RESULTADOS OBTENIDOS POBLACIÓN CON BURNOUT

3.8.1 Inoculation and Incubation

The samples were inoculated onto Butzler-type selective medium which consisted of Brucella medium base (Oxoid CM0169B), supplemented with 10% (v/v) sheep blood, Campylobacter growth supplement (Oxoid SR0232E) and Campylobacter selective supplement (Oxoid SR0085E) and incubated micro-aerobically by candle-extinction jar at 37^oC for 72 hours.²⁸ The plates were checked on the second and third day of inoculation for evidence of Campylobacter growth.

Campylobacter colonies/spp on Butzler-type medium, were flat, moist (like water droplets), translucent and non-haemolytic. The colonies tend to become confluent along the streaks made by the inoculating wire. Suspicious colonies were subcultured onto 2 blood agar plates (Oxoid blood agar base No. 2, CM0271B), supplemented with 5-7% sheep blood, and incubated micro-aerobically at 25^oC and 42^oC respectively for 48 hrs.^25,53

All suspected Campylobacter colonies were also Gram stained, and tested for Oxidase reaction, Catalase production and motility. Antimicrobial susceptibility was also done before being stored at -10^oC in FBP medium (5% Glycerol in Nutrient broth supplemented with

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Campylobacter growth supplement, SR232E, containing Sodium pyruvate 0.125g/l, Ferous sulphate 0.125g/l and Sodium metabisulphate 0.125g/l) for further analysis.

3.8.2 Gram Stain Characteristics

The smears were Gram stained by Preston & Morrell’s method,¹⁴⁶ counter stained with dilute carbol fuchsin for 5 minutes. Campylobacter spp. appeared as Gram negative slender, straight, curved or spiral bacilli, appearing in comma, S-shaped or ‘gull-wing’ forms.

3.8.3 Motility test

This was done by the hanging drop method. A drop of the organism suspension in peptone water hanging from a cover slip placed onto a cavity of the hanging drop slide was examined for the characteristic darting movement of Campylobacter spp.

Positive control – Campylobacter jejuni NCTC 11168 Negative control – Klebsiella spp

3.8.4 Biochemical reactions:

3.8.4.1 Catalase test – It was done by rapid slide technique - as described by Hendrickson.¹⁴⁷ The colony of the organism was emulsified in a drop of 3% Hydrogen peroxide on a glass slide placed in a Petri dish. Positive test was indicated by immediate formation of bubbles.

Campylobacter spp. are catalase positive.

Positive Control: Staphylococcus spp.

Negative control: Streptococcus spp.

3.8.4.2 Oxidase test – This was carried out as described by Kovacs.¹⁴⁸ About 3 drops of freshly prepared oxidase reagent (10g/l solution tetramethyl-p-phenylenediamine dihydrchloride) were added onto a piece of filter paper placed in a Petri dish. With a piece of match stick a colony of

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the organism was smeared on the filter paper. A positive test was indicated by development of a blue-purple colour within a few seconds. Campylobacter spp. is oxidase positive.

Positive Control: Pseudomonas aeruginosa ATCC 27853 Negative control: Escherichia coli ATCC 25922

3.8.5 Antibiotic Sensitivity Testing

The disk diffusion method as described by Modified Kirby-Bauer et al was used.¹⁴⁹ Suspensions of the isolated Campylobacter colonies were made in 2 mls peptone water. The turbidity of the suspensions were standardised by comparison to a 0.5 McFarland standard, to give a semi confluent growth.¹⁵⁰ Then plates of Blood agar were inoculated with swab stick charged with bacteria from the suspensions. To the surface of the pre-inoculated agar media, amoxicillin (AML) 10μg, erythromycin (E) 5μg, ciprofloxacin (CIP) 5μg, tetracycline (TE) 30μg, gentamicin (CN) 10μg, chloramphenicol (C) 30μg, sulphamethoxazole/trimethoprim (SXT) 25μg, nalidixic acid (NA) 30μg Oxoid antibiotic discs were applied, and the plates incubated at 37^oC micro-aerobically for 24 hrs.¹⁵⁰ Standard strain of Campylobacter spp (NCTC 11168) was also tested with the same antibiotics in a similar manner. The diameters of the zones of inhibition were measured to the nearest mm using a ruler and interpretation done according to the Clinical and Laboratory Institute Standard (formally: National Committee of Clinical Laboratory Standard) 1999 recommendations.^151,152

Control: Campylobacter jejuni NCTC 11168

3.8.6 Beta lactamase detection

All the isolates were tested for beta-lactamase activity by the starch paper technique.¹⁵³ Strips of starch paper were soaked for 10 minutes in a solution of Benzyl penicillin containing 10⁵mg/ml

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and then laid smoothly in Petri dishes. The colonies of the isolated bacteria were transferred with bacteriological loop, from the culture plates to the surface of the test paper and spread over an area of 2 – 3 mm. The inoculums were placed at least 1 – 3 cm apart. The plates were incubated at 37^oC for 30 minutes after the papers were flooded with iodine solution (Gram’s iodine diluted 1 in 2). The iodine solution turned the paper uniformly black within 30 seconds.

The discolouration of the blue-black colour surrounding the organisms with the widening of the white-halo within five minutes, and the surface of the inoculum remaining whitish, indicates Beta-lactamase producing strains. Non Beta-lactamase producing Campylobacter strains showed no discolouration around the inoculum.

Positive control: Staphylococcus aureus ATCC 25923 3.8.7 Biotyping Scheme

The isolates were biotyped by Lior Extended biotyping, consisting of rapid hippurate hydrolysis test, the rapid Hydrogen sulphate (H2S) test and DNA hydrolysis.¹⁴³ (Table 3.1)

Table 3.1: Lior’s Extended Biotyping Scheme

Campylobacter jejuni Campylobacter coli Campylobacter lari Biotype I II III IV I II I II

Hippurate hydrolysis + + + + - - - - H2S Production - - + + - - + + DNase Test - + - + - + - +

3.8.7.1 Rapid Hippur ate Hydrolysis Test

Small loopful of 48-hr culture growth from blood culture agar plates were well emulsified in test tubes of thawed 1% sodium hippurate. The tubes were incubated, with frequent

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mixing, for 2 hrs at 37^oC in water bath. After incubation, 0.2 ml of ninhydrin reagent (3.5 g ninhydrin in 100 ml of a mixture of equal parts of acetone and butanol) was slowly overlaid on the side of each of the test tubes. The tubes were returned to the water bath, without mixing, for another 10 minutes. Thereafter, the tubes were examined immediately, without shaking, for colour development. A positive test was indicated by deep purple colour. A pale purple colour or

colourless tubes were considered as negative for hippurate hydrolysis.

Positive control – Streptacoccus agalactiae ATCC 12386

Negative control – Streptacoccus pyogenes ATCC 19615 3.8.7.2 Hydrogen Sulphide Production Test

Pure colonies of the isolates were stabbed with straight inoculating wire into the butt of triple sugar iron agar (TSI) media. The tubes were then incubated micro-aerobically at 37^oC for 18 – 24 hours. Positive H2S production was indicated by blackening reaction in the test tubes, whereas a negative reaction showed no blackening.

Positive control – Proteus spp.

Negative control – Escherichia coli ATCC 25922 3.8.7.3 DNA Hydrolysis Test

Large loopfuls of a 24/48-hr culture grown at 37^oC were heavily inoculated in a circular area of about 1 cm diameter on a well-dried DNase test agar medium (Difco DNase Test agar with Methyl Green). The plates were incubated at 35 - 37^oC in a candle extinction jar, and examined daily for 3 to 5 days. An area of growth surrounded by a clear, colourless zone in the green-blue agar indicates a positive DNA hydrolysis. No change or a narrow hazy zone around the bacterial growth was considered as a negative test.

Positive control – Staphylococcus aureus ATCC 25923 Negative control – Staphylococcus epidermidis ATCC 12228

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