Carbapenem and Colistin Resistance, Integrons and Plasmid Replicon Types in Multi-Drug Resistant Klebsiella Strains Isolated in Turkey.

BACKGROUND: Multidrug-resistant (MDR) Klebsiella is a globally important nosocomial pathogen. In the present study, 101 multidrug-resistant Klebsiella strains isolated from various clinical specimens obtained from two different Medical Faculties' hospitals were involved. We aimed to find out the prevalence of carbapenemase, mobile colistin resistance genes, and integrons in MDR Klebsiella strains. METHODS: The antibiotic susceptibilities of strains were determined by Kirby Bauer disc-diffusion method and resistance to colistin was confirmed by detection of minimum inhibitory concentrations. The prevalence of carba-penemase genes (blaOXA-48, blaNDM, blaIMP, blaVIM, blaKPC), mobile colistin-resistance genes (mcr-1 and mcr-2), and integrons (class I, II and III) were examined in Klebsiella strains by polymerase chain reaction. RESULTS: All strains were resistant to ß-lactam antibiotics, carbapenems, and quinolones. On the other hand, only nine (8.9%) strains were resistant to colistin. The most common carbapenemase genes were blaNDM (64.3%) and blaOXA-48 (53.5%). Besides, 28 (27.7%) strains were found to harbor both blaNDM and blaOXA-48. These 28 strains be-longed to the IncA/C (18.7%), IncL/M (7.7%), and IncFIIs (1.1%) plasmid replicon types. No strain was positive for blaIMP, mcr-1, and mcr-2. Class I and Class II integrons were shown to be harbored in 83.2% and 63.3% of strains, respectively. In total, 63 (63.6%) of strains harbored both classes I and II integrons. Class III integron was not detected. There was a statistically significant relationship between the presence of integrons and antibiotic resistance for cefotaxime (p = 0.024), ciprofloxacin (p < 0.001) trimethoprim/sulfamethoxazole (p < 0.001) and levofloxacin (p = 0.002). To our knowledge, this study represents the first report of a human isolate for the co-presence of blaNDM, blaOXA-48 and both Class I and Class II integrons, from Turkey. CONCLUSIONS: Our findings also highlight the dissemination of integrons and carbapenemases and the importance of surveillance on emerging antibiotic resistance.

Progesterone and Estradiol alter the growth, virulence and antibiotic susceptibilities of Staphylococcus aureus.

In the present study, the effects of progesterone (PRO) and estradiol (EST) on the growth, adhesion, invasion, biofilm and antibiotic susceptibilities of methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-susceptible S. aureus (MSSA) were examined. We also investigated effects of S. aureus infections on the viability of human breast adenocarcinoma (MCF-7) cells in the presence/ absence of hormones. The effects of hormones on the growth, adhesion and invasion of S. aureus were investigated in MCF-7 cells. Growths were assessed spectrophotometrically. Adhesive/invasive bacterial counts were examined by colony counting method. Biofilm was determined using microtiter plate assay. Minimum inhibitory concentrations (MIC) and minimum bactericidal concentrations (MBC) of ciprofloxacin (CIP) and gentamicin (GN) were examined by the microdilution method. Cell viabilities were detected via methylthiazolyldiphenyl-tetrazolium bromide assay. Growths of bacteria were decreased by hormones (p<0.0001). Adhesion was affected differently depending on hormones and strains tested. Hormones reduced the invasion (p≤0.0001) and biofilm (p<0.0001) of both strains. Progesterone increased and estradiol decreased MIC and MBC of CIP for MRSA; however, MICs of MSSA were not affected. S. aureus infected-MCF-7 viabilities were decreased in the presence of hormones except for high-level PRO (p<0.05). Our results showed that these two hormones have different effects on behaviors of S. aureus strains.

Association between active pulmonary tuberculosis and circulating microRNAs: a preliminary study from Turkey

Background/aim: Tuberculosis is a public health problem that still remains significant. For prevention, diagnosis, and treatment of tuberculosis more effective novel biomarkers are needed. MicroRNAs can regulate innate and adaptive immune responses, alter host-pathogen interactions, and affect progression of diseases. The relationship between microRNA expression and active pulmonary tuberculosis (APT) has not yet been investigated in the Turkish population. We aimed to test the potential diagnostic value of some microRNAs whose levels were previously reported to be altered in APT patients. Materials and methods: Using two different references (U6 and miR-93), we compared the expression levels of potentially important microRNAs in serum of APT patients with healthy individuals using quantitative polymerase chain reaction (qPCR). Results: miR-144 expression level was down-regulated in APT patients when either U6 or miR-93 was used for normalization. When data was normalized with miR-93, a statistically significant decrease in miR-125b (0.8 fold) and miR-146a (0.7 fold) expression levels were observed, while no differences were detected for U6. The receiver operating characteristic suggested that miR-144 may be a candidate biomarker for discriminating APT patients and controls (p < 0.05) both for U6 and miR-93. Conclusion: These findings suggest that miR-144 can have potential as a biomarker for APT. Using a single reference may be misleading in evaluation of microRNA expression. U6 and miR-93 can be used in combination as references for normalization of serum microRNA expression data.

Cell-Free Lactobacillus sp Supernatants Modulate Staphylococcus aureus Growth, Adhesion and Invasion to Human Osteoblast (HOB) Cells.

The increase of antibiotic resistance has become a problem. Probiotic bacteria play an important role in preventive/supportive medicine. Therefore, we examined the inhibitory effects of four different Lactobacillus species' (L. acidophilus-La, L. plantarum-Lp, L. fermentum-Lf and L. rhamnosus-Lr) cell-free supernatants (CFSs) on growth, adhesion, invasion, and biofilm formation of Staphylococcus aureus and effects of S. aureus, CFSs, and S. aureus-CFSs co-existence on human osteoblast (HOB) cell viability. Growth alterations were measured spectrophotometrically. Adhesive/invasive bacterial counts were detected by colony counting. Biofilm was evaluated using microtiter plate assay. The MTT assay was used for detection of HOB cell viability. The growth of MSSA significantly (P < 0.01) decreased in the presence of two CFSs (Lf and Lr) (P < 0.01); the growth of MRSA significantly (P < 0.05) reduced in the presence of La CFSs. All tested CFSs were found to reduce adhesion and invasion of MSSA (P < 0.0001). The adhesion of MRSA was enhanced (P < 0.0001) in the presence of all CFSs except La and the invasion of MRSA was decreased (P < 0.01) in the presence of Lr and Lf CFSs. All tested CFSs were shown to inhibit biofilm formation significantly (P < 0.0001). The reduction of S. aureus infected HOB cell viability and exposed to all CFSs except Lr that was found to be significant (P < 0.0001). The viability of HOB cell during co-incubation with MSSA and CFSs was shown to be decreased significantly. However co-existence of MRSA and CFSs did not alter HOB cell viability. These results suggested that lactobacilli as probiotics have low protective effects on MRSA-infected host cells.

The roles of hormones in the modulation of growth and virulence genes' expressions in UPEC strains.

BACKGROUND: Host factors such as hormones are known to modulate growth, virulence and antibiotic susceptibility of bacteria. In the present study, the effect of norepinephrine (NE) and estradiol (Est) on growth and expression levels of virulence genes (usp, sfa/foc, cnf1, aer) of uropathogenic E. coli (UPEC) strains C7 and C149 were investigated. METHODS: E. coli C7 and C149 were grown in serum based SAPI broth with and without three different concentrations of norepinephrine and estradiol. Growths were determined via optical density measurement in a spectrophotometer. Real-time polymerase chain reaction was used to determine gene expression levels. Statistical analyses were performed by one way Anova Tukey's post hoc-test. RESULTS: According to our results it has been shown that, growths of bacteria could be affected in the presence of hormones which are variable according to incubation period and hormones' concentrations. Up regulation of usp, sfa/foc, cnf1 were shown to be statistically significant (p < 0.05) in the presence of low, medium levels NE and all concentrations of Est. The expression of aer was down regulated significantly in the presence of low (p < 0.001) and medium level of Est; but all levels of NE was shown to be increased the expression of aer significantly (p < 0.05). CONCLUSIONS: The results of the present study has shown once more that host factors (norepinephrine and estradiol) could influence the growth of a bacterium as well as gene expressions.

Alterations of growth rate and gene expression levels of UPEC by antibiotics at sub-MIC.

The host is the main environment for bacteria, and they also expose to many antibiotics during the treatment of infectious diseases in host body. In this study, it was aimed to investigate possible changes in growth rate and expression levels of three virulence genes (foc/foc, cnf1, and usp) in a uropathogenic E. coli standard strain within the presence of ciprofloxacin, nitrofurantoin, and trimethoprim-sulfamethoxazole. The UPEC C7 strain was grown on tryptic soy broth-TSB (control), TSB + ciprofloxacin, TSB + nitrofurantoin, and TSB + trimethoprim-sulfamethoxazole for determination of both growth rate and gene expression level. Antibiotics were added according to their sub-minimal inhibition concentrations. E-test was used to determine MIC values of antibiotics. Growth changes were measured in absorbance 600 nm during 24-h period. Total RNA isolations were performed after incubation for 24 h at 37 °C. Gene expression levels were determined by quantitative PCR. Tukey's post hoc test was used for statistical analysis. According to absorbance values, it has been shown that only ciprofloxacin and trimethoprim-sulfamethoxazole have lead significant decrease on growth rate. We also detected statistically significant differences in each gene expression levels for all antibiotics via relative quantification analysis. Fold changes in gene expression was found 0.65, 1.42, 0.23 for foc/foc gene; 0.01, 0.01, 2.84 for cnf1 gene; and 0.1, 0.01, 0.01 for usp gene in the presence of ciprofloxacin, nitrofurantoin, and trimethoprim/sulfamethoxazole, respectively. This investigation has shown that antibiotics can play a role as an environmental factor which may determine the pathogenicity of bacteria in vivo.

The Effects of Insulin and Glucose on Different Characteristics of a UPEC: Alterations in Growth Rate and Expression Levels of some Virulence Genes.

BACKGROUND: Host factors are known to modulate virulence, antibiotic susceptibility, and growth rate of bacteria. The effect of human insulin and glucose on growth rate and expression of virulence genes (usp, sfa/foc, cnf1) of a uropathogenic E. coli (UPEC) strain were investigated in this study. METHODS: E. coli C7 was grown in tryptic soy broth (TSB-control) and TSB containing 20 µU/mL insulin, 200 µU/mL insulin, 0.1% glucose, and 200 µU/mL insulin + 0.1% glucose. Growth rates were determined via optical density measurement in a spectrophotometer. Real-time polymerase chain reaction was used to determine the gene expression levels. Statistical analyses were performed via Tukey's post hoc-test. RESULTS: Differences were found to be not statistically significant for bacterial growth rate in TSB and TSB with insulin and/or glucose. The expression levels of all three virulence genes were shown to be reduced significantly in the presence of insulin and/or glucose. The highest degree of repression was observed in 200 µU/mL insulin added to TSB. Also, the repression level of the gene expression was revealed to be reduced in 0.1% glucose supplemented TSB. CONCLUSIONS: In the present study, it was shown that insulin and glucose can modulate UPEC's gene expression while the growth rate was not affected.