The Antibiotic Effect Of Amoxicillin And Erythromycin Against Streptococcus Pneumoniae In Vitro

ProfaceOne of the experiential therapy of community acquired pneumonia (CAP) is to combine the β - lactam antibiotics with macrolides. Because they can extremely coveraged the main pathogenic bacteria including Streptococcus pneu-moniae, Hemophilus influenza, Moraxelle catarrhalis and some atypical pathogenic bacteria such as Legionella, Mycoplasma and Chlamydozoan. Though this treatment perhaps is not judicious in traditional abstracto. The Macrolides as a baterial inhibitor may inhibite the bactericidal effect of β - lactan agents which are rapid bactericidal agents.S. pneumonia is the most common pathogenic bacteria in CAP, especially when the patients are children and aged people. It also causes the more severe degree disease than other pathogenic bacteria. Once infected with it,the case fatality ratio is very high. And the drug resistance of S. pneumonia has become very serious in current years. The sensitivity to penicillin and macrolide antibiotic has decayed rapidly since 90^th of the last century. The study of drug - resistance detections have showed that drug resistant S. pneumonia continuously increased throughout the whole world.Amoxicillin is a broad - spectrum aminopenicillin, and the erythromycin is a classic macrolide antibiotic. They are both the frequently used agents. As the reports about clinical studies around the world have shown that patients treated with the recommended β - lactam antibiotics and macrolides can be reduced o-verall costs, inpatient days, case fatality rates and the trend of generating the drug - resistance than those receiving alternative treatment. The empirical therapy covered atypical causative organism would change the prognosis of patientswho were with severe bacteremic pneumococcal pneumonia.Several investigators tested combination penicillin and erythromycin in vitro for antimicrobial activity against S. pneumoniae, but they reported the results nonuniformly. One group of investigators reported on the interaction of penicillin and a macrolide and their results showed antagonism. Another report showed indifference.To study possible interactions between amoxicillin and erythromycin against streptococcus pneumonia time - killing experiment and fractional inhibitory combination test were performed. At the same time verify whether the bactericidal action of erythromycin against S. pneumococci is exist.Meterials and MethodsThe clinical isolates of pneumococci and international standard Streptococcus pneumoniae ( ATCC49619) were used. Plated the frozen stock cultures on Columbia blood agar plates,37°C ,5% CO2, incubated 20 hours. The minimum inhibited concentration( MIC) were determined by Agar twofold dilutions, minimal bactericidal concentration(MBC) for amoxicillin and erythromycin were determined by replica plating. And calculated the value of MIC50, MIC^, MBC50 and MBCgo-The combination effect of amoxicillin and erythromycin against clinical isolates were tested by checkerboard microdilution. One antibiotic was placed in the wells of seven rows in descending concentrations starting at four times the MIC and ending at zero MIC. The other was similarly distributed among the seven columns. An inoculum of 0. 01ml S. pneumoniae per well were used at a concentration of about 5 x 105CFU/well. Calculate fractional inhibitory concentration index (FICI) to interpret the results.S. pneumoniae (ATCC49619) , No.4 isolate (the MICs of amoxicillin and erythromycin are both 0. 016 mg/L) and No. 41 ( MIC of amoxicillin is 0.03mg/ L,MIC of erythromycin is ^ 256 mg/L) were incubated for time -killing assay. The 20 hours cultures were incubated in 20ml of MH broth at 35 °C in an incubator - shaker over 7 hours. The isolates were exposed to amoxicillin (7. 6mg/L) , erythromycin (3. lmg/L) alone and in combination respectively. Take the time before the addition of antibiotics and 1,3 ,5 ,7 hours later as X - axis, the viable organism numbers were used as Y - axis to plot the time - illing curves.Results1. MIC of amoxicillin and erythromycin against S. pneumoniaeThere were three different statuses of the sensitivity of all the cilinical isolates. The most were resistant to both amoxicillin and erythromycin. 14 isolates were susceptible to both amoxicillin and erythromycin, and 30 isolates were susceptible to amoxicillin and resistant to erythromycin. No one was resistant to amoxicillin and susceptible to erythromycin. In all the isolates, The range of amoxicillin MIC was ^ 0. 004 mg/L ²mg/L and that of erythromycin was 0. 008 mg/L ^ 256 mg/L. The MIC50 and MIC90of erythromycin against amoxicillin and erythromycin - susceptible isolates was 0. 063 mg/L ,0. 125 mg/L respectively , and those against amoxillin - susceptible, erythromycin - resistant isolates were both ^ 256 mg/L. The MIC50 and MIC90of amoxicillin against amoxicillin and erythromycin - susceptible isolates was 0. 016 mg/L,0. 125 mg/L respectively , and those against amoxillin - susceptible, erythromycin - resistant i-solates was 0.016 mg/L,l mg/L respectively.2. Bactericidal action of amoxicillin and erythromycin against S. pneumo-cocciIn all 46 cilinical isolates, the range of amoxicillin MBC was ^ 0. 004mg/ L ⁴mg/L,and that of erythromycin was 0. 016mg/L ^ 256mg/L. The ratio of amoxicillin MBC and MIC was 1 2, and- that of erythromycin was 1 4 (not included isolates which MIC were ^ 256mg/L). They were both no more than 4, suggesting that both amoxicillin and erythromycin possesed bactericidal effect a-gainst S. pneumococci.3. The result about amoxicillin and erythromycin combination susceptibility testThe MIC values of the most isolates when amoxicillin combined with erythromycin were similar or slightly decreased than those of amoxicillin or erythromy-cin alone. The range of FICI was 1 ². This result showed that amoxicillin and e-rythromycin was indifferent. Though only two isolates showed antagonist, the e-rythromycin MIC alone and combinated with amoxicillin was 0.031mg/L and 0. 016mg/L respectively,and the FICI was 2. 5 and 3 respectively.4. Time - killing curveThe bactericidal effect of amoxicillin, erythromycin alone and combination increased as time went by. The bactericidal effect of erythromycin alone was worse than that of amoxicillin alone and two agents combination. Because No. 41 isolate was an erythromycin - resistant isolate , there was only feeble bactericidal action of erythromycin against it. Treated with agents 7 hours later,the mount of viable organism with amoxicillin and erythromycin combined was changed no more than 0. 51ogi0CFU/ml compared with that of using amoxicillin alone.DiscussionIn this experiment , MBC/MIC value range of all the isolates was 1 4 ,it i-dentified that erythromycin was bactericidal but not bacteriostatic against S. pneumoniae. This was the same as Fernandes et. have reported. When the pathogen was S. pneumoniae, the effect of (3 - lactam antibiotics can' t be decreased as the patients being treated with combination of p - lactam antibiotics and mac-rolides. Otherwise some investigators have reported that azithromycin and clar-ithromycin et. macrolides were all bactericidal against S. pneumoniae. They are all inhibited the synthesis of RNA associated protein through combinating with 50subunit of ribosome. But the pharmacokinetics showed the bactericidal action was slow. And amoxicillin as a bactericidal agent produce a marked effect by way of inhibiting the synthesis of cell wall protein. We also obserbed the bactericidal effect of erythmycin alone was not as well as that of amoxicillin alone and amoxicillin combined with erythromycin through the time -killing curves.We compared amoxicillin alone and amoxicillin with erythromycin combination time - killing curves, the altered quantities of viable organisms were no more than 0. 51og10CFU/ml on every time point. It was almost the same about the two ways. So the erythromycin didn' t affect the bactericidal effect of amoxicillin. Inthe checkerboard microdilution test , we have saw the similar result. The FICI range of the most isolates were at 1 2 except two isolates. It was the same as Deshpande etc. reported. All of these promped that @ - lac tarn antibiotics can be combined with macrolides for experiential therapy.In this experiment chose 30 isolates which were sensitive to amoxicillin and erythromycin - esistant. All the isolates were highly resistant to erythromycin, MIC value were ^ 256mg/L. The resistance rate was highly than which chen mingjun etc. have reported. In our contury,the rate of erythromycin - resitance was about 50% 70% , and the mechanism was also different from that in the north American countries. The report of Zhao Tiemei and Liu Youning showed that the erythromycin -resistant cilinical isolates 79. 1% were erm gene , 10. 1% were erm + mef, only 10. 8% were mef gene. However the resistant gene generally was mef in North America. Erm gene performed resistance by target modificated,and the spectrum is extensive,the degree is high. In spite of this, the combination with macrolides and (3 - lactam antibiotics was commanded in experiential therapy, especially when the infection organism cannot rule out My-coplasma, Chlamydia and Legionella. Because the macrolides can also affect the immune function in vivo, such as enhancing the phgocytosis of reticuloendotheli-al system, macrophage and monocytic. They maybe produce more marked effect.This study was performed all in vitro,but the conditions in vitro was different from the internal environment. In MIC test and time - killing assay test, the concentration of S. pneumoniae cell density is 105 106CFU/ml,whereas micro-bial concentration can be as dense as 108 1010 CFU/g of infected tissues. The similar problem exists in concentration of medicine supplied. Macrolides such as erythromycin can be uptaked into cells by neutrophil or macrophage in vivo, so the concentration can get to 10-20 times than that of extracellular, and the concentration of infection part maybe much higher than concentration used in experiments in vitro. Consequently, we need more experiments in vivo to study the effect and mechanism of the combination of macrolides and (3 - lactam antibiotics.ConclusionErythromycin possesses bactericidal action against S. pneumoniae in vitro. No matter static checkerboard microdilution or time - killl curves which can observe the dynamic interaction of two agents both showed it was indifferent when amoxicillin and erythromycin were combined together. The bactericidal effect of amoxicillin can't be decreased. As experiential therapy, the combination of ma-crolides and (3 - lactam antibiotics can be used in cilinic. More evidences should be proved by more experiments in vivo.