2.1. Site Selection and Semi-Structured Interviews
This study was conducted in two different markets of Mongu (A) and Lusaka (B) districts (15.2736° S, 23.1501° E and 15.3875° S, 28.3228° E respectively), Zambia (Figure 1
). The markets supply a wide range of products from fresh foods to dry foods. Semi-structured interviews were conducted with the traders (comprised of randomly selected males and females) and consumers (including randomly selected males and females), using a simple interview tool, which included the following thematic areas:
How the vendor/consumer feels about the presence of high numbers of flies at fish stalls.
Whether or not the vendor has tried some control measures against the flies
The consumer’s view of a fish stall that appears to have some intervention against flies in place.
What interventions those vendors that have had no means of reducing or getting rid of flies would like to try, given a chance.
What the vendors think about covering the fish with netting material to keep flies away from the fish.
The discussions were held with male and female traders and consumers, to understand their perception of flies and interest in interventions to reduce their numbers. Individual males and females that comprised the groups were randomly selected. The survey was conducted in January and April in Mongu and Lusaka respectively. Because consumers were in a hurry to make their purchase and return home, it was easier to have the five-to-ten minute discussion with traders. Ten consumers were interviewed in the Mongu market while 20 traders participated in the interviews. Twenty traders and twenty consumers were interviewed in Lusaka where consumers were more willing to participate in interviews.
2.3. Culture, Isolation and PCR Detection of Salmonella spp. and Enteropathogenic E. coli
Before analysis the frozen flies were thawed and the contents of each Eppendorf tube were vortexed for 30 s to initiate exterior washing. After washing, 100 µL of PBS fly wash was subjected to total bacterial and coliform counts on Nutrient agar (Oxoid, Basingstoke, UK) and MacConkey agar (Oxoid, Basingstoke, UK) respectively, using the drop plate methodology, as described before [20
spp. and E. coli
were cultured using the PBS fly wash as the primary inoculum. In the case of Salmonella
100 µL of the PBS fly wash was placed in 900 µL selenite broth (Wako Pure Chemical Industries Ltd., Osaka, Japan) for enrichment at 37 °C overnight. The isolation of Salmonella
spp. was made on Xylose Lysine Deoxycholate agar (HiMedia Laboratories, Mumbai, India) following inoculation and culture for 24 h at 37 °C. The identity of suspected Salmonella
isolates was confirmed biochemically and serologically by standard methods [21
]. In the isolation of E. coli
, the PBS fly wash was inoculated on MacConkey agar and colonies suspected to be E. coli
through lactose fermenting observation were selected. Identification of E. coli
lactose-fermenting positive colonies was conducted using phenotypic characteristics and confirmed by the Triple Sugar Iron (TSI) and IMViC tests [21
PCR was used as a further confirmatory test for Salmonella
spp. and E. coli
. In order to perform the PCR, the template DNA for Salmonella
spp. and E. coli
was extracted by using the heat treatment method as previously described [23
]. Briefly, the suspected isolates were inoculated in 1 mL of Brain Heart Infusion (BHI) broth (Nissui, Tokyo, Japan), placed in an Eppendorf tube and incubated at 37 °C for 18 to 24 h. After cultivation, 0.5 mL of the BHI broth was subjected to heat treatment on a heat block (Thermo Alumi Bath Alb-220: Iwaki Glass Co. Ltd., Tokyo, Japan) at 95 °C for 10 min. The heated broth was centrifuged at 13,000× g
for 5 min to obtain the supernatant (DNA sample) which was then stored at −20 °C for further use.
The presence of Salmonella
spp. was confirmed with the invA
gene following amplification using previously used primers [25
], while Enteropathogenic E. coli
was confirmed through the detection of the multi-gene region, as previously indicated [23
]. The reaction conditions for E. coli
were 5 μL Phusion®
Flash High-Fidelity PCR Master Mix (Thermo Fisher Scientific, Vantaa, Finland), 2 μL distilled water, 2 μL primer mix (MORA-Primer Diarrheal pathogens, Takara Bio Inc., Shiga, Japan), and 1 μL of the DNA sample. Ten microliters reaction mixtures were subjected to amplification for 30 cycles (98 °C denaturing for 10 s and 55 °C for 5 s and 72 °C for 15 s) in PIKO Thermal Cycler (Thermo Fisher Scientific, Vantaa, Finland). Similar conditions were used for Salmonella
spp. The only difference was that the annealing temperature was at 60 °C.
After PCR, products were directly added to 2 μL of gel loading dye (New England Biolabs, Ipswich, MA, USA) to prepare samples for electrophoresis. Samples were gently applied (2 μL per well) to agarose gel (1% agarose and 1% synergel, Wako Pure Chemical Industries Ltd., Osaka,, Japan). Electrophoresis was done at 100 V until dye markers migrated to an appropriate distance, depending on the size of DNA to be visualized. A 100 to 1517 bp DNA ladder moleculer weight marker (Quick-Load® 100 bp DNA Ladder, New England Biolabs, Ipswich, MA, USA) was used to identify the corresponding amplified products. Gels were stained with ethidium bromide and observed under ultraviolet illumination at 256 nm. Salmonella strains previously identified originating from chicken carcasses were used as positive controls while E. coli was used as a negative control. In case of E. coli, EDL931 (stx-1/stx-2 positive strain) was used as a positive control and the known Salmonella as a negative control.
2.4. Determination of Extended Spectrum Beta-Lactamase (ESBL) Producing E. coli and Antibiotic Susceptibility Patterns
The detected enteropathogenic E. coli
were inoculated on MacConkey agar (Oxoid, Basingstoke, UK) containing 2 mg/L of cefotaxime (Sigma-Aldrich, Munich, Germany) for preliminary screening of ESBL-producing bacteria [23
]. The plates were later incubated at 37 °C for 24 h and the colonies that grew on MacConkey agar were subjected to genetic determination of ESBL producing. The E coli
isolates were cultured on brain-heart-infusion followed by DNA extraction using the heat treatment method. The E. coli
isolates were then subjected to PCR for confirmation of resistance genes TEM (Temoniera), SHV (Sulphydryl Variable) and CTX-M (Cefotaxime–Munich) using primers previously used by other workers [21
]. The PCR (Finnzymes Oy, Espoo, Finland) was performed in a total reaction volume of 10 µL consisting of 5 µL Phusion master mix, 2 µL sterile distilled water, 2 µL primers (forward and reverse) and 1 µL bacterial DNA template. The PCR was performed using the rapid cycle DNA amplification method comprising of an initial denaturation step at 98 °C for 30 s, followed by 35 cycles of template denaturation at 98 °C for 1 s, primer annealing at 60 °C for 5 s and 72 °C for 1 s with final extension at 72 °C for 10 s. The PCR products were later viewed with ethidium bromide after electrophoresis through 1.5% agarose gel.
The antimicrobial susceptibility testing was done using the Kirby-Bauer disc diffusion method on Mueller Hinton Agar (Becton, Dickinson and Company, Sparks, MD, USA) based on the Clinical Laboratory Standard Institute (CLSI) guidelines [24
]. The antibiotic discs (Becton, Dickinson and Company, Sparks, MD, USA) used included ampicillin (10 μg), sulfamethoxazole/trimethoprim (1.25/23.75 μg), streptomycin (300 μg), ciprofloxacin (5 μg), tetracycline (30 μg), gentamicin (10 μg), nalidixic acid (30 μg), chloramphenicol (30 μg), ceftazidime (30 μg), norfloxacin (10 μg) and cefotaxime (30 μg). The phenotypic confirmation of ESBL isolates was done by the combination of disc approximation method using either ceftazidime (30 μg) or cefotaxime (30 μg) alone followed by over-night incubation at 37 °C for 18 to 24 h. Interpretation of susceptibility patterns on other anti-microbial discs was done using guidelines laid down in the CLSI, which provides break points corresponding to zones of inhibition diameter. Quality control standard laboratory procedures were strictly followed to avoid contamination. Escherichia coli
ATCC 25922 was used as a quality control organism.