The relationship between passive smoking exposure (PSE) and breast cancer risk is of major interest.To evaluate the relationship between PSE from partners and breast cancer risk stratified by hormone-receptor (HR) status in Chinese urban women population.Hospital-based matched case control study.Chinese urban breast cancer patients without current or previous active smoking history in China Medical University 1st Hospital, Liaoning Province, China between Jan 2009 and Nov 2009.Each breast cancer patient was matched 1∶1 with healthy controls by gender and age (±2 years) from the same hospital.The authors used unconditional logistic regression analyses to estimate odds ratio for women with PSE from partners and breast cancer risk.P = 0.002), as were women exposed to passive smoke for 16–25 years , and those exposed to > 4 pack-years . Similar trends were significant for estrogen receptor (ER)/progesterone receptor (PR) double positive subgroup, but not for ER+/PR−, ER−/PR+, or ER−/PR− subgroups.312 pairs were included in the study. Women who endured PSE had significantly increased risk of breast cancer , comparing with unexposed women. Women who exposed to >5 cigarettes/day also had significant increased risk (adjusted OR: 1.99; 95% CI: 1.28–3.10; limitations of the hospital-based retrospective study, lack of information on entire lifetime PSE and low statistical power.Our findings provide further evidence that PSE from partners contributes to increased risk of breast cancer, especially for ER/PR double positive breast cancer, in Chinese urban women. Breast cancer is the most common neoplasm among women, with more than one million newly diagnosed cases and nearly 600,000 cancer death worldwide annually Compared with the western countries, breast cancer in China shows more invasive ductal carcinoma with larger tumor size, later stage, lower ER and PR expression and higher HER2 over-expression. This difference was thought to be associated with the different ethnic background and the modifiable lifestyle risk factors between West and East Unlike most other modifiable lifestyle risk factors, cigarette smoking, including both active smoking and passive smoking exposure (PSE), doesn't actually show consistent association with breast cancer risk in western populations. And the evidence of PSE remains controversial and unclassifiable because of a lack of conclusive mechanistic information Our primary objectives were to determine the relationship between PSE and breast cancer risk, stratified by hormone receptor status in Chinese urban women using data from a hospital-based case-control study.The study's protocol was approved by the hospital human research ethics committee. All the participants provided written informed consent before enrollment.A hospital-based matched case-control study of breast cancer risk was conducted in the First Hospital of China Medical University from Jan 2009 to Nov 2009. All participants were Chinese women residents, living in the Central Liaoning city cluster, aged 18 years or older.Case participants were women histologically diagnosed with primary invasive breast cancer based on the 2003 WHO classification of breast cancer, who received surgery in this hospital and were recruited within 6 months from pathologic diagnosis for their interviews. All information on HR status was verified by pathology reports from the Clinical Pathology Diagnosis Center in this hospital, which classified ER and PR status as negative (IHC −), or positive . Controls were women recruited randomly from Medical Examination Centre in this hospital during the same period according to key inclusion criteria, and anyone who was diagnosed as any benign or neoplastic breast disease, or another malignant disease were excluded. Controls were individually matched to cases as 1∶1 by age (up to 2 years older or younger) and sex.Study participants were interviewed face-to-face by trained personnel using a structured questionnaire. Each interview usually took 40–50 minutes. Participants were asked about a variety of information including: (1) demographic and basic characteristics, such as living area, education, marital status, height and weight; (2) menstrual, contraceptive and reproductive histories; (3) lifestyle factors, such as alcohol consumption, exercise activity and active smoking histories; (4) medical history, such as family history of cancer; (5) for PSE from partner, we asked these questions: (a) How many people living with you smoke cigarettes and/or pipes? (b) How many cigarettes per day were smoked by your partner with whom you live? (c) How long were you exposed to these smokers?All data were checked for completeness and authenticity at the end of each interview by a trained researcher. Those who could not recall their smoking exposure histories or refused to report associated information were excluded. Information bias may influence the association between PSE and breast cancer risk, such as women living with different numbers of family smokers; regular or irregular smokers; or women were active smokers also receiving PSE. We defined the definition of passive smoker as women reported living with a partner, who smoked regularly at home for at least one year, as passive smokers. Women who lived with two or more smokers were excluded from the study. Women who reported smoking 20 packs of cigarettes or more over her lifetime were defined as active smokers and also excluded from this study.Associated PSE variables  were estimated in separate models. Cigarette pack-years were calculated by multiplying the number of cigarette packs per day to which the subject was exposed by the number of years she was exposed. No PSE were taken as the reference; other levels of number of passive cigarettes per day , years of PSE duration  and pack-years  were assessed respectively.2) at five years before interview date was calculated as weight (kg)/height (m2).Women's routine exercise activity was measured in weekly metabolic equivalent task (MET) hours; MET scores 6, 4.5 and 2.5 were categorized as strenuous sports, moderate activity and walking. MET-hour per week for each intensity activity was expressed as MET score multiplied activity time Differences in baseline characteristics were assessed by univariate analysis to select potential confounders, including age at interview, educational level , age (in years) at menarche , parity , oral contraceptive use (no/yes), family history of cancer (no/yes), exercise activity , BMI at five years before interview date, alcohol consumption (no/yes). Confounders were reevaluated in multivariate models to further assess associations between PSE and breast cancer risk. These confounders were selected on the basis of previous established risk factors and potential confounders in this study, including age at interview, age at menarche, menopausal status, oral contraceptive use, family history of cancer, alcohol consumption and BMI.The total samples were then stratified by menopausal status . Associations between PSE and breast cancer risk were further assessed in each subgroup. Additionally, we stratified all cases into ER+ or ER− subgroups, and compared PSE-related risks among these 2 subgroups and controls.t-test for continuous variables and Wald test for trends All statistical analyses were performed with the SPSS 16.0 statistical package . In univariate analyses, statistical comparisons were assessed using two-tailed chi-square test for categorical variables, The study closed on Nov 30, 2009. Out of the 327 eligible case patients, 319 were interviewed, 8 patients refused to participate in the study (non-response rate: 2.4%). Three hundred thirty-nine eligible controls were recruited, and 329 were interviewed finally (non-response rate: 3.0%). The 7(2.2%) cases and 17 (5.2%) controls who could not remember their smoking exposure histories or who had active smoking histories were excluded from our analyses. Finally, data from 312 eligible patients and 312 matched controls as total samples were analyzed. As only 41 ER−/PR+ patients and 20 ER+/PR− patients were included, making them unsuitable for separate analysis, these two kinds of cases were combined to form a single case group in sub-analyses.P = 0.001), to use oral contraceptives , to have first-degree family histories of cancer  and lower BMIs , but fewer were alcohol drinkers . Obvious differences were also seen for PSE. More patients endured PSE at home . However, there were no differences between patients and controls in age at interview, menopausal status, education level, parity, routine exercise activity in terms of MET-hours per week.Comparisons of baseline characteristics and exposure factors inpatients and controls are summarized in P = 0.027) (P = 0.010). Duration of PSE was also correlated with breast cancer risk. Compared with controls, women with PSE less than or equal to 15 years, and those with PSE over 16–25 years had 1.60-fold (95% CI: 0.97–2.64) and 1.87-fold (95% CI: 1.22–2.86) greater risk of breast cancer. However, no association was seen in women with >25 years of PSE history . Cumulative PSE was also significantly associated with breast cancer risk. Compared with controls, adjusted ORs (95% CIs) of women with PSE <4 pack-years, and those with PSE of ≥ 4 pack-years, were 1.12 (0.72–1.76) and 1.71 (1.17–2.50) respectively (P = 0.019). Thus, we found evidence of a dose-response trend in breast cancer risk for amount per day, duration and cumulative amount of PSE.In our study, continuous data of each exposure factor was categorized as mentioned in methods. Category boundaries were shown in = 0.027) . CompareThe association between PSE and breast cancer risk was assessed by menopausal status and further compared in P = 0.009). Relative to controls, adjusted ORs (95% CIs) were 1.91 (1.08–3.38), 1.99 (1.21–3.27), and 1.17 (0.65–2.09) in women having PSE for < 15 years, 16–25 years, and > 25 years, respectively (P = 0.021). Relative to controls, a significant dose-response relationship between cumulative amount of PSE and the risk of HR double positive breast cancer was also shown in women with PSE < 4 pack-years, and those with PSE of ≥ 4 pack-years, whose adjusted ORs (95% CIs) were 1.37 (0.82–2.29) and 1.92 (1.23–3.00) (P = 0.015). On the contrary, there were null associations on all passive smoking factors in ER+/PR−, ER−/PR+, and ER−/PR− patients (P>0.10), but the statistical power was limited by the small sample sizes. Women with PSE ≥25 years had lower risk of breast cancer than did the control group . To sum up, a weak but significant positive relationship between PSE and breast cancer risk was seen for the ER+/PR+ subtype; however, risks to other subtypes are difficult to determine due to limited statistical power.We further explored the relationship between PSE and breast cancer risk by HR status. This trend was only observed in patients with ER+/PR+ group . RelativTo improve the statistical power, we compared PSE-related risks among the ER+ subgroup, the ER−subgroup and controls. In this analysis, PSE remained a significant risk factor when comparing the ER+ subgroup with controls, but was not a significant risk factor when we compared risks in ER– subgroup with its control group, or the ER+ subgroup with the ER− subgroup .Recent studies suggest that PSE is no safer than active smoking These data are consistent with many previous epidemiological studies All the above hinted that there might not existed an identical effect of PSE on breast cancer risk. And we held an opinion that the relationship might depend on ethnicity, breast cancer heterogeneity, and other underlying factors. Therefore future researches should identify high-risk groups among the entire PSE population Moreover, the current data suggest that HR+ breast cancer is particularly associated with PSE, which was consistent with some previous studies of western patients In this study, a weak dose-response trend was seen only in the population who endured ≤25 years of PSE history. Notably those with >25 years of PSE history, the association was null or even slightly inverse  & 3. ThiThe strength of the study was the strict criteria of recruitment and grouping: Participants were all city dwellers, who were presumed to have similar lifestyles. Patients and controls were 1: 1 matched by age (+/− 2 years), which granted intergroup balance as much as possible. We also evaluated PSE quantitatively, which was uncommon compared with the uncertainty of PSE intensity in the previous studies One limitation of the study was its retrospective nature, leading to possible memory bias, especially for recalling partners' behavior among those who were newly diagnosed with cancer. They were more conscious of their disease, and, although they could be more likely to obtain information on their family history and cigarette exposure, were also more prone to exaggerate points that they thought would lead to their disease. This situation may overstate the risk. Additionally, given that each participant reported her husband's smoking behavior, the report of passive smoking, especially as to dose, is a proxy exposure, which might decrease the accuracy. Third, this study was a hospital-based, rather than population-based study, which potentially implies a selection bias. However, recent studies, using the data from this hospital, show that hospital controls are comparable with population controls for most demographic characteristics and lifestyle factors measured To clarify the association between PSE from partners and breast cancer risk in a Chinese urban female population, we performed a case-control study that included 312 breast cancer patients and 312 controls, matched at a 1∶1 ratio by age and residence. We found that PSE was associated with increased risk of breast cancer, with a weak dose-response relationship between PSE and breast cancer, especially in ER+/PR+ cancers. The association between PSE from partners and ER/PR-related breast cancer warrants further study.Table S1Association between passive smoking exposure and breast cancer risk.(DOC)Click here for additional data file.Table S2Relationship between passive smoking exposure and breast cancer risk by ER/PR status.(DOC)Click here for additional data file.


Blood pressure (BP) reduction in hypertensive patients is more difficult to achieve in the elderly or in the presence of comorbidities. We aimed to investigate the efficacy of the single-pill combination (SPC) aliskiren/amlodipine in hypertensive elderly patients, patients with high body mass index (BMI), with at least one metabolic risk factor, and/or type 2 diabetes mellitus (DM).In an open-label non-randomized study, patients not adequately controlled by previous treatment with the SPC olmesarten 40/amlodipine 10 (phase 1) were switched to the SPC aliskiren 300/amlodipine 10 (phase 2). The present post-hoc analysis investigated BP reduction in phase 2 in the named subgroups. The EudraCT identifier was 2009-016693-33, ClinicalTrials.gov identifier NCT01113047.2 or ≥30 kg/m2, respectively), 91 had metabolic risk factors (without DM) and 41 had DM. At the beginning of phase 2, depending on the subgroup, baseline SBP was 168–169 mmHg and DBP 103–104 mmHg. After 4 weeks of treatment with aliskiren 300/amlodipine 10, SBP/DBP was lowered by −5.1/−4.8 mmHg in the total cohort, by −5.5/−5.1 mmHg in elderly patients, by −6.7/−5.5 in overweight and by −4.2/−4.5 mmHg in obese patients, by −6.4/−4.7 mmHg in patients with metabolic risk factors without DM, and by −3.3/−5.0 mmHg in DM patients. Limitations include low sample size, limited treatment duration and the fact that the post-hoc defined groups were not mutually exclusive.Of the 187 patients not adequately controlled in phase 1 and thus treated with the SPC aliskiren 300/amlodipine 10 in phase 2, 69 were of advanced age (≥65 years), 74 or 89 were overweight or obese (BMI 25.0–29.9 kg/mIn this study reflecting clinical practice, the aliskiren/amlodipine combination achieved effective BP reduction in elderly patients or with metabolic comorbidities, including DM that might be more difficult to treat. This consistent BP lowering pattern facilitates everyday care of patients who receive aliskiren/amlodipine. The great majority of hypertensive patients require drug treatment to control their condition, usually in the form of combinations with drugs of different mode of action and synergistic efficacy2–5. Current guidelines advise the use of combination therapy not only in second-line, but also in first-line therapy5–7. In addition, the ESH/ESC Guidelines recommend that in patients at higher risk, goal BP should be achieved promptly, which supports initial combination therapy and quicker adjustment of doses. Common clinical variables that influence prognosis should be used to stratify patient risk, including among others, higher age, abdominal obesity, diabetes mellitus (DM) or other metabolic risk factors.Hypertension is an established major risk factor for an array of cardiovascular complications such as myocardial infarction or stroke, and is associated with a substantial increase in risk of premature death8–10. The basis for approval of the single-pill combination (SPC) olmesartan/amlodipine (Sevikar), was formed by a factorial dose-finding study in patients with mild-to-moderate hypertension11, a long-term safety study12 and two randomized, double-blind studies in patients with moderate-to-severe hypertension who were not adequately treated on one component alone14.The dihydropyridine calcium channel blocker (CCB) amlodipine, administered at doses of 5–10 mg once daily, is a direct peripheral arterial vasodilatator that reduces peripheral vascular resistance, and may reduce left ventricular hypertrophy16. A number of studies with aliskiren investigated the drug in special patient groups  was investigated in a factorial study22, a long-term study23 and in add-on studies in patients not adequately responding to the monotherapy25, which led to the approval of the SPC aliskiren/amlodipine26.Aliskiren and amlodipine have similarly long half-lives , which allows constant BP reduction throughout the entire dosing interval27 we aimed to investigate the BP lowering effect of the SPC aliskiren/amlodipine in special patient groups with hypertension that typically present in everyday practice and may pose a particular challenge to treating physicians owing to comorbidity, comedication or complexity of the disease: patients of advanced age, high body mass index (BMI) , with metabolic risk factors and/or with DM.In the present post-hoc analysis of a previously published prospective non-randomized studyThe AWESOME study (Aliskiren in Combination With Amlodipine in Hypertensive Patients Not Responding to Angiotensin Receptor Blocker Plus Amlodipine) was performed between May and October 2010 as a multicenter, open-label, non-randomized single-arm study in 38 centers in Germany. The study was approved by the ethics committee of the State Chamber of Physicians in Saxony in Dresden and the responsible health authority . All patients provided written informed consent prior to inclusion in the study. The study was conducted according to the ethical principles of the Declaration of Helsinki. It was registered in the ClinicalTrials.gov database under NCT01113047 and in EudraCT under 2009-016693-33.27. Study drugs were to be taken orally in the morning. Compliance was assessed by pill count.The study consisted of three phases, as shown in Male and female patients aged ≥18 years with uncomplicated moderate essential hypertension as defined above were eligible for participation. Patients were excluded from the core study, if they presented with DBP ≥110 mmHg or SBP ≥180 mmHg at any time between visit 1 and baseline, or if they could not discontinue all antihypertensive medication safely for a period of up to 2 weeks.Further important exclusion criteria comprised: controlled BP levels on current antihypertensive medication (DBP <90 mmHg and SBP <140 mmHg) at visit 1; known hypertensive retinopathy; heart failure in NYHA II–IV; secondary hypertension; refractory angina pectoris; significant hepatic or renal disease; DM ; second- or third-degree heart block without a pacemaker; significant arrhythmia or valvular heart disease; history of transient ischemic attack, stroke, hypertensive encephalopathy, myocardial infarction; contraindications for the study medication, and use of concomitant medications known to significantly affect the metabolism of the study drug or BP, pregnant or breast feeding women. Main exclusion criteria for the extension study were premature discontinuation during the core study, hypersensitivity or contraindications to diuretics, in particular to HCT.28. BP values were measured three times at each visit, and the mean of all three sitting measurements recorded.The primary efficacy parameter of this trial was the change in trough msDBP between visit 5 (start of treatment phase 2) and visit 6 (end of treatment phase 2), and in addition further BP parameters were assessed  as well as safety and tolerability. BP was recorded using a calibrated standard sphygmomanometer and appropriate size cuff, in accordance with the 2005 AHA Committee Report on blood pressure measurement2, obesity as BMI ≥30 kg/m2. The condition ‘at least one metabolic risk factor’ was present, if serum glucose was ≥5.56 mmol/l, LDL cholesterol ≥4.16 mmol/l, or triglycerides were ≥2.28 mmol/l.Patient aged ≥65 years were defined as elderly. The diagnosis DM in these hypertensive patients was according to physician’s individual assessment. Overweight was defined as BMI 25.0–29.9 kg/mThe safety information included frequency of adverse events (AE), results of physical examinations, data on body weight, and laboratory evaluations. Vital signs were assessed as part of the efficacy evaluations. All laboratory samples, obtained in a fasting state, were sent to a central laboratory for analysis.The safety population of the respective phase comprised the sample of all patients who took at least one dose of the study medication, the intention-to-treat population (ITT) of all patients from the safety population in phase 2 who had at least one evaluation of the primary efficacy parameter after visit 5 (baseline phase 2).The present post-hoc analysis was performed in subgroups of ITT patients with certain diagnoses as provided by demographic criteria (elderly patients), physician diagnoses (DM) in the context of clinical and laboratory criteria (metabolic risk factors including obesity). Patients suitable for several subgroups were analyzed in all these groups. The main analysis was performed descriptively on the mean change in trough SBP/DBP between visit 5 and visit 6 (treatment phase 2). The interpretation of all secondary parameters was explorative.A total of 439 patients were screened of which 97 were not eligible  and 342 patients were enrolled in phase 1. A total of 187 patients were switched to aliskiren 300 mg/amlodipine 10 mg at the beginning of phase 2. In that phase, 74 patients were overweight, 89 were obese, 69 were aged 65 years and above (elderly), and 132 had at least one metabolic risk factor (91 without DM and 41 with DM).Baseline characteristics in the total cohort and in the subgroups are shown in SBP and DBP at the beginning of the treatment phases are displayed in 27. AE were not analyzed by subgroups in view of the low patient numbers.Overall, 19 patients (10.1%) in phase 2 experienced at least one AE. No deaths or serious adverse events occurred. The most frequently observed AEs were ‘peripheral edema’ in seven (3.7%) patients, ‘edema’ in three (1.6%) patients and ‘eczema’ in one (0.5%) patient. All other AEs occurred only in one or two patients at maximum. Changes in laboratory values generally were infrequent, in accordance with the known profiles of the drugs. Details have been reported elsewhereThe present subgroup analysis shows that treatment with the aliskiren/amlodipine SPC achieves meaningful BP reductions in elderly or obese patients, in metabolic risk patients or DM patients. These BP reductions were in line with the BP reduction in the overall study population.2 29. Overall prevalence of hypertension, defined as BP ≥140/90 mmHg or on antihypertensive medication, was 60.6%, in grade 1 obesity 72.9%, in grade 2 obesity 77.1%, and in grade 3 obesity 74.1%. The odds ratio for good BP control (<140/90 mmHg) in diagnosed and treated patients was 0.8 in overweight patients compared with normal weight, 0.6 in grade 1, 0.5 in grade 2, and 0.7 in grade 3 obese patients29. While patients with overweight/obesity are not specifically considered in the current ESC hypertension guidelines, those with metabolic syndrome are. Although no implication is made that it is a pathogenetic entity, metabolic syndrome is specifically mentioned as it constitutes a cluster of risk factors often associated with high BP which markedly increases cardiovascular risk30–35. At least five definitions with slight or moderate deviations from each other have been proposed by international organizations or by expert groups30–35. In our study, a more general approach was chosen, as patients with at least one ‘metabolic risk factor’ were analyzed in the respective group.Such patients are frequent in everyday care, and they may present a particular challenge to treating physicians. In a cross-sectional point prevalence study of 45,000 unselected consecutive patients in a representative nationwide sample of primary care physicians in Germany (HYDRA), 28% had a BMI ≥30.0 kg/m5.With respect to age, in elderly patients with uncomplicated hypertension, treatment should be initiated gradually, while in those with higher risk, goal BP should be achieved more promptly, which favors initial combination therapy and quicker adjustment of dosesAs high age, high BMI and metabolic problems including DM are highly prevalent, the aliskiren monotherapy and aliskiren/amlodipine combination studies have included substantial fractions of such patients and found conclusive and consistent effects on BP in the various subgroups .36, and  had a substantially lower incidence of hypokalemia (1 vs. 14%) compared with thiazide-based therapy37. In the same study, aliskiren provided a stronger antihypertensive effect compared with HCT37. In the ATTAIN study, the aliskiren/HCT combination was more effective than ramipril38, and a subanalysis of an aliskiren/amlodipine combination study showed similar BP lowering in obese or non-obese patients39. Recently, Boschmann highlighted aliskiren's prolonged BP-lowering effect following discontinuation, and showed that the drug penetrates adipose and skeletal muscle tissue at levels that are apparently sufficient to reduce tissue RAS activity40.In obese patients, aliskiren-based treatment was highly effective and well-tolerated in patients who failed first line-treatment with a thiazideet al. study suggested that aliskiren 300 mg may offer advantages to irbesartan 300 mg41. In the pooled analysis by Gradman et al. no differences in the BP lowering effects between women with or without metabolic syndrome were noted42. Weinberger et al. reported that the aliskiren/amlodipine combination was more effective than amlodipine monotherapy in African-Americans with obesity or metabolic syndrome43. In a subgroup analysis of the ASCENT study, the dual aliskiren/amlodipine combination was as effective as the triple aliskiren/amlodipine/HCT combination in lowering BP among high-risk US minority patients with cardiometabolic syndrome44.With regard to patients with metabolic syndrome, the Krone 46. However, the studies by Uresin et al. 47 and Townsend et al. 48 showed beneficial BP lowering effects of aliskiren in diabetic patients. Also, a pooled analysis of aliskiren depicted the same effect in diabetic versus non-diabetic patients49. Interestingly, prospective registry data from the 3A Registry and the DRIVER study supported this finding in unselected patients51.In patients with DM, studies on aliskiren  mainly focused on the assessment of antiproteinuric/nephroprotective effects rather than the antihypertensive effects52. A significant dose relationship for the BP lowering effect was found in the elderly, with no evidence of dose-related increases in adverse events54. Compared with a ramipril-based regime, aliskiren lowered BP significantly stronger in elderly patients with systolic hypertension55. A substantial BP reduction over the long term has further been substantiated in the 3A Registry with 15,000 unselected patients56.In elderly patients, aliskiren exposure is modestly increasedOverall, the present analyses of the various subgroups in the AWESOME study, taken together with evidence from the aliskiren/amlodipine study program and other randomized studies indicate that the use of the combination leads to comparable efficacy in various subgroups. This could facilitate clinical management of the diverse hypertensive population. The overall results of the study show that the switch of patients not adequately controlled by the SPC olmesartan 40 mg/amlodipine 10 mg once daily to aliskiren 300/amlodipine 10 mg benefits about one-third of patients (36.4%) that can be treated to target at the end of phase 2 (week 8). For non-normalizers in phase 2, the addition of a thiazide diuretic in a SPC further led to a significant reduction of SBP and DBP.57. The multiple subgroup analyses were specified post-hoc and carried out descriptively, and performed to assess the consistency of a treatment effect among various patient characteristics. Thus, the typical problems of subgroup analyses (in particular multiplicity) do not apply here58. However, the sample size of the current study was low and the treatment duration limited. Larger and longer-term studies are needed for definitive conclusions. Also for the assessment of safety, much larger datasets are needed59.Some methodological limitations should be considered when interpreting the results of the present analysis. Patients could be part of more than one subgroup . An open-label study, in particular without a control arm, can be compromised by unknown bias of various typesThe SPC aliskiren 300 mg/amlodipine 10 mg had consistent BP lowering effects in various subgroups of patients with stage 2 hypertension. The BP lowering effect was in line with the achieved BP lowering effect in the total population of this study, and with various reports of other randomized controlled trials on this combination.The study was funded by Novartis Pharma GmbH.C.S. and F.M. are employees of Novartis Pharma GmbH Germany. E.K. is a former employee of Novartis. D.P. has received consultancy honoraria from Novartis.We acknowledge the cooperation and commitment of all investigators and their staff, who made the present trial possible.These results were presented, among others, at the 21st Annual European Meeting of Hypertension 17–20 June 2011, Milan, Italy.


The Epstein-Barr virus (EBV) is an oncogenic virus that may be related to cervical carcinogenesis. In gastric cancer, it has been demonstrated that E-cadherin gene (CDH1) hypermethylation is associated with DNMT1 overexpression by EBV infection. Our aim was to analyze the gene promoter methylation frequency of RB1 and CDH1 and verify the association between that methylation frequency and HPV and EBV infection in cervical lesions.Human papillomavirus (HPV) inactivates the Sixty-five samples were obtained from cervical specimens: 15 normal cervices, 17 low-grade squamous intraepithelial lesions (LSIL), 15 high-grade squamous intraepithelial lesions (HSIL), and 18 cervical cancers. HPV and EBV DNA testing was performed by PCR, and the methylation status was verified by MSP.p = 0.005) but not EBV frequency (p = 0.732). Viral co-infection showed a statistically significant correlation with cancer (p = 0.027). No viral infection was detected in 33.3% (5/15) of controls. RB1 methylated status was associated with cancer (p = 0.009) and HPV infection (p = 0.042). CDH1 methylation was not associated with cancer (p = 0.181). Controls and LSIL samples did not show simultaneous methylation, while both genes were methylated in 27.8% (5/18) of cancer samples. In the presence of EBV, CDH1 methylation was present in 27.8% (5/18) of cancer samples. Only cancer cases presented RB1 promoter methylation in the presence of HPV and EBV (33.3%).HPV frequency was associated with cervical cancer cases  for this article can be found here:  Cervical cancer represents an important public health problem, as it is the fourth most common type of carcinoma in women worldwide. This disease was responsible for 265,000 deaths in 2012, of which 87% occurred in developing countries  r. (2005)  reportedp = 0.005), as corroborated by the literature. In spite of that proved correlation, this infection alone is probably not sufficient to develop the disease. On the other hand, the samples in which HPV was not detected also presented cervical lesions. This molecular event was explained by Han et al. (2006) [We detected HPV with a higher frequency among the case samples (66.0%) than the control (26.6%) in which 77.8% of the cancer group samples presented HPV. We found a very statistically significant association between HPV infection and cervical cancer (. (2006)  and Sotl. (2006) . These s. (2006) -28 about. (2006) , which mEBV infection was found in a series of cases and control groups in which the rate of detection frequency was not considered statistically significant p = 0.732). This finding indicates that EBV cannot be responsible for cervical cancer progression alone. Nevertheless, cancer was revealed to be associated with HPV and EBV coinfection (p = 0.027), showing its possible role as a cofactor in cervical cancer progression. Some preliminary studies found similar results  than in the other lesions and controls (6.7% for RB1 and 13.3% for CDH1 in controls). These data for CDH1 are consistent with findings in the literature , and these groups had the lowest frequencies for the unmethylated status in both genes . Our findings also agree with those of Narayan et al. (2003) , who anaRB1/CDH1 methylation status and HPV/EBV infection. Our findings showed an important increase in the methylation status of both genes, RB1 and CDH1, with a pathological change seen with HPV/EBV infection. It is important to note that the methylated status of RB1 was considered to be associated with HPV infection (p = 0.042), revealing an important role for HPV in cervical cancer epigenetics.We also analyzed the potential association between The elucidation of the molecular relationships between viral and host proteins and their epigenetic modifications could improve the process of cervical cancer screening. Indeed, the detection of possible biomarkers and cofactors for the possibility of cervical cancer could allow for molecular differentiation between initial and precursor lesions.RB1 methylation and cervical cancer. With EBV, the methylation of RB1 was a tumor-associated event, where only the cancer group presented RB1 methylation in the presence of this virus. HPV infection and cancer progression were significantly associated. The global frequency of methylation was higher when HPV was present, showing its important role in this epigenetic mechanism in cervical carcinogenesis. Nevertheless, EBV seems to have a cofactor role in this process, which needs to be further investigated.In conclusion, our results showed that the methylation status of both genes increased with disease progression, revealing a significant correlation between


In France, equality in access to screening has been one of the main thrusts of public policies implemented between 2009 and 2013 . Our aim in this study was to analyse the obstacles to and levers for breast, cervical, and colorectal cancer screening uptake and their trends over time.Based on representative data from the French Health Care and Health Insurance Survey , multivariate logistic regressions were used to model the association between the nonuse of screening for the three cancers and various independent variables. Then, interactions with survey year dummies allowed the changes in the determinants of these cancer screenings over time to be estimated.cervix = 1.64 [1.38-1.95]), individuals without complementary health insurance (ORbreast = 2.05 [1.68-2.51]), or individuals with free complementary health insurance who more rarely use outpatient care. Moreover, individuals reporting either risky behaviours, namely heavy smokers  and high-risk drinkers (ORcervix = 1.42) or very safe behaviours, namely neither smoking nor drinking, underused screenings. Despite the implementation of national programmes for breast and colorectal cancer screenings, the disparities and inequalities in screening uptake did not decrease over the study period.Whereas the incentives for screening were strengthened during the period considered, cervical and breast cancer screenings decreased, and colorectal cancer screenings increased sharply (from 18.2% (95% CI = [17.0-19.4]) in 2006 to 38.9% (95% CI = [37.4-40.5] in 2010. Under-users of the three cancer screenings were primarily unskilled workers (ORThese results demonstrate the need for additional primary prevention efforts targeting the identified under-users by focusing on, for instance, individuals with a very healthy lifestyle. Health authorities could also intensify their efforts to promote increased access to screening for the most disadvantaged individuals. Cancer is one of the leading causes of disease and mortality. Breast 2009–2013 Cancer Plan, with a stated priority of combating inequalities in access to and use of screening. There is no national programme for cervical screening (only experiments are ongoing), but national screening programmes targeting breast cancer and colorectal cancer have been established in France since 2004 and 2009, respectively. The national screening programme for breast cancer targets women between 50 and 74 years of age and consists of sending a voucher through the mail that allows each eligible woman to benefit from a free screening at a radiological centre participating in the programme. The national screening programme for colorectal cancer targets men and women between 50 and 74 years of age and consists of sending a faecal occult blood test (FOBT) (accompanied by directions on how to perform the test) through the mail, for a free laboratory analysis. In addition to these programs, incentives directed towards GPs have been implemented in France, targeting breast cancer screening since 2009 and cervical cancer screening since 2012. These incentives consist of financial bonuses to referral GPs who have screened 80% of their eligible patients. Yet, the impact of these schemes has not been evaluated thus far.Policy mechanisms have been implemented based on these findings, and cancer screening remains one of the main thrusts of the French vs. 2005); however, the inequalities in access for the poorest individuals have persisted) of women reported having received a test for cervical cancer in 2006, this rate decreased significantly to 71.9% (95% CI = [70.5%-73.3%]) in 2010. The screening rate for breast cancer decreased (but not significantly at the 5% level) between 2006 and 2010, from 77.6% (95% CI = [75.8%-79.3%]) in 2006 to 74.0% (95% CI = [72.1%-75.9%]) in 2010. Overall, colorectal cancer screening uptake increased significantly between 2006 and 2010, from 18.2% (95% CI = [17.0%-19.4%]) in 2006 to 38.9% , having a chronic disease (58.5% in 2010 vs. 68.8% in 2006), having seen a specialist a least once (47.1% in 2010 vs. 42.0% in 2008) and never drinking alcohol (19.3% in 2008 vs. 24.2% in 2010).Table The results of the main-effects and interactions models for cervical screening are displayed in Table 45–54 = 1.56 [1.37-1.78], OR55–65 = 2.39 [2.08-2.74]. The negative impact of age on cervical screening has increased over time: compared to women aged 25–34 years in 2006, women aged 45–65 years had a higher likelihood of not being screened in 2010. Compared to women having an intermediate profession, living in a couple, and having private complementary health insurance, women who were farmers, office workers, skilled/unskilled workers, or unemployed; who were single; who did not have private health insurance; who had free complementary health insurance (the CMU-C) were significantly more likely to report not being screened. In addition, women who reported having a chronic disease, having good, fair, or poor self-rated health, and using medical care less often , were more likely to not have received a screening compared to women who reported greater access to medical care, no chronic disease, and very good self-rated health. Finally, regarding so-called risky lifestyles, the results regarding the probability of not being screened for cervical cancer followed a U-shaped curve: compared to not smoking, being an ex-smoker or a light smoker decreased whereas being a heavy smoker increased the likelihood of not being screened . Similarly, compared to safe drinkers, not drinking alcohol or being a high-risk drinker increased the likelihood of not being screened . All else being equal, the survey year had a significant impact on whether women reported being screened: women had a higher probability of not being screened for cervical cancer in 2010 than in 2006 . Compared to younger women (25–34 years), women aged 45–54 years or 55–65 years were more likely not being screened , women who reported not being screened more commonly were unskilled worker (OR = 1.62 [1.26-2.09]) or unemployed (OR = 1.61 [1.14-2.28]), single (OR = 1.45 [1.17-1.80]), and did not have a private complementary health insurance (OR = 2.08 [1.57-2.75]) or received free complementary health insurance (OR = 1.79 [1.36-2.37]). In addition, reporting a chronic disease (OR = 1.29 [1.11-1.59]), self-reporting poor health (OR = 1.87 [1.38-2.54]), having a low number of consultations with a GP (OR = 1.40 [1.21-1.62]) or a specialist  and never drinking alcohol (OR = 1.84 [1.44-2.37]) significantly increased the probability of not being screened. All else being equal, women were more likely to have not been screened for breast cancer in 2010 than in 2006. We performed a complementary analysis including FOBT uptake in the model and found a significant impact of the variable: all else being equal, not having received FOBT increased the odds ratio associated to not having performed a mammogram by 3.82 [3.18-4.60].The results of the main-effects and interactions models for colorectal cancer screening are displayed in Table For the two genders, the determinants of not being screened were being younger (50–54 years of age), being an unskilled worker, having less health care consumption , and being a heavy smoker. All else being equal, the likelihood of not being screened for colorectal cancer was significantly lower in 2010 than in 2008. There were differences regarding the impact of other determinants on FOBT uptake between males and females: females reporting poor health (OR = 1.48 [1.07-2.03]) and never drinking alcohol (OR = 1.22 [1.05-1.42]) had a higher likelihood of not being screened, but it was not the case for males. Conversely, being a light smoker increased the likelihood of not being screened for males (OR = 1.28 [1.03-2.22]) but not for females. One interaction was significant in the model for males: compared to having private complementary insurance, having free complementary health insurance (the CMU-C) was associated with a reduced probability of not being screened for colorectal cancer in 2008 (OR = 0.53 [0.30-0.94]) but a higher probability of not being screened in 2010 (OR = 4.31 [1.90-9.76]). We performed a second analysis for females in which we included whether they had received a mammogram in the past two years into the model and found that this variable had a significant impact: not having received a mammogram increased the odds ratio associated to not having received a FOBT by 3.85 [3.20-4.63].This study aimed to examine the determinants of and trends in the failure to receive breast, cervical, and colorectal cancer screenings in France between 2006 and 2010, a period in which various incentive policies were implemented. We focused on three types of cancer that were either the focus of recent debate or the subject of mass screening policies that were implemented following national and international guidelines. However, the supply context and incentive policies related to these types of screening differ. A national programme for breast cancer screening has been in place since 2004 and highly publicised for many years. Mammography is generally prescribed by a GP or gynaecologist and performed in a radiology centre. A Pap smear is most often performed by a gynaecologist or GP before it is sent to a laboratory for analysis, but a national programme has not been established (only experiments are ongoing). This test has been the focus of a number of economic evaluations aiming to provide recommendations for the vaccine against human papillomavirus. A national programme for colorectal cancer screening that targets the entire population over the age of 50 years was recently implemented. Colorectal cancer screening is most often prescribed by a GP but may also be recommended by a gastroenterologist or anatomopathologist.Cancer Plan 2009–2013). In contrast, the rate of colorectal cancer screening uptake increased substantially between 2006 and 2010. This result can be explained by the introduction of a national cancer screening programme in 2009 that combined two actions: a voucher mailed to eligible individuals on the demand side and a lump sum payment to GPs on the supply side.Our results show that the rates of cervical and breast cancer screening use in France decreased between 2006 and 2010. These results, which are consistent with those of other studies obtained in different contexts and countries, are parAccording to our results, young age, low socioeconomic status, and poor or fair self-rated health had the strongest negative impact on cervical screening uptake relative to breast and colorectal cancer screening uptake. This result could be explained by the absence of a national programme for cervical screening and, as a consequence, reduced media attention on cervical screening and/or by the costs associated with cervical screening . FurtherConcerning breast cancer screening, socioeconomic characteristics had affected the likelihood of undergoing a mammogram, but the effect was less pronounced than that for cervical screening. A possible explanation for this result is that the combination of highly publicised information campaigns for breast cancer in France and the national screening programme indeed targeted a larger population, but economic inequalities have persisted: not having private complementary health insurance (whether free or not) decreased the probability of screening. In France, not having complementary health insurance and, to a greater extent, receiving free complementary health insurance are often associated with low to very low household income. Moreover, even though mammograms are free of charge in the context of organised cancer screening, lower overall access to the health system  may prevent individuals from receiving the test. Thus, even though a national programme was implemented in 2004, i.e., before the study period, inequalities in access to breast cancer screening persist and do not seem to have declined over time.Regarding colorectal cancer screening, despite the introduction of a national programme in 2009, age-related screening disparities have persisted: individuals under 54 years of age were less likely to have been tested. The complementary health insurance variable had no effect on colorectal cancer screening uptake, in contrast to cervical and breast cancer screening uptake, over the study period. However, among males, inequalities related to having free complementary health insurance (the CMU-C) were significant in 2010. Moreover, individuals with less access to health care  were less likely to be tested. Thus, health authorities could intensify efforts to promote increased access to screening for the most disadvantaged populations. For this purpose, a national programme is one mechanism among many and should likely be complemented by targeted primary prevention policies aimed at reducing these inequalities.Another interesting result of our paper concerns the impact of individual risk behaviours and the use or nonuse of other screening tests. The results for individuals who engaged in high-risk behaviours in terms of alcohol and tobacco consumption were consistent with our expectations and the results of previous studies, 24. Onee, and concerning colorectal cancer, 34% of individuals were screened between 2009 and 2010f. Thus, the screening rates in the ESPS samples seem to overestimate INVS’s rates (even though they are not comparable for breast cancer screening because the "inclusion" period is not the same), but they are consistent with other self-reported measures provided for instance by the French National Institute for Prevention and Health Education (INPES)[The main shortcomings of the study are related to the declarative nature of the data. Specifically, so-called social desirability bias may have resulted in an overestimation of screening uptake. In France, the Institute for Public Health Surveillance (INVS) provides an annual estimation of breast cancer screening (since 2004) and colorectal cancer screening (since 2009). Concerning breast cancer, 49.3% of individuals were screened in 2006, 52.5% in 2008 and 52.0% in 2010 (INPES).Other determinants of screening, such as cancer-related family history, beliefsOur results demonstrate that persistent obstacles to screening, which are primarily related to socioeconomic characteristics, health care access, and risky behaviours, remain. However, we could not precisely determine if low screening uptake result from lower patient uptake  or from less access to screening services . Further studies should more specifically investigate the associations among attitudes/beliefs, individual barriers in access to care, and screening uptake.Greater coordination between the different actors involved in cancer screening also seems necessary because, in the context of French national programmes—particularly the breast cancer screening programme—the GP or gynaecologist is not always aware of whether their patients have received a mammogram. Some studies argue that the GP’s role should be to coordinate the patient’s various screenings, 35. IndaFor a complete description of the authorisation:http://legimobile.fr/fr/cnil/del/aut/2010/2010-003/.bIn France, private complementary health insurance is generally purchased to cover reinsurable copayments that are not covered by public health insurance: for any given service, the reimbursement is computed as a percentage of regulated prices, and some providers are also allowed to charge additional fees. Below a certain income threshold, individuals can benefit from a form of free complementary health insurance called the CMU-C. Individuals benefiting from the CMU-C are generally more socially and economically disadvantaged.cWe did not include breast cancer and colorectal cancer screening as predictors of cervical cancer screening uptake because the age groups differed.dWe thank an anonymous referee for offering two other rationales for this stratification: first other factors affecting colorectal screening may differ by sex ; second, this stratification is useful for examining whether breast cancer screening compliance predicts the likelihood of colorectal screening among women.ehttp://www.invs.sante.fr/Dossiers-thematiques/Maladies-chroniques-et-traumatismes/Cancers/Evaluation-des-programmes-de-depistage-des-cancers/Evaluation-du-programme-de-depistage-du-cancer-du-sein.fhttp://www.invs.sante.fr/Dossiers-thematiques/Maladies-chroniques-et-traumatismes/Cancers/Evaluation-des-programmes-de-depistage-des-cancers/Evaluation-du-programme-de-depistage-du-cancer-colorectal.


Sinefungin is bacteriostatic to pneumococci and significantly decreased biofilm growth and inhibited proliferation and structure of actively growing biofilms but did not alter growth or the matrix structure of established biofilms. Sinefungin significantly reduced pneumococcal colonization in rat middle ear. The quorum sensing molecule (autoinducer-2) production was significantly reduced by 92% in sinefungin treated samples. The luxS, pfs, and speE genes were downregulated in biofilms grown in the presence of sinefungin. This study shows that sinefungin inhibits pneumococcal biofilm growth in vitro and colonization in vivo, decreases AI-2 production, and downregulates luxS, pfs, and speE gene expressions. Therefore, the S-adenosylmethionine (SAM) inhibitors could be used as lead compounds for the development of novel antibiofilm agents against pneumococci.Pneumococcal colonization and disease is often associated with biofilm formation, in which the bacteria exhibit elevated resistance both to antibiotics and to host defense systems, often resulting in infections that are persistent and difficult to treat. We evaluated the effect of sinefungin, a nucleoside analogue of S-adenosylmethionine, on pneumococcal  Streptococcus pneumoniae is a major cause of pneumonia, meningitis, bacteremia, and otitis media (OM) in young children and the elderly [ elderly , 2. Pneu elderly , in whic elderly , 5. Bact elderly , 7. Vari elderly , 9. Mark elderly . Therefo S. pneumoniae, quorum sensing (QS) signaling regulates biofilm communities and plays a key role in coordinating the spatial disposition, aggregation of cells, and exopolysaccharide formation [ S. pneumoniae detoxification of SAH is carried out by MTA/SAH nucleosidase (Pfs) and S-ribosylhomocysteine lyase (LuxS), and this pathway is absent in human [The effective antibiofilm strategies should include inhibition of microbial adhesion to the surface and of colonization and interference with the signal molecules modulating biofilm development and the disaggregation of the biofilm matrix , 12. In ormation , 14. Autormation . In AMC,ormation , 17. Furormation . In S. pin human . in vitro biofilm.The SAM is a central molecule of AMC involved in recycling of methionine, methylation of biomolecules, biosynthesis of AI-2, and polyamine biosynthesis , 21. The Pseudomonas acyl homoserine-lactone quorum-sensing signal generation [ S. pneumoniae has not been studied. In this study we examined the effect of sinefungin on S. pneumoniae, particularly on biofilm growth.Sinefungin is a natural nucleoside and a structural analog of SAM. It inhibits transmethylation reactions related to DNA, RNA, proteins, and other molecules –25. Antineration . HoweverS. pneumoniae D-39 strain (NCTC 7466) is an encapsulated, serotype 2 pathogenic strain. It was obtained from the Health Protection Agency Culture Collections . Bacteria were routinely grown in tryptic soy broth  or on blood agar  supplemented with 5% v/v sheep blood at 37°C in an atmosphere of 5% CO2. Vibrio harveyi (V. harveyi) strains MM32 (ATCC BAA-1121) [AA-1121)  were groAA-1121) . SinefunS. pneumoniae were grown with different concentrations of sinefungin  at 37°C in 5% CO2 for different time points (0 h–10 h). And optical density at 600 nm (OD600) was measured after 2 h time interval using a SpectraMax plus 384 automated microplate reader . After 6 h of incubation, 100 μL aliquots was serially diluted and plated on BA for enumeration of colony forming units (CFU). The percentage decrease in planktonic cell growth was calculated by subtracting the values of sinefungin treated samples from control (untreated) samples. The experiment was replicated three times with triplicate samples at each time point.In vitro pneumococcal biofilm growth was carried out in 96-well, flat-bottom, polystyrene microtiter plate  using a static model [ S. pneumoniae grown overnight on BAP was scraped and grown in TSB+1% glucose medium [8 CFU mL−1) was diluted 1 : 100 with fresh sterile TSB+1% medium and 200 μL aliquots were inoculated in wells of 96-well microtiter plates and incubated at 37°C in 5% CO2. After incubation, the medium was discarded and plates were gently washed three times with 200 μL sterile phosphate buffered saline (PBS). Thereafter, plates were air-dried and stained with 50 μL crystal violet  for 15 min. Excess stain was decanted off and plates were washed three times with sterile distilled water. The biofilm was dissolved in 200 μL of 95% ethanol and the OD570 nm was measured using the aforementioned automated spectrophotometer. The data represent the average value of three replicates.ic model . Brieflye medium . The mid S. pneumoniae biofilms were grown with 10–50 μg mL−1 sinefungin for 18 h as described above. Biofilm biomass was detected by CV-microtiter plate assay as described above. The percentage decrease in biomass was calculated by subtracting the biomass of control biofilms grown without sinefungin. Enumeration of biofilm bacteria was done as described above .To study the concentration-dependent effect of sinefungin on pneumococcal biofilms,Pneumococcal biofilms were established in microtiter plate wells for 12 h. The formed biofilms were exposed to different concentrations of sinefungin for 6 h and analyzed by CV-microtiter plate assay and the cell viability was determined by CFU counts.μg mL−1 sinefungin for 6, 12, 18, and 24 h. At each time point, biofilm biomass was determined as described above.Pneumococcal biofilms were generated in the presence of 35 In vitro biofilms grown with 35 μg mL−1 sinefungin and without sinefungin in 24-well tissue culture plates for 18 h were analyzed by SEM. The medium was removed and the plates were gently washed two times with sterile PBS to remove planktonic cells. The samples were prefixed by immersion in 2% glutaraldehyde in 0.1 M phosphate buffer and postfixed for 2 h in 1% osmic acid dissolved in PBS. Samples were treated in a graded series of ethanol and t-butyl alcohol, dried in a model ES-2030 freeze dryer , platinum coated using an IB-5 ion coater , and observed using a S-4700 field emission scanning electron microscopic (Hitachi).V. harveyi MM32 was used as the qualitative reporter strain to detect the changes in the production of AI-2. V. harveyi MM32 (BB120 luxN::Tn5 luxS::Tn5) is an ideal reporter strain as it can sense AI-2 but cannot synthesize AI-2 of its own [ S. pneumoniae biofilms were grown with 35 μg mL−1 sinefungin and without sinefungin as described earlier for 18 h and sonicated for 3 seconds to disperse the adherent cells. Then 1 mL of the cell suspension was centrifuged and filter sterilized. And 10% of the filtered supernatant was added to the 90% reporter stain. Media without bacteria were negative control. The plates were incubated at 30°C for 15 h and the luminescence of the reporter strain was monitored using a luminometer . its own . The rep in vivo colonization study, we used rat OM model. The animal experiment protocol was reviewed and approved by the animal research and care committee at Dongguk University Ilsan Hospital . Twenty pathogen-free, Sprague Dawley rats weighing 150–200 g were obtained from Orient Bio . All rats were examined prior to use by otomicroscopy to document abnormal middle ear and were kept isolated in an infection-free zone for 2 weeks. Rats were assigned randomly to groups that received bacteria (n = 8), bacteria with 35 μg mL−1 sinefungin (n = 8), or no procedure . Fifty microliters of cell suspension containing 3 × 107 CFU S. pneumoniae was injected into the middle ear cavity through the tympanic membrane of the right ear using a tuberculin syringe and a 27-gauge needle [Fore needle . Animals luxS, pfs, and speE genes in biofilms grown for 18 h without and with (35 μg mL−1) sinefungin were quantified by real-time RT-PCR. The biofilms were washed and the adherent cells were scraped and immediately processed for RNA extraction. The cells were lysed by incubation of the cell pellet in 100 μL (3 mg mL−1) lysozyme  for 4 min. The total RNA was extracted using a RNeasy Total RNA Isolation System Kit  according to the manufacturer's instructions with few modifications. On-column DNAse (Qiagen) treatment was performed for 10 min at 20–25°C. RNA quality was assessed spectrophotometrically.Expressions of speE gene were designed by standard procedures from the nucleotide sequence of S. pneumoniae D39 strain  genes were previously reported [μL, consisting of 10 μL of 2X SYBR Green PCR Master Mix , 3 pmol of each forward and reverse primers, 4 μL cDNA, and nuclease-free water. PCR conditions included initial denaturation at 95°C for 10 min, followed by 45 cycles of denaturation at 95°C for 15 sec, annealing at 56°C for 10 sec, extension at 72°C for 15 sec, and final extension at 72°C for 5 min, followed by melting curve analysis from 60 to 95°C. Negative controls containing nuclease-free water and no reverse transcriptase control were included in each RT-PCR experiment. The relative gene expression was analyzed using the 2−ΔΔCT method [ gyrB and the standard condition was biofilms grown without sinefungin.cDNA was synthesized using the ImProm-II Reverse Transcriptase Kit  according to the manufacturer's instructions. Primers for9 strain . The prireported , 34. ReaT method . The reft-test. The statistically significant tests were set at a P value <0.05.The values were calculated as the mean of individual experiments performed in triplicate and compared with those of the control groups. Differences between two mean values were calculated by Student's The growth of pneumococci with different concentrations of sinefungin in time course experiment showed bacteriostatic effect of sinefungin. The growth of bacteria in sinefungin treated samples was slow in comparison to control samples (untreated). And a significant maximum growth difference in sinefungin treated and control samples was detected at mid-log phase (4 h after inoculation). However, at the end of the log phase (6 h of inoculation) there was no significant planktonic bacteria growth inhibition . The CFUP < 0.05) decreased biofilm biomass and CFU counts were detected in the samples grown with sinefungin. The inhibitory effect of sinefungin was concentration-dependent  and 50 μg mL−1 (highest concentration) sinefungin demonstrated a significant (P < 0.05) biomass decrease of 15% and 53%, respectively.Significantly   decrease of biofilm biomass was evident at all time points  of sinefungin were examined by SEM. Highly organized biofilm was produced in the absence of sinefungin. These biofilms were heterogeneous in nature, with cells embedded in the matrix and connected with each other and to the base of the plate. The adherent cells formed a three-dimensional structure of significant depth  and of sinefungin treated group were 1.70 × 103 (SD = 2533). A significant (P < 0.05) 70% less bacteria were recovered in sinefungin treated rat bulla  in the biofilms grown with sinefungin. Expression of the pfs gene was decreased by 2.88-fold (P = 0.006). Similarly, the speE gene was downregulated by 3.22-fold (P = 0.01). Gene annotation revealed the identity of the luxS and pfs genes as methionine pathways genes that recycle cysteine through AMC and the QS molecule AI-2 is the byproduct of AMC  decreased gene expressions oft of AMC . In this luxS and pfs genes expression and limits synthesis of autoinducer-2, and, hence, causes reduction in biofilm formation and may attenuate virulence.SAM is an important nucleoside that serves as an activated methyl group donor in the methylation of macromolecules to yield SAH/MTA which leads to biosynthesis of AI-2 and polyamine. Therefore, inhibition of SAM activity by certain substrate analogs could decrease in vitro and in vivo colonization.In this study, we examined the effect of sinefungin, a nucleoside analogue of SAM and a methytransferase inhibitor, on pneumococcal biofilm growth in vitro biofilms was concentration-dependent. We tested the inhibitory effect of sinefungin with time on in vitro biofilms grown [Planktonic growth of pneumococci with different concentrations of sinefungin demonstrated that sinefungin is bacteriostatic. The significant inhibitory effect of sinefungin was on actively growing cells (log phase). No significant planktonic cell growth inhibition was detected at concentrations that inhibited biofilm growth, which indicates that sinefungin may be a biofilm growth inhibitor. The inhibitory effect of sinefungin onms grown . At all ms grown , 37. No ms grown , 38.The difference in the architecture of the biofilms grown with and without sinefungin was verified with SEM. SEM confirmed that the biofilms grown with sinefungin were significantly different in terms of their connection to substrate and thickness and were unorganised. They also lost their cell-to-cell connections and were devoid of microcolonies, similar to a prior report . in vivo colonization of pneumococci. It is previously reported that mutant bacterial strains defective in QS create less potent infections [ S. pneumoniae infections in mouse were less effective at spreading to the lungs or the bloodstream [Using the rat OM model, we detected significantly low recovery of pneumococci in rat bulla treated with sinefungin. This indicates that sinefungin inhibitsfections . QS-defiodstream . S. pneumoniae. Various studies reported the prevention and treatment of infectious diseases by inhibiting bacterial QS [ luxS mutant strain to elicit bioluminescence in a V. harveyi AI-2 reporter strain.The QS regulates pneumococcal biofilms and AI-2 in the only QS molecule inerial QS . Therefoerial QS  reported luxS and pfs genes. The significantly decreased expressions of luxS and pfs genes could be due to inhibition of SAM-dependent methyltransferase activity. The encoded Pfs and LuxS enzymes are necessary for biofilm formation and production of AI-2 [ S. pneumoniae, luxS is a virulent gene and a central regulator of competence, fratricide, and biofilm formation, and a luxS mutant strain displays a significant decrease in pneumococcal biofilm formation [ luxS and pfs genes may decrease the biosynthesis of AI-2 in sinefungin treated samples [ S. pneumoniae, AI-1 is absent and AI-2 is the only QS molecule involved in expression of the enzymes for biofilm formation, exotoxin synthesis, and antibiotic resistance factors [ luxS and pfs genes leads to decreased biosynthesis of AI-2, which in turn disrupts spatial arrangement and build-up of the organized biofilms. Previous studies reported that cultures of Vibrio cholerae and enterohemorrhagic E. coli (EHEC) strain O157:H7 treated with MTA/SAH nucleosidase inhibitors did not synthesize AI-2 and showed reduced biofilm formation [Next, we detected changes in gene expression of of AI-2 , 41. In ormation , 14, 42. samples . In S. p factors . Thus, tormation . In a prormation . S. pneumoniae, methionine is converted to SAM by SAM synthetase (encoded by metK) and the SAM-dependent methyltransferase produces SAH and methylated products. The detoxification of SAH is carried out by MTA/SAH nucleosidase (Pfs) to produce S-ribosylhomocysteine (SRH) [3 (sulfonium moiety) of SAM is replaced by a C–NH3 (amine). Therefore, sinefungin inhibits SAM-dependent nucleic acid methyltransferases by competing with SAM for occupancy of the methyl donor site on the enzyme [ luxS and pfs genes, resulting in an imbalance of SAH, SRH, and MTA. Similarly the growth defects were observed in the Neisseria meningitidis pfs and luxS mutants either due to the accumulation of toxic SAH and MTA or due to metabolic imbalances within the bacteria [The other reason for inhibitory effect of sinefungin may be accumulation of toxic byproducts of AMC. Recent reports have suggested that substrates of AMC, such as MTA and SAH, are toxic to cells and that bacteria possess MTA/SAH nucleosidase and/or a combination of SAH hydrolase and MTA phosphorylase to remove these inhibitory nucleosides . In S. pne (SRH) , 19. Thee enzyme , 43, 44.bacteria . speE catalyzes the production of spermidine from putrescine and decarboxylated SAM [ speE a virulence gene involved in pneumococcal colonization, pneumonia, and invasive infections [ speE gene in biofilms grown with sinefungin indicates that sinefungin also interfere in spermidine biosynthesis.The decarboxylated SAM is involved in biosynthesis of spermidine, a polyamine involved in biofilms. Spermidine synthase encoded byated SAM . Previoufections . The low in vitro biofilm growth and in vivo middle ear colonization of pneumococci.Only a few molecules have proven effective against pneumococcal biofilms. Domenech et al. reported 80% reduction of pneumococcal biofilms growth with the amidase LytA, and, recently, Sumitomo et al. reported that S-carboxymethylcysteine inhibits pneumococcal adhesion to human alveolar epithelial cells , 49. Sim in vitro pneumococci biofilm growth, decreases formation of organised biofilms, decreases AI-2 production, and downregulates expressions of luxS, pfs, and speE genes. Sinefungin also decreases in vivo colonization of pneumococci in the middle ear. Therefore, the S-adenosylmethionine (SAM) inhibitors can be used as lead compound for the development of novel antibiofilm agents against pneumococci.In conclusion, sinefungin decreases


Cervical lymph node metastases in papillary thyroid cancer are common. Although central neck dissection is indicated in clinically nodal-positive disease, it remains controversial in patients with no clinical evidence of nodal metastasis. The aim of this retrospective study was to determine the outcomes of clinically lymph node-negative patients with papillary thyroid cancer who underwent total thyroidectomy without a central neck dissection, in order to determine the rates of recurrence and reoperation in these patients compared with a group of patients submitted to total thyroidectomy with central neck dissection.Two-hundred and eighty-five patients undergoing total thyroidectomy with preoperative diagnosis of papillary thyroid cancer, in the absence of suspicious nodes, were divided in two groups: those who underwent a thyroidectomy only  and those who also received a central neck dissection .Six cases (2.1%) of nodal recurrence were observed: 4 in group A and 2 in group B. Tumor histology was associated with risk of recurrence: Hürthle cell-variant and tall cell-variant carcinomas were associated with a high risk of recurrence. Multifocality and extrathyroidal invasion also presented a higher risk, while smaller tumors were at lower risk.The role of prophylactic central lymph node dissection in the management of papillary thyroid cancer remains controversial. Total thyroidectomy appears to be an adequate treatment for clinically node-negative papillary thyroid cancer. Prophylactic central neck dissection could be considered for the more appropriate selection of patients for radioiodine treatment and should be reserved for high-risk patients only. No clinical or pathological factors are able to predict with any certainty the presence of nodal metastasis. In our experience, tumor size, some histological types, multifocality, and locoregional infiltration are related to an increased risk of recurrence. The potential use of molecular markers will hopefully offer a further strategy to stratify the risk of recurrence in patients with papillary thyroid cancer and allow a more tailored approach to offer prophylactic central neck dissection to patients with the greatest benefit. Multi-institutional larger studies with longer follow-up periods are necessary to draw definitive conclusions. Papillary thyroid carcinoma (PTC) is the most common thyroid malignancy, comprising approximately 90% of new cases of thyroid cancer in iodine-sufficient areas of the world -6. PatieDespite the excellent prognosis, cervical lymph node metastases are common, occurring in 20 to 50% of patients ,2,6,8-14Although central neck dissection is indicated in clinically nodal-positive disease, it remains controversial in patients with no clinical evidence of nodal metastasis ,21.Some authors recommend routine central neck dissection in order to prevent a future recurrence, citing the high risk of positive lymph nodes, the accuracy of staging, better outcomes, reduced postoperative thyroglobulin (Tg) levels, and a lower morbidity rate associated with the first operation ,19,22-26Because of the risk of complications following prophylactic central neck dissection, recently several authors have introduced a more limited  central neck dissection, as an alternative treatment for patients with unilateral PTC ,25,29.The aim of this retrospective study was to determine the outcomes of clinically lymph node- negative patients with PTC who underwent total thyroidectomy without a central neck dissection, in order to determine the rates of recurrence and reoperation in these patients compared with a group of patients submitted to total thyroidectomy with central neck dissection.The clinical records of patients undergoing total thyroidectomy with or without prophylactic lymphadenectomy, between 2002 and 2010, presenting preoperative cytological evidence of PTC but no signs of enlarged lymph nodes during preoperative ultrasonography and intraoperative inspection and palpation were analyzed. In our unicenter retrospective study, 285 consecutive PTC patients were identified by computerized search. These patients had undergone TT with curative intent in our Department of Surgical Sciences at the University of Cagliari. The data were collected from retrospective chart review. Patients were divided into two groups: those who had received a thyroidectomy without central neck dissection , and those who had received a thyroidectomy with central neck dissection . These patients were considered for central neck dissection on the basis of the surgeon’s judgment of the surgical field, macroscopic appearance of the tumor, tumor size, and suspicion of extra-capsular invasion. In all cases, surgery was performed by three experienced endocrine surgeons. For each patient, a preoperative diagnosis of PTC had been obtained by ultrasound (US)-guided fine-needle aspiration cytology (FNAC). The preoperative work-up consisted of free thyroid hormone , thyrotropin (TSH), Tg and anti-Tg antibody (TgAb) measurements, and high resolution US of the neck by a skilled sonographer. A pre- and postoperative fibrolaryngoscopy was routinely performed in all patients. Postoperative diagnosis of lymph node recurrence was performed by US-guided FNAC and Tg measurement in FNAC fluid wash-out (FNAC-Tg) in the case of enlarged lymph nodes ≥ 1 cm. Patient demographics and postoperative complications were recorded, including neck hematomas requiring reoperation, transient or permanent hypoparathyroidism, transient or permanent vocal cord palsy, and distant and locoregional recurrence detected by postoperative surveillance. Hypoparathyroidism (defined as a parathyroid hormone (PTH) level < 10 pg/ml; normal values range between 10 to 65 pg/ml) was considered permanent when it lasted for more than six months. Permanent recurrent laryngeal nerve injury was defined as vocal fold paralysis, confirmed with laryngoscopic examination, persisting for more than six months. Qualitative data were expressed as percentages, while quantitative data were expressed as the mean value ± standard deviation.The study was approved by the Institutional Ethical committee of the University of Cagliari.FT3, FT4, and TSH were determined by automatic ultrasensitive chemiluminescent assays . Tg and TgAb were detected by chemiluminescent assays .All operations were performed by the same three surgeons, thus the TT and lymph node dissection techniques used were consistent across patients. Recurrent laryngeal nerves were routinely identified and exposed up until their insertion in the larynx, and parathyroid glands were identified and preserved. In cases of suspected devascularized or incidentally removed parathyroid glands, a muscular autoimplantation followed. Serum calcium and intact parathyroid hormone levels were assayed on the first postoperative day, and subsequently on the basis of a clinical evaluation.131I scanning after recombinant human thyrotropin (rhTSH) stimulation and neck US. Diagnosis of disease recurrence in the cervical lymph nodes was based on US-guided FNC, Tg washing of FNC aspirates, and serum Tg level monitoring. The median length of follow-up was 100 months (range 38 to 137 months).Patient follow-up examinations consisted of neck USs and the monitoring of serum Tg and TgAb levels every six months during suppressive L-thyroxine treatment. A serum Tg level ≤ 0.2 ng/ml was taken as undetectable. Surveillance for possible recurrence in patients considered disease-free was achieved by Tg detection or, in those positive for TgAb, by whole-body t-tests were used. Data were reported as the mean value ± standard error of the mean (SEM). All calculations were performed using the software package SPSS 15.0 for Windows . Comparisons were considered statistically significant for P < 0.05.Data were analyzed using descriptive statistics: for categorical variables, the Pearson’s chi-squared (exact) test was used; for quantitative variables independent Student’s P = 0.79). Transient or definitive hypoparathyroidism was observed in 55 (25%) and 10 patients (4.54%), respectively, in group A; while 25 (38.46%) and 7 patients (10.76%) were diagnosed in group B, respectively (P = 0.049 and 0.117). Temporary recurrent laryngeal nerve paralysis was observed in 3 patients in group A (1.36%) and in 2 patients in group B (3.07%) (P = 0.69). No cases of permanent or bilateral recurrent laryngeal paralysis were observed , with a median age of 51.5 years (range 18 to 81), were submitted to TT. Group A consists of 220 patients, 172 female and 48 male (F/M ratio = 3.58/1) with a median age of 53 years (range 18 to 81), who were submitted to TT without lymphadenectomy. Group B consists of 65 patients, 53 female and 12 male (F/M ratio = 4.41/1) with a median age of 46 years (range 20 to 77), who were submitted to TT with prophylactic central neck bilateral lymphadenectomy (see Table Median tumor size was 16 mm (16 mm in group A and 17 mm in group B). A microcarcinoma (<1 cm) was diagnosed in 57 patients (20%): 47 in group A (21.36%) and 10 in group B (15.38%). The histotype was classic papillary in 178 patients , follicular-variant in 86 , Hürthle cell-variant in 9  and tall cell-variant in 12 . Eighty patients (28.07%) had multifocal tumors: 60 in group A (27.27%) and 20 in group B (30.76%). Seventy-three patients (25.61%) had a locoregional infiltration (T3): 46 in group A (20.9%) and 27 in group B (41.53%)  and nodal micrometastases in 3 cases (4.61%). The median number of removed lymph nodes was 7.5. In patients with metastases, the median number of metastatic nodes was 3.5.131I). Apart from lymph node involvement, indications for postoperative 131I treatment were: a tumor > 1 cm, extra-capsular thyroid invasion or locoregional extension, unfavorable histological subtype , multifocal disease, or BRAF-positive tumor specimens. Twenty-six patients (9.12%) with tumor < 1 cm and without risk factor  were not submitted to adjuvant radioiodine ablation. To obtain adequate levels of endogenous TSH (>30 mU/ml), that are associated with an increased radioiodine uptake, patients stopped L-T4 replacement three to four weeks before radioiodine treatment; when L-T4 withdrawal was not indicated, TSH stimulation was achieved using rhTSH , according to standard protocols. Post-therapy whole-body scanning was performed four to seven days after radioiodine treatment.After surgery, 259 patients (90.87%) underwent adjuvant radioiodine ablation . Median dose administered was 2,420 MBq-131I in group A and 2,600 MBq-131I in group B; so prophylactic neck dissection did not statistically correlate with radioiodine dose.Radioiodine ablation was performed in 194 patients in group A and all 65 patients in group B. In five patients of group B, two or more radioiodine administrations were needed; in all the other patients only one radioiodine administration was performed. Differences between the two groups were not statistically significant . Only one central (VI) node recurrence was observed in group A (0.45%). The demographic characteristics of patients with recurrence were the following: 2 male (47 and 34 years) and 4 females . The mean elapsed time between intervention and lymph node recurrence was 15 months in group A (range 6 to 24) and 7.5 months in group B (6 and 9). Two patients had classic-variant, 1 had follicular-variant, 1 Hürthle cell-variant and 2 patients had tall cell-variant papillary carcinomas. In particular, this last variant was strongly associated with recurrence  recurrence was observed in 6 cases (2.1%): 4 in group A (1.81%) and 2 in group B 3.07%)  ,17,22, wet al.  reportedet al.  and in oet al. .The low rate (2.1%) of lymph node recurrence following TT observed in the present study is in line with data reported by others . In the P = 0.049 and 0.117). Temporary recurrent laryngeal nerve paralysis was observed in 3 patients in group A (1.36%) and in 2 patients in group B (3.07%) (P = 0.69). In particular, the differences in the rate of transient hypoparathyroidism were statistically significant while the incidence of definitive hypoparathyroidism and temporary recurrent laryngeal nerve paralysis are approximately double in group B, even if the differences do not reach statistical significance, probably because of the limited number of cases.The complication rates observed following prophylactic central neck dissection (group B) are, in our experience, high: a transient or definitive hypoparathyroidism was observed in 55 (25%) and 10 patients (4.54%), respectively, in group A; while 25 (38.46%) and 7 patients (10.76%) were diagnosed in group B, respectively . Multifocality  and extrathyroidal invasion  also presented a higher risk of recurrence, in line with other reports in the literature [Of consequence, the current indications for performing a prophylactic central neck dissection are still a matter of debate. Our low recurrence rate 2.1%) combined with the non-negligible incidence of complications  leads us to sustain that prophylactic central lymph node dissection should not be carried out on a routine basis in the treatment of PTC. Instead, it may be more useful to develop criteria for the identification of high risk patients for whom central neck dissection could be of benefit. The incidence of recurrence depends on numerous factors, such as tumor size, patient age, sex , and extra-capsular spread .1% combi,22,42. Iterature .The problem that remains is how to define the assessment criteria of high-risk patients, considering the fact that only the size of the tumor can be assessed preoperatively, while the type and the histological characteristics  can usually only be identified after surgery. We believe that a prophylactic central neck dissection should not be routinely recommended for smaller tumors (≤1 cm) while it may be advisable for larger tumors (>2 cm), especially if cytological suspicion of a high risk subtype arises or if there are intraoperative signs of extra-capsular spread. A wider use of immunocytochemical and genetic markers could prove useful in better defining the high-risk population. For example, patients with RET/PTC oncogene expression have a higher rate of lymph node metastases , and thiThe role of prophylactic central lymph node dissection in the management of PTC remains controversial. There is no convincing evidence that prophylactic central neck dissection leads to an improvement in recurrence rate, overall survival, or any clinically significant variable when applied indiscriminately to all PTC patients. The recurrence rate found in this study of 2.1% confirms the rarity of lymph node recurrence and leaves many doubts regarding the usefulness of prophylactic central neck dissection. The lateral neck was mainly involved in recurrence regardless of initial central neck dissection, suggesting the clinical benefit of central neck dissection may be small.Our study confirms that prophylactic central neck dissection is associated with increased morbidity, even when performed by experienced surgeons: in particular, it is associated with a higher rate of transient complications, mostly hypoparathyroidism.Therefore, in our opinion, until conclusive evidence emerges of the actual benefit of prophylactic central neck dissection procedure in the treatment of PTC without suspicious enlarged nodes, it may be avoided.In conclusion, TT appears to be an adequate treatment for clinically node-negative PTC. Prophylactic central neck dissection could be considered for the more appropriate selection of patients for radioiodine treatment and should be reserved for high-risk patients only. Unfortunately, no clinical or pathological factors are able to predict with any certainty the presence of nodal metastasis. However, in our experience, tumor size is related to an increased risk of recurrence, as are some histological types , multifocality, and locoregional infiltration.The potential use of molecular markers will hopefully offer a further strategy to stratify the risk of recurrence in patients with PTC and allow a more tailored approach to offer prophylactic central neck dissection to patients with the greatest benefit. Multi-institutional larger studies with longer follow-up periods are necessary to draw definitive conclusions.FNAC: fine-needle aspiration cytology; PTC: papillary thyroid carcinoma; PTH: parathyroid hormone; rhTSH: recombinant human thyrotropin; TgAb: anti-Tg antibody; Tg: thyroglobulin; TSH: thyrotropin; TT: total thyroidectomy; US: ultrasound.The authors declare that they have no competing interests.PGC conceived of the study and drafted the manuscript. GP participated in the design of the study and operated some of the patients. FM participated in the design of the study and performed the statistical analysis. JM helped to review the literature and collected patient follow-up data. LG collected patient follow-up data and helped to review the literature. EE participated in the design of the study and helped to draft the manuscript. AN participated in the design and coordination of the study and helped to draft the manuscript. All authors read and approved the final manuscript.


A total of 51 Salmonella isolates were detected from 44.8% (47/105) of the total samples from 15 poultry slaughterhouses examined, among which 27 (52.9%) NaR isolates were detected while ciprofloxacin (Cip) resistance was not present in the isolates. These 27 NaR isolates of DNA sequencing revealed that it contained three types of gyrA mutations in only D87 codon. Mutations in the D87 codon resulted in substitutions to G in most of the isolates, but D87Y and D87N exchanges were also detected. Although Cip resistance was absent, reduced susceptibility characterized by mutations in gyrA was apparent among Salmonella isolates from poultry slaughterhouses in Korea.This study was undertaken to identify and characterize amino acid substitutions in  Salmonellae are Gram-negative bacteria that are found worldwide in both cold- and warm-blooded animals as well as the environment. These bacteria are the main cause of salmonellosis in mammals and birds. Quinolone resistance in Salmonellae has developed over the last three decades since the introduction of Na, the first synthetic lone antimicrobial agent [Salmonella spp. have been documented including point mutations in the quinolone resistance-determining region (QRDR) of DNA gyrase (gyrA and gyrB) or topoisomerase IV (parC and parE), expression of efflux pumps on the outer membrane, and plasmid-mediated quinolone resistance [gyrA and parC mutations associated with quinolone resistance in 27 NaRSalmonella isolates collected from poultry slaughterhouses in South Korea.al agent . Severalsistance . The goaSalmonella isolates were recovered from 105 samples  collected at two out of nine duck slaughterhouses as well as 13 out of 41 chicken slaughterhouses located in different regions of South Korea. The first chilling water, the last chilling water, and five carcasses from each slaughterhouse were sampled. Bacteria were isolated from the samples according to the standard International Standardization Organization (ISO)-6579 method [A total of 51 9 method . SerotypSalmonella isolates were evaluated using a disc diffusion test with the following discs (Difco): amikacin , ampicillin , chloramphenicol , ceftazidime , cephalothin , ciprofloxacin , cefotaxime , cefazolin , cefepime , cefoxitin , gentamicin , imipenem , kanamycin , nalidixic acid , norfloxacin , streptomycin , trimethoprim/sulfamethoxazole , and tetracycline . The results were evaluated according to the Clinical and Laboratory Standards Institute (CLSI) guidelines [Escherichia coli ATCC 25922 was used as the control strain.Antimicrobial resistance of all 51 idelines . EscheriSalmonella isolates from the present study were further tested for amino acid changes in the QRDR, and screened for the presence of the gyrA and parC genes. Minimal inhibition concentrations (MICs) were determined for two antimicrobials, Na and Cip, belonging to the quinolone and fluoroquinolone classes of antimicrobials, respectively, using an agar dilution method according to CLSI guidelines [All 51 idelines .gyrA  and parC  genes containing the QRDR associated with quinolone resistance were amplified by PCR and sequenced as previously described [http://blast.ncbi.nlm.nih.gov/Blast.cgi). DNA sequences of the gyrA and parC genes were compared to those of native gyrA DNA (GenBank accession number X78977) and native parC DNA (GenBank accession number M68936), respectively.DNA was isolated for further molecular studies using a generation capture column kit  and stored at -70°C before use. Fragments of the escribed . The PCRSalmonella isolates to 18 antimicrobials is shown in Table Salmonella isolates, resistance to Na (52.9%), S (35.3%), and Am and Te (21.6%) was frequently observed. None of the isolates were resistant to An, Cip, Fox, Imp, Nor, or Sxt. Seven out of eight S. Typhimurium isolates remained susceptible to all the tested antimicrobials. S. Enteritis and S. Hadar isolates showed the highest rates of antimicrobial resistance in this study. Nine out of 18 S. Enteritidis isolates were resistant to at least three antimicrobial agents while the remaining nine isolates were resistant to one antimicrobial compound. All five S. Hadar isolates were resistant to more than two antimicrobial agents. A previous report determined that S. Hadar is one of the most resistant Salmonella serotypes [Resistance of the 51 erotypes .Salmonella isolates. Twenty-seven NaR isolates were serotyped as S. Enteritidis (n = 18), S. Montevideo (n = 5), S. Senftenberg (n = 2), and S. Newport (n = 2). Na resistance does not seem to have decreased during the last several years in South Korea and rates of resistance remain higher compared to those reported in other countries. Na resistance rates among Salmonella strains originating from chickens were found to range from 37% to 94.3% over the last 6 years [Salmonella isolates from poultry meats from several sources in Canada were shown to lack Na resistance [In the present study, Na resistance (52.9%) was predominant among the 51 dis n = 1, S. Mont 6 years . In contsistance  while vesistance  and the sistance .R (MIC ≥ 512 μg/mL) while the remaining 24 (47.1%) were susceptible to Na (MIC = 4–16 μg/mL). Interestingly, all 51 isolates were susceptible to Cip (MIC ≤ 0.5 μg/mL). In addition, all 18 S. Enteritidis isolates collected in the present study were highly resistant to Na (MIC ≥ 512 μg/mL) while all eight S. Typhimurium isolates were susceptible (MIC = 8–16 μg/mL).MICs for Na and Cip are presented in Table gyrA and parC found in the 27 NaR isolates. These results indicate that mutations only at position 87 in gyrA conferred both Na resistance and Cip susceptibility. All 27 NaR isolates possessed three different types of point mutations only at position 87 in gyrA and carried a D87 to G, Y, or N substitution. Among these 27 NaR isolates, the most common gyrA mutation was D87G (51.9%) followed by D87Y (37.0%) and D87N (11.1%). No mutations at S83 of gyrA were observed in any isolate from the present study. No mutation in parC that affected resistance or susceptibility was identified. However, silent mutations at V67, H75, H77, V100, D101, and G102 were found within the QRDR of parC.Table gyrA alone can be sufficient to induce Na resistance and Cip susceptibility. Additionally, results from the present study imply that parC mutations are not necessary to obtain a high level of Na resistance as hypothesized in previous reports [Our data may confirm that the mutations at S87 of  reports ,17.et al.[et al. [Salmonella isolates from animals contained a S83 mutation. Mutations at S83 of gyrA have been described in Salmonella isolates from humans and animals [Our findings contrast with those generated by Griggs et al. and Lieb.[et al.  showing  animals , and areSalmonella spp. encountered in human and veterinary medicine is increasing. Other previous investigations [gyrA gene is associated with Cip susceptibility. In contrast, parC mutations can result in a high level of fluoroquinolone resistance [Piddock  also repigations -22 demonsistance .gyrA gene are sufficient for conferring Na resistance and Cip susceptibility. Our findings also suggest that mutations in codon S83 of the gyrA gene are not a main factor of Na resistance, and parC mutations are not essential for quinolone resistance. Therefore, prudent use of antimicrobials and continued surveillance are absolutely needed to inhibit the increasing prevalence of quinolone resistance.In conclusion, results from the present study showed that mutations in codon D87 of the QRDR: Quinolone resistance-determining region; ISO: International standardization organization; CLSI: Clinical and laboratory standards institute; MICs: Minimal inhibition concentrations; BLAST: Basic local alignment search tool; NCBI: National center for siotechnology information.The authors declare that they have no competing interests.DHB analyzed the samples, performed the statistical analysis and wrote the manuscript. HJB and SJJ helped collect and analyse the samples. YJL provided the basic format of the study, obtained the funding and acted as study team leader. All authors read and approved the final manuscript.