque ME, Barua A, Chowdhury T, et al. (2014) Isolation and Identification of non-plasmid Multidrug Resistant E.coli from Poultry Wastes in Chittagong Region, Bangladesh. J Bacteriol Parasitol 5:182.

Copyright: © 2014 Khan MS, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
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