SciELO - Scientific Electronic Library Online

 
vol.84 número1The sero-prevalence and sero-incidence of African horse sickness and equine encephalosis in selected horse and donkey populations in ZimbabweA retrospective study of anthrax on the Ghaap Plateau, Northern Cape province of South Africa, with special reference to the 2007-2008 outbreaks índice de autoresíndice de materiabúsqueda de artículos
Home Pagelista alfabética de revistas  

Servicios Personalizados

Articulo

Indicadores

Links relacionados

  • En proceso de indezaciónCitado por Google
  • En proceso de indezaciónSimilares en Google

Compartir


Onderstepoort Journal of Veterinary Research

versión On-line ISSN 2219-0635
versión impresa ISSN 0030-2465

Onderstepoort j. vet. res. vol.84 no.1 Pretoria  2017

http://dx.doi.org/10.4102/ojvr.v84i1.1425 

ORIGINAL RESEARCH

 

Epidemiological studies based on multi-locus sequence typing genotype of methicillin susceptible Staphylococcus aureus isolated from camel's milk

 

 

Alsagher O. Ali; Hassan Y.A.H. Mahmoud

Division of infectious Diseases, Animal Medicine Department, South Valley University, Egypt

Correspondence

 

 


ABSTRACT

One hundred milk samples were collected from camel's milk for the isolation of Staphylococcus aureus. Thirty-one isolates were S. aureus, 45 were other forms of staphylococci and 24 represented other bacteria. Five isolates from S. aureus were methicillin resistant S. aureus (MRSA) and 26 samples were methicillin susceptible S. aureus (MSSA). The whole genome sequence of S. aureus was annotated and visualised by rapid annotation using subsystem technology (RAST) which is a fully-automated service for annotating complete or nearly complete bacterial genomes. Four isolates from MSSA strains were subjected to multi-locus sequence typing (MLST). Three multi-locus sequences types or sequence types (MLST/ST) were found, namely ST15, ST1153 and ST130. The phylogenetic analysis of the concatenated sequences of the seven genes forming the MLST profile of S. aureus classification revealed a high degree of similarity and close relationship between the ST15 and ST1153 while the third ST (ST130) was located in a different cluster.


 

 

Introduction

Staphylococcus aureus is a prominent pathogen causing a wide array of diseases in different animal species as well as in human beings. It has been subjected to numerous studies from different countries all over the world, from Egypt (Aly & Abo-Al-Yazeed 2003), Sudan (Shuiep et al. 2009), Saudi Arabia (Zahran & Al-Saleh 1977), Ethiopia (Semereab & Molla 2001), Morocco (Benkerroum et al. 2003; Khedid, Faid & Soulaimani 2003) and the USA (Solyman et al. 2013). Several molecular procedures have been established and used to identify and evaluate S. aureus isolates. Coagulase gene (coa gene) typing is considered a simple and effective method for typing S. aureus isolates from human patients and bovine mastitic milk (Aarestrup, Dangler & Sordillo 1994; Da Silva & Da Silva 2005; Talebi-Satlou, Malahat & Habib 2012; Weese & Van Duijkeren 2010). Epidemiological studies based on analysis of this gene have shown that S. aureus isolates could be divided into a number of subtypes (Aslantaş et al. 2007). The multi-locus-sequence typing (MLST) has been used widely to outline the population genetic structure of S. aureus as well as other bacterial species. The MLST genotyping could identify different strains, track the spread of methicillin resistance and identify epidemic clones (Holden et al. 2013; Kurt et al. 2013; Nübel et al. 2010). Whole-genome sequencing provides the best discriminatory power between closely related bacterial isolates; hence, because of the rapidly decreasing cost and time required for this technology, it could conceivably become a viable tool in diagnostic laboratories in the near future to reconstruct intercontinental and local transmission of S. aureus lineages (Harris, Feil & Holden 2010; Köser, Holden & Ellington 2012).

This present study aimed to investigate the effectiveness of MLST as a method of typing S. aureus isolates from camel origin as well as the implementation of multiple sequence alignment and phylogenetic analysis to clarify the molecular characterisation of MLST genes.

 

Materials and methods

A total of 100 raw milk samples were collected from dairy she-camels in Red Sea Governorate (Al-shalateen area), Egypt. Animals were examined for body temperature, pulse rate and respiratory rate. Animals were apparently healthy with no local or systemic infection. The milk samples were collected and stored in sterile plastic tubes.

Isolation and culturing of Staphylococcus aureus

Staphylococcus aureus was isolated on Baired-Parker media. Three to four typical colonies were harvested and picked up by a sterile metal bacteriological loop then immersed in glycerol and kept at -70 °C - -80 °C for further studies.

Biochemical tests

The coagulase test was performed by two different methods, namely the slide coagulase test and the tube coagulase test (Field & Smith 1995).

Detection of mecA gene

The mecA gene was utilised to affirm the classification of S. aureus into methicillin resistant S. aureus (MRSA) or methicillin susceptible S. aureus (MSSA). The mecA gene was analysed by multiplex PCR in addition to the mecC gene which was identified recently as a homologue for mecA. The femB gene was used as a corroborative gene for S. aureus (Alsagher 2016; Nakagawa et al. 2005; Paterson et al. 2014).

Whole genome sequencing and analysis

Staphylococcus aureus DNA was used to obtain the genomic sequence of S. aureus by shotgun sequencing (Sanger institute, UK).

Artemis was used to manipulate the genome sequence of S. aureus. This is a free genome browser and annotation tool that permits visualisation of sequence features, next generation information and the results of investigations inside the context of the sequence and furthermore its six-frame translation (Alsagher 2016; Rutherford et al. 2000). Sequence alignments, translations and comparisons were done utilizing BIOEDIT (Version 7.0.9.0) (Hall 1999). The BLAST algorithm was used to search the NCBI Gen Bank (http://www.ncbi.nlm.hih.gov/) databases for homologous sequences. Neighbour-joining trees (Saitou & Nei 1987) were constructed on the basis of genetic distances and estimated by two-parameter method (Kimura 1980) using MEGA6 (http://www.megasoftware.net) (Tamura et al. 2013). The reliability of the trees was estimated by 500 bootstrap confidence values. To construct the phylogenetic tree, we used 15 STs retrieved from the MLST websites (http://www.mlst.net/) (7, 8, 9, 11, 17, 19,126, 127, 128, 132, 1146, 1147, 1148, 1149 & 1150). In addition, 3 STs (15, 130 & 1153) were extracted from the bacterial chromosome of local isolates. These STs were used to construct the phylogenetic tree.

 

Results

Standard culturing methods and the application of coagulase tests for 100 milk samples of she-camels revealed that 31 isolates were S. aureus, 45 isolates were other forms of staphylococci, and 24 isolates were other bacteria. According to the presence or absence of the mecA gene in S. aureus isolates, five isolates were MRSA and 26 were MSSA.

The whole genome sequence of S. aureus was annotated by using subsystem technology (RAST) which is a fully-automated service for annotating complete or nearly complete bacterial genomes (http://rast.nmpdr.org/) and Artemis (Rutherford et al. 2000). Four isolates from S. aureus were sequenced by the shotgun sequencing technique and the whole genome sequence was obtained. The MLST genes were annotated, extracted and subjected to further analysis to predict the sequence typing by using the MLST websites (http://www.mlst.net/). One local isolated (A11) gave ST 15 (CC 5), local isolates (A12 & A13) gave ST 130 (CC 130) and the local isolate (A15) gave ST1153 (CC 1153) (Table 1).

 

 

The seven genes used in S. aureus MLST genotyping (yqil, aroe, glpf, gmk, pta, tpi & arcc) were highly polymorphic and they were divided into numerous numbers of different alleles. The allelic composition of each sequence type (ST), similarity and length were shown for ST15, ST130 and ST1153 in (Tables 1, 2 and 3) respectively.

 

 

 

 

The multiple sequence alignment of the translated amino acid sequences of the concatenated MLST genes were shown and aligned with selected other sequence types of S. aureus (Figure 2).

 

The evolutionary history was inferred by using the maximum likelihood method based on the Tamura-Neimodel (Tamura & Nei 1993). The analysis involved 18 nucleotide sequences. All positions containing gaps and missing data were eliminated. There were a total of 3186 positions in the final data set. The phylogenetic analysis revealed that ST15, ST130, ST1153 and other sequence types used to construct the phylogenetic tree were divided into two main clusters. The ST15 and ST1153 were closely related to each other and gathered in one cluster away from the third ST130 (Figure 1).

 

 

Discussion

Classification and characterisation of S. aureus isolates is an essential and preliminary step to understanding the epidemiological status of this contagious bacterium. Nowadays, MLST genotyping is widely used to investigate the dynamic nature of S. aureus. The implementation of the new techniques such as the whole genome sequence can potentially facilitate the mining of bacterial chromosomes for all the S. aureus genes. In our study, traditional culturing methods and the coagulase test revealed that 31% of the isolates were S. aureus, 45% isolates were other forms of staphylococci and 4% were other pathogens. These results were higher than the 14% reported by Abdel Hameid et al. in 2004. Using the mecA gene to classify S. aureus isolates into 16.12% MRSA and 83.87% MSSA, our results contradict those obtained by Monecke et al. (2011b) which showed that absence of mecA gene and the rarity of antibiotic resistance related genes in S. aureus isolates from camel's milk.

The MLST analysis of four isolates of MSSA revealed that three sequence types were recorded (ST15, ST130 and ST1153) in (A11, A12, A13 and A15) local isolates respectively (Table 4, Figures 1 and 2). The presence of ST15 in MSSA isolates was in agreement with another study in livestock and humans (Sanz 2013). ST130 was found in two local isolates (A12 and A13), both of which were MSSA strains. This finding was in agreement with the findings of Shore et al. (2011) and Le Maréchal et al. (2011) in sheep and human isolates respectively. The ST1153 found in A15 local isolate was in agreement with a study of Enany et al. (2010) and Moneck et al. (2011a) which indicated that the PVL+MSSA had hallmark characteristics from PVL+MRSA strains in that it possessed a novel MLST (ST1009) with a single variant (ST1153) as well as double locus variants (ST1216) (Table 4, Figure 2).

 

 

Conclusion

MLST genotyping is a useful way to classify S. aureus which can also provide a highly efficient molecular epidemiological investigation. Three sequence types (ST15, ST130 and ST1153) were obtained in this study from MSSA isolated from she-camel's milk. Phylogenetic analysis revealed close similarity between ST15 and ST1153, while the other ST130 is located in a separate cluster of other sequence types. Further investigations using this approach are needed to make a clear molecular epidemiological survey on S. aureus from camel's milk.

 

Acknowledgements

Competing interests

The authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article.

Authors' contributions

Both authors contributed equally to the writing of this article.

 

References

Aarestrup, F.M., Dangler, C.A. & Sordillo, L.M., 1994, 'Prevalence of coagulase gene polymorphism in Staphylococcus aureus isolates causing bovine mastitis', Canadian Journal of Veterinary Research 59, 124-128.         [ Links ]

Abdel Hameid, K., Sender, G., Prusak, B. & Ryniewicz, Z., 2004, 'Multiplex PCR protocol for the diagnosis of cow udder infection with Staphylococcus aureus', Animal Science Papers and Reports 22, 679-685.         [ Links ]

Alsagher, O.A., 2016, 'Molecular epidemiology based on SPA genotyping of Staphylococcus aureus isolated from cattle and camels in Egypt', Alexandria Journal of Veterinary Sciences 48, 62-68.         [ Links ]

Aly, S.A. & Abo-Al-Yazeed, H., 2003, 'Microbiological studies on camel milk in NorthSinai, Egypt', Journal of Camel Practice and Research 10, 173-178.         [ Links ]

Aslantaş, Ö., Demir, C., Türütoğlu, H., Cantekin, Z., Ergün, Y. & Doğruer, G., 2007, 'Coagulase gene polymorphism of Staphylococcus aureus isolated from subclinical bovine mastitis', Turkish Journal of Veterinary and Animal Sciences 31, 253-257.         [ Links ]

Benkerroum, N., Boughdadi, A., Bennani, N. & Hidane, K., 2003, 'Microbiological quality assessment of Moroccan camel's milk and identification of predominating lactic acid bacteria', World Journal of Microbiology and Biotechnology 19, 645-648. https://doi.org/10.1023/A:1025114601811        [ Links ]

Da Silva, E.R. & Da Silva, N., 2005, 'Coagulase gene typing of Staphylococcus aureus isolated from cows with mastitis in southeastern Brazil', Canadian Journal of Veterinary Research 69, 260-264.         [ Links ]

Enany, S., Yaoita, E., Yoshida, Y. & Enany, M., 2010, 'Molecular characterization of Panton-Valentine leukocidin-positive community-acquired methicillin-resistant Staphylococcus aureus isolates in Egypt', Microbiological Research 165, 2152-2162. https://doi.org/10.1016/j.micres.2009.03.005        [ Links ]

Field, H. I. & Smith, W., 1995, 'The coagulase test for staphylococci', The Journal of Comparative Pathology and Therapeutics 55, 63-69. https://doi.org/10.1016/S0368-1742(45)80007-3        [ Links ]

Hall, T.A., 1999, 'A user-friendly biological sequence alignment editor and analysis program for Windows 95/98/ NT', Nucleic Acids Symposium Series 41, 95-98.         [ Links ]

Harris, S.R., Feil, E.J. & Holden, M.T., 2010, 'Evolution of MRSA during hospital transmission and intercontinental spread', Science 327, 469-474. https://doi.org/10.1126/science.1182395        [ Links ]

Holden, M.T., Hsu, L.Y., Kurt, K., Weinert, L.A., Mather, A.E., Harris, S.R. et al., 2013, 'A genomic portrait of the emergence, evolution and global spread of a methicillin resistant Staphylococcus aureus pandemic', Genome Research 23, 653-664. https://doi.org/10.1101/gr.147710.112        [ Links ]

Khedid, K., Faid, M. & Soulaimani, M., 2003, 'Microbiological characterisation of one humped camel milk in Morocco', Journal of Camel Practice and Research 10, 169-172.         [ Links ]

Kimura, M., 1980, 'A simple method for estimating evolutionary rate of base substitutions through comparative studies of nucleotide sequences', Journal of Molecular Evolution 16, 111-120. https://doi.org/10.1007/BF01731581        [ Links ]

Köser, C.U., Holden, M.T.G. & Ellington, M.J., 2012, 'A neonatal MRSA outbreak investigation using rapid whole genome sequencing', The New England Journal of Medicine 363, 2267-2275. https://doi.org/10.1056/NEJMoa1109910        [ Links ]

Kurt, K., Rasigade, J.P., Laurent, F., Goering, R.V., Žemličková, H., Machova, I. et al., 2013, 'Subpopulations of Staphylococcus aureus clonal complex 121 are associated with distinct clinical entities', PLoS One 8, e58155. https://doi.org/10.1371/journal.pone.0058155        [ Links ]

Le Maréchal, C., Hernandez, D., Schrenzel, J., Even, S., Berkova, N., Thiery, N. et al. 2011, 'Genome sequences of two staphylococcus aureus ovine strains that induce severe (Strain O11) and mild (Strain O46) mastitis', Journal of Bacteriology 193, 2353-2354. https://doi.org/10.1128/JB.00045-11        [ Links ]

Monecke, S., Coombs, G., Shore, A.C., Coleman, D.C., Akpaka, P., Borg, M. et al., 2011a, 'A field guide to pandemic, epidemic and sporadic clones of methicillin-resistant S. aureus', PLoS One 4, e17936. https://doi.org/10.1371/journal.pone.0017936        [ Links ]

Monecke, S., Ehricht, R., Slickers, P., Wernery, R., Johnson, B., Jose, S. et al., 2011b, 'Microarray-based genotyping of Staphylococcus aureus isolates from camels', Veterinary Microbiology 150, 309-314. https://doi.org/10.1016/j.vetmic.2011.02.001        [ Links ]

Nakagawa, S., Taneike, I., Mimura, D., Iwakura, N., Nakayama, T., Emura, T. et al., 2005, 'Gene sequences and specific detection for Panton-Valentine leukocidin', Biochemical and Biophysical Research Communications 328, 995-1002. https://doi.org/10.1016/j.bbrc.2005.01.054        [ Links ]

Nübel, U., Dordel, J., Kurt, K., Strommenger, B., Westh, H., Shukla, S.K. et al., 2010, 'A timescale for evolution, population expansion, and spatial spread of an emerging clone of methicillin-resistant Staphylococcus aureus', PLoS Pathogens 6, e1000855.         [ Links ]

Paterson, G.K., Ewan, M.H. & Mark, A.H., 2014, 'The emergence of mecC methicillin-resistant Staphylococcus aureus', Trends in Microbiology 22, 42-47. https://doi.org/10.1016/j.tim.2013.11.003        [ Links ]

Rutherford, K., Parkhill, J., Crook, J., Horsnell, T., Rice, P., Rajandream, M.A. et al., 2000, 'Artemis: Sequence visualization and annotation', Bioinformatics 10, 944-945.         [ Links ]

Saitou, N. & Nei, M., 1987, 'The neighbor-joining method: A new method for reconstructing phylogenetic trees', Molecular Biology and Evolution 4, 406-425.         [ Links ]

Sanz, E.G., 2013, 'S. aureus and S. pseudintermediusin animals', PhD thesis, Universidad De La Rioja, Spain.         [ Links ]

Semereab, T. & Molla, B., 2001, 'Bacteriological quality of raw milk of camel (Camelusdromedarius) in Afar region (Ethiopia)', Journal of Camel Practice and Research 8, 51-54.         [ Links ]

Shore, A.C., Deasy, E.C., Slickers, P., Brennan, G., O'Connell, B., Monecke, S. et al., 2011, 'Detection of staphylococcal cassette chromosome mec type XI carrying highly divergent mecA, mecI, mecR1, blaZ, and ccr genes in human clinical isolates of clonal complex 130 methicillin-resistant Staphylococcus aureus', Antimicrobial Agents and Chemotherapy 55, 3765-3773. https://doi.org/10.1128/AAC.00187-11        [ Links ]

Shuiep, E.S., Kanbar, T., Eissa, N., Alber, J., Lämmler, C., Zschöck, M. et al., 2009, 'Phenotypic and genotypic characterization of Staphylococcus aureus isolated from raw camel milk samples', Research in Veterinary Science 86, 211-215. https://doi.org/10.1016/j.rvsc.2008.07.011        [ Links ]

Solyman, S.M., Black, C.C., Duim, B., Perreten, V., Van Duijkeren, E., Wagenaar, J.A. et al., 2013, 'Multilocus sequence typing for characterization of Staphylococcus pseudintermedius', Journal of Clinical Microbiology 51, 306-310. https://doi.org/10.1128/JCM.02421-12        [ Links ]

Talebi-Satlou, R., Malahat, A., & Habib, D.S., 2012, 'Restriction fragment length polymorphism genotyping of human staphylococcus aureus isolates from two hospitals in Urmia Region of Iran using the coa gene', Jundishapur Journal of Microbiology 5, 416-420. https://doi.org/10.5812/jjm.3522        [ Links ]

Tamura, K. & Nei M., 1993, 'Estimation of the number of nucleotide substitutions in the control region of mitochondrial DNA in humans and chimpanzees', Molecular Biology and Evolution 10, 512-526.         [ Links ]

Tamura, K., Stecher, G., Peterson, D., Filipski, A. & Kumar, S., 2013, 'Molecular evolutionary genetics analysis version 6.0', Molecular Biology and Evolution 30, 2725-2729. https://doi.org/10.1093/molbev/mst197        [ Links ]

Weese, J.S. & Van Duijkeren, E., 2010, 'Methicillin-resistant Staphylococcus aureus and Staphylococcus pseudintermediusin veterinary medicine', Veterinary Microbiology 140, 418-429. https://doi.org/10.1016/j.vetmic.2009.01.039        [ Links ]

Zahran, A.S. & Al-Saleh, A.A., 1977, 'Isolation and identification of protease-producing psychrotrophic bacteria from raw camel milk', Australian Journal of Dairy Technology 52, 5-7.         [ Links ]

 

 

Correspondence:
Alsagher Ali
alsagher.ali@vet.svu.edu.eg

Received: 27 Jan. 2017
Accepted: 10 Aug. 2017
Published: 22 Sept. 2017

Creative Commons License Todo el contenido de esta revista, excepto dónde está identificado, está bajo una Licencia Creative Commons