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Investigation of Virulence Properties of Staphylococcus Species Isolated from Goats with Subclinical Mastitis
Donmez, E. and Kirkan, S.
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Abstract
Identification of coagulase-negative staphylococci (CNS) is tedious and sometimes cannot be done satisfactorily since many biochemical tests are required in routine diagnostic laboratories. CNS, which play an important role in the aetiology of mastitis, are important members of the skin and mucous membranes and cause infections especially in immunocompromised individuals. The increasing incidence of CNS, especially in the aetiology of mastitis, requires the correct identification of Staphylococci isolates at the species level. In recent years, tuf gene analysis has been frequently preferred for species identification of CNS due to its practicality and reliable results. The aim of this study was to identify Staphylococcus species by conventional and molecular methods from Saanen goat milk with mastitis and to determine the virulence genes and the antibiotic susceptibility of the isolates. In this study, 41 (37.2%) Gram-positive cocci were isolated from the 110 goat milk samples with subclinical mastitis. Bacterial identification was carried out using conventional methods. All Gram-positive cocci were examined with the BD PhoenixTM 50 device for identification and antibiogram susceptibility analyses. Polymerase chain reaction (PCR) was used to confirm the species level identification of the isolates, and to identify virulence genes. After the identification of the Staphylococcus isolates, analysis for the presence of 16S rRNA, nuc, tuf, coa genes, virulence genes, enterotoxin genes were performed for genotypic verification by PCR. The results were as follows: 5 (12.2%) S. pettenkoferi; 5 (12.2%) S. aureus; 4 (9.7%) S. epidermidis; 3 (7.3%) S. equorum; 3 (7.3%) S. warneri; 3 (7.3%) S. caprae; 2 (4.9%) S. capitis; 1 (2.4%) S. simulans and 15 (36.6%) Streptococcus spp. were identified using BD PhoenixTM. tuf gene sequencing results of Staphylococcus isolates (n=26) showed that; 5 (19.2%) S. aureus, 4 (15.4%) S. caprae, 4 (15.4%) S. succinus, 4 (15.4%) S. devriesei, 3 (11.5%) S. chromogenes, 1 (3.8%) S. heamolyticus, 1 (3.8%) S. petracii sp. jettensis. The resistance to antimicrobial drugs ampicillin, penicillin and amoxicillin-clavulanic acid was 88.4%, 88.4%, 84.4%, respectively. The prevalence of the studied virulence genes was sea 3.8%, seb 30.7%, sec 11.5%, PVL 3.84%, Hla 53.8%, Hlb 34.6%, and BAP 7.7%. Our results confirmed the predominance of S. caprae and S. succinus among CNS isolates. Tuf genes are considered as an accurate tool for the identification of CNS strains. Finally, the use of advanced diagnostic methods in goat mastitis cases and the determination of antibiotic susceptibility of Staphylococcus isolates will preclude the occurrence of disease that may harm public health.
Key words: Mastitis, Staphylococcus spp.; Sequencing; Virulence Genes; Antibiogram.
Introduction
The Staphylococcus spp., Streptococcus spp., Enterobacteriaceae and Mycoplasma spp. are the most widespread pathogens that cause mastitis in goats. Staphylococcus aureus and coagulase negative Staphylococcus (CNS) play an important role in goat mastitis (1). Many methods have been developed for the identification of Staphylococcus species. Staphylococcal species can be rapidly identified using a variety of systems based on biochemical reactions (i.e., tube tests, miniature biochemical tests, and computerized identification systems) (2). Several polymerase chain reactions (PCR), sequencing- based methods, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS), the macrorestriction analysis of DNA-fragments (REA-PFGE), whole genome sequencing (WGS) enables rapid identification of organisms through molecular analysis which have been developed for the identification of staphylococci (3).
S. aureus, the major pathogen of the Staphylococcus genus, causes a wide variety of clinical infections in humans and animals (4). The distinction between S. aureus from most staphylococci is primarily made by evaluating its ability to produce the enzyme coagulase, which promotes coagulation of plasma. This activity is the basis of routine identification testing and aids in the distinction between S. aureus and CNS, long considered harmless but emerging as important opportunistic pathogens (5). The coa (coagulase) gene in Staphylococcus results in a secretory protein that causes blood coagulation through the activation of prothrombin inorder to overcome the host defence system (6). It is one of the major virulence proteins used as a basis for differentiation of coagulase positive staphylococci (CPS) from coagulase negative staphylococci (CNS) species. The nuclease (nuc) gene sequence is responsible for the thermonuclease activity specific for Staphylococcus aureus. The enzyme thermonuclease (TNase), which has exo- and endonuclease activity, cleaves DNA and RNA into nucleotides by hydrolysing the phosphodiester bonds in their structure (7).
The elongation factor Tu (tuf) gene sequence plays a role in peptide chain formation in Staphlyococcus species. The tuf gene cluster, which is located in the short tandem repeat region on the bacterial chromosome, shows a significant diversity among members of staphylococci. Its small size and its conserved location in bacterial chromosome enhances its superiority in DNA sequencing compared with 16S rRNA for construction of phylogenetic trees on species and genus level in staphylococci, enterococci, and streptococci isolates. It has been recognized that the tuf gene is a more specific gene than the 16S rRNA gene in the identification of CNS species (8).
Staphylococcus exotoxins play a role in mastitis infections and are consequently found in dairy products. Staphylococcal food poisoning (SFP) outbreaks occur as a result of human consumption of these foodstuffs. Rapid, sensitive and accurate detection of exotoxins in the mammary glands and indeed in food is crucial both to determine the best treatments for S. aureus infections and to prevent economic loss in the dairy industry (9).
S. aureus enterotoxins include toxic shock syndrome toxin 1 (TSST 1) and staphylococcal enterotoxins (SEs). Hemolysins attack cell membranes as β, α and δ toxins and cause platelet damage, lysosome destruction and necrosis. Hla (α hemolysin) is a toxin that disrupts the cell membrane, causes irreversible osmotic changes and causes cell death through apoptosis. Hla can damage the membranes of a variety of cells, such as lymphocytes, red blood cells, platelets, and endothelial cells (10). Beta hemolysin (Hlb) is encoded by a lysogenic bacteriophage and in itself it cannot destroy most cell types, but exposes sensitive cells to other lytic proteins such as Hla and leukocidins (11). Also known as sphingomyelinase, this toxin can also damage keratinocytes, helping bacteria to colonize mammalian skin. The different susceptibility of different types of erythrocytes to Hlb may be due to the amount of sphingomyelin present in the cells (10). Leucocidins are also pore-forming bicomponent toxins that attack immune cells. There are seven different types of leukocytes, and Panton-Valentine leukocidin (PVL) is the toxin with the strongest effect on immune cells (12).
To survive, bacteria have developed, various strategies such as development of biofilms to colonize and cause infections. Biofilm production is an important virulence factor. Biofilm-producing isolates can remain in the milk environment for a long period of time, transfer genetic material to other bacteria. Biofilm-associated protein (BAP) is a cell wall-bound surface protein encoded by the BAP gene, increasing the dispersal of virulence factors (13).
The aim of this study is to determine the important Staphylococcus species that play a role in the aetiology of goat mastitis using molecular methods, and to investigate the important virulence genes (enterotoxin genes, hemolysin genes, TSST, BAP, PVL) and antibiotic susceptibility of the isolates. [...]
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