| PartⅠAnalysis on antimicrobial resistance of Shigella isolated in Anhui,ChinaObjective:To investigate the prevalence of Shigella serotype in Anhui province and the resistance of antimicrobial agentsMeterials and Methods:From September 2011 to September 2015,a total of 525 non-duplicate Shigella isolates were collected from 34 hospitals in Anhui,China.Escherichia coli ATCC 25922 was used as a quality control strain for antimicrobial susceptibility testing.The minimal inhibitory concentrations(MICs)of 18 antimicrobial agents were tested by agar dilution method for 525 Shigella isolates.Results:Among the 525 non-duplicate Shigella isolates,S.flexneri was the most popular(n=449,85.5%),followed by S.sonnei(n=68,13.0%).Among the S.flexneri,238 strains(53%)were from the northern region,126 strains(28%)were from the central region and 85strains(19%)were from the southern region.Among the S.sonnei,34 strains(49%)were from northern regions,20 strains(30%)were from central regions and 14 strains(21%)were from southern regions.The proportion of Shigella isolates from children under 5 years old was highest in all ages(n=331,63.1%).In addition,the proportion of male patients infected with S.flexneri was higher than that of female patients(57%vs.43%;P<0.0001).Among the S.flexneri,the antimicrobial resistance rate of ampicillin(97.3%)was the highest,followed by nalidixic acid(96.9%),tetracycline(93.8%)and chloramphenicol(86.6%).Sensitivity to piperacillin/tazobactam was the highest(99.3%),followed by imipenem(98.2%)and amikacin(98%).Among the S.sonnei,the antimicrobial resistance rate of ampicillin was also the highest(100%),followed by nalidixic acid(98.5%),tetracycline(94.1%),trimethoprim/sulfamethoxazole(92.6%)and gentamicin(92.6%).Sensitivity to piperacillin/tazobactinand and amikacin(100%)were the highest,followed by imipenem(98.5%)and cefepime(98.5%).417 S.flexneri isolates(92.9%)were multidrug-resistant strains,and the most common multidrug-resistant phenotype was ampicillin-nalidixic acid-tetracycline(90.2%).66 S.sonnei isolates(94.1%)were multidrug-resistant strains,and the most common multidrug-resistant phenotype was also ampicillin-nalidixic acid-tetracycline(92.6%).The resistance rates to chloramphenicol,ciprofloxacin and levofloxacin for S.flexneri were significantly higherthanthatforS.sonnei.However,thesensitivitiesto trimethoprim/sulfamethoxazole and gentamicin for S.flexneri were significantly higher than that for S.sonnei.Conclusions:1.S.flexneri is still a popular subgroup of Shigella spp.in Anhui,China.2.The most common multidrug-resistant phenotype of Shigella isolates in Anhuiprovince is ampicillin-nalidixic acid-tetracycline.The resistance to fluoroquinolonefor S.flexneri was significantly higher than that for S.sonnei.PartⅡIn vitro and in vivo ciprofloxacin combined with fosfomycin for shigella isolatesObjective:The antibacterial activity of ciprofloxacin combined with fosfomycin in vitro was studied by using the microtitre plate chequerboard assay and time–kill methodology.The model of Galleria mellonella was used to assess the in vivo activity of antimicrobial agents.Meterials and Methods:80 S.flexneri with ciprofloxacin resistance were selected for antimicrobial combination test by microtitre plate chequerboard assay.In the time–kill test of ciprofloxacin combined with fosfomycin,two strains of S.flexneri were screened,among which one strain(GN120471)was resistant to both ciprofloxacin and fosfomycin,one strain(GN120454)was resistant to ciprofloxacin and sensitive to fosfomycin.According to the CLSI standard in 2017,the MIC value of fosfomycin(≥256μg/mL)against S.flexneri was defined as antimicrobial resistance.In addition,25μg/mL 6-phosphate glucose was supplemented when the sensitivity of fosfomycin was detected.According to the plasma drug concentration after receiving the standard dose of antimicrobial agents,the plasma drug concentration is low,mid and high.The low concentration of ciprofloxacin is 0.5μg/mL,the mid concentration is 1μg/mL,and the high concentration is 2.5μg/mL.The low concentration of fosfomycin is 30μg/mL,the mid concentration is150μg/mL,and the high concentration is 300μg/mL.Different concentrations of antimicrobial agents were combined,and the colony counts were conducted at 3h,5h,8h and 24h,respectively.The model of Galleria mellonella was used to assess the in vivo activity of antimicrobial agents.Two strains of S.flexneri infected respectively the larvae by different concentrations,and the virulence of the bacteria was detected.The optimal inoculation concentrations of the two strains were determined according to the concentration of bacteria that could cause 80%of the larvae to die at 96h.Take 10μL ciprofloxacin with the concentration of 100μg/mL and inject it every 12h for 4 days(the peak concentration of ciprofloxacin 2.5μg/mL).Take 10μL fosfomycin with the concentration of 1000μg/mL and inject it every 6h for 4 days(the peak concentration of fosfomycin 150μg/mL).The survival rates of Galleria mellonella were observed at 24h,48h,72h and 96h,and the antibacterial effect of the combined treatment was determined.Results:Of the 80 S.flexneri resistant to ciprofloxacin,31 strains(38.75%)were FICI≤0.5 and49 strains(61.25%)were 0.5<FICI≤4.There were 36 colony counting points in the vitro time-kill test.For the strain GN120471(CIP~R-FOS~R),22 points(61.1%)showed the synergy and 3 points(8.3%)showed the additivity.For the strain GN120454(CIP~R-FOS~S),35 points(97.2%)showed the synergy and 1 points(2.8%)showed the additivity.For the strain GN120471(CIP~R-FOS~R),the bactericidal effect was most obvious when ciprofloxacin 2.5μg/mL was combined with fosfomycin 150μg/mL or300μg/mL.For the strain GN120454(CIP~R-FOS~S),ciprofloxacin 0.5μg/mL combined with fosfomycin had obvious bactericidal effect.The concentration of bacteria was~10~5 CFU in the body that could cause 80%of the larvae to die at 96h(bacteria suspension concentration for~10~7 CFU/mL).When ciprofloxacin(the peak concentration 2.5μg/mL)was combined with fosfomycin(the peak concentration 150μg/mL)for the bactericidal activity in vivo,the survival rates of the larvae in 96h infected with GN120471 and GN120454 were 68.75%and 81.25%,respectively,and were significantly higher than that of monotherapy.Conclusions:1.For S.flexneri with ciprofloxacin resistance,the bactericidal activity ofciprofloxacin combined with fosfomycin was better than that of monotherapy.2.The time-kill test and survival curves showed that the strain(CIP~R-FOS~R)needed to select a higher concentration for drug combination,and the strain(CIP~R-FOS~R) could select a low concentration of ciprofloxacin for antimicrobial combination,which further verified the obvious synergistic effect of ciprofloxacin combined with fosfomycin in the treatment of bacterial dysentery.Part Ⅲ Prevalence of plasmid-mediated azithromycin resistance determinants for Shigella isolatesObjective:To investigate the distribution of plasmid-mediated azithromycin resistance genes for Shigella isolates in Anhui province and the correlation with antimicrobial agents.Meterials and Methods:449 non-duplicate S.flexneri and 68 non-duplicate S.sonnei were collected from September 2011 to September 2015.According to the CLSI standard in 2017,the azithromycin resistance for S.flexneri was defined as the epidemiological cutoff≥16μg/mL,the azithromycin resistance for S.sonnei was defined as the epidemiological cutoff≥32μg/mL.The bacterial DNA was extracted by boiling method and the plasmid DNA was extracted by plasmid mini kit.Azithromycin resistance genes(mphA/B,ermA/B/C/F/T/X,ereA/B,mefA and msrA)were identified by polymerase chain reaction(PCR).All purified PCR products were directly sequenced,and sequence alignments were compared with the GenBank nucleotide database to clarify the plasmid-mediated azithromycin resistance genotype.Conjugation experiments for the plasmid-mediated azithromycin resistance isolates were carried out with the recipient Escherichia coli J53Az~R as previously described.The minimum inhibitory concentration(MIC)of wild-type isolates,recipient strains and transconjugants were tested by agar dilution method.The homology of plasmid-mediated azithromycin resistance isolates was analyzed by pulsed field gel electrophoresis(PFGE).Results:The azithromycin resistance rate for S.flexneri was 33.6%,and that for S.sonnei was39.7%.There was no significant difference in gender and age between the bacteria with azithromycin resistance.The mphA gene was detected in 93(61.6%)of 151 S.flexneri with azithromycin resistance,and in 11(40.7%)of 27 S.sonnei with azithromycin resistance.The genes of mphB,ermA,ermB,ermC,ermF,ermT,ermX,ereA,ereB,mefA and msrA were not detected in all strains.There were 89 transconjugants from 93 mphA-positive S.flexneri and 10 transconjugants from 11 mphA-positive S.sonnei.Compared with recipient strains,the azithromycin MIC of transconjugants had increased in various degrees.All the Shigella isolates with azithromycin MIC>256μg/mL carried the mphA gene.There were 13 different types(P1-P13)of 93 mphA-positive S.flexneri by PFGE analysis,including 41(44.1%)P1 and 28(30.1%)P2.There were 5 different types(P1-P5)in 11 mphA-positive S.sonnei by PFGE analysis,including 7(63.6%)P1.10strains had homology from 13 mphA-positive S.flexneri with high-resistance to azithromycin(MIC>256μg/mL).Of 10 multidrug-resistant strains,8 strains were from children(<5 years old),and 2 strains were from the elderly(>60 years old).There were7 strains from male patients and 3 strains from female patients.Conclusions:1.Plasmid-mediated azithromycin resistance genes can be transplanted horizontally, causing the same or different strains to azithromycin resistance.2.The mphA-positive S.flexneri with high-resistance to azithromycin were all multidrug-resistant bacteria,most of which had homology.The homologous strains were all from children and the elderly,which should be paid great attention. |
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