MATERIALS AND METHODS

Sampling Area

This study was carried out in Iligan City, Philippines – a highly urbanized

city situated in Northern Mindanao (Region 10) that has a total land area of

81,337 hectares (813.37 sq. km.) and a total population of 342,618 people as of

2015, comprised mostly of Roman Catholics, followed by Muslims, which

comprises 11.48% of the population (reference).

Due to cultural and religious grounds, poultry production and consumption

is relatively high, thus, commodities consumed by the locals are commonly

bought from the large markets in the city. However; only one market was selected

in this study to obtain samplesfrom, the Pala-o Wet Market(Figure1), which is the

only market selling chicken intestines. In this marketplace,only one meatshop and

seven stalls were vending chicken intestines.

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Figure 1. Geographical Location of Pala-o Wet Market in Iligan City, Philippines

(Google, n.d.) Comment [LJCF1]: please adjust
figure title, it ahould be aligned with
the figure

Sample Collection

Sample collection was done once a month, for three months, from

September to November 2018 at Pala-o Wet Market. A total of 24 fresh raw

chicken samples were randomly purchased, with one sample obtained from each

of the eight stalls. Samples were bought early in the morning, around 06:00 A.M.

– 07:00 A.M. to ensure that the collected samples are fresh. Chicken intestine

was taken from each retail market stall aseptically, with each vendor handling the

chicken intestine using a clean plastic cellophane, then the sample was placed in a

sterile glass bottle covered with aluminum foil. It was transported to the

laboratory immediately in an insulating foam box with ice to ensure that the

samples are fresh once tested for analysis.

Broth Enrichment

To obtain a considerable number of bacterial isolates, broth enrichment

was done after sample collection. A loopful of chicken intestine sample was

swabbed using sterile inoculating loop then soaked in 10-mL lactose broth. The

broth tubes were then incubated at room temperature for 24 hours and were used

for culture and isolation of the chicken intestine bacteria.

Culture of Bacteria and Isolation of Escherichia coli from Chicken Intestines

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To isolate E. coli,one mL of pre-enriched lactose broth solutions were

transferred to a test tube containing nine mL of sterile distilled water and was

serially diluted to obtain a dilution of 10-2. One hundredmicroliters of enriched

solution aliquot was pipetted and cultured on Eosin Methylene Blue agar plates,

employing the spread-plating technique and was then incubated at room

temperature for 48 hours. Presumptive colonies of E. coli displaying a green

metallic sheen property were isolated and purified twice in the same agar medium

at the same length of incubation period and temperature to obtain pure colonies.

SuspectedE. coli isolates were stored at 4℃ in EMB agar slants until used.

Presumptive Identification of E. coli through Gram Staining

and Biochemical Testing

The suspected E. coli colonies were classified through cellular

morphological characterization and biochemical testing or growth-dependent

identification methods (Adejuwon et al.,2011). Morphological characterization

included observation of bacterial cell shape, arrangement, colorand Gram stain

reaction; while biochemical tests employed were indole, methyl red, and citrate

utilization test (Elumba 2018). The schematic diagram for the identification of E.

coli is shown in Figure 2.

All suspected E. coli isolates that wereGram negative, rod-shaped, and

arranged ____________and tested positive for indole and methyl red tests while Comment [LJCF2]: arrangement of
E. coli cells
negative for citrate utiilization assay were presumptively-identified as E.coli.
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Pure cultures

Positive growth in Eosin Methylene

Blue agar (green metallic sheen
colonies)

Gram staining (-)

Biochemical test results

Indole (+), Methyl Red (+), Citrate (-)

Escherichia coli

Figure 2. Schematic diagram used for the identification of presumptive E. coli

isolates.

Antibiotic susceptibility testing

Kirby-Bauer disc diffusion method was employed to assess antibiotic

sensitivity of the72 presumptively-identifiedE. coli isolates. Twenty-four-hour old

isolatebacterial suspensions were tested and prepared based on 0.5 McFarland

turbidity standard (1.5x108 bacterial cells/mL). One hundred microliters of each

suspensionwere spread-plated on Mueller Hinton Agar (MHA) plates to create

bacterial lawn and where the antibiotic discs were aseptically placed (Sivaraman,

2017). Five antibiotics were used, namely: Amoxicillin with Clavulanic Acid (30

µg), Aztreonam (30 µg), Ceftazidime (30 µg), Ceftriaxone (30 µg), and
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Ciprofloxacin (5 µg). A 6-mm blank paper disk pipetted with 10-µL sterile

distilled water served as the negative control. The MHA plates were then

incubatedat room temperature and after 18-24 hours of incubation, inhibition

zones were measured and isolates were categorized as susceptible, intermediate-

resistant, or resistant to each antibiotic according to the criteria set by the Clinical

Laboratory Standard Institute (CLSI) (Hudzicki 2013), as shown in the table

below (Table 1).

Table 1. Zone diameter interpretative standards for Escherichia coliagainst

different antibiotics(CLSI, year).
Interpretative Categories and Zone
Diameter Breakpoints, mm
Intermediat Resistan
Susceptible
Antibiotics e t
Amoxicillin with
≥18 14-17 ≤13
clavulanic acid (AUG)
Aztreonam (ATM) ≥21 18-20 ≤17
Ciprofloxacin (CIP) ≥21 16-20 ≤15
Ceftazidime (CAZ) ≥21 18-20 ≤17
Ceftriaxone (CRO) ≥23 20-22 ≤19

Moreover, E. coli Biot (include the reference number or code) was used as

a reference strain to validate identity and compare antibiotic sensitivity.

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Figure 3. E. coli(code) reference strain.

Statistical Analysis

Test results were documented using a Microsoft Excel 2016 spreadsheet.

A cross tabulation was performed to test if there was a relationship between the

months and resistance levels of the isolated E. coli. Further analysis was done

through Pearson’s Chi-Square Test to provide stronger claims to the

aforementioned relationship of months vs. resistance levels. Lastly, Analysis of

Variance was used to compare the significant difference between the computed

means to determine which antibiotic E.coli showed the least to greatest resistance

and lastly Post-Hoc to test antibiotic efficacy.