In Vitro Antitumor and Antioxidant Capacity as well as Ameliorative Effects of Fermented Kefir on Cyclophosphamide-Induced Toxicity on Cardiac and Hepatic Tissues in Rats.

Fermented prebiotic and probiotic products with kefir are very important to slow down and prevent the growth of tumors and to treat cancer by stimulating the immune response against tumor cells. Cyclophosphamide (CPx) is widely preferred in cancer treatment but its effectiveness in high doses is restricted because of its side effects. The aim of this study was to investigate the protective effects of kefir against CPx-induced heart and liver toxicity. In an experiment, 42 Wistar albino rats were divided into six treatment groups: the control (Group 1), the group receiving 150 mg/kg CPx (Group 2), the groups receiving 5 and 10 mg/kg kefir (Groups 3 and 4) and the groups receiving 5 and 10 mg/kg kefir + CPx (Group 5 and 6). Fermented kefirs obtained on different days by traditional methods were mixed and given by gavage for 12 days, while a single dose of CPx was administered intraperitoneally (i.p.) on the 12th day of the experiment. It was observed that alanine transaminase (ALT), aspartate transaminase (AST), alkaline phosphatase (ALP), lactate dehydrogenase (LDH), creatinine kinase-MB (CK-MB), ischemia modified albumin (IMA) and Troponin I values, which indicate oxidative stress, increased in the CPx-administered group, and this level approached that of the control in the CPx + kefir groups. Likewise, as a result of the kefir, the rats' CPx-induced histopathological symptoms were reduced, and their heart and liver tissue were significantly improved. In conclusion, it was observed that kefir had a cytoprotective effect against CPx-induced oxidative stress, hepatotoxicity and cardiotoxicity, bringing their biochemical parameters closer to those of the control by suppressing oxidative stress and reducing tissue damage.

Examination of the effects of kefir on healing factors in a mice burn model infected with E.coli, S.aureus and P.aeruginosa using qRT-PCR.

Burn areas are susceptible to bacterial growth and infections, particularly in cases with lengthy periods of hospital stay. Burn wound healing, which involves various molecular and cellular mechanisms, continues to be a significant problem. Growth factors and cytokines play an active and vital role in wound healing. In the present study, the effects of kefir on wound healing in a 2nd-degree mouse burn model infected with e.coli, s.aureus and p.aeruginosa were investigated in vitro. In order to clarify the effects of kefir in the wound healing process, the macroscopic changes in kefir-applied scar tissue as well as wound depth and width were examined and IL-1α, IL-1ß, IL-6, IL-8, IL-10 and TNF-α, VEGF, TGF-ß protein levels were determined using the qRT-PCR method. The findings of the present study show that kefir has a positive impact on the factors playing a role in wound healing and accelerates the healing process.

Efficacy of antimicrobial peptide LL-37 against biofilm forming Staphylococcus aureus strains obtained from chronic wound infections.

The antimicrobial peptide LL-37 showed inhibitory effects against Staphylococcus aureus strains, which often responsible for wound infections. Understanding the molecular mechanisms of biofilm-containing wound infections is important. Thus, this study aimed to investigate both the antimicrobial and biofilm efficacy of LL-37 against biofilm-positive methicillin-susceptible S. aureus (MSSA) strains and biofilm-positive methicillin-resistant S. aureus (MRSA) strains obtained from chronic wound infections and its effect on different quorum sensing and virulence genes at suboptimal concentrations. Fifteen biofilm-forming MRSA and 15 biofilm-forming MSSA strains were included in this study. The minimum inhibitory concentration (MIC) values and biofilm formation were tested by microdilution methods. Real-time PCR was performed to determine gene expression levels. MIC values for LL-37 were 89.6 mg/L and 132.3 mg/L for MSSA and MRSA strains, respectively. No statistically significant difference was found between MRSA and MSSA strains in terms of the effect of LL-37 on biofilm formation. A statistically significant difference was found between MRSA and MSSA strains for atlA, RNAIII, and agrA gene expression levels following exposure to a suboptimal concentration of LL-37. Ultimately, the required LL-37 antimicrobial concentration was quite high; however, LL-37 antibiofilm concentration may be acceptable for use in humans against biofilm-forming MRSA and MSSA strains. This is the first study to investigate to effect of a suboptimal LL-37 concentration on gene expression levels of biofilm-forming MSSA and MRSA strains. LL-37 affected quorum sensing and biofilm producing mechanisms, even at suboptimal MIC concentrations.

Presence of biofilm and adhesin genes in Staphylococcus aureus strains taken from chronic wound infections and their genotypic and phenotypic antimicrobial sensitivity patterns.

The purpose of this research was to examine biofilm (icaA, icaD and bap) and adhesin (clfA, fnbA, cna) genes, and also assess the genotypic and phenotypic antimicrobial resistance patterns of Staphylococcus aureus strains taken from wound specimens in Mardin, Turkey. A total of 220 wound specimens were investigated. The biofilm forming ability and resistance pattern for eleven antimicrobial agents were investigated by conventional and multiplex PCR methods. S. aureus were taken from 112 (50.9%) of 220 wound specimens. Moreover, biofilm production was found in 79 (70.5%) of the 112 S. aureus isolates. 97 (86.6%) strains of all isolates were positive for icaA and icaD, and 15 (13.4%) for bap. The adhesin genes, cna, fnbA and clfA were detected in 98 (87.5%), 87 (77.7%), and 75 (66.9%) strains, respectively. The numbers of MSSA and MRSA bearing antimicrobial resistance genes were 19 (16.96%) and 32 (28.57%) for blaZ, 9 (8.04%) and 17 (15.18%) for tetK, 6 (5.36%) and 14 (12.5%) for ermC, 2 (1.79%) and 7 (6.25%) for tetM, 0 (0%) and 5 (4.46%) for mecA, 2 (1.79%) and 4 (3.57%) for ermA, 1 (0.89%) and 2 (1.79%) for both tetK and tetM, respectively. Our findings indicate that multiplex PCR is a suitable way for identifying biofilm and adhesin producing S. aureus. Our data also provided a country-wide oversight of the S. aureus antimicrobial resistance gene profiles for the properly therapy of patients and to control the spreading of the resistance genes.

Protective properties of kefir on burn wounds of mice that were infected with S. aureus, P. auroginasa and E. coli.

Burns and burn wounds are very sensitive to infections and cause a large amount of death worldwide. Although burn wound is sterile at the beginning, because of the risk factors such as prolonged hospital stay, immune suppression and burn affecting large surface area, colonisation with Staphylococcus aureus, Pseudomonas aeruginosa and Escherichia coli occur. For the burn therapy, one of the most important ways is to control bacterial infections. A probiotic fermented milk product kefir has antioxidant, antimicrobial, antiinflammatory, anticancer and various health promoting features. This study aims to examine possible protective properties of kefir which was used on the burn wounds that were infected with S. aureus, P. auroginasa and E. coli.  Swiss albino / Balb-c mice were seperated into four groups: (1) used as control group, (2) second-degree burn model+ burn wounds were infected with P.aeruginosa + S.aureus + E.coli, (3) second-burn wounds were treated with sterile pads dressed with kefir and (4) second-degree burn+burn wounds were infected with P. aeruginosa + S.aureus +E.coli before being treated with sterile pads dressed with kefir. The serum biochemical results verified the histopathological results and our findings showed that kefir is an effective product with cell-protecting properties.

Hepato-preventive and anti-apoptotic role of boric acid against liver injury induced by cyclophosphamide.

This study aims to examine cyclophosphamide (CP) exsposure associated toxicity on rat livers and the likely defensive effects of boric acid (BA). The rats used in this study were divided into four groups: control group, CP group, BA group, and BA + CP group. The present study was carried out using routine histological H&E stain, immunohistochemical stain caspase-3 as apoptotic marker, serum biochemical analysis for liver function markers (alanine transaminase (ALT), aspartate transaminase (AST) and alkalen phosphatase (ALP)), oxidative stress markers (total oxidant status (TOS), oxidative stress index (OSI) and total antioxidant capacity marker (TAC)). In the CP group, the levels of ALT, AST, ALP, TOS, OSI and caspase-3 increased whereas TAC levels decreased compared with the control group. In the BA + CP group, the levels of ALT, AST, ALP, TOS, OSI and caspase-3 decreased whereas TAC levels increased compared with the CP group. The histopathological evaluation of light microscope images and immunohistochemical caspase-3 activity in the BA + CP group were found to be decrease compared with those in the CP group. In conclusion, BA was successful in defending the liver against apoptosis and histopathological changes that are attributable to CP.

Investigation of the antibiotic resistance and biofilm-forming ability of Staphylococcus aureus from subclinical bovine mastitis cases.

A total of 112 Staphylococcus aureus isolates obtained from subclinical bovine mastitis cases were examined for antibiotic susceptibility and biofilm-forming ability as well as genes responsible for antibiotic resistance, biofilm-forming ability, and adhesin. Antimicrobial susceptibility of the isolates were determined by disk diffusion method. Biofilm forming ability of the isolates were investigated by Congo red agar method, standard tube method, and microplate method. The genes responsible for antibiotic resistance, biofilm-forming ability, and adhesion were examined by PCR. Five isolates (4.5%) were identified as methicillin-resistant Staph. aureus by antibiotic susceptibility testing and confirmed by mecA detection. The resistance rates to penicillin, ampicillin, tetracycline, erythromycin, trimethoprim-sulfamethoxazole, enrofloxacin, and amoxicillin-clavulanic acid were 45.5, 39.3, 33, 26.8, 5.4, 0.9, and 0.9%, respectively. All isolates were susceptible against vancomycin and gentamicin. The blaZ (100%), tetK (67.6%), and ermA (70%) genes were the most common antibiotic-resistance genes. Using Congo red agar, microplate, and standard tube methods, 70.5, 67, and 62.5% of the isolates were found to be biofilm producers, respectively. The percentage rate of icaA, icaD, and bap genes in Staph. aureus isolates were 86.6, 86.6, and 13.4%, respectively. The adhesion molecules fnbA, can, and clfA were detected in 87 (77.7%), 98 (87.5%), and 75 (70%) isolates, respectively. The results indicated that Staph. aureus from sublinical bovine mastitis cases were mainly resistant to ß-lactams and, to a lesser extent, to tetracycline and erythromycin. Also, biofilm- and adhesion-related genes, which are increasingly accepted as an important virulence factor in the pathogenesis of Staph. aureus infections, were detected at a high rate.

Lacrimal Gland Fistula following Severe Head Trauma.

We aim to present a unique case with discharging lacrimal gland fistula secondary to severe head trauma by an animal. A 9-year-old girl presented with serous fluid discharge from a cutaneous fistula in the left orbital region. The patient had history of surgery for traumatic frontal bone fracture and skin laceration in the superior orbital rim three weeks earlier. She underwent a complete ophthalmological examination and there was no anterior segment or fundus pathology. The orifice of the fistula was detected in mediolateral part of the left superior orbital rim and fluid secretion was increasing with irritation of the left eye. Neurosurgical complications were excluded and radiological assessment was nonremarkable. The patient's legal representatives were informed and lacrimal gland fistulectomy was planned. However, the fistula was self-closed one week after initial ophthalmological examination, and the patient had no symptoms. In conclusion, traumatic injuries of superior orbital region should be carefully evaluated and wounds should be well closed to prevent consecutive lacrimal gland fistula.

Association with leptin gene C.-2548 G>A polymorphism, serum leptin levels, and body mass index in Turkish obese patients.

Leptin is a protein hormone which plays a critical role in the regulation of both body-weight through reducing food intake and stimulating energy expenditure. Several polymorphisms in leptin gene (LEP), which encodes for leptin, have been described. However, its association with obesity is still controversial. Therefore, in the present study, we aimed to investigate whether LEP c.-2548 G>A polymorphism was associated with serum leptin levels, lipid parameters, and body mass index in Turkish obese patients. Forty-seven obese patients and 48 healthy individuals were included in the study. Blood samples were collected for DNA extraction. LEP c.-2548 G>A polymorphism were detected using polymerase chain reaction-restriction fragment length polymorphism technique. Serum leptin levels and lipid parameters were measured by ELISA and enzyme colorimetric assay techniques, respectively. GA or AA genotypes and A allele carrier frequencies of the c.-2548 G>A polymorphism in the LEP were higher in obese (38.3, 34.0 and 72.3 %) when compared with controls (14.6, 12.5, and 27.1 %; p = 0.011, 0.016, and 0.002, respectively). On the other hand, AA or AG genotypes were also related to increased serum leptin levels (p < 0.001) and body mass index (p < 0.0001). All these consequences showed that LEP -2548 AA or AG genotypes are important predictors for increased levels of leptin and BMI in Turkish obese patients and it may be a useful marker for obesity risk in our population.

Isolation and molecular characterization of methicillin-resistant staphylococci from horses, personnel and environmental sites at an equine hospital in Turkey.

The present study was carried out to assess the frequency of methicillin-resistant staphylococci (MRS) among racehorses (n=209) and veterinary personnel (n=13) as well as environmental surfaces (n=14) at an equine hospital in Adana, Turkey. In addition, species distribution, antimicrobial susceptibility, resistance genes, staphylococcal chromosomal cassette mec (SCCmec) type and clonality of these isolates were also investigated. MRS were identified by 16S rRNA sequencing, and typed by pulsed-field gel electrophoresis (PFGE). As a result, MRS was isolated in horses (48.3%), clinic staff (92.3%) and environmental samples (71.4%). Of the 123 MRS isolates, 118 isolates were identified as Staphylococcus lentus, and the remaining ones were found to be S. sciuri (n=3), S. intermedius (n=1) and S. fleuretti (n=1). All isolates were found to be susceptible against vancomycin, quinupristin-dalfopristin and rifampicin. Additionally, single or various combinations of resistance genes were detected among MRS isolates. SCCmec type II was identified in all isolates. Similar PFGE patterns were observed among MRS isolated from horses, humans, and environmental samples. Since MRS were concurrently isolated from horses and humans it is suggested that cross-transmission of MRS between horses and humans might occur. However, it cannot be ruled out that transmission is human to animal or animal to human.

Investigation of the antibiotic resistance and biofilm formation of Staphylococcus aureus strains isolated from gangrenous mastitis of ewes.

In this study, Staphylococcus aureus strains (n = 110) isolated from seven ewe flocks in Sanliurfa, Turkey were screened for antibiotic resistance and biofilmforming ability as well as for genes associated with antibiotic resistance and biofilm-forming ability. All isolates were found to be susceptible to oxacillin, gentamicin, clindamycin, cefoxitin, tetracycline, vancomycin, amoxicillin-clavulanic acid, ciprofloxacin and sulphamethoxazole-trimethoprim. The percent proportions of strains resistant to penicillin G, ampicillin and erythromycin were 27.2% (n = 30), 25.4% (n = 28) and 6.3% (n = 7), respectively. Regarding the antibiotic resistance genes, 32 (29%) isolates carried the blaZ and 8 (7.2%) the ermC gene. Other resistance genes were not detected in the isolates. All isolates showed biofilm-forming ability on Congo red agar (CRA), while 108 (98.18%) and 101 (91.81%) of them were identified as biofilm producers by the use of standard tube (ST) and microplate (MP) methods, respectively. All isolates carried the icaA and icaD genes but none of them harboured the bap gene. The results demonstrated that S. aureus isolates from gangrenous mastitis were mainly resistant to penicillins (which are susceptible to the staphylococcal beta-lactamase enzyme), and less frequently to erythromycin. Furthermore, all of the S. aureus isolates produced biofilm which was considered a potential virulence factor in the pathogenesis of staphylococcal mastitis.

Antibiotic resistance genes & susceptibility patterns in staphylococci.

BACKGROUND & OBJECTIVES: This study was carried out to evaluate the association between the antibiotic susceptibility patterns and the antibiotic resistance genes in staphylococcal isolates obtained from various clinical samples of patients attending a teaching hospital in Hatay, Turkey. METHODS: A total of 298 staphylococci clinical isolates were subjected to antimicrobial susceptibility testing. The genes implicated in resistance to oxacillin (mecA), gentamicin (aac(6')/aph(2''), aph(3')-IIIa, ant(4')-Ia), erythromycin (ermA, ermB, ermC, and msrA), tetracyclin (tetK, tetM), and penicillin (blaZ) were amplified using multiplex PCR method. RESULTS: Methicillin resistance rate among 139 Staphlococcus aureus isolates was 16.5 and 25.9 per cent of S. aureus carried mecA gene. Of the 159 CoNS isolates, methicillin resistance rate was 18.9 and 29.6 per cent carried mecA gene. Ninety four isolates identified as gentamicin resistant phenotypically, contained at least one of the gentamicin resistance genes [aac(6')/aph(2''), aph(3')-IIIa, ant(4')-Ia], 17 gentamicin-susceptible isolates were found as positive in terms of one or more resistance genes [aac(6')/aph(2''), aph(3')-IIIa, ant(4')-Ia] by multiplex PCR. A total of 165 isolates were resistant to erythromycin, and contained at least one of the erythromycin resistance genes (ermA, ermB, ermC and msrA). Phenotypically, 106 staphylococcal isolates were resistant to tetracycline, 121 isolates carried either tetK or tetM or both resistance genes. The majority of staphylococci tested possessed the blaZ gene (89.9%). INTERPRETATION & CONCLUSIONS: The present results showed that the phenotypic antibiotic susceptibility patterns were not similar to those obtained by genotyping done by multiplex PCR. Rapid and reliable methods for antibiotic susceptibility are important to determine the appropriate therapy decisions. Multiplex PCR can be used for confirmation of the results obtained by conventional phenotypic methods, when needed.

Detection of superantigenic toxin genes in Staphylococcus aureus strains from subclinical bovine mastitis.

The aim of this study was to determine the presence of genes encoding enterotoxins (sea-sej) and toxic shock syndrome toxin-1 (tst) of Staphylococcus aureus strains (n = 130) isolated from subclinical bovine mastitis in Turkey by polymerase chain reaction. Sixty-one (46.9%) isolates were found to contain one or more toxin genes. The most frequently found enterotoxin genes were seg (16.2%) and sei (16.2%), followed by sec (15.4%), sed (10.8%), and sej (10.8%), respectively. The tst gene was detected in seven (5.4%) isolates. None of S. aureus strains harbored sea, seb, see, and seh genes. Since these toxins are recognized agents of staphylococcal food poisoning, it must be considered that the consuming raw milk and raw milk products would pose public health risk as high prevalence of toxigenic S. aureus was found in this study.

Detection of slime and methicillin resistance genes in Staphylococci isolated from nasal samples of patients with orthopaedic implants.

BACKGROUND: The purposes of the present study were (1) to determine the prevalence of mecA and femA genes, (2) to investigate the presence of icaA and icaD genes responsible for slime synthesis, and (3) to search in vitro slime synthesis by staphylococcal strains isolated from the nares of patients with orthopaedic implants using the Congo red agar (CRA) plate test. MATERIAL/METHODS: Staphylococci strains were defined by multiplex polymerase chain reaction (PCR) technique to determine intercellular adhesion genes icaA and icaD. Slime production capability was searched by the CRA plate test, phenotypically. Also, the presence of mecA and femA genes was determined by PCR in all strains. RESULTS: The presence of icaA and icaD was detected in 101 isolates of 134 (75.4%) strains. This ratio was 74.8% (89 of 119) among the Staphylococcus epidermidis and 80% (12 of 15) among the Staphylococcus aureus isolates. A total of 63.4% of all the strains were found to be icaA and icaD positive as well as slime-forming on the CRA plate test. The percentage of icaA- and icaD-negative strains was 36.6%, and all of them were negative on the CRA plate test. Although femA presence was detected in all 15 (11.2%) S. aureus isolates, a total of 5 (3.7%) isolates carried the mecA gene. CONCLUSIONS: The frequency of icaA and icaD genes was determined to be of high prevalence among staphylococcal isolates. The staphylococcal strains that were found in the nasal flora of patients with orthopaedic implants may be important potential sources of infection for these patients.