Mechanisms In The Replacement Of Major Methicillin-resistant Staphylococcus Aureus Epidemic Clones

Staphylococcus aureus is an opportunistic pathogen. This pathogen can commensallycolonise the human skin, nose, throat, armpit and groin, and cause numerous hospital-andcommunity-acquired infections, including mild skin and soft tissue infections, as well aslife-threatening pneumonia, bacteremia, and sepsis. Treatment failure is mostly due to thewidespread antibiotic resistance among S. aureus isolates, particularly themethicillin-resistant S. aureus (MRSA), which has become a major global public healthconcern. In the United States, the death caused by MRSA infection was greater than the sumof HBV and AIDS in2005. In China, the highest bacterial isolation rate from samplesderived from patients suffered bacterial infections was awarded to S. aureus that occupied15.1%of the isolated bacteria in2011. MRSA, HBV and HIV were considered to the threebigger infectious pathogen in the world.Several molecular methods can be used to type MRSA isolates, includingstaphylococcal cassette chromosome mec (SCCmec), pulsed-field gel electrophoresis(PFGE), multilocus sequence typing (MLST) and staphylococcal protein A (spa) typing. Ina particular region only a small number of advantage clonal complexes (CCs) cause themajority of MRSA infections. In Europe countries, in the UK for example, the major clonesare CC22(ST22) and CC30(ST36), whereas in Asian countries the predominant clone isCC8(ST239). Liu et al (2009) reported that ST239-MRSA-III is the most dominant clonein China, which accounted for approximately80.8%of MRSA isolates. In our previousstudy, MRSA strains recovered from nine hospitals of six cities (Urumqi, Shenyang, Beijing,Shanghai, Chongqing and Guangzhou) between2009and2012, ST239-MRSA-III-t030accounted for56.6%(90/159) and ST239-MRSA-III-t037accounted for30.2%(48/159) of the ST239isolates. The major clonal replacement among MRSA lineages has been observedworldwide. In the UK, the CC30clone was gradually replaced by CC22from2001to2007.In a German university hospital, CC45-MRSA-IV and CC5/ST228-MRSA-I were replacedby CC22-MRSA-IV and CC5-MRSA-II within11years. In Hungary, the ST239clone,which was predominant in1994to1998, was replaced by ST228and ST5from2001to2004. In China, ST239-MRSA-III-t037was the most dominant clone prior to2000;however, ST239-MRSA-III-t030rapidly replaced ST239-MRSA-III-t037and became themost predominant clone after2000. The clonal replacement occurrence in the same clonalcomplex was first discovered in our country.A clearer understanding of S. aureus population dynamic changes might contribute tothe development of effective approaches to control this major pathogen. The replacement ofMRSA clones is very complex, MRSA strains particular biological properties such asantimicrobial resistance, growth rate, adaptability to environment and fitness cost may be animportant reason for the replacement of MRSA clones. In France, gentamycin-susceptibleMRSA (GS-MRSA clone) replaced the gentamycin-resistant MRSA (GR-MRSA clone), itmaybe due to more fitness cost associated with antimicrobial resistance (Laurent et al.2001).SCCmec elements also affect the replication of S. aureus cells. The SCCmec I element isassociated with higher glucose consumption and higher growth rate compared withSCCmecIV (Lee et al.2007). In China, ST239-MRSA-III-t030and ST239-MRSA-III-t037are the most dominant clones, and the mechanisms underlying the clonal replacementwithin strains carrying the same SCCmec element may be very complicated. In our previousstudy, we surprisingly found that most ST239-MRSA-III-t030strains isolated from ourcountry are rifampicin-resistant and sulfamethoxazole/trimethoprim (SXT)-sensitive,whereas most ST239-MRSA-III-t037strains are SXT-resistant and rifampicin-sensitive.Whether the special drug-resistance profile is associated with clonal replacement? In thisstudy,24ST239-MRSA-III-t030and ST239-MRSA-III-t037isolates recovered fromChongqing, Guangzhou, and Shanghai were randomly collected and divided into12t030/t037pairs, to investigate the main mechanisms underlying the clonal replacement ofST239-MRSA-III-t037by ST239-MRSA-III-t030. The independent growth rates of allisolates were determined, the competition assay in vitro and in a mouse model,cross-inhibition experiments, drug-resistance profile determination, drug determinants sequencing and the related gene expression levels determination were also conducted. Ourresults showed that ST239-MRSA-III-t030strains are fitter than the ones ofST239-MRSA-III-t037, the advantage fitness of ST239-MRSA-III-t030clone mayassociate with their rifampicin-resistant phenotypes and the disadvantage fitnesscompetitive of ST239-MRSA-III-t037strains may associate with SXT resistance. The maincontents and results are as following:1. MRSAantibiogram redetectionBroth dilution method was used to detect the drug resistances of all24selected strainsto linezolid, vancomycin, teicoplanin, oxacillin, clindamycin, rifampicin, andsulfamethoxazole/trimethoprim. The result showed that all strains are linezolid, teicoplanin,vancomycin sensitive and oxacillin resistant,23strains are clindamycin resistant; allST239-MRSA-III-t030strains are rifampicin-resistant and SXT-sensitive and allST239-MRSA-III-t037strains rifampicin-sensitive and SXT-resistant (except SH02).2. Growth curve determinationThe growth curves of ST239-MRSA-III-t030and ST239-MRSA-III-t037isolates weredetermined by measuring the optical density (OD) at600nm at1h intervals for24h at37°C. To achieve the same initial inoculation, the overnight culture of each strain was1:500diluted with fresh bacterial medium and then subjected to growth curve determination.ST239-MRSA-III-t030strains in11pairs had shorter lag phases and reached significantlyhigher OD600levels after24h of culturing in an independent growth assay. The strains ofthe remaining pair recovered from Shanghai (SH05/SH39) exhibited a similar growth rate inthe logarithmic phase. However, SH05(t030) grew faster than SH39(t037) from the latestage of the logarithmic phase and reached a significantly higher OD600level after24h ofculturing.3. In vitro competition and cross inhibition experimenti) The strains of each ST239-MRSA-III-t030/ST239-MRSA-III-t037pair were diluteto0.5MCF and mixed at a1:1ratio, and then cultured at37°C for24h with shaking. Theappropriate diluted samples were plated in triplicate onto selective (4mg/L rifampicin) andnonselective brain heart infusion agar (BHIA; Oxoid) plates and c ultured at37°C for24h.The colony counts on the selective plate yielded the ST239-MRSA-III-t030bacterial levels,whereas the difference between the colony counts of the selective and nonselective plates indicated the ST239-MRSA-III-t037bacterial levels. All ST239-MRSA-III-t030strains innine replicates outcompeted the ST239-MRSA-III-t037strains by reaching higher averageviable counts after24h of culture. The result suggests that ST239-MRSA-III-t030grewfaster than ST239-MRSA-III-t037. ii) Proteins in the culture supernate of t030and t037strains were precipitated with a7.5%trichloroacetic acid (TCA). The culture supernatantproteins were highly similar within the same clone but differed between the t030and t037strains. To determine whether the different secreted proteins or the undetected factors of oneclone could inhibit the growth of the other, we performed a cross-inhibition experimentusing the filtrate prepared from a7h or4h culture supernate, the results show that there areno significant inhibition observed between030and t037strains.4. In vivo competition experimentIn order to investigate the competitive advantage of ST239-MRSA-III-t030strains invivo infection. Two pairs strains (CQ29/CQ40, SH08/SH02) were randomly selected,ST239-MRSA-III-t030and ST239-MRSA-III-t037strains were separately grown overnightand diluted to1×108CFU/ml, mixed at a1:1ratio. Total of100μL of the bacterialsuspension of each strain pair was intravenously injected into56-week-old to8-week-oldfemale BALB/c mice. Three days after the challenge, the mice were killed. Kidneys wereremoved and homogenised in1ml of PBS with1%Triton X-100. Appropriate dilutions ofthe samples were plated in triplicate onto selective and nonselective BHIA plates. Theresults showed that the mean CFU of ST239-MRSA-III-t030that infected the mouse were(9.06±0.40)×106CFU/kidney (CQ29) and (9.01±0.42)×106CFU/kidney (SH08), and thesevalues were significantly higher than that of ST239-MRSA-III-t037strains with (3.7±0.80)×106CFU/kidney (CQ40) and (4.48±0.69)×106CFU/kidney (SH02),respectively.These in vivo data strongly suggest that ST239-MRSA-III-t030has a competitive fitnessadvantage over ST239-MRSA-III-t037during infections.5. Drug resistance determinants sequencing and the related gene expression levelsdetectionIn our country, the antibiograms of ST239-MRSA-III-t030and ST239-MRSA-III-t037strains are different when against rifampicin and SXT. SXT is recommended as the first lineagent to cure skin and soft tissue infection coursed by MRSA. However, rifampicin isproposed to be used in combination with vancomycin to prevent bacterial infections during operation. i) The RNA polymerase βsubunit mutations always associate with MRSAresistance to rifampicin. We sequenced the rpoB genes of all ST239-MRSA-III-t030strains,and found these strains exhibited the same double mutations, H481N and L466S. As a result,the MIC values of ST239-MRSA-III-t030strains against rifampicin were increased to512μg/ml (5strains) even to1024μg/ml (7strains). SXT interferes with the bacterialtetrahydrofolic-acid pathway. Tetrahydrofolic acid is a critical cofactor in the conversion ofdUMP to dTMP, which is essential in DNA synthesis. MRSA resistance to SXT is toocomplex. We sequenced the whole genes encode thymidylate synthase (thyA), dihydrofolatereductase (dfrA) and dihydropteroate synthase (dhps) of all ST239-MRSA-III-t037strains.Unfortunately, we didn’t find any mutations in these genes, indicating that the resistancemechanisms of ST239-MRSA-III-t037strains to SXT in our county are different to that ofthe strains isolated from other countries and the actual mechanisms underlyingST239-MRSA-III-t037resistance to SXT in our county need further investigation. ii)Intragenic compensatory mutations generally facilitate the growth of resistant bacteria byeliminating or reducing the fitness costs. The most common clinical genotypic mutations inS. aureus RpoB (H481N+S529L) does not exhibit any fitness cost in vitro (O’Neill et al.2006). S. aureus RpoB double mutants always increased some genes expression levels toeliminate fitness cost. So we randomly selected two pairs of strains (CQ29/CQ40，SH50/SH31) to detect RNAIII gene and α-haemolysin gene expression levels. The resultshowed that the RNAIII and α-haemolysin gene expression levels ofST239-MRSA-III-t030strains were significantly higher than that of ST239-MRSA-III-t037strains. Although, no mutations in the genes that associate with SXT resistance in allST239-MRSA-III-t037strains were detected, however, we found thatST239-MRSA-III-t037strains in8pairs exhibited small colony varients (SCVs) on BHIbroth plates, which may due to the increasing fitness costs of ST239-MRSA-III-t037strainsto deal with SXT resistance. To form SCVs may be an important reason for the successfulreplacement of ST239-MRSA-III-t037clone by ST239-MRSA-III-t030in our country.In summary,12pairs of t030/t037strains recovered from Chongqing, Shanghai, andGuangzhou were analyzed in this study. The antibiogram analysis, growth curvedetermination, cross inhibition experiment, in vitro and in vivo competition experiment,drug resistance determinants sequencing and the related gene expression levels detection were performed. The results show that the higher growth rate may facilitateST239-MRSA-III-t030to replace ST239-MRSA-III-t037as the most predominant clone inChina. The fitness competitive disadvantage of ST239-MRSA-III-t037could be associatedwith SXT resistance. Our findings may be helpful in the understanding of the replacementof MRSA clones in China, and may be useful in searching new strategies to control MRSAinfections.