Antimicrobial susceptibility and biofilm forming ability of staphylococci from subclinical buffalo mastitis.

The starting objective of this research communication was to determine the prevalence of subclinical mastitis in buffalo in Turkey. We also seeked to isolate and identify staphylococci, determine their antimicrobial susceptibilities and biofilm-forming abilities as well as investigating the presence of biofilm-related genes and microbial surface components recognizing adhesive matrix molecules. A total of 107 (66.9%) staphylococci (28 S. aureus and 79 coagulase-negative staphylococci, CoNS) were isolated from 160 mastitic milk samples collected from 200 lactating water buffalos. The staphylococci were especially resistant to beta-lactams except for cefoxitin but were less resistant to the other antimicrobials that were tested. Based on the Congo red agar method, 92.9% of the S. aureus and 70.9% of the CoNS isolates were positive for biofilm-forming ability, while all S. aureus and 97.5% of CoNS isolates were positive by a microtiter plate analysis. The presence of icaA and icaD genes was not always correlated with biofilm synthesis, and even in the absence of these genes, the isolates were able to synthesize biofilm.

The Investigation of the Association of Cutaneous Leishmaniasis in Biopsy Specimens of the Patients with Granulomatous Disease and Skin Cancer Using the Molecular Method.

BACKGROUND: Clinically, cutaneous leishmaniasis (CL) can be confused with granulomatous diseases and skin cancers, and it may lead to erroneous diagnosis and treatment. Diagnosis based and histopathology can have some difficulties due to low number of parasites, especially in chronic CL cases. We aimed to emphasize the necessity of considering CL in the differential diagnosis for cases of granulomatous diseases and basal cell carcinoma, particularly in areas where CL is endemic. METHODS: One hundred and seven paraffin-embedded tissue biopsy specimens were selected from the archive, as of 2002, of Pathology Department, School of Medicine, University of Hatay Mustafa Kemal in Hatay, Turkey. After DNA isolation, performed with the samples were used for PCR analysis with specific 13A, 13B primers targeting kinetoplastid DNA (kDNA) found in all Leishmania species. Another PCR was performed with LITSR and L5.8S primers targeting ITS-1 internal-transcribed-spacer-1 (ITS-1) region to subtype positive samples. Then these samples were further analyzed for subtyping with PCR-RFLP using HaeIII enzyme (BsuRI). RESULTS: Ten out of 107 tissue specimens were positive via kDNA-PCR. Lupus vulgaris, sarcoidosis, skin lymphoma and Leishmania cutis appeared in 9 out of 10 positive specimens. One of the cases presented with a mass on the cheek and was pre-diagnosed with hemangioma, but leishmaniasis did not appear. All of 10 specimens were diagnosed as granulomatous dermatitis. Two out of 10 samples, found positive with kDNA-PCR, were analyzed with ITS-1-PCR and identified as L. infantum/donovani after RFLP. CONCLUSION: Molecular methods should be utilized in the differential diagnosis of CL to eliminate false diagnoses of granulomatous diseases and skin cancers.

Phylogenetic Group/Subgroups Distributions, Virulence Factors, and Antimicrobial Susceptibility of Escherichia coli Strains from Urinary Tract Infections in Hatay.

INTRODUCTION: Nosocomial and community acquired urinary tract infections (UTIs) are one of the most encountered infections in the world. METHODS: This study aimed to determine the antibiotic susceptibility, phylogeny, and virulence genes of 153 Escherichia coli strains isolated from UTIs. Antimicrobial susceptibility of the isolates to different classes of antimicrobials was determined by the VITEK-2 automated system. Presence of virulence genes and phylogenetic groups were investigated by PCR. RESULTS: Regarding susceptibility to antimicrobials, ampicillin resistance was most abundant (67.3%), followed by amoxicillin-clavulanic acid (50.9%); least abundant was resistance to amikacin (1.3%) and nitrofurantoin (1.3%). Multi drug resistance (MDR) was observed in 34.6% of the isolates, and all isolates were found to be susceptible to imipenem, meropenem and fosfomycine. The majority of the isolates belonged to the phylogenetic group B23 (35.9%), followed by A1 (20.9%), D1 (18.9%), D2 (12.4%), A0 (%5.9), B1 (3.9%) and B2 (1.9%). Among E. coli strains examined, 49% had iucD, 32.7% papE-F, 26.1% papC, 15% cnf2, 11.1% sfa, 7.8% cnf1, 1.3% afaE, 1.3% afaD, 1.3% hlyA, 0.7% f17a-A, 0.7% clpG and 0.7% eaeA genes. CONCLUSIONS: Our research demonstrated that virulence factors were distributed among different phylogroup/subgroups, which play a role in UTIs pathogenesis in humans. For this reason, complex and detailed studies are required to determine the relationship between virulence factors and specific E. coli strains that cause UTIs in humans.

High occurrence of CMY-2-type beta-lactamase-producing Escherichia coli among broiler flocks in Turkey.

In this study, the prevalence of ESBL/pAmpC-producing Escherichia coli and their molecular characterization from cloacal swab samples were investigated. All samples were obtained from broiler flocks that are located in Hatay, Adana, and Mersin provinces of Turkey. Antimicrobial susceptibilities of the isolates were determined by disk diffusion method following the CLSI criteria. Genetic mechanisms mediating resistance in ESBL/pAmpC-producing E. coli isolates were identified by polymerase chain reaction (PCR) and followed by DNA sequencing. Phylogenetic groups and plasmid replicon types of the isolates were also investigated by PCR. The clonal relationship of selected isolates was investigated by enterobacterial repetitive intergenic consensus (ERIC)-PCR and multilocus sequence typing (MLST) method. Of 430 cloacal swab samples, 154 (35.8%) were positive for ESBL/pAmpC-producing E. coli. The ESBL/pAmpC type beta-lactamases were as follows: CMY-2 (n = 46), CMY-2 + TEM-1b (n = 63), SHV-12 (n = 5), SHV-12 + TEM-1b (n = 12), CTX-M-3 (n = 14), CTX-M-3 + TEM-1b (n = 1), CTX-M-15 (n = 4), CTX-M-15 + TEM-1b (n = 4), and CTX-M-1 (n = 3). Moreover, various rates of resistance to different antimicrobials were determined such as nalidixic acid (92.9%), ciprofloxacin (76%), sulfamethoxazole-trimethoprim (78.6%), tetracycline (73.4%), streptomycin (52.6%), chloramphenicol (44.2%), kanamycin (27.9%), tobramycin (24.7%), gentamicin (19.5%), and amikacin (0.6%). Furthermore, 148 (96.1%) isolates were found to be MDR. The ESBL/pAmpC-producing isolates were distributed into the following phylogroups: E (n = 61), B1 (n = 30), F (n = 20), A (n = 19), B2 (n = 11), D (n = 10), and C (n = 3). ERIC-PCR analysis showed 51 unrelated patterns. Out of the 28 selected isolates, the following sequence types (STs) were detected: ST354 (n = 3), ST114 (n = 3), ST5696 (n = 2), ST156 (n = 2), ST174 (n = 2), ST362 (n = 2), ST157 (n = 2), ST5114 (n = 2), ST6635, ST539, ST457, ST1640, ST95, ST5843, ST1158, ST10, ST648, and ST4248. The results of the current study revealed that broilers in Turkey are important reservoir of ESBL/pAmpC-producing E. coli, which suggest that these agents have a great potential of transmission to humans by food chain or direct contact.

Phylogenetic Group/Subgroups Distributions, Virulence Factors, and Antimicrobial Susceptibility of Escherichia coli Strains from Urinary Tract Infections in Hatay

Abstract INTRODUCTION: Nosocomial and community acquired urinary tract infections (UTIs) are one of the most encountered infections in the world. METHODS: This study aimed to determine the antibiotic susceptibility, phylogeny, and virulence genes of 153 Escherichia coli strains isolated from UTIs. Antimicrobial susceptibility of the isolates to different classes of antimicrobials was determined by the VITEK-2 automated system. Presence of virulence genes and phylogenetic groups were investigated by PCR. RESULTS: Regarding susceptibility to antimicrobials, ampicillin resistance was most abundant (67.3%), followed by amoxicillin-clavulanic acid (50.9%); least abundant was resistance to amikacin (1.3%) and nitrofurantoin (1.3%). Multi drug resistance (MDR) was observed in 34.6% of the isolates, and all isolates were found to be susceptible to imipenem, meropenem and fosfomycine. The majority of the isolates belonged to the phylogenetic group B23 (35.9%), followed by A1 (20.9%), D1 (18.9%), D2 (12.4%), A0 (%5.9), B1 (3.9%) and B2 (1.9%). Among E. coli strains examined, 49% had iucD, 32.7% papE-F, 26.1% papC, 15% cnf2, 11.1% sfa, 7.8% cnf1, 1.3% afaE, 1.3% afaD, 1.3% hlyA, 0.7% f17a-A, 0.7% clpG and 0.7% eaeA genes. CONCLUSIONS Our research demonstrated that virulence factors were distributed among different phylogroup/subgroups, which play a role in UTIs pathogenesis in humans. For this reason, complex and detailed studies are required to determine the relationship between virulence factors and specific E. coli strains that cause UTIs in humans.

Molecular and phenotypic characterization of enterococci isolated from broiler flocks in Turkey.

The aim of this study was to determine the antimicrobial resistance, resistance mechanisms implicated, and virulence genes (asa1, gelE, cylA, esp, and hyl) of Enterococcus spp. isolated from broiler flocks in Turkey. In addition, clonality of ampicillin and vancomycin-resistant enterococci was also investigated using pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). Out of 430 cloacal swab samples investigated, 336 (78.1%) Enterococcus spp. was isolated. The most frequently identified species were E. faecalis (87.8%), E. faecium (8.3%), E. durans (2.4%), E. casseliflavus (0.9%), and E. hirae (0.6%). The most common resistance was against tetracycline (81.3%), erythromycin (77.1%), ciprofloxacin (56.8%), and chloramphenicol (46.4%). Fifty (14.9%) isolates showed high-level gentamicin resistance (HLGL) phenotype. Ampicillin and vancomycin resistance were observed in 3.3% and 1.5% of the isolates, respectively. Two hundred eighty-three isolates were positive for the presence of virulence genes. Among the virulence genes tested, only gelE, asa1, esp, and cylA genes were detected. The most prevalent virulence gene was gelE (234, 69.6%), followed by asa1 (160, 47.6%), esp (37, 11%), and cylA (2, 0.6%). In conclusion, this study revealed that commensal enterococci from broiler flocks showed high rate of resistance to antimicrobials including clinically important antimicrobials for humans. The main underlying reason for high resistance could be attributed to the inappropriate and widespread use of antimicrobials. Therefore, there is an urgent need to develop control strategies to prevent the emergence and spread of antimicrobial resistance.

Antimicrobial resistance and underlying mechanisms in Staphylococcus aureus isolates.

OBJECTIVE: To investigate the antimicrobial susceptibility of 97 clinical Staphylococcus aureus (S. aureus) strains against 14 antimicrobials and corresponding resistance mechanisms. METHODS: The antimicrobial susceptibility of the isolates was determined using a disk diffusion method and antimicrobial resistance genes were screened by polymerase chain reaction. Mutations responsible for ciprofloxacin and rifampicin resistance were investigated by polymerase chain reaction and DNA sequencing. RESULTS: All isolates were found to be susceptible to vancomycin. Various rates of resistance to penicillin (83.5%), ampicillin (77.3%), erythromycin (63.9%), tetracycline (16.5%), amoxicillin/clavulanic acid (16.5%), ciprofloxacin (15.5%), trimethoprim/sulfamethoxazole (15.5%), oxacillin (13.4%), fusidic acid (12.4%), rifampin (6.2%), clindamycin (6.2%), gentamicin (6.2%) and mupirocin (5.2%) were determined. In addition, different combinations of resistance genes were identified among resistant isolates. Ciprofloxacin resistant isolates had mutations in codon 84 (Ser84Leu) and 106 (Gly106Asp) in the gyrA gene. Mutations in grlA were mostly related to Ser80Phe substitution. Leu466Ser mutation in the rpoB gene was detected in all rifampin resistant isolates. All methicillin resistant S. aureus isolates were SCCmec type V. CONCLUSIONS: In conclusion, it was determined that the isolates were resistant to different classes of antimicrobials at varying rates and resistance was mediated by different genetic mechanisms. Therefore, continuous monitoring of resistance in S. aureus strains is necessary to control their resistance for clinically important antimicrobials.

Prevalence and molecular characterization of extended-spectrum ß-lactamase (ESBL) and plasmidic AmpC ß-lactamase (pAmpC) producing Escherichia coli in dogs.

This study aimed to determine the prevalence of fecal carriage of extended spectrum ß-lactamase (ESBL) and/or plasmidic AmpC ß-lactamase (pAmpC) producing Escherichia coli among dogs (n=428) in Turkey. Polymerase chain reaction (PCR) and sequencing were used to characterize genes encoding ß-lactamase and plasmid mediated quinolone resistance (PMQR). Antimicrobial susceptibility testing and PCRs for virulence genes and phylogenetic groups were also performed. Cefotaxime resistant E. coli isolates were detected in 95 (22.2%) of the swab samples. Sequencing analysis results showed occurrence of various ß-lactamase genes: blaCTX-M-15 (62), blaTEM-1b (42), blaCMY-2 (22), blaCTX-M-3 (16), blaCTX-M-1 (15), blaOXA-1 (9) and blaSHV-12 (3) alone or in combination. The most frequently encountered phylogenetic group was group A1 (35.8%), followed by group D2 (22.1%), B1 (15.8%), D1 (9.5%), A0 (7.4%), B22 (5.3%) and B23 (4.2%), respectively. PMQR genes, aac(6')-Ib-cr, qnrS1 and qnrB10 were detected in 25.3, 10.5 and 1.1% of the isolates, respectively. While all isolates were susceptible to imipenem and amikacin, resistance rates to non-ß-lactam antibiotics ranged from 20.0% for tobramycin to 56.8% for tetracycline. The virulence genes were only detected in 34 (36.2%) of the isolates and this isolates carried single or various combination of virulence genes of iucD, papC, papE, f17a-A and eaeA. Four isolates were identified as human virulent pandemic CTX-M-15 producing E. coli clone O25b:ST131/B2. To the best of our knowledge, this is the first study to show fecal carriage of ESBL/pAmpC type ß-lactamase producing E. coli isolates among dogs in Turkey.

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.

Characterisation of Phenotypic and Genotypic Antibiotic Resistance Profile of Enterococci from Cheeses in Turkey.

The aim of this study was to determine the prevalence of enterococci in cheese samples and to characterize their antimicrobial resistance profiles as well as the associated resistance genes. A total of 139 enterococci were isolated from 99 cheese samples, the isolates were identified as E. faecalis (61.2%), E. faecium (15.1%), E. gallinarum (12.9%), E. durans (5.0%), E. casseliflavis (2.9%) and E. avium (2.9%). The most frequent antimicrobial resistance observed in enterococci isolates was to lincomycin (88.5%), followed by kanamycin (84.2%), gentamycin (low level, 51.1%), rifampin (46.8%) and tetracycline (33.8%). Among the isolates, the frequencies of high level gentamycin and streptomycin resistant enterococci strains were 2.2% and 5.8%, respectively. Apart from the mentioned antibiotics, low levels of resistance to ciprofloxacin, erythromycin and chloramphenicol were found. Moreover no resistance was observed against penicillin and ampicillin. The antimicrobial resistance genes including tetM, tetL, ermB, cat, aph(3')-IIIa, ant(6)-Ia and aac(6')-Ieaph(2")-Ia were found in enterococci from Turkish cheese samples. In the current study, we provided data for antibiotic resistance and the occurrence of resistance genes among enterococci. Regulatory and quality control programs for milk and other dairy products from farms to retail outlets has to be established and strengthened to monitor trends in antimicrobial resistance among emerging food borne pathogens in Turkey.

Prevalence of ß-Lactamase Producing Escherichia coli from Retail Meat in Turkey.

Extended spectrum ß-lactamase (ESBL) and plasmid-mediated AmpC ß-lactamase (pAmpC) producing Escherichia coli have been shown to be present in humans and animals representing a significant problem worldwide. This study aimed to search the presence of ESBL and/or AmpC-producing E. coli in retail meats (chicken and beef) in Turkey. A total of 88 ß-lactamase-producing E. coli were isolated from chicken (n = 81/100) and beef meat (n = 7/100) samples and their susceptibility to several antimicrobials were tested using disc diffusion method. E. coli isolates were further characterized for their phylogenetic groups. ß-Lactamase encoding (blaTEM , blaSHV , blaOXA , blaCTX-M , and blaAmpC ) and quinolone resistance genes (qnrA, qnrB, qnrS, qepA, and acc(6')-Ib-cr) were also secreened by polymerase chain reaction (PCR). However, in regard to ß-lactamase genes, 84 of 88 isolates were positive for blaCTX-M-1 (n = 39), blaCTX-M-3 (n = 5), blaCTX-M-15 (n = 4), blaTEM-1b (n = 2), blaSHV-12 (n = 1), blaCTX-M-1 /blaTEM-1b (n = 10), blaCTX-M-1 /blaTEM-1b /blaSHV-5 (n = 1), blaCTX-M-1 /blaCMY-2 (n = 1) and blaTEM-1b /blaCMY-2 (n = 6), blaCTX-M-15 /blaSHV-12 (n = 1), blaCTX-M-15 /blaTEM-1b (n = 1), blaTEM-1b /blaSHV-12 (n = 1), and blaCMY-2 (n = 12) genes. Resistance to cefuroxime (75.6% and 85.7%), nalidixic acid (89% and 85.7%), tetracycline (91.4% and 100%), streptomycin (40.2% and 100%), and trimethoprim-sulfamethoxazole (36.6% and 85.7%) was observed among strains isolated from chicken and beef, respectively. However, all isolates were found to be susceptible to amikacin, imipenem, and cefepime. Resistance to ampicillin and cefoxitin was significantly linked to blaCMY-2 gene, while there was a significant correlation between CTX-M type ESBL and antimicrobial resistance to cefuroxime and streptomycin (P < 0.05). The results of this study suggest that raw chicken retail meats are highly contaminated with ESBL-producing E. coli implementing a great risk to human health in Turkey.

Silencing of PrP C (prion protein) expression does not affect Brucella melitensis infection in human derived microglia cells.

Cellular prion proteins (PrP(C)) are mainly expressed in the central nervous system where they have antioxidant effects and a role in the endocytosis of bacteria within cells. These proteins also have some crucial biological functions including roles in neurotransmission, signal transduction and programmed cell death. However, the role of prion proteins in neuronal Brucella infection, specifically in the interaction of the pathogen and the host cell is controversial. In the present study, the silencing of PrP(C) mRNA by small interfering RNA (siRNA) transfection was investigated in human microglia cells infected with Brucella melitensis. More than 70% of prion proteins were down-regulated in microglia by siRNA transfection and this caused a slight decrease in the cellular viability of the control cells. Silencing of PrP(C) suppressed the antioxidant systems, though it led to an up-regulation of pro-inflammatory cytokines such as IL-12 and TNF-α as demonstrated by qRT-PCR analysis. B. melitensis infection of prion protein-silenced cells led to increase host viability, but had no effect on bacterial phagocytosis. According to the present study, there is no significant effect of prion proteins on phagocytosis and intracellular killing of B. melitensis in microglia cells.

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.

Prevalence and molecular mechanism of macrolide and lincosamide resistance in staphylococci isolated from subclinical bovine mastitis in Turkey.

Macrolide and lincosamide (ML) resistance and the related resistance genes of staphylococci were assessed from cases of bovine subclinical mastitis. Of the 104 Staphylococcus aureus and 62 coagulase negative staphylococcus (CoNS) isolates, 26 (25%) and 12 (19.4%) were resistant to ML, respectively. While constitutive ML resistance phenotype accounted for 15.4% (16/104) of S. aureus and 8.1% (5/62) of CoNS, inducible ML resistance phenotype accounted for 2.9% (3/104) of S. aureus and 3.2% (2/62) of CoNS. Among erythromycin-resistant isolates, single or various combination of different resistance genes were detected. The results of this study showed that ML resistance was prevalent among staphylococci from subclinical bovine mastitis cases in Hatay, Turkey. Therefore, a continuous surveillance is necessary to minimise the spread of antimicrobial-resistant staphylococci.

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.

Cisplatin reduces Brucella melitensis-infected cell number by inducing apoptosis, oxidant and pro-inflammatory cytokine production.

Brucella species are able to survive and replicate within the phagocytic cells and cause chronic infections in domestic animals and humans. Modulation of programmed cell death by Brucella spp. may be one of the reasons of the chronicity of the infection. In this study, whether cisplatin treatment, an apoptotic anticancer agent, would enhance the host resistance against Brucella melitensis-infected human macrophage-like cells was investigated. The infection neither induced inflammation nor oxidative stress. But, Brucella caused a decrease in infected macrophage viability of 36% at 48 h postinfection (p.i.) as compared with uninfected cells. Treatment of infected macrophages with 20 microM cisplatin for 48 h caused a large increase in nitric oxide (NO) levels in a time-dependent manner via induction of iNOS transcription. Cisplatin also enhanced glutathione peroxidase, myeloperoxidase and xanthine oxidase activities, providing evidence of generation of reactive free radicals. N-acetylcysteine was able to decrease cisplatin-induced NO, and prevented the agent-induced apoptosis, similar to effects found in l-NAME (N(G)-nitro-l-arginine methyl ester) treatment. Cisplatin stimulated inflammation through the induction of TNF-alpha and IL-12 secretion, and down-regulated Brucella-stimulated IL-10 transcription. The number of infected cells and their viability were decreased by 80% at 48 h p.i. by cisplatin in comparison with infected cells. Similar to this result, cisplatin treatment resulted in reduced intracellular CFU of B. melitensis being reduced by 80% at 48 h p.i. These findings demonstrate that pharmacological agents such as cisplatin may be considered to influence immune responses and apoptosis to help decrease Brucella-infected cell number.

Preventive effect of rolipram, a phosphodiesterase 4 enzyme inhibitor, on oxidative renal injury in acute ascending pyelonephritis model in rats.

OBJECTIVES: To evaluate the effects of rolipram, a phosphodiesterase 4 enzyme inhibitor, on Escherichia coli-induced renal oxidative damage in an acute pyelonephritis (PYN) rat model. METHODS: A total of 35 male Wistar albino rats were randomly divided into 7 groups (n = 5) as follows: control (uninfected), PYN 24 hours, PYN 48 hours, PYN 72 hours, PYN + rolipram 24 hours, PYN + rolipram 48 hours, and PYN + rolipram 72 hours. Ascending PYN was induced in the study groups by E. coli inoculation into the bladder, and the urethras were then occluded by collodium for 4 hours. Rolipram injections (1 mg/kg) were started before bacterial inoculation and repeated at 24-hour intervals in the PYN + rolipram groups until death. The rats were killed at the indicated times. Malondialdehyde and nitric oxide levels and superoxide dismutase and catalase activities were determined in kidney homogenates. Histopathologic examinations were also performed. RESULTS: Tissue malondialdehyde and nitric oxide levels and superoxide dismutase and catalase activities were significantly increased in the kidneys from the PYN groups. However, rolipram administration reduced renal malondialdehyde and nitric oxide levels and enhanced superoxide dismutase and catalase activities. The histopathologic examinations demonstrated that rolipram treatment reduced the inflammation grade in the kidney specimens. CONCLUSIONS: The results of our study have shown that rolipram has a protective effect on renal tissue from E. coli-induced oxidative injury. Therefore, phosphodiesterase 4 inhibitors might be a novel therapeutic option for the prevention and/or management of acute PYN.

The effects of increased cAMP content on inflammation, oxidative stress and PDE4 transcripts during Brucella melitensis infection.

Cyclic AMP (cAMP) is a key intracellular second messenger which at increased levels has been shown to have anti-inflammatory and tissue-protective effects. Its concentration is determined by the activities of both adenylate cyclase (AC) and the phosphodiesterase (PDE) enzymes. The aim of this study was to compare the effects of increased cAMP and glucocorticoid dexamethasone administration on B. melitensis-induced lipid peroxidation, Brucella suppressed antioxidant enzyme activities and PDE4 transcripts in rats. Intracellular cyclic AMP level was elevated by two different approaches; activation of AC and inhibition of PDE activities. Rats were inoculated with B. melitensis for seven days then a single dose of nonselective PDE inhibitor 3-isobutyl-1-methylxanthine (IBMX), the adenylate cyclase activator forskolin and dexamethasone were administrated to each infected group, and animals were challenged for 48 h. Brucella-induced lipid peroxidation was significantly reduced by the cAMP elevating agents as well as dexamethasone administration in plasma, liver and spleen. The antioxidant enzymes glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD) activities were significantly decreased by the pathogen. Whilst suppressed GSH-Px activity was reversed by cAMP elevating agents, SOD activity was not restored. Superoxide generating enzyme xanthine oxidase activity was not altered at the end of the infection period. Brucella infection increased plasma IL-12 level and this effect was also suppressed by the cAMP elevating agents, whereas TNF-alpha, IFN-gamma and IL-10 levels were unchanged. Intracellular cAMP levels are entirely hydrolyzed by cAMP-specific PDE 4 isozymes (PDE4s) in inflammatory and immunocompetent cells. Brucella reduced mRNA transcript levels for PDE4A by 40%, though PDE4B and 4D transcriptions were being unaffected in spleen. It was concluded that B. melitensis infection decreased activity of the antioxidant defence system, induced lipid peroxidation and suppressed PDE4A transcription. Administration of cAMP elevating agents exhibited similar affect with dexamethasone on lipid peroxidation, IL-12 production and antioxidant enzyme activities in Brucella infection.

Antibacterial activity of propolis against MRSA and synergism with topical mupirocin.

OBJECTIVES: The aim of the present study was to investigate the activity of the propolis and its combinations with mupirocin against methicillin-resistant Staphylococcus aureus (MRSA) in nasal carriage. METHODS: This study was carried out between June and August 2005. To infect nares of the rabbits, MRSA (ATCC 33591) strain was used. Minimum inhibitory concentration was determined according to National Committee for Clinical Laboratory Standards. Each inoculum was prepared in the same medium at a density adjusted to a 0.5 McFarland turbidity standard (10(5) colony-forming units [cfu]/mL) and diluted 1:100 for the broth microdilution procedure. Ten microliters (10 microL) (10(5) cfu/mL) of the bacterial suspension containing approximately 1000 cfu of MRSA was administered with sterile microsyringe through both nostrils of each rabbit. Ninety-six (96) hours after inoculation, the presence of infection was confirmed by using bacterial cultures. Twenty-six young New Zealand rabbits were randomly divided into 4 groups. Each treatment group (1, 2, and 3) included 7 rabbits and control group (group 4) included 5 rabbits. Group 1 was treated with topical mupirocin + ethanolic extract of propolis drops, group 2 received topical mupirocin, group 3 was administered ethanolic extract of propolis drops, and the control group (group 4) was only treated with phosphate-buffered solution drops for 7 days. At the end of study, nasal cultures and smears were obtained for bacterial count and cytologic examination. RESULTS: The colony numbers of bacteria in group 1 were determined to be significantly lower than in group 2 (p = 0.0001), group 3 (p = 0.0001), and group 4 (p = 0.0001). The mean bacterial cell counts of groups 1-4 were 360.2 +/- 52.4 cfu/mL, 4120.6 +/- 860.4 cfu/mL, 5980.8 +/- 1240.6 cfu/mL, and 11500.0 +/- 2568.4 cfu/mL, respectively. Mupirocin + propolis administration (group 1) resulted in a significant reduction in the polymorphonuclear leukocyte (PMNL) count in the mucous membranes of rabbits compared with the other treatment groups (p < 0.05). CONCLUSIONS: Propolis addition to mupirocin regimen was found to result in more profound reduction in bacterial cell count and inflammatory response compared with the rest of the treatment modalities.