Sak Gene Research Articles

Cloning & expression and production SAK enzyme from staphylococcus aureus and transfer in bacteria E. coli

Staphylococcus aureus is one of the most dangerous organisms that threatens the life of humans and animals alike. It has been found that people who suffer from chronic diseases such as diabetes, blood vessels, pulmonary and cancerous diseases are more susceptible to infection, in addition to ability resist antibiotics, which made fierce and dangerous. This staphylococcus aureus possessed the SAK gene, which acts as a bio stimulant that plays an important role in the process of analyzing blood coagulation, as it acts as an anti-plasmogen activator and is in the form of an equal compound of human plasmin. SAK consists of a chain of amino acids numbering 136 amino acids. The study aimed to isolate the SAK gene using amplification technique PCR and insert it into an ideal vectorPET21a. And he transferred it to one of the strains of the E.coli(BL21DE3) , stimulating the bacteria by adding IPTG, the purpose of obtaining the highest level of protein expression in an ideal time, then purifying the protein by affinity chromatography.

Detection and molecular characterisation of methicillin-resistant Staphylococcus aureus isolated from raw meat in the retail market

This study aimed to detect and characterise methicillin-resistant Staphylococcus aureus (MRSA) from retail meat in the Czech Republic. Isolates were identified by PCR detection of the S. aureus-specific fragment Sa442 and mecA gene. spa typing, MLST, detection of genes encoding staphylococcal enterotoxins, Panton-Valentine leukocidin (pvl), exfoliative toxins A and B (eta and etb), toxic shock syndrome toxin (tst) and staphylokinase (sak), detection of φSa3 prophage and antimicrobial susceptibility testing were performed. Of 65 raw meat samples examined (poultry, beef, pork and rabbit), 23 (35.4%) were positive for MRSA. Twelve positive samples originated from poultry (12/33; 36.4%), while the remaining eleven came from pork (9/9; 100%) and pork/beef mixed minced meat (2/5; 40.0%). Eight spa types belonging to five different sequence types (STs) were identified. ST398 was the most frequent (28/36; 77.8%), presenting spa types t011, t034, t2576, t4132, t588 and t899. Other livestock-associated MRSA STs (ST9-t899, ST5-t002, ST692-t8646 or the newly described ST4034-t899) were also sporadically identified. In seven isolates (19.4%), one or more staphylococcal enterotoxin genes were detected, with sea, seg and sei prevailing. Three isolates from turkey [ST398-t899 (n=2) and ST398-t011] harboured the sak gene, and the latter also harboured the sea gene. Seven isolates from poultry harboured the φSa3 prophage and were resistant to tetracycline. Specific kinds of meat appear to be a possible source of MRSA, although the risk to humans is hard to define. Therefore, surveillance of MRSA in meat as well as hygienic practices should be improved.

Genetic Diversity and Virulence Factors of S. aureus Isolated from Food, Humans, and Animals.

Staphylococcus aureus is a commensal bacterium in humans and animals able to adapt to multiple environments. The aim of this study was to compare the genetic diversity and virulence profiles of strains of S. aureus isolated from food (29 strains), humans (43 strains), and animals (8 strains). 80 lipase-producing strains belonging to a biobank of 360 isolates, identified phenotypically as S. aureus, were selected. Confirmation of the species was made by amplifying the spA gene and 80% (64/80) of the strains were confirmed within this species. The virulence profile of each of the isolates was determined by PCR. The seA gene coding for enterotoxin A was found in 53.1% of the strains, the saK gene, which codes for Staphylokinase, was amplified in 57.8% of the strains, and, finally, the hlB gene coding for β-Hemolysin was amplified in 17.2%. The profile of antimicrobial resistance was determined by the Kirby Bauer method showing that the strains from food presented greater resistance to erythromycin (40.7%) and ciprofloxacin (18.5%) while in strains isolated from humans were to erythromycin (48.4%) and clindamycin (21.2%). Also, in strains from animals, a high resistance to erythromycin was observed (75%). The frequency of MRSA was 12.5% due to the presence of the mec gene and resistance to cefoxitin. Of the total strains, 68.7% were typed by PCR-RFLP of the coa gene using the AluI enzyme; derived from this restriction, 17 profiles were generated. Profile 4 (490 bp, 300 bp) was the most frequent, containing a higher number of strains with a higher number of virulence factors and antimicrobial resistance, which is associated with greater adaptation to different environments. In this study, a wide genetic diversity of strains of S. aureus from different foods, humans, and animals was found. This demonstrates evolution, genetic versatility, and, therefore, the adaptation of this microorganism in different environments.

Mutagenicity Acridine Orange Mutagen On The Biological Activity of S.aureus Isolated From Tonsillitis

Study design of cases is a cross-sectional study that descriptive study design of 280 isolates collected from tonsillitis, the identification achieved by using Vitek2-GP. The biological activity of antimicrobial sensitivity test of all 280 isolates against antibiotics including Ceftazidine 70(25%)resistance, 196(70%) sensitive, 14(5%) intermediate; Azithromycin 154(55%) sensitive, 28(10%) intermediate., 98(35%) resistance; Oxacillin 266(95%) sensitive, 14(5%) resistance; Cephalexin 238(85%) sensitive, 42(15%) resistance; Gentamycin 266(95%) sensitive, 14(5%) resistance; Ceftriaxone 238(85%) sensitive, 42(15%) resistance;Norfloxacin 252(90%) sensitive, 28(10%) resistance; Cefoxitin 266(95%) sensitive, 14(5%) resistance; Erythromycin 42(15%)sensitive, 238(85)% resistance; Cloxacillin 126(45%) resistance, 154(55%) sensitive; Imipenem 266(95%) resistance, 14(5%) sensitive; Vancomycin 266(95%) resistance, 14(5%) sensitive and Methicillin 280(100%) resistance, 0 (100%) sensitive) compared with CLSI(2013). Radial caseinolysis assay and Fibrinolysis assay of Staphylococcal fibrinlysin achieved by quantitative screening of staphylokinase on plasma agar plate and semi-quantitative screening of staphylokinase on skimmed milk agar plate to limit fibrinolysis zone and caseinolysis zone around well on plat. Results showed variable degree of production staphylokinase, some isolates positive for production, another negative for production staphylokinase (staphylococcal fibrinolysin). Induced mutation achieved by chemical mutagenesis when exposition S.aureus to different mutage of Acridine Orange (AO) chemical mutagen in dose (10,20,30,40,50,60) mg, the results of mutagenesis in optimum mutage (dose of mutagen) with increase production of staphylococcal fibrinolysin when occurs random mutagenesis was 75 colony (viable cells) viability in 30 mg. Genotypic analysis of sak gene encoded for staphylokinase accomplished by using PCR, all results of S.aureus isolated from tonsillitis positive for possessing sak gene (400bp).

Detection and Molecular Characterization of Methicillin-Resistant Staphylococcus aureus Isolated from Bulk Tank Milk of Cows, Sheep, and Goats.

This study is aimed at detecting and characterizing methicillin-resistant Staphylococcus aureus (MRSA) from bulk tank milk samples of cows, sheep, and goats collected from dairy farms in the Czech Republic. All MRSA isolates were identified using PCR detection of the Staphylococcus aureus-specific fragment SA442 and mecA gene. The staphylococcal chromosomal cassettes mec (SCCmec), spa, and multilocus sequence types (MLST) were determined. The presence of genes encoding enterotoxins (ses), Panton-Valentine leukocidin (pvl), exfoliative toxins A, B (eta, etb), and toxic shock syndrome toxin (tst) were assessed. To differentiate human and animal origin, the presence of staphylokinase (sak) gene, ϕSa3 prophage, and susceptibility to tetracycline was tested. Out of 49 bulk tank milk samples examined, 14 (28.6%) were MRSA-positive. Eleven positive samples came from cow's milk (38%) and the remaining three from goat's milk (33%). All samples of ewe's milk were negative. In MRSA isolates three sequence types containing seven spa types were identified. Twelve isolates (85.7%) belonged to ST398 spa types t011/SCCmec IVa, t011/SCCmec V, t034/SCCmec V, t1456/SCCmec IVa, t1255/SCCmec V, and t2346/SCCmec V. Another two isolates belonged to ST5/t3598/SCCmec IVa and ST8/t064/SCCmec IVNT. In six isolates, one or more ses genes (seb, sed, seg, sei, and sej) were confirmed. One isolate from cow's milk harbored the tst gene. Another two isolates (ST398/t1456/SCCmec IVa and ST5/t3598/SCCmec IVa) harbored the sak gene and ϕSa3 prophage, and the latter was the only tetracycline-susceptible isolate in this study. However, none of the isolates was positive for pvl or eta, etb. These results suggest that there is the wide geographical spread of ST398 across different regions of the Czech Republic with no host preference among dairy cattle and goats. Therefore, when evaluating the occupational and foodborne risks, MRSA carriage and infection should be taken into account.

Characterization of Livestock-Associated Methicillin-Resistant Staphylococcus aureus Isolates Obtained From Commercial Rabbitries Located in the Iberian Peninsula.

Infections caused by methicillin-resistant Staphylococcus aureus (MRSA) have been a growing problem in human medicine since the 1960s, and more recently in veterinary medicine with the appearance of livestock-associated MRSA (LA-MRSA). Nevertheless, information about the presence of MRSA in rabbits is quite scarce since only one LA-MRSA identification has been previously reported. The present study aimed to determine genotypic characterization by verifying the presence of resistance determinants, virulence, and toxin genes of different S. aureus strains that cause lesions in rabbits, and their phenotypic traits based on the antimicrobial susceptibility profile. The analysis of 240 S. aureus isolates obtained from different lesion types collected from 89 Spanish and Portuguese rabbit commercial farms in the last 4 years (2014–2017) was performed. The methicillin-resistant gene mecA was found in 11.25% of the studied isolates (27 of 240) from 19 farms (13 Spanish and 6 Portuguese). Staphylococcal cassette chromosome mec (SCCmec) typing predominantly revealed type III (n = 15). Additionally, three MRSA isolates carrying the mecC gen were detected in samples from three different farms (two Spanish and one Portuguese). None of the 30 MRSA isolates was PVL-positive or tst-positive. After the multilocus sequence typing (MLST) procedure, 16 belonged to ST2855, 6 to ST146, 6 to ST398, and 2 ST4774. No ST121 isolate was mec-positive. ST398 and ST4774 isolates lacked the immune-evasion-cluster (IEC) genes. ST2855 strains were associated with the presence only of the sak gene, and ST146 isolates were ascribed to IEC type E. Therefore, this is the first description of LA-MRSA from rabbits belonging to ST2855. Interestingly, one ST2855 and two ST4774 isolates were mecC-positive, which could act as a mecC-MRSA reservoir. More studies are needed to further characterize these isolates and their relationship with humans and other animal species.

Detection of Sak Gene and Expression of Staphylokinase in Different Clinical Isolates of Staphyloccocus spp.

This study had investigated the presence of Staphylokinase (SAK) phenotypically and genetically. A total of 100 Staphylococcus spp. samples were isolated from different clinical cases. Morphological, biochemical tests and for further conformation; Viteck2 technique were adopted to isolate and diagnose the staphylococcal samples. Phenotypic assay tests including hydrolysis of casein and plasma agar were performed to check for the production of Staphylokinase from producing Staphylococcus spp. Out of 100, only 32 isolates had exhibited (SAK) activity whereby 29 of them represented the species S. aureus while 3 belonged to others. All isolates were subjected to Polymerase Chain Reaction (PCR) to determine the possessed sak gene. This investigation certainly had shown that not all isolates of S. aureus has the sak gene, only the lysogenic one has it and that it is not species committed but also other staphylococcus species could have it.

Staphylococcus aureus Complex in the Straw-Colored Fruit Bat (Eidolon helvum) in Nigeria.

Bats are economically important animals and serve as food sources in some African regions. They can be colonized with the Staphylococcus aureus complex, which includes Staphylococcus schweitzeri and Staphylococcus argenteus. Fecal carriage of S. aureus complex in the straw-colored fruit bat (Eidolon helvum) has been described. However, data on their transmission and adaptation in animals and humans are limited. The aim of this study was to investigate the population structure of the S. aureus complex in E. helvum and to assess the geographical spread of S. aureus complex among other animals and humans. Fecal samples were collected from E. helvum in Obafemi Awolowo University, Ile-Ife, Nigeria. The isolates were characterized by antimicrobial susceptibility testing, spa typing and multilocus sequence typing (MLST). Isolates were screened for the presence of lukS/lukF-PV and the immune evasion cluster (scn, sak, chp) which is frequently found in isolates adapted to the human host. A Neighbor-Joining tree was constructed using the concatenated sequences of the seven MLST genes. A total of 250 fecal samples were collected and 53 isolates were included in the final analysis. They were identified as S. aureus (n = 28), S. schweitzeri (n = 11) and S. argenteus (n = 14). Only one S. aureus was resistant to penicillin and another isolate was intermediately susceptible to tetracycline. The scn, sak, and chp gene were not detected. Species-specific MLST clonal complexes (CC) were detected for S. aureus (CC1725), S. argenteus (CC3960, CC3961), and S. schweitzeri (CC2463). STs of S. schweitzeri from this study were similar to STs from bats in Nigeria (ST2464) and Gabon (ST1700) or from monkey in Côte d'Ivoire (ST2058, ST2072). This suggests host adaptation of certain clones to wildlife mammals with a wide geographical spread in Africa. In conclusion, there is evidence of fecal carriage of members of S. aureus complex in E. helvum. S. schweitzeri from bats in Nigeria are closely related to those from bats and monkeys in West and Central Africa suggesting a cross-species transmission and wide geographical distribution. The low antimicrobial resistance rates and the absence of the immune evasion cluster suggests a limited exposure of these isolates to humans.

Genome comparisons of two Taiwanese community-associated methicillin-resistant Staphylococcus aureus ST59 clones support the multi-origin theory of CA-MRSA

Sequence type (ST) 59 is an epidemic lineage of community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) in Asia. Two ST59 clones are prevalent in Taiwan: the Taiwan clone (TW) causes severe infections, whereas the Asian-Pacific clone (AP) is usually commensal. In this study, we sequenced the genome and transcriptome of the representative strains of these two clones and found their differences to focus on three mobile genetic elements: TW carries SCCmec Type VT, Panton-Valentine leucocidin (PVL)-encoding prophage ΦSa2, whereas AP carries SCCmec Type IV and staphylokinase (SAK)-encoding prophage ΦSa3. The anti-virulent role of SAK was confirmed using murine skin and bloodstream infection models. ΦSa3 usually integrates into the hlb gene, but in AP was found to be integrated at the genomic island νSaβ. The mutation of the attB site “TGTATCCAAACTGG” to “TGTATCCGAATTGG” led to a failure in the integration of ΦSa3 in hlb, prompting atypical integration at other sites. The sak gene possessed remarkably different patterns of distribution among the different STs of S. aureus. We conclude that the atypical integration of ΦSa3 may help S. aureus adapt to the human host habitat and that the subsequent loss of ΦSa3 contributes toward the development of a virulent CA-MRSA lineage for wider horizontal transmission.

Pathogenic Characteristics of Staphylococcus aureus Endovascular Infection Isolates from Different Clonal Complexes.

Staphylococcus aureus is a major cause of bacteremia and, even with appropriate clinical management, causes high morbidity, and mortality due to its involvement in endovascular complications and metastatic infections. Through different pathogenic in vivo and in vitro models we investigated the behavior of S. aureus most relevant clonal complexes (CCs) causing endovascular complications. We analyzed 14 S. aureus strains representing CC5, CC8, CC15, CC30, and CC45 that caused endovascular complications, including methicillin susceptible and resistant isolates and strains with different functionality of the agr global regulator. Their adherence to collagen, interaction with the endothelium, resistance to immune attack, capacity to form biofilm and virulence in the Galleria mellonella model were analyzed. CC30 and CC45 showed greater adhesion to collagen and CC8 showed a trend towards higher rate of intracellular persistence in endothelial cells. All CCs exhibited similar tolerance to neutrophil antimicrobial peptide hNP-1 and were capable of forming biofilms under static conditions. The virulence assay in the G. mellonella model demonstrated that CC15 and CC30 were the most and least virulent, respectively. The analysis of the genomic sequences of the most relevant virulence genes identified some CC15 specific gene patterns (absence of enterotoxins and sak gene) and variants (mainly in leucocidins and proteases), but did not reveal any gene or variant that could be responsible for the increased virulence detected for CC15 strains. Even though all the CCs were capable of causing endovascular complications, our results showed that different CCs are likely to produce these complications through different mechanisms which, if confirmed in more sophisticated models, would indicate the need to more specific management and therapeutic approaches.

Expression of Staphylokinase Gene S. aureus Strains Isolated from Breast Milk and Clinical Outcomes in Breastfed Infants

This study aimed to look for the presence of the immune evasion gene cluster in the strains of Staphylococcus aureus isolated from breast milk of women and to estimate their possible role in development of infant’s staphylococcal infections. Strains of S. aureus are isolated by bacteriological examination of breast milk in a hospital (24 strains) and an ambulatory (11 strains) basis. Breastfed infants had staphylococcal infections (42.9 %), intestinal infections (5.7 %), and respiratory infections (20 %), and 11 (31.4 %) infants were intestinal carriers of S. aureus. PCR analysis of 35 strains S. aureus was carried out to detect genes of staphylokinase (sak), inhibitor of the system of complement (scn), inhibitor of chemotaxis of leukocytes (chp), and β-hemolysin (hlb). Sak gene was found in 20 (57 %) S. aureus isolates. Scn, chp, and hlb genes in S. aureus isolates were not detected. Infants received breast milk infected with sak positive S. aureus strains more frequently had S. aureus intestinal colonization than infants who received breast milk infected with sak negative S. aureus (OR 4.5 (1.02; 20.30)). Presence of sak gene in S. aureus strains from breast milk was not associated with development of staphylococcal infection in infant (OR 1.2 (0.3–4.7)). In conclusion, sak is an important factor in avoiding immune defense by S. aureus strains in infected breast milk. Expression of sak gene by S. aureus in infected breast milk may be an important factor for S. aureus intestinal colonization of infants, but not the development of staphylococcal infections in breastfed infants.

Poultry-Like pA+ Biotype of Staphylococcus aureus CC346/084 Clone in Human Population.

The aim of the study was (1) to analyse the prevalence of P-like pA+ biotype of S. aureus in material from healthy and diseased individuals, not employed at slaughterhouses or meat processing plants, and (2) to analyse the relatedness of these strains and their genetic variability. The study included 344 strains of Staphylococcus aureus isolated from hospitalized patients with staphylococcal infections and from healthy carriers. The biotypes of S. aureus were determined on the basis of fibrinolysin and β-haemolysin production, coagulation of bovine plasma, and type of growth on crystal violet agar. Additionally, the strains were tested for the synthesis of protein A in order to distinguish between P-like pA+ and poultry biotypes. Fibrinolysin gene (sak) and methicillin resistance (mecA) were detected by means of PCR. The clonal structure of studied strains was analysed using pulsed field gel electrophoresis and sequencing of spa gene. Finally, the strains were typed with a basic set of 23 bacteriophages. The strains belonging to P-like pA+ biotype corresponded to nearly 20 % of all the studied strains. In contrast to the human biotype, they formed one clonal complex, spa-CC346/084. The P-like pA+ biotype strains did not synthesize fibrinolysin, lacked the sak gene, and showed susceptibility to methicillin. In contrast to the human biotype strains, they belonged mostly to phage group II. The P-like pA+ biotype strains, previously described solely in meat products and meat industry workers, can be also present in hospitalized patients and extra-hospital carriers. These strains form a single, fibrinolysin-negative, clonal complex t084/CC346.

Sources of Contamination of Bovine Milk and Raw Milk Cheese by Staphylococcus aureus Using Variable Number of Tandem Repeat Analysis

Milk and dairy products are frequently implicated in food-borne infections caused by Staphylococcus aureus and infected animals may contaminate bulk milk. In addition, human handlers, milking equipment, the environment, the udder and the teat skin of dairy animals are possible sources of bulk milk contamination. The main objective of this study was to identify and prioritize the sources of S. aureus contamination of bulk milk and raw milk cheese, and secondly to investigate the diversity of strains involved in bovine mastitis, and test the controversial hypothesis of cross-infection between humans and animals. Four farms manufacturing cheese from raw milk with a total of 135 dairy cows were selected. Bacteriological analyses were performed on quarter milk samples, swabs of udders and teats, the milking machine, bulk milk, cheeses, swabs taken from staff members’ hands and nasal cavities. Typing of S. aureus isolates was carried out using the Multiple Locus Variable Tandem Repeat Analysis [MLVA] including five genes [clfA, clfB coa, fnb and SAV1078] combined with the investigation of the presence of staphylokinase gene [sak]. A total of 537 isolates were genotyped. The genotyping results confirmed that most intramammary infections in each farm were due to a prevalent genotype. The majority of genotypes present on the teat skin were also isolated from quarter milk samples. These isolates are the main sources of the contamination of bulk milk and cheese. The identity of certain genotypes characterized in both humans and animals was sometimes associated with the presence of the sak gene and suggests the existence of cross-contamination and also the occasional involvement of human handlers in the contamination of milk and cheeses.

Cloning, high-level expression, purification and characterization of a staphylokinase variant, SakC, from Staphylococcus aureus QT08 in Escherichia coli BL21

The staphylokinase (Sak) is emerging as an important thrombolytic agent for the treatment of patients suffering from cardiovascular disease. Hence in this study, we reported the cloning, high-level expression, purification and characterization of the Sak variant SakoC from Staphylococcus aureus QT08 in Escherichia coli Bl21. The sak gene of 489 bp encoding a protein (163 amino acids) with a predicted molecular mass of 18.5 kDa and pI 7.28 showed 99.8 to 99.6% identity with corresponding sequences from S. aureus strains deposited in GenBank (AF332619, X00127, EF122253 and M57455). The DNA sequence (411 bp) encoding the mature Sak (15.5 kDa) truncated 27 N-terminal amino acids was expressed in E. coli BL21/pESak under the control of the strong promoter tac in the presence of isopropyl-β-D-1-thiogalactopynoside (IPTG) as inducer. The expression level of rSak was estimated at about 42% of the total cellular proteins by densitometry scanning, which is the highest expression level of rSak expressed in any E. coli system. The recombinant staphylokinase was purified by Ni 2+ - ProBond TM column to a single homogeneous 16-kDa band on sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) with a specific activity of 15175 U/mg protein, a recovery yield of 58% and a purification factor of 2.56. The optimal pH and temperature for the rSak activity was 9 and 37°C, respectively. rSak was stable over a temperature range of 25 to 50°C and at pH range of 7 to 9. Metal ions and detergents also showed an inhibitory effect on rSak, especially Zn 2+ and Cu 2+ which completely inhibited the enzymatic activity. Key words: Staphylococcus aureus QT08, staphylokinase, cloning, high-level expression, purification, characterization.

High-level expression, purification and properties of a fully active even glycosylated staphylokinase variant SakfC from Staphylococcus aureus QT08 in Pichia pastoris

Staphylokinase (Sak) (EC 3.4.99.22) is a 136-aa protein secreted by the lysogenic phase of Staphylococcus aureus and functions as potential clot dissolving agent for treating blood-clotting disorders. Sak converts the zymogen human plasminogen (hPg) to its active serine protease form, plasmin (hPm) and forms 1:1 complex with hPm, which activates other hPg molecules. In this report, we described high-level expression, purii¬cation and characterization of a fully active even glycosylated staphylokinase variant SakfC from S. aureus QT08 in a eukaryotic system Pichia pastoris GS115. The sak gene of 411 bp encoding a mature staphylokinase (136 aa, 15.5 kDa) was amplified from S. aureus QT08 genomic DNA, inserted into the expression vector pPICZαA under the control of the AOX1 promoter using methanol as an inducer and expressed in P. pastorisGS115 with a yield of 19 mg/L culture broth. The recombinant staphylokinase (rSak) was purified to homogeneity using ProBondO Ni2+-affinity chromatography with a specific activity of 20658 U/mg. The enzyme was expressed as an N-glycosylated protein in P. pastoris with a molecular mass of approximately 24 kDa on SDS-PAGE. rSak showed an optimum temperature of 30 to 37°C and optimum pH 7.5 (phosphate buffer) and pH 8 (Tris-HCl buffer). rSak was stable in a temperature range of 15 to 37°C and pH of 4 to 9. Additives (metal ions and detergents) all inhibited slightly or strongly the rSak activity. For the first time, a fully active staphylokinase from S. aureus was expressed in P. pastoriseven it was glycosylated. Key words: Staphylococcus aureus QT08, Pichia pastoris, fully active staphylokinase,high-level expression, purification, characterization, glycosylation

Differentiation of toxigenic Staphylococcus aureus strains isolated from retail meat products

Staphylococcus aureus is a saprophyte and commensal of the skin and mucous membranes in both animals and humans. As a pathogen, it can cause a number of diseases ranging from minor skin infections to fatal sepsis. Toxigenic strains of S. aureus are currently among the leading causes of food-borne intoxication (staphylococcal enterotoxicosis). Food contamination sources can be humans, raw materials, environment, technological equipment, etc. The identification of the origin of S. aureus would be helpful in the detection of the sources and routes of contamination. The aim of our study was to determine the probable origin of the selected S. aureus isolates coming from retail meat products intended for direct consumption with the use of phenotypic and genotypic methods. A set of 45 S. aureus isolates producing staphylococcal enterotoxins (SEs) with the potential to cause food-borne intoxication were selected for the study. These isolates were producers of the following enterotoxins: SEA (n = 10), SEB (n = 8), SEC (n = 10), SED (n = 7), SEH (n = 9), and SEB along with SED (n = 1). The phenotypic method used was based on the assessment of the growth on crystal violet agar (CV agar). A PCR-based genotypic method enabled the screening of the isolates for the sak gene encoding the enzyme staphylokinase typically found in human S. aureus isolates. As can be inferred from the type of growth on CV agar and the presence of the sak gene, all the isolates analysed were probably of human origin. These results confirm that humans are a major source of the bacteria S. aureus in both the food industry and retail sale of food products.

Bacteriophage analysis of staphylokinase‐negative Staphylococcus aureus strains isolated from people

Staphylokinase is one of virulence factors produced by Staphylococcus aureus strains. Producing it is associated with the presence of prophage in a bacterial cell which carries the sak gene. In this study, we examined the ability to produce staphylokinase by 257 S. aureus strains isolated from the inpatient and the outpatients (carriers) and the fact whether there is a correlation between sensitivity to bacteriophages and the lack of producing staphylokinase by strains. The results of this study showed, that staphylokinase-negative strains of S.aureus are widely common inpatients and among outpatients and their percentage does not usually exceed 20% of staphylococci. All staphylokinase-negative S. aureus strains were negative for sak gene. The correlations between a type of clinical material and the frequency of occurrence of staphylokinase-negative strains has not been stated. Typing with the use of the basic set of phages has shown that these strains do not constitute a homogenous group and they belong to different phage types. The same phage patterns occur both in staphylokinase-negative strains and in staphylokinase-positive strains, and the differences in phage types have the quantitative character. S.aureus not producing staphylokinase are more frequently common in the phage group II than other phage groups (p < 0.0001) and are typed mainly by 3C and 55 phages. Staphylokinase-negative strains of S. aureus do not occur among the phage group II of strains sensitive to phage 71.

Activation of Plasminogen by Staphylokinase Reduces the Severity ofStaphylococcus aureusSystemic Infection

Staphylokinase (SAK) is produced by the majority of Staphylococcus aureus strains. It is an extracellular protein that activates the conversion of human plasminogen (plg) to plasmin. The role played by SAK in staphylococcal infection is unclear. Wild-type S. aureus strain LS-1, which lacks the ability to produce SAK, was modified by an insertion of the sak gene into its chromosome. The sak gene was integrated in 2 forms--(1) linked to its own promoter and (2) fused to the promoter of the protein A gene--which resulted in the overexpression of SAK. SAK is highly specific for human plg and exhibits almost no activity toward murine plg. To investigate the role played by SAK in a murine infection model, human plg transgenic mice and their wild-type counterparts were inoculated intravenously with congenic S. aureus strains differing in SAK production. Human plg transgenic mice inoculated with SAK-expressing strains displayed significantly reduced mortality, less weight loss, and lower bacterial loads in kidneys than did the wild-type mice. No difference in the severity of sepsis was observed between transgenic and wild-type mice infected with a SAK-deficient strain. The results suggest that expression of SAK followed by activation of plg alleviates the course of S. aureus sepsis.

Novel preparation protocol for the expression and purification of recombinant staphylokinase

Staphylokinase (Sak), produced by lysogenic strains of Staphylococcus aureus, can convert plasminogen into its proteolytic form, plasmin, and thus is widely used to dissolve pathological clots in clinical applications. In the present paper, we report a novel approach to produce r-Sak (recombinant Sak) using an engineered Escherichia coli expression system. The expression plasmid was constructed by placing the Sak gene into the expression vector pET32(a), resulting in the expression of 35% fusion protein. Subsequently, a rapid and simple chromatographic procedure was developed for the large-scale purification of therapeutic-grade r-Sak from E. coli, which includes Ni(2+)-affinity chromatography, ultrafiltration and Q-Sepharose Fast Flow chromatography. This method led to the production of highly pure r-Sak (>99%), according to SDS/PAGE and HPLC analysis.

Quadruple or quintuple conversion of hlb, sak, sea (or sep), scn, and chp genes by bacteriophages in non-β-hemolysin-producing bovine isolates of Staphylococcus aureus

In 13 of 43 non-β-hemolysin-producing bovine isolates of Staphylococcus aureus, two truncated β-hemolysin ( hlb) genes were demonstrated by PCR and sequencing, and one truncated hlb gene was located beside the integrase ( int) gene of phage origin. The staphylokinase ( sak) gene was detected in all 13 isolates in which the truncated hlb genes were detected by PCR. Enterotoxin A ( sea) and enterotoxin P ( sep) genes were also detected in 5 and 2 of the 13 isolates, respectively. Moreover, the scn and chp genes encoding staphylococcal complement inhibitor (SCIN) and chemotaxis inhibitory protein of S. aureus (CHIPS) were detected in 13 and 4 of the 13 isolates, respectively. The bacteriophage induced by mitomycin C treatment was able to lysogenize one β-hemolysin-producing isolate of S. aureus, and the sak and scn genes were detected from the lysogenized isolate. These results suggest quadruple or quintuple conversion of hlb, sak, sea (or sep), scn, and chp genes by bacteriophages among non-β-hemolysin-producing bovine isolates of S. aureus.