Intrauterine environment plays a pivotal role in the origin of fatal diseases such as the metabolic syndrome. Diabetes is associated with low-grade inflammatory state and dysregulated adipokines production. The aim of this study is to investigate the effect of maternal diabetes on adipocytokines  production in F1 offspring in rats.CF1-CD), F1 offspring of control mothers under high caloric diet (HCD) (CF1-HCD), F1 offspring of diabetic mothers under CD (DF1-CD), and F1 offspring of diabetic mothers under HCD (DF1-HCD). Every 5 weeks post-natal, 10 pups of each subgroup were culled to obtain blood samples for biochemical analysis.The offspring groups were as follows: F1 offspring of control mothers under control diet (CD) (p > 0.05). DF1-CD and DF1-HCD groups had high hepatic and muscular depositions of TGs. The significant elevated NEFA level only appeared in offspring of diabetic mothers that was fed HCD. DF1-CD and DF1-HCD groups demonstrated low serum levels of adiponectin, high levels of leptin, and elevated levels of TNF-α compared to CF1-CD (p > 0.05). These results reveal the disturbed metabolic lipid profile of offspring of diabetic mothers and could guide further characterization of the mechanisms involved.The results indicate that DF1-CD and DF1-HCD groups exhibited hyperinsulinemia, dyslipidemia, insulin resistance and impaired glucose homeostasis compared to CF1-CD (Dysregulated adipocytokines production could be a possible mechanism for the transgenerational transmittance of diabetes, especially following a postnatal diabetogenic environment. Moreover, the exacerbating effects of postnatal HCD on NEFA in rats might be prone to adipcytokine dysregulation. Furthermore, dysregulation of serum adipokines is a prevalent consequence of maternal diabetes and could guide further investigations to predict the development of metabolic disturbances. There is a compelling clinical and epidemiological evidence suggests that prenatal environment plays a pivotal role in the origin of fatal diseases such as the metabolic syndrome and its components: hypertension, insulin resistance, and dyslipidemia . The altAdiponectin have insulin-sensitizing, anti-diabetic, antioxidant, anti-inflammatory and anti-atherogenic properties through its action on adiponectin receptors, e.g., AdipoR1 and AdipoR2 . Leptin Low-grade inflammation is a hallmark of type 2 diabetes and metabolic syndrome . Tumor nIntrauterine epigenetic modifications, deterioration of glucose tolerance and oxidative stress could be a possible mechanisms for the transgenerational transmittance of diabetes , 10; howThe study was done in accordance with the ethical guidelines of the Medical Research Institute, Alexandria University, Egypt. Wistar rats were housed 4 per cage at an ambient temperature of 23 ± 1 °C with 12/12 h light/dark cycles and 45 ± 5% humidity.Female rats were allocated randomly in two groups: control and diabetic. Diabetes was instigated via neonatal injection of streptozotocin (100 mg/kg) at the day 5 postnatally. After 12 weeks, chronic hyperglycemia was confirmed; where rats exhibited fasting blood glucose (FBG) level >200 mg/dl were considered diabetic. 78 hyperglycemic female rats were obtained and maintained under normal control diet (CD) throughout the experiment.Pregnancy was established by overnight mating the females (control and diabetic) with normal healthy males. Pregnancy was confirmed next morning by the presence of vaginal mucus plug. Pregnancies were completed to term. After delivery, the offspring were weaned to control diet (CD) or high-caloric diet (HCD) and followed up for 30 weeks. Therefore, the offspring groups were as follows: F1 offspring of control mothers under CD (CF1-CD), F1 offspring of control mothers under HCD (CF1-HCD), F1 offspring of diabetic mothers under CD (DF1-CD), and F1 offspring of diabetic mothers under HCD (DF1-HCD). Pregnancy outcome and composition of diets used were previously published . Every 5FBG level was measured using Glucometer . Lipid profile was assessed by using a commercial diagnostic kit (Randox (UK)) according to the manufacturer instructions.Plasma insulin was assayed using ELISA kit (Mercodia). Serum levels of adiponectin, leptin, NEFA and TNF-α were assessed using ELISA kits  according to the manufacturer instructions.t test was employed to compare the mean values of females and those of males of the same group at the same age. The results are presented as mean ± SEM or mean ± SD and values of p > 0.05 were considered non-significantly different, while those of p < 0.05 were considered significant.All statistical analyses were performed using SPSS statistical software version 18 . The data were analyzed using the one-way analysis of variance (ANOVA) followed by LSD test to compare the mean values from the offspring of diabetic mothers and the offspring of control mothers. th week  using fasting serum levels of insulin (μIU/ml) and glucose levels (mmol/l) indicated that males and females of DF1-HCD group were insulin resistant compared to CF1-CD group  level compared to CF1-CD and CF1-HCD groups throughout the 3 time points, except the females of DF1-CD at the 5eek Fig. .Fig. 2Lith week. Furthermore, there was a significant rise in NEFA level in females of CF1-CD at the 25th and 30th weeks as well as females of DF1-CD at the 5th week, compared to their male counterparts  had a significant elevated muscular TGs content compared to CF1-HCD. Furthermore, females of DF1-CD (except at the 30th week) and DF1-HCD had significantly lower muscular TGs content than their male counterparts  levels, all diabetic groups, except females of DF1-CD at the 5nce Fig. .th and 30th weeks, except females of DF1-CD at the 30th week, showed high levels of low-density lipoprotein-cholesterol levels (LDL-C) compared to CF1-CD group. Moreover, all members of DF1-HCD at the 15th week as well as males of diabetic groups at the 30th week showed a significant elevated LDL-C level than CF1-HCD group. Significant sex difference appeared only in DF1-HCD at the 15th week and in DF1-CD at the 30th week  throughout the study period. Males of CF1-HCD and DF1-CD showed significantly lower serum adiponectin levels than CF1-CD from the 20th week to the 30th week. Males of DF1-HCD (from weeks 10 till 30) and females of the same group (at the 25th and 30th weeks) exhibited significantly lower serum adiponectin levels than CF1-CD. Males of DF1-CD (at weeks 25 and 30), males of DF1-HCD  and females of DF1-HCD (at weeks 25 and 30) had a significantly lower adiponectin levels compared to CF1-HCD , all members of DF1-CD  and all members of DF1-HCD  had a significantly lower leptin levels compared to CF1-CD. Males of DF1-CD (except at weeks 5 and 10) had significantly lower leptin levels than CF1-HCD, while females of the same group at weeks 5 and 10 had significantly higher leptin levels compared to CF1-HCD. Females of DF1-HCD (except at week 5) showed significantly higher leptin levels compared to CF1-HCD  throughout the study period. All members of DF1-HCD and all males of DF1-CD and CF1-HCD had significantly higher serum TNF-α levels compared to CF1-CD. Also, females of CF1-HCD  and females of DF1-CD (at weeks 10 and 15) showed significantly higher serum TNF-α levels compared to CF1-CD. Moreover, all males of DF1-CD at every time point and females of the same group at week 10 exhibited significantly higher serum TNF-α levels compared to CF1-HCD. All males of DF1-HCD at every time point and females of the same group at weeks 10,25 and 30 exhibited significantly higher serum TNF-α levels compared to CF1-HCD (Fig. Females had significant higher serum TNF-α levels than their male counterparts in all study groups (except CF1-HCD and DF1-CD at the 5According to the hypothesis of fetal origin of adult diseases, the intrauterine environment may have a critical influence on long-term health and disease of the offspring. Therefore, intrauterine milieu may alter the metabolic status of the fetus, driving an insulin resistance state and facilitates the development of type 2 diabetes mellitus and metabolic syndrome in the adult life, especially following a postnatal obesogenic environment . In suppDyslipidemia is one of the major risk factors for the development of cardiovascular diseases in the context of diabetes mellitus. The cardinal features of diabetic dyslipidemia are: a high plasma TGs level, low HDL-C level and elevated level of small dense LDL-C particles. These lipid changes are attributed to the increased NEFA flux secondary to adipose tissue insulin resistance . The incHyperinsulinemia has a pivotal role in the development of hepatosteatosis and hepatic insulin resistance . ConsistOur data indicates that in-utero maternal diabetes is sufficient for alteration the adipocytokines production in the offspring during their adult life, and the diabetogenic environment exaggerates this effect. Offspring of diabetic mothers showed low levels of adiponectin, high levels of leptin, and elevated levels of TNF-α. Assessment of the serum adiponectin in diabetic patients is useful for evaluating the metabolic status and for determining the risk of cardiovascular diseases .It has been shown that inflammatory markers are increased in the offspring of type 2 diabetic patients –21. AlsoIn conclusion, dysregulated adipocytokines production could be a possible mechanism for the transgenerational transmittance of diabetes, especially when the offspring encounter diabetogenic environment. Furthermore, serum adipokines level could be a powerful tool to predict the development of metabolic disturbances in offspring.


Antimicrobial resistance may be promoted by divergent routines and lack of conformity in antibiotic treatment, especially regarding the practice of antibiotic prophylaxis. The aim of the present study was to assess differences in gallstone surgery regarding antibiotic use in Sweden.The study was based on data from the Swedish Register for Gallstone Surgery and ERCP  2005–2015. Funnel plots were used to test impact of grouping factors, including, hospital and surgeon and to identify units that deviated from the rest of the population.After adjusting for cofounders including age, gender, ASA classification, indication for surgery, operation time, gallbladder perforation and emergency status, there were 0/21 (0%) at the regional level, 18/76 (24%) at the hospital level and 128/1038 (12%) at the surgeon level outside the 99.9% confidence interval (CI). The estimated median odds ratios were 1.13 (95% CI 1.00–1.31) at the regional level, 1.93 (95% CI 1.70–2.19) at the hospital level and 2.38 (95% CI 2.26–2.50) at the surgeon level.There are significant differences between hospitals and surgeons, but little or no differences between regions. These deviations confirm the lack of standardization in regards to prescription of antibiotic prophylaxis and the need more uniform routines regarding antibiotic usage. Randomized controlled trials and large population-based studies are necessary to assess assessing the effectiveness and safety of antibiotic prophylaxis in gallstone surgery. In the absence of firm evidence regarding the effectiveness of antibiotic prophylaxis in gallstone surgery, there is a risk of non-stringent practice and arbitrary use of antibiotics. This may eventually lead to an increasing antimicrobial resistance. The increasing drug resistance is strongly promoted by divergent routines, especially regarding the attitude towards antibiotic prophylaxis –3.Whereas there are several studies confirming the lack of benefit of antibiotic prophylaxis in routine laparoscopic cholecystectomy , there aThe adherence to evidence-based routines regarding antibiotic prophylaxis in Greece , the NetThe aim of the present study was to explore the use of antibiotic in gallstone surgery in Sweden and to determine, whether any deviation from the average could be explained by lack of conformity at the regional, hospital or surgeon level.This study is based on data from the Swedish Register for Gallstone Surgery and ERCP . GallRiks is a national register covering all open and laparoscopic surgery of the gallbladder performed in Sweden. It is continuously validated and has been shown to have 98% matches between the medical records and the database. Between 2006 and 2008, the register grew to cover more than 90% of Swedish surgical units of by the end of 2008 .This study is based on all cholecystectomies registered in GallRiks 2005–2015. The variables used were gender, age, calendar year, method of approach, ASA classification, indication for surgery, operation time, accidental gallbladder perforation, antibiotic treatment and emergency status. Antibiotics were registered as none, prophylactic or therapeutic in GallRiks. In the present study, antibiotics given as prophylaxis (duration not intended to be longer than 24 h) and antibiotics given as therapy (duration longer than 24 h) were merged to one category since it was impossible to retrospectively determine the purpose of administrating antibiotics in each case. Choice of drug was also not registered. Only units where at least 25 cholecystectomies were performed were included in the analysis.The following patients were excluded:Patients for which data on antibiotic prophylaxis, age, gender, ASA, gallbladder perforation, emergency status or surgeon were missing.Patients undergoing common bile duct exploration (post cholecystectomy status).Patients undergoing cholecystectomy for indications other than gallstone disease.Procedures with duration unknown or recorded as being longer than 24 h (assumed to be erroneous recordings).Patients with indication vital indication was also removed.Hospitals and surgeon with fewer patients than 25 were also excluded.The number of excluded in each category is given in Table The study was approved by Stockholm ethical review board. All participants gave informed consent to inclusion. The study was conducted in accordance with the Helsinki declaration.0 ± 1.96 xp0 is the proportion of patients receiving antibiotics in the whole population and n is the number of patients in the unit.The funnel plots provide a graphical presentation of which units have antibiotic use outside the confidence intervals (the “funnel”), thus representing units deviating more from the general population than what would have been expected if there have been an underlying uniform approach and random variation at each level . Three lWe also constructed multi-level funnel plots, with the indicators derived from a Bayesian multilevel regression model, adjusted for other levels . As an example, imagine a hospital where all surgeons have high antibiotics prescription rates. This will be interpreted by the model as normal surgeons in a high-prescribing hospital. Therefore, the hospital will look extreme (outside the funnel), whereas the surgeons will not, in their respective funnel plots. In the multi-level plots, adjustments were also made for potential confounding factors i. Patients with missing data were removed from the analysis . The underlying statistical method has been described in detail in a previous report .Altogether 113,209 cholecystectomies were performed 2005–2015. From this group, 14,108 were excluded according to the exclusion criteria described above, leaving 99,101 patients in the study group. Baseline data of the study group are given in Table Plain funnel plots for regions, hospitals and surgeons are shown in Figs. In Table 3, estimates and credibility intervals for the random effect median odds ratios  achievedThis study shows a lack of conformity at the surgeon and hospital levels in Sweden, whereas there is no evidence of disparity at the regional level. How this affects the development of antibiotic resistance and the consequences for the individual patient is not fully understood. Nevertheless, good quality in health care is generally considered to include adherence to common routines.Although resistant pathogens are not as widespread in Sweden as in other parts of Europe, standardized guidelines are necessary in order to prevent further spread across borders . The disStandard common routines may be one of the best ways of reducing the risk for surgical site infections , 9. By aA way of improving antibiotic prescribing practice is through antimicrobial stewardship programmes , 19. TheIn a Cochrane review, it was concluded that several interventions aimed at reducing inappropriate use of antibiotics in hospitals might be effective in reducing the incidence of antimicrobial-resistant pathogens and Clostridium difficile-associated diarrhea . The natIn 1994, the Swedish Strategic Programme for the Rational Use of Antimicrobial Agents and Surveillance of Resistance (STRAMA) was started , 23. TheIn a previous study, the incidence of infectious complications within thirty days after planned surgery for gallstones was found to be 4.3% . Some ofThe present study is limited by the difficulties in defining objective criteria for antibiotic prophylaxis and antibiotic use. Even if indication for surgery was included as covariate, there may have been circumstances related to the procedure that may have had an impact on the decision to give antibiotics, including previous or ongoing jaundice, concomitant ERC, degree of cholecystitis and co-morbidity. As many of these factors may have been unevenly distributed between the units, there may have been variations that could be explained by varying indications and not lack of conformity. Furthermore, no studies have been able to provide definite evidence for an association between local variations in antibiotic use and the ecology of microbial drug resistance, even if there are many reasons to assume such a relationship.The data are assembled from GallRiks 2005–2015. Although there has been an ongoing debate regarding the use of antibiotics for prophylaxis in surgery since 2010, the routines have not changed much since then .In conclusion, the present study shows that the antibiotic usage routines are relatively uniform at the regional level, but vary at the hospital and surgeon levels in Sweden. This calls for standard antibiotic regimens and the development of national guidelines for use of antibiotic prophylaxis in gallstone surgery. The present study shows that uniform routines are necessary, although the level of evidence regarding antibiotic prophylaxis is insufficient. At present, there are ongoing randomized controlled trials and register-based studies with the aim of assessing the effectiveness and safety of AP in surgery for acute cholecystitis.


According to scientific literature, antibacterials are prescribed for common pediatric conditions that do not benefit from antibiotic therapy. The link between antibiotic use and bacterial resistance is well known. Antibiotic overprescribing generates high social costs and severe consequences for children. Our objectives were to analyze antibiotic prescription patterns in pediatric outpatients residing in the Lazio region , to identify physicians’ characteristics associated with increased propensity for prescription, to identify the priority axes for action to improve the rational use of antibiotics.We enrolled all children aged 13 years or less in 2014. Antibiotic prescription patterns were analyzed during a one-year follow-up period. The main outcome measures were the antibiotic prescription prevalence, and the geographic variation in antibiotic prescribing. Multilevel models were performed to analyze variation. Variation was expressed as Median Odds Ratios (MORs). If the MOR is 1.00, there is no variation between clusters. If there is considerable between-cluster variation, the MOR will be large.p < 0.001) variation was observed between physicians working in the same LHD: MORs were equal to 1.52 (1.48–1.56) and 1.46 (1.44–1.48) in the 0–5 and 6–13 age groups, respectively. The probability of prescribing antibiotics was significantly (p < 0.001) lower for more-experienced physicians.We enrolled 636,911 children. Most of them were aged 6–13 years (57.3%). In 2015, the antibiotic prescription prevalence was 46% in the 0–13, 58% in the 0–5, and 37% in the 6–13 age group. Overall, penicillins were the most prescribed antibiotics, their consumption increased from 43% to 52% during the 2007–2015 period. In 2015, the antibiotic prescription prevalence ranged from 30% to 62% across local health districts (LHDs) of the region. Moreover, a significant (Pediatric antibiotic use in the Lazio region is much higher than in other European countries. The intra-regional drug prescribing variability underlines the lack of therapeutic protocols shared at regional level and raises equity issues in access to optimal care. Both LHD managers and individual physicians should be involved in training interventions to improve the targeted use of antibiotics and mitigate the effect of contextual variables, such as the spatial-related socioeconomic status of the patient/parent binomial. Antibiotics are the most widely prescribed therapeutic agents in children. The prevalence of antibiotic prescriptions differs across age, with preschool children being more likely to be prescribed antibiotics . It is wThe objectives of this study are as follows: 1) to measure the antibiotic prescription prevalence in pediatric outpatients of the Lazio region, Italy; 2) to describe the distribution of prescriptions by antibiotic class; 3) to identify physicians’ characteristics associated with increased propensity for antibiotic drug prescriptions; 4) to identify the priority axes for action aimed at improving the rational use of antibiotic drugs.This is a retrospective, observational, population-based study, conducted on the population living in the Lazio region of Italy, which comprises approximately 5 million persons. Patients were recruited from individuals registered with the LHDs of the region, who were eligible over the study period. The information on patients and their assigned physicians was retrieved from the regional Health Assistance Register. Drug information was collected from the regional Drug Claims Register, which was described in detail elsewhere . Data avindex date). Antibiotic (ATC code J01) prescription patterns were analyzed during a one-year follow-up period, starting from the index date. Therefore, the main analysis was performed using data from 2015, however, the same enrollment criteria were applied to the period 2007–2014, to describe and compare the “cross-sectional” antibiotic prescription patterns over time. Drug exposure was measured by the prescription prevalence, expressed as the number of people who received at least one prescription during follow-up, per 100 individuals in the population [The study included all children aged 13 years or less on December 31, 2014  was used to determine the model that provided the best account of the data. In fact, AIC deals with the trade-off between the goodness-of-fit and the complexity of the model. The “best” model is the one with minimum AIC value [Continuous variables were presented as mean value ± standard deviation A global chi-square test was performed in order to check for differences in antibiotic prescription prevalence during the 2007–2015 period. A map of the Lazio region was produced to show and compare the antibiotic prescription prevalence by LHD in 2015. The classes used in the map were calculated applying the Jenks natural breaks optimization algorithm , which rIC value . All anaWe analyzed 636,911 children, 51.4% of them were male. A total of 271,780 children were aged 0–5 years (42.7%), whereas 365,131 were aged 6–13 years (57.3%).p-value <0.001) and 0–5  age groups. The highest values were observed in 2009: almost 56% in the 0–13 age group, more than 66% in preschool children, and 47% in the 6–13 age group. Figure p-value <0.001). In the 0–5 age group, for each 10-percentage-point increment in antibiotic prescription prevalence, the propensity of prescribing cephalosporins increased by 2 percentage points. In the 6–13 age group, for each 10-percentage-point increment in antibiotic prescription prevalence, the propensity of prescribing cephalosporins increased by 2.5 percentage points. The 6–13 age group is particularly interesting. In fact, within this age range, parents can choose to remain with the pediatrician or switch to a general practitioner. This allows comparing differences in antibiotic prescribing habits between pediatricians and general practitioners. In the study period, the antibiotic prescription prevalence in the 6–13 age group was systematically higher for general practitioners (Fig. In 2015, the antibiotic prescription prevalence in the Lazio region was about 46% in the 0–13 age group, 58% in the 0–5, and 37% in the 6–13 age group. Figure In this study of about 637,000 children, we found that, in 2015, the antibiotic prescription prevalence in the Lazio region was about 46% in the 0–13 age group, 58% in preschool children, and 37% in the 6–13 age group. Pediatric antibiotic use in Lazio was in line with other Italian regions, and was much higher than in other European countries , despiteThere are some study limitations to be considered. First, our health information systems do not collect data on indications for which antibacterials are prescribed. Second, the results come from a single region in Italy and may be not generalizable to other geographic areas. However, our findings are in line with results of other studies carried out in different Italian regions , 15, 16.Despite international and national guidelines aimed at reducing antibiotic prescribing in pediatric outpatients, antibiotic use in the Lazio region of Italy is still much higher than in other European countries. Moreover, the use of second-choice antibiotics is common. The intra-regional drug prescribing variability underlines the lack of therapeutic protocols shared at regional level and raises equity issues in access to optimal care. Both LHD managers and individual primary care physicians should be involved in training interventions, aimed to improve the targeted use of antibiotics and mitigate the effect of contextual variables, such as the spatial-related socioeconomic status of the patient/parent binomial.


All pregnant females realize 3 successive pregnancies. Plasma results showed that Met excess increased Hcy, raised CRP in NPMet and decreased it in PMet, enhanced significantly proMMP-2 and proMMP-9 activities in NPMet, and reduced them in PMet. Aorta showed a rise in collagen level, essentially in PMet, a reduction of elastin content in both PMet and NPMet, and a significant decrease in lipid content in PMet, with histological changes that are more pronounced in NPMet than PMet. Met excess enhanced proMMP-9 activities in NPMet while it decreased them in PMet. PMet newborn presented increase in uremia and CRP and significant rise of active MMP-2 and MMP-9 forms. In aorta, media and adventitia thickness increased, total lipids content decreased, proMMP-9 activity decreased, and proMMP-2 activity increased.This study investigated whether a high Met diet influences biochemical parameters, MMPs activities in plasma, and biochemical and histological remodeling in aorta, in both pregnant female rabbits and their offspring. Four female rabbit groups are constituted (each  Met enriched diet causes hyperhomocysteinemia (Hhcy), which is associated with the metabolic syndrome, oxidative and nitrosative endoplasmic reticulum stress , inflamm2O2 [α, IL-6, and IL-1β potentially stimulate the expression of MMPs in macrophages and vascular SMCs [Methionine, a sulfur amino acid with essential roles in intermediary metabolism, is a universal methyl donor for more than hundred reactions ; this am2O2  with red2O2 . Akasaka2O2  noted th2O2 , 20. Hhc2O2 . Excess 2O2  and rais2O2 . Under e2O2 . Proinfllar SMCs . Met enrlar SMCs . The MCPlar SMCs .α and ER β receptor; estrogen that stimulates the release of NO can modulate endothelial function by receptor activation, gene transcription, and dependent MAPK endothelial NOS (eNOS) activation [Endothelial cells contain the two types of ER tivation . Chikanitivation  showed ttivation  indicatetivation  reportedtivation . A beneftivation . The E2 tivation .Extensive experimental studies indicate that a suboptimal environment during fetal and neonatal development in both humans and animals may program offspring susceptibility to later development of chronic diseases . Some stRabbit is a suitable model to analyse the impact of fetal development in offspring and adult health because of its lipid metabolism which is similar to that of humans and its sensitivity to develop atherosclerosis . The maiIf maternal alteration is responsible for increasing fetal lesion formation and susceptibility to atherosclerosis later in life, then pharmacological or nutritional interventions in mothers during pregnancy that prevent or minimize the onset of these pathogenic events in the fetus would be implemented to improve the health of offspring. (NPC) (n = 8) was fed a normal commercial chow containing alfalfa , oat bran (28%), barley (23%), corn , soja  equivalent to 30,46% protein, 8,26% lipids, 44,05% total carbohydrate, 11% moisture, 5% crude ash, 1% vitamin mixture, and 0,1% methionine (Met) content; (b) the high-methionine (NPMet) group (n = 8) received a normal commercial chow supplemented with 0,35% L-methionine (w/w) for 3 months (500 mg/day); (c) the pregnant control (PC) group received normal commercial chow; and (d) the pregnant group fed with high Met diet (PMet) is submitted to the same conditions as NPMet group. All the females of the groups PC and PMet realize 3 successive pregnancies. D-L-Methionine and other chemicals were obtained from Sigma Chemical Co. . The chows were stored at 4°C. Food intake was controlled periodically.This study involved female domestic white rabbits  obtained from Algiers agricultural cooperative. The animals were allowed free access to diet and water and kept in wire-bottomed stainless steel cages. Our study is made on nonpregnant (control) and on pregnant rabbits that realized 3 successive pregnancies during this experimentation period. They were housed with normal temperature and light cycles. Experimental period extended from February to May (22–28°C). Female rabbits were divided into the four groups: (a) the control groupNewborns obtained from control and experimented females are weighted and their blood and aortic tissue collected for biochemical and histological studies.All experiments were carried out in compliance with the guidelines of the Federation of European Laboratory Animal Science Associations (FELASA) following approval by the local Ethical Committee of the Sciences and Technology, Houari Boumediene University, Algeria.Every week, during a period of 3 months, all the rabbits are weighted and the blood was harvested  after overnight fasting, into dry, citrate, and heparin-containing tubes. Plasmas and serum obtained by centrifugation at 1500 ×g for 10 min were stored at −80°C. After 90 days of diet administration, the rabbits were killed by decapitation. Aorta  was immediately sampled; one fraction is snap-frozen in liquid nitrogen and stored at −80°C for biological evaluation, another was introduced in Folch solution for lipid determination, another was fixed in adequate aqueous solutions and embedded in paraffin for histological analysis, and one fraction was cut into small segments, performed under sterile conditions, and designed for zymographic analysis.Serum Hcy levels were determined by FPIA method in IMX apparatus . Serum gThe C reactive protein (CRP) was determined by turbidimetry (at 552 nm) according to Eda et al.  method. (1) Lipid Content. Lipid content of aorta was estimated by method of Folch et al. [h et al. , after s(2) Activity of Diffusible Gelatinases. A fraction of aorta was taken rapidly after animal dissection, cut in small fragments, and incubated, in sterile conditions, in 96-well microplate (100 μL/hole), with DMEM  during 24 h, at 37°C. The middle containing enzyme was removed and frozen at −80°C for activity estimation and the fragments of aorta are weighted; enzyme activity was expressed by mg protein.(3) Collagens and Elastin Determinations. Frozen aortas are homogenized in ice cold Tris/Triton buffer  and submitted to cold centrifugation (12 000 ×g for 30 min); collagens level was estimated by the reaction of OH Pro with chloramine T [ramine T . Measure(4) Histopathological Study. Aorta from female and newborn rabbit was fixed in Bouin's solution and in 10% buffered formaldehyde, dehydrated in increasing concentrations of ethanol, cleared in toluene, and finally embedded in paraffin wax. Sections 5 μm thick were stained with Masson's trichrome, PAS, Picro-Indigo-Carmine, and Sudan Black B for histological studies. Mean cross-sectional areas of aorta were calculated by measuring at least 50 areas for each aortal sample.Statistical Analysis. All data were expressed as mean ± standard error of the mean (SEM). Statistical analyses were performed using StatView 5.0 Software. Differences between groups were determined by performing one-way analysis of variance (ANOVA). Comparisons were made with Student's t-test. A p value of p < 0.05 was considered statistically significant.p < 0.05). The newborn rabbits submitted to Met have a smaller body weight than controls in the first pregnancy, but those of the second pregnancy have a body weight more important  and a reduction in the length of gestation (28 days instead of 30) according to Dasarathy et al. , who indmportant .p < 0.0001), while in PMet the elevation reaches 13,8%; pregnancy alone, without Met diet reduces Hcy in 30% (p < 0.01) while decreasing this setting in P rabbit .Met diet administered to NP during 12 weeks increases plasma Hcy over 900% the initial value (y in 30% . Plasma y in 30%  and Met β/α which is 2.17 in NPC increases significantly under the effect of Met (p < 0.01)  .p < 0.01)  and in PMet versus PC (p < 0.01)  . Met red < 0.01) .p < 0.01) and reduction of cholesterol (p < 0.05).In thoracic aorta, total fat does not seem to be changed during pregnancy  with siglesterol . Met enrp < 0.01), while it causes a decrease of this proMMP-9 activity in PMet (p < 0.01)  but a nonsignificant decrease in the PMet. It appears that supplemented-Met diet causes a rise in proMMP-9 and active MMP-9 activities of rabbit aorta, but this diet raises the proMMP-2 activity in NP and has no effect on pregnant female aorta; the active MMP-2 form appears only in NPMet aorta  and a significant elevation of adventitia size (p < 0.01) , infiltration of SMCs and blood elements, presence of microthrombi fixed to the intima side, signs of impaired antithrombotic endothelium function Figures , and loc < 0.01) . The obs < 0.01)  and the  < 0.01) .p < 0.0001)  .p < 0.0001) under the Met effect  with important reduction of the inactive forms  (p < 0.01) (p < 0.05)  . It seem < 0.05) .p < 0.0001) after a month of diet, although some authors [The current study demonstrates that excess Met diet affects the metabolism of Hcy; for Refsum and Ueland , it caus authors , 52 did  authors , on rabb authors , on sandp < 0.0001); this increase is much greater than that obtained by Rolland et al. [p < 0.05). The administration of Met causes a significant increase in CRP levels among NP (p < 0.01); this protein produced by the liver, adipose tissue, and monocytes/macrophages in injured areas would be responsible for oxidized LDL opsonization [Administration of this diet, during 3 months, allows the development of important Hhcy in NPMet group   ; this isExcess Met in the NPMet causes variations in cholesterol which slightly exceeds that observed in NP control. For Sugiyama and Muramatsu , this efAccording to Voutilainen et al. , plasma Similarly, we notice, in NPMet, a lipoprotein (VLDL + LDL/HDL) ratio of 3.07 (limit 3) indicating that the NPMet rabbits are in atherosclerotic state; for PMet rabbits the ratio is 2.44 which underlines the protective effect of sex steroid hormones, mainly estradiol, on the vascular wall. This hormone increases in fact the plasma membrane fluidity, promoting exchanges between endothelial cells and lipoproteins. The HDL has antiatherogenic effects (cholesterol reverse transport), protects against the LDL oxidation (HDL 3), and presents anti-inflammatory, antiapoptotic, and antithrombotic actions .The Met enriched diet administered to rabbits leads to a significant elevation of proteinemia in the NPMet and a moderate rise in PMet, suggesting that the Met excess is mainly drawn to the embryo-fetal development. The measured uremia shows an inverse evolution to that of total protein with significance for the NPMet.Met administered in the diet appears to stimulate protein synthesis demonstrating the leading role played by the Met, donor of methyl groups in various synthetic ways , especiaExcess Met ingested is not metabolized in urea ; in humaIn plasma, the proMMP-2 and proMMP-9 are highly expressed in NPMet compared to PMet, and the active form MMP-2 is the predominant circulating form in PMet. This result suggests that excess of Met, and consequently the Hhcy, stimulates the expression of diffusible metalloproteinases produced by the cells blood and by the fibroblasts of different tissues including the aorta, in accordance with the rearrangements that we have observed.It appears clearly that the diet supplemented with Met (500 mg/kg/d) causes disruption of the plasma biochemical parameters, contrary to the results obtained by Rolland et al.  and AugiFor Yang et al. , Hcy migMet excess reduces aorta total lipids especially in pregnant cases. While a significant reduction in TG and cholesterol in the aorta of PMet occurs, we note TG accumulation in the NPMet aorta, according to histochemical results (Sudan Blacks staining), which shows high chromophilic tissues . Thus, excess Hcy inhibits methylation of lipids and proteins of LDL, increasing their endocytosis ; secondlHhcy causes methylation of L Arg to dimethyl Arg, an inhibitor of NO synthase (NOS). According to Cayatte et al. , lipid aThe intimal thickening, characterized by hypertrophy of the endothelial cells (lunar shaped cores) and an accumulation of subendothelial material, is also observed by Ichikawa et al.  in rabbiThe increase of vascular adhesion, observed by us in rabbits fed with Met excess, is consequent to the expression and production of adhesion molecules by endothelial cells, such as VCAM-1 , E-selec via the p30 protein; their migration contributes to enlarging the intima. The SMCs change from quiescent contractile state to synthetic proliferative state, producing the MMPs and accelerating matrix remodeling.We also noted hypertrophy of SMCs, their subendothelial infiltration, and their reorientation in the media; this hypertrophy was already noticed by Fanapour et al.  in vitroOur results showed an increase in collagen and a diminution in elastin as demonstrated by Steed et al.  indicatiReduction of elastin content measured at NPMet aorta reflects the fenestration we observed by histological analysis in aorta elastic strips of this rabbit group. The reduction of elastin level was also observed in chick aorta subjected to diet enriched with 2% of Met . It seemThe analysis of aortic MMP-2 and MMP-9 activities consolidates the biochemical and histological results. Thus, the proMMP-2 and MMP-9 that are absent in the two groups of control appear as a consequence of Met, with accentuation to NP indicating atherosclerosis status, their expression being stimulated in macrophages and vascular SMCs. The presence of aortic active MMP-2 form, which is observed only in the NPMet, indicates that elastin itself, by structural change, activated proMMP-2 as MMP-2 (contact activation) leading to its own autolysis with significant reduction in its content as a consequence.For Narayanan et al. , Hhcy inIt is demonstrated that MMP-2 and MMP-9 facilitate cell movement by breaking down the basement membrane and degrading type IV collagen; many MMPs seem to control, with precision, processes of immunity, cell migration, and angiogenesis via a variety of extracellular effector proteins, including cytokines, chemokines, and some ECM components .γ) by T and NK cells [For Chaussalet et al. , the endNK cells . BischofNK cells  indicate2). Pregnant rabbits subjected to the Met exhibit low Hcy and so stimulation of these MMPs is reduced, and more these enzymes exhibit a significant reduction in their activity.The proMMP-9-NGAL complex we get on NPMet aorta reflects the significant vascular remodeling by these metalloproteinases modulated by neutrophil gelatinase; similar cases described by Hemdahl et al.  occur du via inhibition of NF-КB and inhibiting the formation of neointima [In pregnant rabbits, the Met excess has little effect on the plasma biochemistry and on the components of the target tissues; this is the consequence of high circulating estradiol levels that reduce hyperhomocysteinemia as demonstrated by Lacut et al. , reducineointima .In addition, these hormones exert their neutralizing function on reactive oxygen species through the phenol cycle, as scavengers of free radicals. These hormones exert also their effect by stimulating the synthesis of glutathione and antioxidant systems, SOD, catalase, thioredoxin reductase, GPX, and GST, the latter being in the respective control of estradiol and progesterone .Met overload has also an impact on the rabbit offspring. Maternal nutritional status may result in alteration of the epigenetic state of fetal genome  leading to lethal changes resulting in litter size reduction as we have observed. We note that some females present a reduction of pregnancy duration, from 30 to 27 days and others present outcomes. This result is in agreement with that found by Eskes  who showThe histomorphological changes observed in aorta of NP rabbit, fed with Met enriched diet, such as intima hyperplasia, media remodeling, collagen accumulation, elastic blades disorganization, and fragmentation, have been mentioned by other authors , 78, butMet appears to influence body weight of newborn rabbits, with a significant reduction of this parameter in the first pregnancy and an increase in the second one. Alteration in the glucose availability could endanger fetal development and could explain the reduction of fetal birth weight . On the p < 0.0001). With glucose and amino acids being preferred metabolites in the fetus, it seems that excess Met is transferred to the fetal compartment. Thereafter, Met or Hcy are converted to Glc via gluconeogenesis with significant production of urea.The plasma parameters are unchanged except uremia which is significantly high  and the reduction of proMMP-9 rate mean that the anabolic process exceeds the catabolic phase.Although lipemia is unchanged, we find a very significant reduction of total fat and cholesterol in aorta; Met seems to prevent absorption and integration of lipid in this tissue.The histomorphological, histochemical, and enzymatic studies of rabbit aorta, submitted to Met confirmed the development of atherosclerosis process in rabbits NPMet. Therefore, in plasma of Met fed model the evolution of proMMP-2 and proMMP-9 is similar, that is, highly expressed in NPMet; they are underexpressed in PMet; this reduction can be explained in part by inhibition of NOS, as noted by some authors . In factThis study shows that Met-supplemented diet causes large plasma variations  and ECM connective tissues remodeling in aorta  of pregnant and nonpregnant rabbits, with a more pronounced effect in nonpregnant cases. This means that the NO bioavailability or content is little or not reduced by pregnancy. During pregnancy the significant rise of estrogen level increases the NO production by different ways, via the eNOS and iNOS of the vascular endothelium luteum  and thosp < 0.05); their offspring are leaner than those of control dam but their agility is not altered, with normal behavior, indicating a normal maturation of their brain, since Baydas et al. [Maternal nutritional status may result in an alteration of the epigenetic state of fetal genome (steady changes in gene expression) induced by alteration of the DNA methylation and histone modification . The rabs et al.  mentione


Knowledge about the elution from antibiotic-loaded cement spacers is an indispensable premise for guarantee of clinical success. A systematic literature search was performed through PubMed. Search terms were “antibiotic elution” and “antibiotic release” in combination with “spacer,” “hip spacer,” and “knee spacer,” respectively. A total of 11 studies could be identified. Seven studies reported on the release of antibiotics after spacer implantation, three studies at spacer removal, and one study on both time points. Seven studies reported on hip spacers, one study on knee spacers, and three studies on both. In eight studies, custom-made spacers have been implanted and in three prefabricated ones. In the majority of the studies, the cement has been loaded with an antibiotic combination, mostly consisting of aminoglycoside (either gentamicin or tobramycin) and vancomycin. Measured concentrations exceeded the minimal inhibitory concentration of the particular pathogen organisms in each case. However, large discrepancies were observed with regard to the height of the antibiotic concentration depending on the antibiotic combination and the antibiotic ratio used. Current literature data indicate a sufficient elution of antibiotics after spacer implantation and at spacer removal, respectively. Future studies are required to optimize the local antibiotic therapy at the site of spacer implantation. Periprosthetic joint infections pose a great challenge for the orthopaedic surgeon. At the site of late infections, the implantation of antibiotic-loaded cement spacers is the treatment of choice in Europe and North America with success rates exceeding 90% , 2.Cement spacers have several advantages such as high local antibiotic therapy, preservation of joint mobility in case of articulating ones, less formation of scar tissue, and ease of prosthesis reimplantation , 2. The The release of antibiotics from cement spacers has been well studied in vitro –5. HowevThe aim of the present work is to summarize the current knowledge about the elution of antibiotics from hip and knee spacers in vivo and distinguish between the release kinetics after spacer implantation and at spacer removal, respectively.A literature search was performed through PubMed until November 2016 . Search A total of 11 studies –16 couldµg/ml  [µg/ml, respectively, when 1 g gentamicin and 4 g vancomycin (both powder) were impregnated into 80 g cement for spacer production. Hsieh et al. reported mean local levels of gentamicin at 58.3 mg/l and of vancomycin at 485.5 mg/l on day 1 when liquid gentamicin was combined with vancomycin powder  [All studies determined the local antibiotic concentrations in the joint fluid collected in the redon drain. The time period of measurement varied between the first 24 postoperative hours and seven postoperative days after spacer implantation. In the studies with a longer measurement period, the antibiotic elution demonstrated a biphasic profile, consisting of initial high concentrations, which then constantly decreased over time. The highest values determined were observed in a study where vancomycin and aztreonam were incorporated into 40 g bone cement with initial values exceeding 1,000 Three studies determined the antibiotic concentrations in the local joint fluid found at spacer removal, whereas one study measured them in the local tissue. The length of spacer implantation varied from six weeks to several months. All measured concentrations were above the minimal inhibitory concentration of the particular causative organism in each study despite an apparent trend toward decreasing levels over time seen in two studies , 14. At Knowledge about the antibiotic elution from cement spacers is an indispensable premise for guarantee of infection eradication in the management of periprosthetic hip and knee infections. The present work tried to systematically review the current literature about this topic and differ between the pharmacokinetic properties after spacer implantation and at spacer removal.The evaluation of the efficacy of spacers with different antibacterial loads based on published reports is difficult . As aforBased on these considerations, it is essential to study and understand the pharmacokinetic properties of cement spacers in vivo. Here, specific factors such as place and length of measurement should be critically evaluated. In the majority of the cases among the identified studies, the elution of hip spacers was studied. After spacer implantation, the determination of antibiotic concentrations occurred in joint fluids collected from the drains during the first postoperative days. However, these concentrations are not fully representative for the whole pharmacokinetic properties from the spacers but mostly for the intra-articular spacer part. Especially at the site of hip spacers, this accounts only for the pharmacokinetic properties of either the spacer head alone or the spacer head in combination with a spacer cup. The antibiotic release from the spacer stem in the femoral cavity cannot be determined in the joint fluid. Therefore, the measured concentrations represent only a part of the true antibiotic elution in vivo, and this has to be born in mind.Moreover, the elution properties might depend on the fluid amount that washes the antibiotics out from the bone cement. It could be possible that the initially high antibiotic elution causes a quick saturation of the surrounding tissue. The diffusion gradient that supports the antibiotic release at the beginning is decreased while the tissue saturation is increasing. This could lead to a severe time-dependent reduction of the antibiotic release from spacers . In contBy a constant decrease of the fluid amount during the early postoperative period, the antibiotic release shows then a normal decrease. If the spacers would be again exposed to “fresh” fluids, the elution of antibiotics could be restarted. This phenomenon could be observed in the studies of Bertazzoni Minelli et al.  and KelmThe type of antibiotics themselves used for cement impregnation plays also an important role with regard to release kinetics. In the present evaluation, all studies except for one solely used antibiotics in powder form. Antibiotics in powder form have a lower impact on the mechanical properties of bone cement at a ratio of up to 10% , whereasThe choices of bone cement and incorporated antibiotics are probably the two most important factors at the site of cement spacers. Palacos® has been regarded to be the cement type with the best pharmacokinetic properties for many years , 32. HowAlthough it is known that commercially available antibiotic-impregnated bone cements have superior elution properties than those with a manual impregnation of an antibiotic due to the more homogenous distribution of the incorporated antibiotics in the cement powder, orthopaedic surgeons frequently need to add other antimicrobial agents to the bone cement of spacers due to the sensitivity profile of the causative pathogen organism. Therefore, knowledge about the synergism between the incorporated antibiotics is important for the clinical performance. Despite the fact that the precise synergism mechanism is not completely understood, it seems that this mechanism can be attributed to the so-called passive opportunism . The secDue to the decrease of the antibiotic elution from spacers over time, some concerns have been expressed in the past years about the possible growth of bacteria on spacers, hence leading to clinical infection persistence. The present literature shows some partly contradictory results about this topic. Some studies determined the bacteria growth on spacers after their explantation by sonication and could show a bacteria growth on spacers to different percentages, respectively –38. In sDespite numerous studies about hip and knee spacers, several topics still remain unclear with regard to the antibiotic impregnation. None of the identified studies investigated whether custom-made or prefabricated spacers release more antibiotic amounts or higher concentrations, over a prolonged period. Moreover, the properties of antibiotic combinations other than the combination of an aminoglycoside and a glycopeptide are not known. At present, some antibiotic combinations are potential because the choice is limited by safety issue , stability, and compatibility with cement. Last but not least, the ideal ratio for impregnation of bone cement in vivo is also unclear.The management of late periprosthetic infections by means of a spacer implantation is an established method. Current literature data indicate a sufficient elution of antibiotics after spacer implantation and at spacer removal, respectively. However, some large discrepancies with regard to height and length of the sufficient antibiotic release are evident among the identified studies. Future studies are required to optimize the local antibiotic therapy at the site of spacer implantation.


Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, and Fusobacterium nucleatum was developed. The biofilms were exposed to AMX + MET and PV + MET in two different concentrations. Bacterial viability in biofilms following antibiotic exposure was assessed by viable counts and by confocal microscopy. No live colonies of P. gingivalis nor F. nucleatum were retrieved from biofilms exposed to AMX + MET or PV + MET. The amount of A. actinomycetemcomitans was 4–5 logs reduced following antibiotic treatment; no statistical significance was achieved between AMX + MET or PV + MET treated biofilms. Replacement of AMX with PV at the same concentration, in combination with MET, resulted in similar effect on bacterial viability in this in vitro model. The option of using PV + MET instead of AMX + MET deserves further investigation, as this may contribute to reduce the risk of antibiotic resistance development.A combination of metronidazole (MET) and amoxicillin (AMX) is commonly used as adjunct to mechanical therapy of periodontal disease. The use of broad spectrum antibiotics such as AMX may contribute to development of antibiotic resistance. The aim was to evaluate the in vitro effect of replacing AMX with penicillin V (PV) in combination with MET on a biofilm model. A biofilm model consisting of  Bacteria from frozen stock cultures were first grown on fastidious agar medium  for 48–72 hr, then transferred in liquid growth medium. Brain‐heart infusion  supplemented with hemin  and menadione  was the liquid growth medium used in all experiments, referred to as BHI further in the text. Bacteria were precultured for 24 hr in liquid growth medium. Then an aliquot of the bacterial suspension was inoculated in fresh BHI and further incubated overnight and allowed to reach late log‐phase. All incubations were done in anaerobic conditions  at 37°C .2.2P. gingivalis, A. actinomycetemcomitans, and F. nucleatum in volumes as 1:2:1. Each well was inoculated with 300 μl bacterial mix and 1,200 μl fresh BHI. The amount of bacteria in the inoculum were calculated as: 1.83 × 107 ± 5.79 × 106 CFU/ml F. nucleatum, 2.82 × 107 ± 7.2 × 106 CFU/ml P. gingivalis, and 4.43 × 107 ± 1.44 × 107 CFU/ml A. actinomycetemcomitans (mean and standard deviation).Biofilms were prepared in 24‐well plates  by adapting a previously described protocol . The biofilm biomass absorbance was measured at 600 nm immediately after complete growth medium removal.2.3The viability of bacteria in biofilm was assessed by culture plate counting of CFUs. First, the growth medium and planktonic cells were removed from the 3‐day old biofilms, each biofilm was scrapped off the well by use of a sterile cell scraper , suspended in 1 ml fresh BHI, and disrupted by vigorous pipetting. The biofilm suspensions were 10‐fold diluted on FAA plates in order to obtain plates containing CFUs in range of 1–500 colonies. After 5–7 days of incubation in anaerobic conditions, CFUs were enumerated on FAA plates by help of a stereomicroscope, distinguished by the colony morphology. All experiments were run in triplicate.™ LIVE⁄DEAD Biofilm Viability kit . Fully hydrated biofilms were stained for 20–30 min in the dark, in anaerobic conditions, thereafter immediately examined by CLSM  equipped with an oil‐immersion of 63× or 100× objective  with beam path settings at 488 nm and 543 nm, respectively. Fluorescence intensity thresholds were manually set for each fluorescent color when examining a negative control biofilm and not further modified. At least three different areas on each biofilm were investigated. Image stacks were analyzed with the Leica Confocal Lite® software (Leica Microsystems).The biofilm development and structure were assessed by confocal laser scanning microscopy (CLSM). Prior to examining under CLSM, the biofilms grown for 3 days on 4‐wells chamber slides were stained with FilmTracer2.4Antibiotic cocktails to be used in assays were freshly prepared in BHI prior to each experiment. The antibiotic concentrations tested in this study were defined as “high concentration” when combinations used were AMX + MET  and PV + MET ; and “low concentration” when AMX + MET (360 μg/ml + 720 μg/ml), respectively, PV + MET (360 μg/ml + 720 μg/ml) were used.Growth medium together with planktonic bacteria were carefully removed and antibiotic solutions in BHI at given concentrations were gently placed in each well, in order not to induce biofilm disruption. Biofilms exposed to chlorhexidine (CHX) 0.2%  were used as positive control. Fresh BHI alone was used for the negative controls. The challenged biofilms in 24‐well plates were incubated anaerobically for 2 hr. Then, the growth medium containing antibiotics or CHX were removed and each biofilm was harvested in 1 ml fresh BHI and disrupted as described above. Viable counts were recorded on FAA plates incubated for 5 days.2.5t test was used to investigate significant differences between the two different combinations of antibiotics. A p value less than .05 was considered statistically significant.Paired 33.1F. nucleatum and P. gingivalis, which showed a nearly two‐log increase in numbers during the 3 days incubation. Viable counts of A. actinomycetemcomitans declined in the biofilm compared to the initial inoculum per well, as depicted in Figure n = 7) was 0.568 ± 0.075 AU.All three types of bacterial strains were harvested from 3‐day old biofilms. The viable bacteria in biofilms were mainly represented by A mature biofilm with three‐dimensional structure with mushroom‐like shapes and nutrient channels was formed after 3 days incubation in anaerobic conditions. Both live and dead bacterial‐like structures could be observed by CLSM as shown in Figures 3.2Exposure of biofilms to CHX for 2 hr resulted in eradication of all three bacterial strains, as no CFU could be detected at plate counting. A shorter exposure time than 2 hr to CHX 0.2% resulted in incomplete elimination of live bacteria from the biofilms; while exposure for 2 hr to CHX 0.2% diluted in 1:2 or 1:4 volumes resulted also in detection of CFU after harvesting of positive‐control biofilms (results not shown).F. nucleatum and P. gingivalis were completely eliminated from the biofilms by antibiotic treatment, both AMX + MET and PV + MET in high and low concentrations, as presented in Figure A. actinomycetemcomitans was five logs reduced following exposure of biofilms to high concentration of antibiotics and four logs following low concentration. There was no statistical difference (paired t test) between the AMX + MET and PV + MET combinations with respect to growth inhibition of A. actinomycetemcomitans neither in the high concentration (p = .95) nor in the low concentration (p = .17). The combination PV + MET in high concentration had significantly stronger effect in eliminating A. actinomycetemcomitans than the low concentration (p = .041). No significance was achieved when comparing between the effect of low and high concentrations of AMX + MET (p = .076).Both The antibacterial effect of AMX + MET and PV + MET combinations on biofilms was obvious by CLSM imaging, as an increased proportion of red bacterial‐like structures were observed throughout the biofilms' three‐dimensional structure, in contrast to the few remaining green structures, located mainly at the bottom of the biofilms Figure c,d.4F. nucleatum and P. gingivalis, as previously reported in other in vivo and in vitro studies .A biofilm consisting of three bacterial strains, one known as intermediate colonizer and two as late colonizers in subgingival plaque, was developed in this study. The two late colonizers are periodontal pathogens commonly targeted by systemic antibiotic treatment adjunct to mechanical therapy in periodontal disease (Slots, Research, & Therapy, A. actinomycetemcomitans is present. The antibiotic combination eliminates or markedly suppresses the periodontal pathogens that remain after subgingival periodontal instrumentation to a level manageable by the host organism, supporting the host defense in overcoming periodontal infections and reducing the risk for recurrent disease progression (Slots et al., AMX + MET treatment administrated systemically, in individuals who continue to experience loss of periodontal attachment following mechanical debridement, is effective particularly in cases where Infections caused by biofilms are difficult to treat as bacteria in biofilms are 100 to 1,000 times more resistant to antimicrobials compared to the same type of bacteria in a planktonic state (Ceri et al., A. actinomycetemcomitans was drastically reduced following AMX + MET treatment, both in high and low antibiotic concentrations, but not completely eliminated as in case of F. nucleatum and P. gingivalis, despite the high dose antibiotics used. This might be explained by the short exposure time to antibiotics that did not fully affect A. actinomycetemcomitans, which is more slowly growing. It is also possible that in our model, the nutrients are quickly consumed by the two other species, more rapidly growing, resulting in stationary phase‐like dormant A. actinomycetemcomitans cells. It is known that antibiotics affect growing cells, thus they are less efficient on bacteria found in a low metabolic activity located in nutrient‐deficient areas in biofilm (Olsen, A. actinomycetemcomitans in biofilm to six different antibiotics decreases as the biofilm matures (Takahashi, Ishihara, Kato, & Okuda, In our study, m Olsen, . AnotherA. actinomycetemcomitans resistant to AMX and MET collected from periodontal patients have previously been reported (Eick, Pfister, & Straube, Subgingival strains of A. actinomycetemcomitans in vitro using an agar dilution method and checkerboard titrations. Others found synergy between AMX and MET only on a few of the tested A. actinomycetemcomitans and F. nucleatum strains (Kulik Kunz, Lenkeit, Waltimo, Weiger, & Walter, A. actinomycetemcomitans. The combination PV + MET seemed to be dose‐dependent, as exposure of the biofilms to a three times lower antibiotic concentration resulted in one log higher amount of live CFU retrieved. However, these results should be interpreted with caution, because in our study, the antibiotic concentrations were high, combined with a short exposure time.A multitude of studies have demonstrated clinical effect of administration of the AMX + MET in combination (Sgolastra, Gatto, et al., It is timely to recall that although even the commensal bacteria in dental biofilm may cause oral pathology, the aim for periodontal treatment is not a complete elimination of resident periodontal species, but to obtain a microbial profile compatible with health by reducing the proportion of pathogenic species (Marsh, Head, & Devine, In this study, CHX 0.2% was used as a positive control. The plaque inhibiting effect and antibacterial properties of chlorhexidine are well documented in vivo and in vitro Addy, . CHX hasF. nucleatum and P. gingivalis and strongly reducing A. actinomycetemcomitans. The option of using PV + MET instead of AMX + MET deserves further investigation, as this may contribute to reduce the risk of antibiotic resistance development.Concluding, in this in vitro biofilm model, the replacement of AMX with PV at the same concentration, in combination with MET, resulted in similar effect on bacterial viability, by eliminating The authors have no conflict of interest.


Gammarus fossarum over 24 days. Exposure scenarios involved an antibiotic mixture  at 0, 2 and 200 µg/L to assess impacts resulting from exposure to both water and food. The antibiotics had no effect on either leaf-associated fungal biomass or bacterial abundance. However, modification of leaf quality  may have triggered faster growth of gammarids (assessed in terms of body mass gain) at the low antibiotic concentration relative to the control. At 200 µg/L, however, gammarid growth was not stimulated. This outcome might be due to a modified ability of the gut microflora to assimilate nutrients and carbon. Furthermore, the observed lack of increases in the diameter of the gammarids’ peduncles, despite an increase in gammarid mass, suggests antibiotic-induced effects in the moulting cycle. Although the processes responsible for the observed effects have not yet been identified, these results suggest a potential role of food-quality, gammarid gut microflora and alteration in the moulting cycle in mediating impacts of antibiotics on these detritivores and the leaf decomposition process in streams.Pharmaceuticals contribute greatly to human and animal health. Given their specific biological targets, pharmaceuticals pose a significant environmental risk by affecting organisms and ecosystem processes, including leaf-litter decomposition. Although litter decomposition is a central process in forest streams, the consequences of exposure to pharmaceuticals remain poorly known. The present study assessed the impact of antibiotics as an important class of pharmaceuticals on the growth of the leaf-shredding amphipod  Pharmaceuticals contribute greatly to human and animal health and welfare. As a result, the global production of pharmaceuticals has rapidly grown over the last four decades , prefers leaf material conditioned during exposure to an antibiotic mixture over unexposed control leaves  and indirect  exposure pathways on the growth of the shredder G. fossarum in a 24-d bioassay. The selection of this antibiotic mixture at the two concentration levels was motivated by an earlier study  Gaertn.) were collected shortly before leaf fall in October 2005 to serve as food for the test species. Leaves were picked from a group of trees near Mannheim, Germany , and stored frozen at −20 °C until used . Placing the unconditioned leaves in mesh bags (1.0 mm mesh size) allowed us to identify the individual conditioning runs and separate the newly conditioned leaves from those serving as inoculum . Changes in the dry and fresh mass of gammarids were determined according to Pöckl . Each spThree leaf discs per replicate aquarium were taken after every second conditioning run to quantify bacterial abundance and fungal biomass. This resulted in seven independent replicates per sampling date and antibiotic concentration. Bacterial abundance was determined according to Buesing : FollowiFungal biomass was estimated as ergosterol following the procedure described in Gessner . Briefly2O as described in McArdell et al.  was purchased from Molekula . Roxithromycin , sulfamethoxazole , trimethoprim , and erythromycin  were obtained from Sigma-Aldrich . Erythromycin was transformed to erythromycin-Hl et al. . All oth2O:acetonitrile 90:10 (v/v) and solvent B was solvent A:acetonitrile 20:80 (v/v). The injection volume was 100 µl and the column temperature was 20 °C. Electrospray ionization with positive ionization was used for the mass-spectrometric detection of all antibiotics  to assess whether the nominal concentrations were achieved. Briefly, 50-mL subsamples were taken from each experimental unit both initially and immediately before water was exchanged . The water samples were stored at −20 °C until they were analysed without further sample concentration. The LC system used for the analyses was an HP 1100  equipped with a degasser (G1322A), pump (Agilent 1100 G1311A), autoinjector (Agilent 1100 G1329A), and column oven (Agilent 1100 G1316A). This system was connected to a liquid chromatography tandem mass spectrometry 4000 QTrap  detector. The HPLC column was a Chromolith performance RP-18e . Antibiotics were separated along a nonlinear gradient starting with 100% solvent A and 0% solvent B and ending after 40 min with 0% A and 100% B. The flow rate was 400 µl/min. Solvent A was HG. fossarum (C) was individually calculated for each replicate and week  but not the high concentration of the antibiotic mixture  and at a similar concentration (250 µg/L). Such impacts on the gut microflora could explain the lack of growth observed for G. fossarum exposed to 200 µg/L of the antibiotic mixture, if the ability of G. fossarum to profit from a potentially higher food quality was lost at this high antibiotic concentration. In livestock production, antibiotics are often used as promoters that increase the rate and efficiency of growth  pathway, the gammarids were directly exposed to the antibiotic mixture dissolved in water during the entire 24-day test period. Antibiotics can alter the microflora in the gut of invertebrates and consequently their efficiency to assimilate nutrients and energy from the ingested food. For instance, Gorokhova et al.  observedCromwell  at ratheCromwell .Despite increased growth at the low antibiotic concentration, the peduncle diameter of gammarids did not change at either concentration, whereas it increased in the control organisms Tables , 4. ThisG. fossarum. Nonetheless, our finding that G. fossarum gained mass  and mechanisms responsible for the impacts observed at the physiological level  of the leaf-shredding model species ass Fig.  at a totass Fig. , togetheass Fig. , deserveass Fig.  or antidass Fig. . Besidesass Fig. , impactsSupplementary Information