| Stenotrophomonas maltophilia, an opportunistic pathogen, could infect both human and animals.I It is resistant to many antibiotics and disinfectants. The production of two P-lactamases by this bacterium makes it resistant to many antibiotics, especially β-lactam related ones. The two β-lactamase enzymes are L1and L2. L1, a metallo-β-lactamase can hydrolyse penicillins, cephalosporins, P-lactamase inhibitors and majority of carbapenems. L2hydrolyse cephalosporins, a single ring cephalosporins. The two enzymes can be induced and expressed in high levels in response to antibiotics stresses, resulting increased resistance to antibiotics. The expression of L1, L2enzyme induction depends on the existence of regulatory genes AmpR.To investigate the variation and induction of L1, L2and AmpR, resistance profiles of clinical isolates and sequences of the three genes were analyzed, and their correlations were defined. Isolates of S. maltophilia were induced by with two antibiotics, and then the transcription of AmpR and L2before and after the induction were analyzed. The enzymatic activities were tested and compared to define the induction characteristics by antibiotics. Sequence of the two genes were also defined to determine whether the induction cause mutations in the sequence.1. Resistance profile and polymorphism of L1, L2and AmpR from S. maltophiliaA total of99clinical isolates were analyzed. The resistances were defined by K-B disk diffusion and Etest. Results showed that the99isolates had different resistance to antibiotics.96.97%of them are resistant to ceftriaxone, piperacillin resistance, above90%resistant to cefotaxime, aztreonam resistance.85.86%and69.70%of the strains are sensitive to levofloxacin and ciprofloxacin respectively. Most of these strains are multi-drug resistant, over93.9%are resistant to at least two antibiotics. Sequences of L1, L2and AmpR were PCR amplified and analyzed to test their relations with resistance. Of the99isolates,22were L1positive. Sequences of L1of the22strains were conservative and only a few sites were mutated. All the L1containing strains are highly resistant to many antibiotics.78strains were positive for AmpR and L2. Their sequences were compared to those in Genebank, and according to the sequence polymorphisms,3groups were defined. The3groups showed different antibiotics resistance profiles.2. Transcriptional induction of L2and AmpR from S. maltophilia by antibioticsUnder selection pressures from different antibiotics, S. maltophilia will increase expression of P-lactamase. In this section, the induction of L2and AmpR were analyzed. S. maltophilia were treated with IP (Imipenem) or PTC (Piperacillin/tazobactam) of different concentrations, and then, transcription of L2and AmpR were analyzed. The results showed that after induction, transcriptions were increased, and furthermore, PTC induced higher increase of transcription. When induced with two antibiotics, transcription level of AmpR was lower than that of L2.3.Effects of antibiotics induction on enzyme characteristics, resistance and sequence variation of L1and L2from S. maltophiliaTo test the enzyme induction of L1and L2, the activity of the enzymes were analyzed. After induction, the MICs for other antibiotics were tested. The results showed that, when induced with4xMIC, both IP and PTC could induced the highest enzyme activities. Higher concentration of antibiotics induced higher enzyme activity. PTC induced increase in L1and L2, while IP induced increased L1, but decreased L2. Under stresses of different antibiotics, the production of LI and L2will be induced. The strain induced by IP are more resistant to TZ (Ceftazidime), but for TLC (Ticarcillin/clavulanate), an increase followed by subsequent decrease. Seuqence analysis showed that, induction by the selected antibiotics for6hours did not lead to any changes in the sequences, implying that short time of induction did not induce mutations. |
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