The urine samples were cultured on

Cystine-Lactose-Electrolyte-Deficient CLED agar for primary isolation.

Presumptiv E. coli li, which appeared

as yellow colonies, smooth, round, and moist after 24 hours of incubation were

kept in nutrient broth to await secondary isolations.  For secondary isolation, the suspec E. coli coli isolates were cultured on

MacConkey and Blood Agar media (OXOID, Hampshire, England). The Blood agar base

was supplemented with 10% horse blood. A loop full of each sample from the

transport media was introduced on the media plate and was streaked

appropriately with a sterile inoculating loop. The media plates were

appropriately labeled and incubated at 37ºC for 24 hours. The following

biochemical tests were performed to confirm further the suspected isolates:

Indole test, Citrate test, oxidase test, and catalase test.

Biochemical Tests

Peptone water suspensions were prepared in

a bottle according to the manufacturer’s protocol. Three to five pure isolates

were then cultured in suspensions and grown overnight. Two to three drops of

Kovac’s reagent were added to the suspension and the bottle was shaken. The formation

of a pink-colored ring that rose to the surface was observed, indicating a

positive result.

The Simon’s citrate agar was prepared

according to the manufacturer’s protocol. Pure isolates of the organisms on

nutrient agar were inoculated into the citrate agar and incubated for 24

hrs.  The citrate agar was green before

inoculation. There was no color change beca E. coli coli is citrate-negative.

A

drop of oxidase reagent, which contains tetramethyl-p-phenylenediamine was

placed on a pure colony of th E. coli li. No color change occurred indicatin E. coli oli exhibits a negative oxidase reaction.

A

few drops of hydrogen peroxide were added to pure colonies of E. coli i. There was an immediate release

of oxygen bubbles, indicating a positive reaction.

Antibiogram of E.coli isolates; Kirby-Bauer disc

diffusion test

The Kirby-Bauer antimicrobial sensitivity

test method was used to determine the antibiogram of the E. coli i isolates (Bauer et al., 1966). Ten

antimicrobial drugs were used. These were imipenem

(IPM, 10 µg), ertapenem (ERT, 10 µg), aztreonam (AZM, 15 µg), cefepime (FEP, 30

µg), nitrofurantoin (F, 50 µg), cefuroxime (CXM, 10 µg), gentamycin (CN, 10

µg), amikacin (AK, 30 µg), ciprofloxacin (CIP, 5 µg) and levofloxacin (LEV, 10

µg).

Mueller-Hinton agar was prepared according

to the manufacturer’s protocol. The organisms were cultured on nutrient agar

overnight. Between 4 and 5 isolated colonies of the organisms were suspended in

about 2 ml of sterile saline by use of inoculating loop. The saline tube was

vortexed to create a smooth suspension. The turbidity of the suspension was

adjusted to a 0.5 McFarland standard. 200 ml of the suspension was introduced onto

the Mueller-Hinton plate. A sterile glass spreader was used to spread the

organisms on the plate. The surface of the plate was allowed to dry for 5 minutes

before the antibiotic discs were placed on them. Sterile forceps were used to

remove the antibiotic discs from the dispensers. After placing the discs on the

agar, each disc was gently touched with the inoculating loop to ensure their

contact with the agar surface. The plates were then incubated upside down for

24 hours at 37ºC.

PCR Screening for ESBL Genes

The DNAs

of the 41 E. coli i isolates were

extracted using a zymogen extraction kit, based on the manufacturer’s protocol.

The isolates were then screened to determine the types of beta-lactamas bla bla) genes they harbored. A total of forty-o E. coli coli isolates were screened for

the presence o bla 0 bla genes. The

protocols employed by Kiiru and colleagues (Kiiru et al.,

2012. were used with slight modifications. The

reactions were carried out in a 10 µl reaction volume. This consists of 5 µl of

2X

SYBR green master mix, 0.2 µl

each of the primer sequence, 2.6 µl of the Nuclease free water, and 2 µl of the

DNA template. The primer concentration was 0.2M. The PCR cycle

conditions were as follows: 3 mins of initial denaturation at 94 ºC, (94 ºC of

denaturation for 30 seconds, annealing for 30 seconds, elongation at 68 ºC for

30 seconds) x 30 cycles, and final elongation at 68 ºC for 10 minutes. The

annealing temperatures were different for the different primers. The primer sequence

and the annealing temperatures are listed in Table 1

Table 1: Primer sequences of the ESBL genes, amplicon sizes, and annealing temperatures s

Phylogenetic Grouping by PCR

As described by Clermont et al. , 2000, the phylogenetic grouping

of the isolated E. coli i was

determined. The positiv E. coli i strains

were investigated for various genes that would determine their phylogenetic

grouping by multiplex PCR (Clermont et al., 2000). The procedures were

performed in a 10 µl reaction mixture. The reaction included 5 µl of 2X SYBR

green master mix, 0.2 µl each oligonucleotide primer, 2.6 µl of nuclease-free

water, and 2 µl of template DNA. Primer concentration was 0.2 M. Conditions of

the reaction mixtures were 3 mins at 94 ºC initial denaturation, (94 ºC of

denaturation for 30 seconds, annealing at 59.2 °C for 30 seconds, elongation at

68 ºC for 3 minutes) x 30 cycles, and final elongation at 68 ºC for 10 minutes.

The marker-specific primer sequences and their amplicon sizes are listed in

Table 2. Based on the presence or absence of spec chuA, yjaA, TspE4.C2, , T arpA C2 and arpA), the isolates were clustered in group A,

  , B2, or D.  Both group B1 and group chuA acks the chuAgene. However, group B1

TspE4.C2 the TspE4.C2 gene, which is abse t in chuA up A. The chuA

gene is present in both B2 and D isolates. The difference between the two is

t yjA roup B2 has yjA genes while group D lacks it

Table 1: Primer sequences of the ESBL genes, amplicon sizes, and annealing temperatures s

SKS