Molecular Basis Of Hypermutator And Its Role In The Evolution And Transfer Of Antibacterial Resistance In Escherichia Coli

With the enlargement of process scale and application area of antibacterials, bacterial resistance, especially multi-resistance have emerged. As a result, it makes antibacterial therapeutic effect fall-off and bring about tremendous hazard in diagnosis and therapeutics clinically. There are several mechanisms in antibacterial resistance: target site change of antibacterial action, inactivation of modification enzyme to antibacterial, deletion of membrane protein and efflux pump in membrane decreasing effective concentration of intracellular antibacterial. In the past twenty years, people concentrated on gene mutation in studying bacterial drug resistance and thought accumulation of gene mutation was important mechanism of bacterial resistance. Lately, people found that pathogenic bacteria which didn't touch antibacterial had resistance to antibacterial. So resistance have transfer characteristic and endogenous or exogenous. At present, resistance concept concentrate that bacterial resistance was produced by bacteria having normal mutation frequency and make bacteria adapt antibacterial circumstance and survive. But in Escherichia coli, Salmonella, Pseudomonas aeruginosa and Streptococcus intracellularis, the strains that was autogenetic and had higher mutation frequency than normal strains were found. These strains were definited as hypermutator. In some circumstance, the mutation frequency of hypermutator was more 1000 times than that of normal strains. These hypermutator quantity account for 20% of strains isolated from clinic. Appearance of hypermutator, on the one hand, may increase frequency of antibacterial resistance emerging, on the other hand, can make bacteria adapt easily to antibacterial concentration that was produced by therapeutic dose and accelerate emergence of endogenous resistance.In bacterial, the ability to maintain the genetic integrity of bacterial cells is balanced by the need to adapt to rapidly changing environments. The mismatch repair system plays a key role in maintaining this balance by recognizing and correcting mismatched bases that arise in duplex DNA as a result of replication error, DNA damage, and recombination between partially divergent, so-called homologous, DNA. A key component of the mismatch repair system is MutS, which initiates the process by recognizing and binding to mismatched bases in double stranded DNA. The importance of MutS in maintaining the stability of the cellular genome is underscored by its ubiquity in the biological world. MutS homolog have been identified in members of all three biological kingdoms, and with the advent of genome sequence analysis, it has become evident that most organisms encode at least one MutS homolog.The presence of permanent and transient mutators in natural populations of bacteria is now reasonably well established. We will therefore turn attention to establishing whether they are a risk factor for the emergence of antibiotic resistance in the clinical setting. We will consider this in relation to resistance that arises both by mutation and horizontal gene transfer. Mutation plays a central role in the evolution of bacterial resistance to antibiotics by refining existing resistance determinants, by altering drug uptake systems or by giving rise to variant drug targets with reduced affinity for antibiotics. What is the evidence that mutators enhance generation of resistance by these routes? In addition to mutation of genomic nucleotide sequences, horizontal transfer of genetic material is an important phenomenon in genetic variation and acquisition of antibiotic-resistance genes. MMR is a major barrier to interspecies recombination events and if this barrier is removed, as in the case of MMR defective mutators, the frequency of horizontal gene transfer is increased. Direct studies on horizontal transfer of resistance genes in mutators have yet to be performed.To evaluate mechanisms that may be involved in the emergence of a imitator phenotype, we determined the molecular basis for MMR deficiencies in natural isolates. To do this, we analyzed wild-type isolates of E. coli for deletion of MutS proteins and determined the molecular defects in mutators identified among E. coli isolates. We also determined the roles of hypermutator in development and transfer of antibacterial resistance.Firstly, 318 E.coli strains from different regions and animals susceptibility to 12 antibacterial [amikacin (AM), ciprofloxacin (CIP), amoxicillin (AMO), spectinomycin (SPE), tetracycline (TET), chloramphenicol (CHL), streptomycin (STR), gentamycin (GEN), norfloxacin (NOR), rifampicin (RIF), ofloxacin (OFL) and enrofloxacin (ENR)] were determined using agar double dilution method. The results were judged according to National Committee for Clinical Laboratory Standards (NCCLS) guidelines. In 12 antibacterial, AM had strong bacteriostasis because it was used very little in veterinary clinic. Its resistant rate was 0.94%. OFL, ENR, CIP, RIF and GEN had finite susceptibility and their resistant rates were 36.16%, 42.77%, 46.86%, 43.08% and 53.14%, respectively. But NOR, AMO, SPE, TET, CHL and STR showed very high resistance and their resistant rate were 70.13%, 92.13%, 71.70%, 98.43%, 82.39% and 84.59%, respectively. In different resistant phenotype, the strains resistant to 6 antibacterial were much more, and added up to 88. Strains resistant to 3, 4, 5, 7 and 8 antibacterial reached 40-70. There were a few strains resistant to 1 and 12 antibacterial. The strains resistant to 2, 9, 10 and 11 antibacterial were about 20-30.Experimentally, MPC has been taken as the drug concentration that allows no mutant to be recovered from a susceptible population of more than 10¹⁰ cells. For MPC to be therapeutically useful, it must be below the concentration achievable in serum or tissue with safe doses of antibiotic. The MPCs of ENR and CIP against ATCC25922 were lowest (range:0.25～0.5μg/mL). MPCs of SPE, AMO, TET, CHL, STR and RIF were highest (range: 64～>512μg/mL) and other drugs MPCs ranged from 2 to 4μg/mL. The MSW of AMO, TET and STR against ATCC25922 were very high (above 500μg/mL), but ENR MSW were very low (0.22μg/mL). The resulting MPC/MIC ratio of AM was very low, it's 4, but that of NOR, AMO, TET and STR were high (range: 64～256). These results suggested that AM had lowest ability to select resistant mutant ATCC25922, but NOR, AMO, TET and STR had possibility to select resistant mutator. Differing from ATCC25922, the strains from clinic had high MPC₉₀ to 12 antibacterial and exceeded that of ATCC25922. The MPC90 of AMO, TET, CHL and STR exceeded 512 ug/mL and can't be determined. Resulting MPC₉₀/MIC₉₀ ratios were also high and exceeded to 64. MPC₉₀ and MPC₉₀/MIC₉₀ of ENR were lowest, next were AM, CIP and OFL. This suggested that these several drugs had small ability to select resistant mutator and were possibly limited their use because of their high MSW. The MPC₉₀/MIC₉₀ ratios of GEN, NOR and SPE were ranged from 16 to 32 and suggested that these drugs have high ability to select mutator and can't be used lonely. Clinically, AMO, TET, CHL and STR must adopt combination therapy because these drugs hadn't therapeutic effect if they were used as mono-therapy.24 E.coli strains that were different phenotype (susceptible, intermediate resistant) were screened from 318 clinical strains and their mutation frequencies were determined. Mutation frequencies of most strain-antibacterial ranged from 10 to 60 per 10⁸CFU. A few strains mutation frequency was 0. That of CE3101, CE5116 and CE7301 to 10 antibacterial exceeded to 100, some strains were up to about 800～900. The reference strains mutation frequency was very low, it's about 3. According to definition of hypermutator, the mutation frequencies of CE3101, CE5116 and CE7301 to 10 antibacterial exceeded to 100, so these strains were hypermutators. PFGE was used to type 24 E.coli strains. K12 had different banding pattern and its genetic relationship was farthest compared to other strains. It belongs to independent type. CE5120, CE2219 and CE2202, CE7311 had duplicate banding pattern. The wild-type strains CE1117 and CE1304, CE2113 and CE3125, CE1305 and CE5103, CE6102 and CE6107, CE6210 and CE6209, CE7303 and ATCC25922 had near banding pattern. The banding pattern of three hypermutators was fairly close to each other and that of CE5116 and CE7301 was most similar. Other strains had different banding pattern.The MutS (important constituent of MMR) of wild-type E.coli and hypermutators was investigated using long PCR, Western-blotting and plasmid complement essay. in order to determine possible deletion of mutS, we designed three pairs primers in two sides and middle of mutS according to fhlA-mutS-rboS gene cluster and carried out long PCR. K.12 was type strain and its each fragment of mutS was identical with anticipant fragment. Three fragment lengths were 12,045bp, 10,688bp and 8,477bp, respectively. Compared to K12, three fragment length of hypermutator CE3101 and CE5116 existed different extent deletion: first band had deletion of 3,500bp and 7,000bp, second band had 3,600bp and 7,400bp and third band had 2,500bp and 7,500bp, respectively. The MutS protein of K12, normal strains and hypermutators were determined by Western blotting. K12 had a very clear band and its MutS was most complete. CE1112, CE1305, CE2219 and CE307 had fairly band and their MutS were fairly complete, too. But CE5103 and CE5120 had vague band and CE2205 had only tenuous band, their MutS proteins were incompletely integrated. The hypermutator CE3101 and CE5116 nearly had no band and their MutS were not integrated. Using pBR322 as a control, we carried out plasmid complement essay used pGW1811 plasmid constructed from pBR322. CE1117, CE3101, CE5116, CE6107, CE2313, CE7301 and K12 were transformed successfully. Mutation frequencies of CE1117, CE1305, CE6107, CE2313 and K12 to RDF changed very little and their change rate was about 40%. But hypermutator CE3101, CE5116 and CE7301 mutation frequency changed very large and their change rate was above 95%. These results suggested that the MutS of hypermutator E.coli existed defection compared to wild-type E.coli.Resistant phenotype and gene of 24 strains E.coli were determined. 24 strains were all resistant to AMO. The bla_TEM-1b genes were identified in all 24 strains, whereas none of the bla_SHV and bla_OXA genes were found. In 7 GEN-resistant strains, the aac(3)-II were found, but aac(3)-I, aac(3)-III were not detected. In 24 strains that were all resistant to STR, we identified aphA1, aphA2, aadA1. The tetA and tetB that were efflux system genes of TET were distributed in all strains. The genes of GyrA, ParC, MarR, AcrR and marO were sequenced. Apart from K12 and ATCC25922, other strains all existed mutations. In GyrA, CE6209 and CE2313 had no mutation, but other strains had Asp-87→Gly, Asp-87→Asn, Asp-87→Tyr and Ser-83→Leu in QRDR. Only one strain had mutation Ser-80→Ile in QRDR in ParC. In MarR, except one strain had one site mutation (Gly-103→Ser), other strains existed two site mutations (Gly-103→Ser, Tyr-137→His). In 12 strains (included 3 hypermutators), there were mutations of Glu-215→Ala, Asn-214→Thr and Lys-146→Thr. All 24 strains had A1332C nucleotide mutation in marO gene. The OMP of 8 strains were determined by SDS-PAGE. Two strains had complete OMP and were found OmpC, OmpF and OmpA. Five strains (included 2 hypermutators) OmpF and one strains OmpC were absence. All strains had no deletion in OmpA. In results of resistant gene and OMP determination, the hypermutators had no essential difference from other strains. This was because these strains had similar resistant phenotype and correspond to "phenotype determine genotype" inheriting rule. But hypermutators had high value in MPC, MSW, MPC/MIC and mutation frequencies and had defection in MMR, so they can easily develop new mutations and obtain resistant genes from outside, thus they may bring about antibacterial resistance. In spite of the hypermutators had low-level resistance to antibacterial, they can survive in host in therapeutic session and provide "platform" in developing high-level resistance. Thus we concluded that the hypermutator increased antibacterial-resistance emerges.Using CE5103, CE2113, CE3125 and CE2219 (containing I or II type integron) as donator strains and hypermutators CE3101 and CE5116 as receptor strains (CE6212 and CE2205 as control, not containing I or II type integron), we carried out filter membrane conjugation essay and determined transformation frequency. The transformation frequency of transconjugant using hypermutator as receptor was about 10^-4. In control essay, Only CE2113-CE6212 and CE2219-CE2205 transformation frequency were about 10^-6 and other transconjugant were zero. We identified integron in transconjugants by PCR. I type integron were found in CE5103-CE3101 and CE2113-CE3101 transconjugants and II type integron were found in CE3125-CE5116 and CE2219-CE5116 transconjugants. These results corresponded to integron type contained in donor strains. These essays suggested that conjugation were taken place easily in hypermutators and hypermutators facilitated resistant gene transfer in different strains.In short, there existed difference in hypermutators and normal strains in MPC, mutation frequencies and PFGE type. Molecular basis of hypermutator was defection in MutS that was important constituent of MMR. Hypermutator facilitated resistance evolution and transfer.