Characterization of Staphylococcus aureus isolates from faecal samples of the Straw-Coloured Fruit Bat (Eidolon helvum) in Obafemi Awolowo University (OAU), Nigeria
© Akobi et al.; licensee BioMed Central Ltd. 2012
Received: 26 June 2012
Accepted: 16 November 2012
Published: 26 November 2012
Bats (Chiroptera) are one of the most diverse groups of mammals which carry out important ecological and agricultural functions that are beneficial to humans. However, they are increasingly recognized as natural vectors for a number of zoonotic pathogens and favourable hosts for zoonotic infections. Large populations of the Straw-Coloured Fruit Bat (Eidolon helvum) colonize the main campus of the Obafemi Awolowo University (OAU), Ile-Ife, Nigeria, but the public health implications of faecal contamination and pollution by these flying mammals is unknown. This study characterized S. aureus obtained from faecal samples of these migratory mammals with a view to determining the clonal types of the isolates, and to investigate the possibility of these flying animals as potential reservoir for zoonotic S. aureus infections.
One hundred and seven (107) S. aureus isolates were recovered from 560 faecal samples in eleven roosting sites from January 2008 to February 2010. A large proportion of the isolates were susceptible to antibiotics, and molecular characterization of 70 isolates showed that 65 (92.9%) were assigned in coagulase type VI, while accessory gene typing classified 69 isolates into the following: type I (12; 17.1%), type II (3; 4.3%), type III (1; 1.4%) and type IV (53; 75.7%). On the whole, the isolates were grouped in five (A-E) main genotypes. Of the ten representative isolates selected for multilocus sequence typing (MLST), nine isolates were assigned with new sequence types: ST1725, ST1726, ST1727, ST2463-ST2467 and ST2470. Phylogenetic analysis provided evidence that S. aureus isolates in group C were closely related with ST1822 and associated clones identified in African monkeys, and group D isolates with ST75, ST883 and ST1223. The two groups exhibited remarkable genetic diversity compared to the major S. aureus clade.
Antibiotic resistance in faecal S. aureus isolates of E. helvum is low and multiple unique S. aureus lineages co-existed with E. helvum. The Straw-Coloured Fruit Bat in Ile-Ife, Nigeria is colonized predominantly by ST1725, ST1726, ST2463 and ST2470 with distinct genotypic characteristics that are rarely found in humans. This study has demonstrated on the possible existence of a reservoir of indigenous and anciently-divergent S. aureus clones among mammals in Africa.
KeywordsStaphylococcus aureus Eidolon helvum ST1725 ST1726 ST2463 ST2470 Anciently-diverged S. aureus
Bats (Order: Chiroptera) are the only mammals capable of true sustainable flight and one of the most diverse and species rich mammals on earth . They assist in the regulation of insect populations in their habitats, pollination of flowers and dispersal of seeds of economically important tress, and these ecological roles support forest regeneration and maintenance . However, they roost near human habitation and their association with emerging infections has increased attention on these flying mammals as vectors of zoonotic pathogens [3–5]. The bat species Eidolon helvum is grouped under the suborder Megachiroptera, and it is the most widely distributed Straw-Coloured Fruit Bat which is found in the forest and savannah zones of sub-Saharan Africa [6, 7]. The prime habitats for E. helvum are the tropical forest and typically roost in colonies on tall trees like Eucalyptus saligna and Cocos nucifera.
Staphylococcus aureus is part of the normal flora of the skin and mucous membrane of a wide variety of mammals and birds, and recent studies have indicated that animals could be a source of S. aureus infections in humans [9–11]. The main campus of the Obafemi Awolowo University, Ile-Ife (OAU) Nigeria, is colonized by a large population of E. helvum[12, 13], but faecal contamination and pollution of the environment by these migratory mammals is a problem, moreover, the public health implications of their activities are not known. This study characterized S. aureus obtained from faecal samples of bats that colonize the main campus of the institution, with a view to understanding the clonal nature and diversity of the isolates, and to determine the possible risk of dissemination of S. aureus from bats to humans in the community through faecal shedding.
Results and Discussion
Antibiotic susceptibility of 107 S. aureus isolates from faecal samples of E. helvum in Nigeria
Antibiotics (disk content in μg)
Number of isolates
Resistance rate (%)
Penicillin (10 units)
Oxacillin (1 μg)
Cefoxitin (30 μg)
Erythromycin (15 μg)
Clindamycin (2 μg)
Tetracycline (30 μg)
Ciprofloxacin (5 μg)
Chloramphenicol (30 μg)
Fusidic Acid (10 μg)
Gentamicin (10 μg)
Mupirocin (5 μg and 200 μg)
Genotypes identified in 70 S. aureus isolates from faecal samples of E. helvum in Nigeria
Representative isolate ID
No of isolates (%)
arcC, aroE, glpf, gmk, pta, tpi, yqiL
This study isolated S. aureus from faecal samples of E. helvum, a migratory mammal with an abundant population in OAU, Ile-Ife, Nigeria, and represents the first molecular study on S. aureus colonization of bats in Africa. The isolates were largely susceptible to a number of antibiotics. The combination of coagulase gene type VI and agr type IV are rare among S. aureus isolates associated with humans [28–31], and the evidence that isolates in group C were closely related with divergent ST1822-related clones identified in African monkeys, and group D isolates with ST75, ST883 and ST1223 indicate that there is the possible existence of a reservoir of indigenous and anciently-diverged clones among mammals in Africa.
A total of eleven roosting sites located in the academic area and the students’ hostel in OAU, Ile-Ife were identified for the study (Figure 1), and the duration for sample collection was from January 2008 to September 2008, February to May 2009, and February 2010. The faecal samples were obtained once a month in a designated sampling site between 6-7am by a non-invasive method in which three sterilized piece (36 × 45 inches) of cotton material were spread under the roosting trees. Fresh faecal samples were collected with sterile swab sticks and conveyed promptly to the Department of Microbiology Laboratory (OAU) for microbiological analysis.
Isolation and identification of S. aureusisolates
The swab stick was inserted into a test tube containing 3 ml of sterile nutrient broth (Biolab, supplied by Merck, Johannesburg, South Africa), swirled briefly to discharge the contents into the medium, and the culture was incubated at 37°C overnight. Thereafter, a loopful was streaked on mannitol salt agar (MSA) (Biolab, supplied by Merck, Johannesburg, South Africa) and incubated at 37°C for 48 hours. Preliminary identification of S. aureus was based on positive Gram stain, and positive results for catalase, coagulase (tube method) and DNase tests. The procedure described previously  was employed for DNA isolation. In summary, a single colony was suspended to a McFarland 1.0 standard in 100 μl of TE buffer (10 mM Tris, 1 mM EDTA, pH 8.0) with 10 U of achromopeptidase (Wako Chemical, Co. Ltd.), and the suspension was incubated at 55°C for 10 min. The supernatant was used as crude DNA for PCR. Molecular identification and confirmation of the isolates was based on sequencing analysis of the hsp60 gene as previously reported . PCR products were sequenced by using a Big Dye Terminator (version 3.1) cycle sequencing kit (Applied Biosystems, Foster City, CA) with an ABI Prism 3100 genetic analyzer (Applied Biosystems).
Antibiotic susceptibility testing
The susceptibility testing of the isolates to 11 antibiotics was performed using the disk diffusion method and the following antibiotics were tested: penicillin (10 units), oxacillin (1 μg), cefoxitin (30 μg), erythromycin (15 μg), clindamycin (2 μg), tetracycline (30 μg), ciprofloxacin (5 μg), chloramphenicol (30 μg), fusidic acid (10 μg) gentamicin (10 μg) and mupirocin (5 μg and 200 μg). S. aureus ATCC 25923 was the control strain for the susceptibility testing. The result was interpreted as resistant or susceptible based on the interpretative standard according to the Clinical Laboratory Standards Institute (CLSI) manual for bacterial isolates from animals . Interpretative zone diameter for resistance and susceptibility breakpoints to fusidic acid and mupirocin which are not stated in the CLSI guidelines were considered as described previously [35, 36]. The D-test for determining inducible resistance of clindamycin using erythromycin was performed. A truncated or blunted clindamycin zone of inhibition (D-Shape) indicated inducible resistance. Constitutive resistance was recognized by a clindamycin zone diameter of ≤14 mm .
Molecular characterization of the S. aureusisolates
Characterization of 70 isolates was determined by detection of the Panton Valentine Leukocidin (PVL) gene , agr and coa gene typing . The MAFFT program was used for multiple alignment of the hsp60 partial sequences, and a phylogenetic tree was constructed by the neighbor-joining and bootstrap methods, using MEGA ver. 5.05 . Furthermore, MLST  was carried out on representative S. aureus isolates (based on hsp60 allelic type, coagulase and agr typing). The amplified PCR products were sequenced, and STs were determined for each isolate based on the alleles identified at each of the seven loci using the S. aureus MLST database (http://www.mlst.net). For six representative isolates (AC10, F9, P1, F16, Q15 and R13), we were unable to amplify the aroE and or glpF genes using the standard MLST primers. Therefore degenerate primers CC75dege-aroE-F (5’-WTGCAGTWATHGGWRRYCC-3’), CC75dege-aroE-R (5’-GGWWTATAAAYAATRT CACT-3’), CC75aroEseq-F (5’-CCAATTGAGCATTCYTTATC-3’), CC75dege-glpF-F (5’-GCWGAATTYHT DGGWACWGC-3’), CC75dege-glpF-R (5’-ATWGGYA AWATHGCATGWGC’), and CC75glpF-seq-R (5’-GCAT GTGCAATTCTTGGDC’), were designed by multiple alignment of amino acid sequences of each gene with complete genomes of S. aureus, S. epidermidis, S. haemolyticus and S. lugdunensis from the KEGG database (http://www.genome.jp/kegg/). Sequences of arcC, aroE, glpf, gmk, pta, tpi and yqiL in S. simiae, which was used as an outgroup, were obtained from the draft genome sequence of S. simiae CCM7213 . A phylogenetic tree was constructed based on concatenated arcC, aroE, glpF, gmk, pta, tpi and yqiL sequences using the neighbor-joining method, using MEGA ver. 5.05.
Obafemi Awolowo University
Panton Valentine Leukocidin
Accessory gene regulator
Multilocus sequence typing
- E. helvum :
- S. aureus :
Mannitol salt agar
Clinical Laboratory Standards Institute
Methicillin resistant Staphylococcus aureus.
We acknowledge the comments and suggestions of Professor Iruka Okeke in the preparation of the manuscript, and the kind assistance of Professor Johnson Lin, Dr. Stella Smith and Dr. Solayide Shittu.
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