Ovine clone ST1464: A predominant genotype of Staphylococcus aureus subsp. anaerobius isolated from sheep in Sudan

Background: The aim of the present study was to examine the phenotypic and genotypic relatedness of 17 Staphylococcus aureus subsp. anaerobius isolates recovered from sheep abscesses in Khartoum state, Sudan, during the period 2007-2008. Methodology: This sample was characterised using antibiogram typing, biochemical typing with the commercial PhenePlate system (PhPCS) and multilocus sequence typing (MLST). Results: Low levels of resistance were noted to the 11 antimicrobial agents tested. All the isolates corresponded to a single PhP type, and to a single, novel, multilocus sequence type, designated ST1464. Conclusion: These results demonstrate that the vast majority of cases of sheep abscess disease in Khartoum state are caused by a single novel clone of S. aureus subsp. anaerobius. J Infect Dev Ctries 2010; 4(4):235-238. (Received 30 October 2009 Accepted 03 February 2010) Copyright © 2010 Elbir et al. This is an open-access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Introduction Staphylococcus aureus subsp. anaerobius is the etiological agent of a disease in sheep known as Morel's disease, which was first described by Morel in 1911 in France. The disease has been reported from Ethiopia, Kenya, Sudan, Saudi Arabia, Hungary, Spain, and Denmark [1,2,3,4]. The first report of S. aureus subsp. anaerobius from a human was in a patient presenting with septicemia, septic arthritis, and multiple pulmonary abscesses to an emergency department in South Australia [5]. Morel's disease is characterized by a large abscess (up to 70 cm [1]) formed adjacent to or within lymph nodes, mainly in the head, neck and shoulder region. The disease is endemic in nature with high morbidity, but no mortality is directly attributed to it [2]. As sheep constitute a significant proportion of the exported livestock from Sudan, understanding the epidemiology of this disease and implementing effective control measures is of high economic importance. Evidence concerning the diversity of strains causing the disease is currently lacking, but such studies are required for vaccine design. To address this issue we genetically and phenotypically characterised 17 S. aureus subsp. anaerobius strains isolated from sheep abscesses from two distinct locations in Khartoum state. Material and Methods A total of 17 isolates of S. aureus subsp. anaerobius were isolated from two different geographic locations in Khartoum state, Sudan, during the period 2007 2008. The first location was a checkpoint for routine examination of animals entering Khartoum, and the second location was Kadaro Export Abattoir. These two locations represent the main entrance and exit for animals. The bacteria were recovered from the prescapular region. Samples from abscesses were taken aseptically and streaked on sheep blood agar and incubated at 37°C in microaerophilic atmosphere for 48 hours. The isolates were verified by Gram staining and biochemical test [6] and sequence analysis of rpoB gene [7]. Elbir et al. Ovine clone ST1464 J Infect Dev Ctries 2010; 4(4):235-238. 236 Antimicrobial susceptibility testing In vitro antimicrobial susceptibility testing was conducted using a commercially available broth microdilution method (VetMic system; National Veterinary Institute, Uppsala, Sweden). All MIC determinations were performed according to methods described in the Clinical and Laboratory Standards Institute document (CLSI, 2008; formerly NCCLS) [8]. Antimicrobials used in this study were penicillin (0.03-4 μg/ml), Cefalothin (0.06-8 μg/ml), Erythromycin (0.25-32 μg/ml), Chloramphenicol (0.5-64 μg/ml), Clindamycin (0.2-32 μg/ml), Tetracycline (0.5-64 μg/ml), Fusidic acid (0.06-8 μg/ml), Gentamicin (0.5-64 μg/ml), Kanamycin (0.25-32 μg/ml), Oxacillin (0.12-16 μg/ml) and Ciprofloxacin (0.06-4 μg/ml). Typing of isolates with the PhenePlate (PhP) system Biochemical typing of the isolates was performed with the commercial Pheneplate system (PhP-CS and PhP-48 plates; PhPlate Microplate Techniques AB, Stockholm, Sweden). These tests were performed according to instructions from the manufacturer. The test is based on measurements of the kinetics of 24 biochemical reactions [9] performed in microplates. The system generates a quantitative result from each of the 24 tests (a biochemical fingerprint), and can discriminate between hundreds of phenotypes. Briefly, a few colonies were suspended in PhPsuspending media, inoculated onto the PhenePlates, and incubated at 37°C. The plates were read with an optical microplate reader connected to a computer after 16, 40 and 64 hours of incubation. For the cluster analysis of the PhP-data, the unweighted-pair group method using average linkages (UPGMA) was employed. Isolates with a similarity index greater than 0.975 were considered to be of the same PhPsubtype. Multilocus Sequence Typing (MLST) Genomic DNA was extracted using the boiling method [10]. Seven housekeeping genes were used as described previously [11]. DNA sequencing was performed by Macrogen Inc. (Seoul, South Korea) using BigDye (Applied Biosystems) on a ABI3730XL DNA sequencer (Applied Biosystems). Forward and reverse sequences were aligned using the DNA Baser V2 program. Sequences were then submitted the MLST database (http:/www.mlst.net) for the generation of an allelic profile and sequence type (ST). Concatenated MLST data from representative STs were downloaded from http:// saureus.mlst.net/ and a neighbour-joining tree was reconstructed using the MEGA v.4.2 program. Genebank accession number The DNA sequences of each allele at the seven loci used in this study have been deposited in GenBank under accession number GQ468500 GQ468506 and GQ370468 for rpoB gene. Results and Discussion Here we describe the characterisation of 17 isolates of S. aureus subsp. anaerobius isolated from sheep abcesses in the Sudan. The use of a sequencebased typing method, like MLST, facilitated rapid comparisons with other S. aureus strains previously typed using this method via the MLST web site (http://saureus.mlst.net). The clustering of isolates obtained by MLST, and the level of discrimination, is largely consistent with that obtained by PFGE [11,12,13]. All the isolates were identical by MLST; thus we found no differences between the isolates recovered from the two sites. All isolates corresponded to a novel allelic profile assigned ST1464 (102-219-204122-13-177-160). The novelty of this clone was not unexpected given the fact that both animal-derived strains, and in particular strains from Africa, are seriously under-represented in the current MLST database. In order to examine the phylogenetic position of this clone within the context of the wider S. aureus population, we constructed a neighbourjoining tree using the concatenated sequence ST1464, 18 representative STs from humans (including ST152 which was commonly recovered from human carriage in Mali [14]), and four STs previously characterised from sheep samples (Figure 1). These extra STs were downloaded from the MLST web site. Previous studies have shown that MLST data divide S. aureus into two main clades (group 1 and group 2) [15], and these major groups are evident in the tree in Figure 1. Our analysis suggests that ST1464 is a divergent genotype which loosely clusters with Group 2. The other samples isolated from sheep in the database are divergent both from each other and from ST1464. As reported previously, the Malian clone ST152 also occupies a divergent position and does not belong to either of the main two groups (Figure 1). Elbir et al. Ovine clone ST1464 J Infect Dev Ctries 2010; 4(4):235-238.


Introduction
Staphylococcus aureus subsp. anaerobius is the etiological agent of a disease in sheep known as Morel's disease, which was first described by Morel in 1911 in France. The disease has been reported from Ethiopia, Kenya, Sudan, Saudi Arabia, Hungary, Spain, and Denmark [1,2,3,4]. The first report of S. aureus subsp. anaerobius from a human was in a patient presenting with septicemia, septic arthritis, and multiple pulmonary abscesses to an emergency department in South Australia [5]. Morel's disease is characterized by a large abscess (up to 70 cm [1]) formed adjacent to or within lymph nodes, mainly in the head, neck and shoulder region. The disease is endemic in nature with high morbidity, but no mortality is directly attributed to it [2].
As sheep constitute a significant proportion of the exported livestock from Sudan, understanding the epidemiology of this disease and implementing effective control measures is of high economic importance. Evidence concerning the diversity of strains causing the disease is currently lacking, but such studies are required for vaccine design. To address this issue we genetically and phenotypically characterised 17 S. aureus subsp. anaerobius strains isolated from sheep abscesses from two distinct locations in Khartoum state.

Material and Methods
A total of 17 isolates of S. aureus subsp. anaerobius were isolated from two different geographic locations in Khartoum state, Sudan, during the period 2007 -2008. The first location was a checkpoint for routine examination of animals entering Khartoum, and the second location was Kadaro Export Abattoir. These two locations represent the main entrance and exit for animals. The bacteria were recovered from the prescapular region. Samples from abscesses were taken aseptically and streaked on sheep blood agar and incubated at 37°C in microaerophilic atmosphere for 48 hours. The isolates were verified by Gram staining and biochemical test [6] and sequence analysis of rpoB gene [7].

Typing of isolates with the PhenePlate (PhP) system
Biochemical typing of the isolates was performed with the commercial Pheneplate system (PhP-CS and PhP-48 plates; PhPlate Microplate Techniques AB, Stockholm, Sweden). These tests were performed according to instructions from the manufacturer. The test is based on measurements of the kinetics of 24 biochemical reactions [9] performed in microplates. The system generates a quantitative result from each of the 24 tests (a biochemical fingerprint), and can discriminate between hundreds of phenotypes. Briefly, a few colonies were suspended in PhPsuspending media, inoculated onto the PhenePlates, and incubated at 37°C. The plates were read with an optical microplate reader connected to a computer after 16, 40 and 64 hours of incubation. For the cluster analysis of the PhP-data, the unweighted-pair group method using average linkages (UPGMA) was employed. Isolates with a similarity index greater than 0.975 were considered to be of the same PhPsubtype.

Multilocus Sequence Typing (MLST)
Genomic DNA was extracted using the boiling method [10]. Seven housekeeping genes were used as described previously [11]. DNA sequencing was performed by Macrogen Inc. (Seoul, South Korea) using BigDye (Applied Biosystems) on a ABI3730XL DNA sequencer (Applied Biosystems). Forward and reverse sequences were aligned using the DNA Baser V2 program. Sequences were then submitted the MLST database (http:/www.mlst.net) for the generation of an allelic profile and sequence type (ST). Concatenated MLST data from representative STs were downloaded from http:// saureus.mlst.net/ and a neighbour-joining tree was reconstructed using the MEGA v.4.2 program.

Genebank accession number
The DNA sequences of each allele at the seven loci used in this study have been deposited in GenBank under accession number GQ468500 -GQ468506 and GQ370468 for rpoB gene.

Results and Discussion
Here we describe the characterisation of 17 isolates of S. aureus subsp. anaerobius isolated from sheep abcesses in the Sudan. The use of a sequencebased typing method, like MLST, facilitated rapid comparisons with other S. aureus strains previously typed using this method via the MLST web site (http://saureus.mlst.net). The clustering of isolates obtained by MLST, and the level of discrimination, is largely consistent with that obtained by PFGE [11,12,13].
All the isolates were identical by MLST; thus we found no differences between the isolates recovered from the two sites. All isolates corresponded to a novel allelic profile assigned ST1464 (102-219-204-122-13-177-160). The novelty of this clone was not unexpected given the fact that both animal-derived strains, and in particular strains from Africa, are seriously under-represented in the current MLST database. In order to examine the phylogenetic position of this clone within the context of the wider S. aureus population, we constructed a neighbourjoining tree using the concatenated sequence ST1464, 18 representative STs from humans (including ST152 which was commonly recovered from human carriage in Mali [14]), and four STs previously characterised from sheep samples (Figure 1). These extra STs were downloaded from the MLST web site. Previous studies have shown that MLST data divide S. aureus into two main clades (group 1 and group 2) [15], and these major groups are evident in the tree in Figure 1. Our analysis suggests that ST1464 is a divergent genotype which loosely clusters with Group 2. The other samples isolated from sheep in the database are divergent both from each other and from ST1464. As reported previously, the Malian clone ST152 also occupies a divergent position and does not belong to either of the main two groups (Figure 1).  238 The MLST database is based largely on isolates from Europe, the United States of America, Australia and Japan, whereas Africa and mainland Asia are under-represented. Furthermore, although an increasing number of isolates from non-human hosts have been characterised in recent years (mostly bovine isolates), there remain very few data for isolates from sheep. More representative samples are therefore required to shed light on the global diversity of this species. Although MLST is a very useful method for global epidemiology and population studies, it is possible that other typing methods may reveal variation within single MLST genotypes. In order to check for variation within the ST1464 clone, we further typed the isolates by biochemical typing (e.g. PhP) and antibiogram typing. All the isolates were susceptible to 11 antimicrobial agents tested listed in Table 1, and biochemical typing revealed that all the isolates belong to a single PhP type. These observations support the view that the majority of sheep abscesses in Khartoum are caused by a single clone which is genetically and phenotypically invariant. This may reflect the recent introduction and rapid spread of this clone, though a high rate of sheep-to-sheep transmission within a single herd, and it is possible that this clone is well adapted to persist in the ovine host. This finding also raises the possibility of the development of a high specificity vaccine against this clone.
In conclusion ST1464 is a dominant clone from sheep abscesses in Khartoum. This study contributes to the understanding of the epidemiology of the disease which is important for control of sheep abscess and for vaccine design.