儿童呼吸道感染肺炎链球菌耐药性及pbp2B与TEM基因的研究

目的：了解广州地区儿童呼吸道感染肺炎链球菌(Streptococcus pneumoniae，SP)的耐药情况以及SP中青霉素耐药相关基因TEM与pbp2B的流行分布及突变情况。方法：采用E-test和K-B纸片法对44株SP分离株进行药敏试验；PCR扩增SP中的TEM基因及pbp2B，并对pbp2B基因进行测序，结果与SP青霉素敏感株R6进行序列比对分析。结果：①44株SP对青霉素的敏感率仅为11.4％，不敏感率高达88.6％。对红霉素耐药率已达100％，对克林霉素、复方新诺明的耐药性也在90％以上。但对头孢曲松、阿莫西林、亚胺培南仍敏感，耐药率分别为0，2.6%和3.9%。未发现对氧氟沙星、万古霉素耐药菌株。②44株SP的pbp2B基因扩增序列与R6敏感株相比较，5株青霉素敏感株99%以上的核苷酸序列相同，未发生氨基酸的替换。39株青霉素不敏感株均发生核苷酸序列的改变，核苷酸序列突变率为13.2％～23.1%，约6.5％～10.9%的氨基酸发生了替换。根据氨基酸在Ser391-Thr492片段之间的突变情况，可将39株青霉素不敏感株分为四型，其中Ⅰ型突变30株，Ⅱ型突变7株，Ⅲ型和Ⅳ型各1株。44株SP均未检出TEM型β-内酰胺酶耐药基因。结论：广州地区儿童呼吸道感染SP多重耐药情况较严重，青霉素、红霉素已不适宜作为SP感染的临床一线用药，阿莫西林及第三代头孢菌素可作为SP感染的经验用药。pbp2B基因突变是广州地区儿童SP对青霉素耐药的主要机制之一。［中国当代儿科杂志，2009，11（8）：623-626］

Antimicrobial resistance and penicillin resistance-associated genes of Streptococcus pneumoniae isolated from children with respiratory tract infection

Objective To investigate the antimicrobial resistance and penicillin resistance-associated genes(TEM and pbp2B)of Streptococcus pneumoniae(S.pneumoniae)isolated from sputum specimens of Guangzhou children with respiratory tract infection.Methods E-test and Kirby-Bauer methods were applied to detect the antibiotic susceptibility of 44 strains of S.pneumoniae.PCR was used to detect resistance genes pbp2B and TEM,followed by DNA sequence analysis of pbp2B gene.The sequence results were compared to those of penicillin-susceptible S.pneumoniae R6.Results Of the 44 isolates of S.pneumoniae,only 5(11.4%)were susceptible to penicillin.All strains were resistant to erythromycin but susceptible to ofloxacin and vancomycin.The resistance rate of the isolates to clindamycin and trimoxazole was more than 90%.The S.pneumoniae isolates showed a high susceptibility to amoxicillin,imipenem and ceftriaxone,with a resistance rate of 0,2.6% and 3.9%,respectively.The sequence analysis showed that more than 99% nucleotide sequence of pbp2B gene of five penicillin-susceptible isolates was the same as penicillin-susceptible S.pneumoniae R6,without any amino acid replacement.Site mutation was found in the remaining 39 penicillin-nonsusceptible isolates with a nucleotide mutation rate ranging from 13.2% to 23.1% and amino acid replacement rate from 6.5% to 10.9%.The 39 penicillin-nonsusceptible isolates were classified into 4 types according to the mutation site between Ser391 and Thr492 of pbp2B:type I(n=30),type II(n=7),type III(n=1)and type IV(n=1).No TEM gene was detected in all the 44 S.pneumoniae isolates.Conclusions The S.pneumoniae isolates from Guangzhou children with respiratory tract infection are resistant to penicillin and erythromycin.Amoxicillin and the third generation cephalosporin may be recommended for treating S.pneumoniae infection.The mutation of pbp2B gene plays an important role in the development of S.pneumoniae resistance to penicillin.