The green bottle fly, Phaenicia (Lucilia) sericata (Diptera:Calliphoridae),
is among the cohort of flies that frequently visit filthy sites within and around
human dwellings. The vectorial competences of such flies in passive or mechanical
transmission of pathogens have been widely documented (Kobayashi
et al., 1999; Sukontason et al., 2007).
Although, the external surface of the insects body (e.g., wings, spines,
etc.), regurgitation of salivary contents and feacal deposits have been highlighted
as the probable routes of pathogen transmission among flies (Rosef
and Kapperud, 1983; Nazni et al., 2005),
the most important route still remains controversial. This might explain why
diarrhea, food poisoning and other preventable infections are still plaguing
man especially in the developing countries and could be ascribed to the indiscriminate
dumping of (domestic) waste, which serves as breeding grounds for these flies
and pathogens (Holt et al., 2007).
Given the relatively low level of hygiene around human dwellings in developing nations and its attendant health effects, the crux of this study was to identify the cohort of microbes associated with P. sericata in Benin City and to discuss the possible health implications of such microbes.
MATERIALS AND METHODS
The specimens used for this study were collected from three locations namely: abattoir in the city centre; restaurant and hostel both within the precinct of the University of Benin, Ugbowo campus. These specimens were collected using an aspirator and transferred to sterile vials for ex situ studies. Crystal violet, distilled H2O, Lugols iodine, Safranin dye and H2O2 were some of the reagents used. Glass wares were cleaned and sterilized at 160-170°C for 45-60 min. Inoculating loops were heat-sterilized over a bursen burner until red hot before use. Nutrient agar, McConkey agar, potato-dextrose agar and blood agar (autoclaved for 15 min at 120°C and cooled to 50°C). The media were aseptically dispensed into the Petri dishes containing the samples. The Petri dishes were gently rotated for evenness and homogenous spread to prevent confluence growth. The media were left to solidify and incubated at 37°C for 24 h.
Isolation and enumeration of bacteria: Using pour plate method, the
total viable bacteria were enumerated. A stock sample was prepared by the introduction
of an adult L. sericata into normal saline and 1 mL of this solution
was serially diluted into 1:10, 1:100 and 1:1000. The serial dilutions of 1:100
were then plate on nutrient agar, McConkey agar, blood agar and potato dextrose
agar. All cultures were incubated at 37°C for 48 h. Using streak techniques,
bacteria colonies were purified by sub-culturing on fresh nutrient agar.
Isolation and enumeration of fungi: Isolates were established using Pour Plate method on potato dextrose agar. To discourage the growth of bacteria 50 mg mL-1 of streptomycin was added to the medium.
Bacteria: Isolates were identified using biochemical and morphological
tests as described in Bergers manual of determinative bacteriology (Buchaman
and Gibbons, 1974).
Fungi: Spores staining carried out on a heat fixed smear of lactophenol cotton and blue green reagent was added for allowed to stand for a minute before rinsing with distilled water. Then 95% ethanol was added for 30 sec and rinsed. Sample was then identified using microscopic and macroscopic features of the hyphal mass and morphology of cells and spores.
Gram staining: On glass slides, smear of each of the bacteria isolate was made and heat fixed. Then treated with crystal violet (0.3% w/v) and rinsed off with distilled H2O after 1 min; in similar manner, the isolates on the slide were also treated with iodine (0.4% w/v) and rinsed, followed by ethanol (95% w/v) for 30 sec and then stained with a secondary stain, Safranin (0.4% w/v) for a minute, rinsed and air dried. Slides were then examined under oil immersion lens (magnification, x100).
Oxidase test: This was used to differentiate between Pseudomonas and other gram -ve rod shaped bacteria. Each isolate from the culture was smear on filter paper using sterile wire loop. A drop of oxidase reagent (1.0% aqueous tetramethyl-B-phenylenediamine dihydrochloride) was added. The appearance of purple colour within 10 sec is indicative of positive oxidase.
Citrate-utilization test: Simmons citrate medium was used to identify microbes utilizing citrate as their sole source of energy for growth. Twenty three gram of Simmons citrate agar was dissolved in 1 L of distilled H2O and dispensed in a sterile McCartney bottle, followed by sterilization at 120°C for 15 min. The test organism was introduced into the medium and incubated at 37°C for 24 h. A colour change from light green to bright blue indicates a positive result, while turbidity in the medium and no colour change indicate a negative citrate test.
Catalase test: This was used to delineate catalase-producing bacteria from non-catalase-producing bacteria by adding 3 mL of H2O2 (3% v/v) into a test tube. Using sterile glass rod, colonies of the test organism were introduced into the H2O2 solution. Immediate release of air bubbles indicates a positive test (Staphylococcus species) and no bubbles shows negative test (Streptococcus species).
Fungal identification: Spore staining was carried out on a heat-fixed smear of lactophenol cotton; blue green reagent was added and rinsed after a minute. Ethanol (95%) was then added and rinsed after 30 sec. Sample was then viewed under the microscope.
RESULTS AND DISCUSSION
Different bacteria were isolated from the external body parts of Phaenicia sericata. The bacteria were Micrococcus species, Klebsiella species, Lactobacillus species, Staphylococcus aureus, Escherichia coli, Corynebacteria species and Streptococcus pyogenes (Table 1). Some bacteria were present on P. sericata across three different sampling sites. Lactobacillus sp., Staphylococcus aureus and Escherichia coli were such ubiquitous microbes whereas Corynebacteria sp. and Micrococcus sp. were only isolated from P. sericata collected around the restaurants.
Staphylococcus aureus and Lactobacillus spp. were the most abundance species found on P. sericata with incidence of 35.29 and 20.57%, respectively and collectively had an incidence of >50% (Table 2). Corynebacteria dipththeria, Klebsella sp. and Corynebacteria sp. were relatively low with an incidence of 2.94% each (i.e., 8.82% collectively).
Eight genera of fungi were isolated from Phaenicia sericata. Of these fungi, Aspergillus species, Trichoderma, Geotrichum were ubiquitous across the studied sites. Others are Fusarium species, Penicillium species and Rhizopus species (Table 3).
In a study carried out in 2008 by Vazirianzadeh et al.
(2009) the most abundant microbes isolated from the external surfaces of
Musca domestica were Escherichia coli, Staphylococcus aureus
and Pseudomonas sp., whereas earlier, (Olsen and
Hammack, 2000) isolated Salmonella enteritidis, S. infantis,
S. heidelberg from housefly. Cases of dermatophytes such as Microsporium
gypseum and Trichophyton mentagrophytes have also been recorded (Zarrin
et al., 2007).
||Morpho-characteristics and identity of bacteria isolated from
Phaenicia sericata in three different sites in Benin city
|+: Present, -: Absent
||Abundance and prevalence of bacteria recovered from P.
sericata in Benin city
|| Fungi isolated from Phaenicia sericata in Benin city
|*1: Within the precinct of the hall or hostel, 2:Abattoir,
Staphylococcus aureus is generally recognized as one of the causative
agent of food poisoning while Streptococcus species are mainly human
pathogen associated with local systemic invasion and post streptococcal disorder.
In congruent with other findings, Phaenicia sericata contributes to
the epidemiology of pathogenic infections around cafeteria and hostels within
the precinct of the university and also abattoir. And the presence of E.
coli on P. sericata collected from these sites, with an incidence
of 8.8%, is symptomatic of an environment lade with human feacal deposits. Escherichia
coli has also been isolated from the body surface of Musca domestica
(Sasaki et al., 2000), a frequent visitor of
In a comparative account houseflies and American cockroaches have been reported
to have similar vectorial competence in the mechanical transmission of pathogens
(Lamiaa et al., 2007). This study highlights
the nuisance value of P. sericata in and around the studied sites with
8 genera of bacteria isolated, precisely a total of nine species.
Aspergillus niger has been isolated from Cochliomyia megacephala
(Kontoyiannis and Lewis, 2010). Similarly, our findings
revealed that Phaenecia sericata can also transmit A. niger.
Worthy of note is the medical importance or pathogenic status of A. niger
in nosocosmic infections (Kontoyiannis and Lewis, 2010).
Aspergillus fumigates has been incriminated in all forms of invasive
and non-invasive aspergillosis. Although, acclaimed to be cosmopolitan, the
fungus, Fusarium species are also regarded as rare opportunistic fungi,
which have been implicated in cutaneous and sub-cutaneous infections (Srivoramas
et al., 2012).
Conclusively, in order to circumvent the possible health issues that might arise from infections associated with these pathogens, the environment should be kept clean; and the agencies and/or departments in charge of the sanitary conditions of these places should brace up to their responsibilities.