Streptococcus suis is a pathogen of zoonotic diseases. Moreover, the emergence of fluoro-quinolones (FQs) resistance in this pathogen has severe consequences for pigs and human health. In this study, the molecular mechanism of FQs resistance in S. suis type 2 (SS2) sensitive strains isolated from pigs was assessed after in vitro induction of resistance against the most frequently used FQs: ciprofloxacin, norfloxacin, and enrofloxacin. Proteome analysis, sequencing and real-time RT-PCR results strongly established an overexpression of an ABC transporter protein (other than SatAB) and topoisomerase mutations in GyrA (Ser81Arg), GyrB (Glu354Lys), and ParC (Ser79Phe) in contributing to high level ciprofloxacin resistance in SS2. Due to the overexpression of the ABC transporter, intracellular ciprofloxacin concentrations were significantly lower in the resistant strains than those of sensitive strains after 20, 35, and 60 min exposures to ciprofloxacin (p < 0.05). It was concluded that improper use of FQs is one of the main causes of the emergence of this zoonotic pathogen as a multiresistant organism against commonly used antibiotics. The existence of an efflux-like protein is an incentive to find new drug targets to avoid the spread of FQs-resistant S. suis isolates in pigs and the human population.

Six drugs were used to perform the antimicrobial susceptibility test in 38 strains of Streptococcus suis serotype 2(SS2).Three parent strains of SS2 were chosen for stepwise exposure to increasing levels of erythrocin and ciprofloxacin followed by selection of resistant mutants.Clinical categorization of isolates as susceptible or resistant was based on minimal inhibitory concentration(MIC) interpretative standards of the Clinical Laboratory Standards Institute(CLSI).Reserpine was used to determine the effect of efflux pump inhibitor on the MICs of erythrocin and ciprofloxacin against susceptible and induced resistant strains of the same parents.The result of MICs tested by microdilution indicated that resistance to sulfamonomethoxine and tetracycline(100%) was most frequent,and then erythromycin(39.5%),ciprofloxacin(28.9%).However,100% strains were susceptible to florfenicol and ampicillin.The MICs of multidrug resistant strains induced in vitro reached 128 mg/L from the initial 0.001 8 mg/L,and the MICs of erythromycin and ciprofloxacin against some resistant strains were decreased to different degree when reserpine was used.The study indicated that stepwise increasing concentration of erythromycin and ciprofloxacin can easily select resistant strains of SS2 and the active efflux pumps may play an important role in drug resistant mechanisms.

To perform a systematic analysis of point mutations in the quinolone resistance determining regions(QRDRs)of the DNA gyrase and topoisomerase genes of Streptococcus suis type 2 strain after in vitro exposure to stepwise increasing concentrations of enrofloxacin and ciprofloxacin.Four parent strains of S.suis type 2 were chosen for stepwise exposure to increasing levels of enrofloxacin and ciprofloxacin followed by selection of resistant mutants.The QRDRs of gyrA,gyrB,parC and parE correlating to mutants with increased MICs were analysed for point mutations.Multiple mutants with significantly increased MICs(128 mg·L-1)were generated from each parent strain.Most of the amino acid substitutions identified were Ser79Phe of ParC,Ser81Arg of GyrA,Asp315Asn,Ser285Leu,Glu354Lys of GryB and Pro278Ser of ParE,which were consistent to the mechanisms of resistance reported in clinical isolates of S.pneumoniae.However,it has some mutable points Gln118His of GyrA,Asn297Tyr of ParE as well as the deletions 288-291 amino acids of GyrB and 62 amino acid of ParC,which haven′t been documented and whether these nuleotide mutations associated with fluoroquinolone resistance are not clear.The number of induction/selection cycles required for the emergence of key point mutations in gyrA and parC was variable among strains.The results indicate that stepwise increasing concentration of enrofloxacin and ciprofloxacin can easily select resistant strains of S.suis.

This study was performed to investigate the distribution of macrolide and tetracycline resistance genes of 48 Streptococcus suis clinical isolates.The microdilution test was used to determine the susceptibility of 48 clinical S.suis isolates to nine antibiotics according to the National Committee for Clinical Laboratory Standards.PCR was used to detect the macrolide resistance genes ermA/B/C,mefA/E,msrD,mphB,23S rRNA,L4,L22,tetracycline resistance genes tetM,tetO,tetL,tetK and transposon related genes int,xis.The resistance rate to macrolides of 31 isolates of S.suis serotype 2 was 3.23%.Of 17 isolates of S.suis serotype 9,the resistance rate to erythromycin was 88.24%,and were all 70.59% to Tylosin,Phosphate Tilmicosin and Azithromycin.All of the clinical isolates were resistant to tetracycline,and sensitive to penicilin,amoxicillin,ceftriaxone sodium and ampicillin.ErmB gene,which accounted for 75%(12/16),was the main resistant gene,while both mefA/E and msrD genes accounted for 25%(4/16).Of 16 macrolide resistant strains,the detection rates of tetM,tetO,int and xis were 25%(4/16),62.5%(10/16),31.25%(5/16)and 31.25%(5/16),respectively.All of the macrolide resistant strains were negative to ermA,ermC,mphB,tetL and tetK,and have not been found the mutation of 23S rRNA,L4 and L22.

A number of 38 strains were surveyed for minimal inhibitory concentration(MIC) in vitro to 7 drugs by microdilution test to find out the resistance present situation among Streptococcus suis isolated from clinic.Staphylococcus aureus ATCC25923 was used as reference strain for MIC quality control.Clinical categorization of isolates as susceptible or resistant was based on MIC interpretative standards of the Clinical Laboratory Standards Institute(CLSI).Carbonylcyanide-p-chlorophenyl hydrazone(CCCP) and reserpine was used respectively to determine the effect of efflux pump inhibitor on the MIC of erythromycin and tylosin against Streptococcus suis resistant isolates.The result of MIC tested by microdilution indicated that none of the strains were susceptible to all antimicrobial compounds tested and eleven resistant patterns were detected.Resistance to sulfamonomethoxine(100%) was most frequent,and then erythromycin(39.5%),tylosin(31.6%),enrofloxacin(28.9%),ciprofloxacin(28.9%),while 100% strains were susceptible to florfenicol and ampicillin.The MICs of erythromycin and tylosin against some resistant strains were decreased to different degree when efflux pump inhibitor was used suggesting that the active efflux pumps inducing resistance to antimicrobial agents become much stronger in Streptococcus suis resistant strains.This study demonstrated a significant increase in resistance to most commonly used antimicrobial agents among Streptococcus suis strains isolated from clinic and the resistant sensitivity must be paid attention to in the clinic.