| Objective:Escherichia coli is the most predominant causative agent of urinary tract infection,bacteraemia,pneumonia and neonatal cephalomeningitis.The aim of our study was to explore the influence of biofilm formation of E.coli on antibiotic sensitivity,and to analysis the correlation between the area and thickness of biofilm of E.coli and single drug resistance and multi-drug resistance.Methods:We collected 110 E.coli clinical isolates.The 110 clinical isolates were divided into three groups according to the drug sensitivity test:the drug sensitive group which was sensitive to all of the antibiotics?n=29?;the single drug resistant group which was resistant to one or a class of antibiotics?n=21?;the multi-drug resistant group which was resistant to three or more than three classes of antibiotics?n=60?.The 110 E.coli clinical isolates were cultured with three copies of medical biological membrane?1.5mm?under the same condition.The three copies of membrane were processed and analyzed with Scanning electron microscope?SEM?,Silver-staining and Confocal laser scanning microscopy?CLSM?respectively.?1?SEM:We sprayed the surface of bacteria with gold and observed surface structure of the biofilm using SEM,and images were captured at 1000-fold magnification.We used the Image-Pro Plus 6.0 to analyze the area of BBF.We calculated the25thh percentile(P25),the 50thh percentile(P50)and the 75thh percentile(P75)of the area of BBF by SPSS version 17.0 statistical software package,and P25,P50and P755 equaled 480?m2,1290?m2,and 1700?m2 respectively.H-scores were evaluated as followed:?1?The score of positive degree?A?:The A score was defined according to the area of the maximum BBF.And the A score of 0,1,2,3,4 meant blank or black spots which were scattered throughout a microscopic field,small block which were smaller than 480?m2,the medium black block which were ranged from 481?m2 to 1290?m2,larger black block which were ranged from 1291?m2 to 1700?m2,big black block which were larger than1701 um2.The positive degree of BBF are expressed as averaged scores of three random microscopic field.?2?The percentage score of BBF was defined as 1?<5%?,2?5%-35%?,3??35%?.The positive degree score?A?was multiplied by the percentage score?B?to obtain a final‘H-score'.H-score 0-3,3-6,6-9,9-12 was defined as"+","2+","3+","4+"respectively.?2?Silver-staining:The BBFs were stained with silver and observed using an optical microscope,and images were captured at 200-fold magnification?20times of the objective,10 times of the eyepiece?.We used the Image-Pro Plus6.0 to analyze the area of BBF.We calculated the 25thh percentile(P25),the 50thh percentile(P50)and the 75thh percentile(P75)of the area of BBF by SPSS version 17.0 statistical software package,and P25,P500 and P755 equaled 525?m2,1320?m2,and 1750?m2 respectively.H-scores were evaluated as followed:?1?The A score was defined according to the area of the maximum BBF.And the A score of 0,1,2,3,4 meant blank or black spots which were scattered throughout a microscopic field,small block which were smaller than 525?m2,the medium black block which were ranged from 526?m2 to 1320?m2,larger black block which were ranged from 1321?m2 to 1750?m2,big black block which were larger than 1751 um2.The positive degree of BBF are expressed as averaged scores of three random microscopic field.?2?The percentage score of BBF was defined as 1?<5%?,2?5%-35%?,3??35%?.The positive degree score?A?was multiplied by the percentage score?B?to obtain a final‘H-score'.H-score 0-3,3-6,6-9,9-12 was defined as"+","2+","3+","4+"respectively.?3?Confocal laser scanning microscopy?CLSM?:We scanned the thickness of BBF using CLSM after stained by SYTO9/PI fluorescence.For each microscopic field we scanned 8?32 layers and the thickness of BBF are expressed as averaged thickness of three random microscopic field??m?.?4?Statistical analyses:The rank sum test was applied to analyze the rank of H-score and the H-score of BBF which detected by SEM and silver-staining.LSD?Fisher'Least Significant Difference?was used to compare the H-score of each group.For the results of CLSM,the thicknesses of BBF are expressed as means±standard deviation?x?s?.The one-way ANOVA was adopted to analyze difference of thickness among groups,and LSD was applied to compare the thickness of each group.The Spearman rank correlation was used to analyze the correlation between drug resistance of E.coli and the positive degrees of BBF which were evaluated by SEM,silver-staining or CLSM.Results:?1?The results of SEM:There were 97 BBF-positive strains among the 110 E.coli strains we tested,and the positive rate was 88.2%.There were 7strains of"-",14 strains of"+",7 strains of"2+",1 strain of"3+",0 strains of"4+"in the drug sensitive group,and the mean H-score was 2.38±2.19.There were 3 strains of"-",6 strains of"+"10 strains of"2+",1 strain of"3+",1strains of"4+"in the single drug resistant group,and the mean H-score was2.86±2.88.There were 1 strains of"-",7 strains of"+",15 strains of"2+",16strains of"3+",21 strains of"4+"in the multi-drug resistant group,the mean H-score was 7.62±3.23.For the rank data,the results of statistic analysis showed that the obtained?2 equaled 45.96?P<0.05?.We used the rank sum test to analyze H-score of BBF and the results showed that the?2 equaled49.82?P<0.05?.Then LSD were applied to compare the H-score of BBF of each group:?There was no significant difference between the drug sensitive group and the single drug resistant group?P>0.05?.?The difference of H-score between the drug sensitive group and the multi-drug resistant group was statistically significant?P<0.05?.?The difference of H-score between the single drug resistant group and the multi-drug resistant group was statistically significant?P<0.05?.?2?The results of silver-staining:There were 101BBF-positive strains among the 110 strains bacteria we tested,and the positive rate was 91.8%.There were 5 strains of"-",15 strains of"+",8 strains of"2+",1 strain of"3+",0 strains of"4+"in the drug sensitive group,and the mean H-score was 2.45±1.88.There were 3 strains of"-",6 strains of"+",10 strains of"2+",1 strain of"3+",1 strains of"4+"in the single drug resistant group,and the mean H-score was 3.14±2.47.There were 1 strains of"-",7 strains of"+",15 strains of"2+",16 strains of"3+",21 strains of"4+"in the multi-drug resistant group,the mean H-score was 7.53±3.38.We used the rank sum test to analyze the rank data of H-score evaluated by silver-staining,and the results showed that the?2 equaled 40.57?P<0.05?.We used the rank sum test to analyze H-score of BBF,and the results showed that the?2 equaled 45.57?P<0.05?.Then LSD were applied to compare the H-score of BBF of each group:?there was no significant difference between the drug sensitive group and the single drug resistant group?P>0.05?.?The difference of H-score between the drug sensitive group and the multi-drug resistant group was statistically significant?P<0.05?.?The difference of H-score between the single drug resistant group and the multi-drug resistant group was statistically significant?P<0.05?.?3?The results of CLSM:The mean BBF thickness of the drug sensitive group,the single drug resistant group and the multi-drug resistant group were 25.91±5.84?m,31.84±4.91?m,47.97±9.53?m respectively.The statistic results of the one-way ANOVA showed that the F value was 48.70?P<0.05?.Then LSD were applied to compare the thickness of BBF of each group:?there was no significant difference between the drug sensitive group and the single drug resistant group?P>0.05?.?The difference of thickness between the drug sensitive group and the multi-drug resistant group was statistically significant?P<0.05?.?The difference of thickness between the single drug resistant group and the multi-drug resistant group was statistically significant?P<0.05?.?4?The results of the multi-drug resistant group:We analyzed the three classes of drug resistant group?7 strains?,the four classes of drug resistant group?26 strains?,the five or more than five classes drug resistant group?27 strains?.SEM:There were 1 strain of"-",3strains of"+",2 strains of"2+",1 strain of"3+",0 strain of"4+"in the three classes of drug resistant group,and the mean H-score of BBF was 3.86±2.67.There were 1 strain of"-",1 strain of"+",7 strains of"2+",7 strain of"3+",10strains of"4+"in the four classes of drug resistant group,and the mean H-score of BBF was 7.85±3.14.There were 0 strain of"-",1 strains of"+",6strains of"2+",8 strain of"3+",12 strains of"4+"in the five or more than five classes of drug resistant group,and the mean H-score of BBF was 8.37±2.72.Silver-staining:There were 1 strain of"-",3 strains of"+",2 strains of"2+",0strain of"3+",1 strains of"4+"in the three classes of drug resistant group,and the mean H-score of BBF was 4.14±3.63.There were 0 strain of"-",2 strains of"+",7 strains of"2+",9 strain of"3+",8 strains of"4+"in the four types of drug resistant group,and the mean H-score of BBF was 7.61±2.94.There were0 strain of"-",2 strains of"+",6 strains of"2+",7 strain of"3+",12 strains of"4+"in the five or more than five classes of drug resistant group,and the mean H-score of BBF was 8.13±3.07.The mean BBF thickness of the three classes of drug resistant group,the four classes of drug resistant group and the five or more than five classes of drug resistant group were 35.69±6.03?m,46.73±9.55?m,52.38±13.81?m.We used the rank sum test to analyze the data of the multi-drug resistant strains group by SEM,silver-staining,and the results showed that the obtained?2 equaled 11.22?P<0.05?and 8.20?P<0.05?respectively.Then we used the rank sum test to analyze the H-score of BBF,which were evaluated by SEM and silver-staining,in the multi-drug resistant group,and the results showed that the obtained?2 equaled 9.57?P<0.05?and6.70?P<0.05?respectively.The one-way ANOVA was applied to analyze BBF thickness and the results showed that the F value equaled 6.19?P<0.05?.Then LSD are applied to compare the thickness of BBF of each group:?The difference of thickness between the three classes of drug resistant group and the four classes of drug resistant group was statistically significant?P<0.05?.?The difference of thickness between the three classes of drug resistant group and the five classes of drug resistant group was statistically significant?P<0.05?.?there was no significant difference between the four classes of drug resistant group and the five or more than five classes of drug resistant group?P>0.05?.?5?The correlation of the drug resistance and the positive degree of BBF?area and thickness?:There was a significant positive correlation between the drug resistance and the positive degree of BBF which was evaluated by SEM and silver-staining,and the correlation coefficient were0.64?P<0.05?and 0.63?P<0.05?respectively.Moreover,there was a significant positive correlation between the drug resistance and the H-score of BBF which was evaluated by SEM and silver-staining,and the correlation coefficient were 0.65?P<0.05?and 0.64?P<0.05?respectively.The same as the results of SEM and seliver-staining,the thickness of BBF was significantly and positively correlated with the drug resistance,and the correlation coefficient was 0.86?P<0.05?.Conclusion:?1?The phenomenon of BBF formation is universal in bacteria:The positive rate of BBF formation was 88.2%when detected by SEM;The positive rate of BBF formation was 91.8%when analyzed by silver-staining.?2?There is no significant correlation between BBF formation and single drug resistance in the 110 E.coli isolates.So we hypothesize that BBF production is not the main cause which leads to the single drug resistance of E.coli.?3?BBF formation is positively correlated with the drug resistance of E.coli.Although the mechanism of antibiotic resistance is complicated,our results showed that the capacities of BBF formation of E.coli were significant difference among the drug sensitive group,the single drug resistant group and the multi-drug resistant group.?4?the drug resistance of E.coli is positively correlated with the BBF area and thickness.The correlation coefficient of the areas which detected by SEM and silver-staining were 0.64?P<0.05?and 0.63?P<0.05?respectively.The correlation coefficient of the H-score which analyzed by SEM and silver-staining were0.65?P<0.05?and 0.64?P<0.05?respectively.The correlation coefficient of thickness which assayed by CLSM was 0.86?P<0.05?.?5?The results which were analyzed with three different methods were consistent.SEM,which can observe the surface of BBF,is more intuitive and more accurate.The reagents of silver-staining are cheap and optical microscope is accessible to most researchers,so silver-staining is suitable for most laboratory.CLSM,the results of which are quantitative,is more suitable for comparing the difference between groups. |
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