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首页> 外文期刊>Journal of Medical Microbiology: An Official Journal of the Pathological Society of Great Britain and Ireland >Emergence of a novel β-lactamase in food of animal origin: characterization of SHV-111, from a cheese isolate of Klebsiella pneumoniae, with an amino acid substitution in a strictly conserved position (P174S) in the Ω-loop
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Emergence of a novel β-lactamase in food of animal origin: characterization of SHV-111, from a cheese isolate of Klebsiella pneumoniae, with an amino acid substitution in a strictly conserved position (P174S) in the Ω-loop

机译：动物源性食物中新型β-内酰胺酶的出现：Shv-111的表征，来自克雷斯氏菌菌的奶酪孤立，在ω环中严格保守位置（P174s）中的氨基酸取代

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The Ω-loop is a conserved structural element in all class Aβ-lactamases. Amino acid substitutions in this loop appearto change the conformational flexibility of the catalyticregion, resulting in the evolution of b-lactamases. The step-wise accumulation of mutations observed during the emer-gence of extended-spectrum b-lactamases (ESBLs) makes ithighly probable that higher-order combinations of muta-tions occurred in clinical settings potentially after exposureto antibiotics [1]. One mutation may change the active sitetopology, another may enhance catalytic activity and somemay be compensatory. The current hypotheses for the evo-lution of b-lactamases are drawn mainly from human clini-cal studies and in vitro evolution experiments. However, asstated by Martínez et al. [2] ‘an accurate prediction of evolu-tionary trajectories, all the steps in evolution that arerequired to produce a given phenotype, requires bettermeasurements and a more complete understanding of allselection pressures in both human and animals, environ-mental variations and evolutionary constraints that consti-tute the real determinants for the correct naturalevolutionary pathways’. Therefore, understanding the effectof novel amino acid substitutions on the phenotypic charac-teristics of SHV b-lactamases that keep the catalytic siteaccessible for even traditional b-lactam antibiotics is ofutmost importance. In light of the scarcity of data on therole of non-clinical isolates in the evolution of b-lactamases,we characterized a novel SHV b-lactamase, SHV-111, froma cheese isolate (KP-DC6-2), which was identified duringmonitoring of traditional Egyptian Domiati cheese forESBLs using conserved primers [3]. The bla SHV-111 gene wasamplified by PCR using the primer pair SHV-F2 (CGGCCTTCACTCAAGGATGTA) and SHV-R2 (GTGCTGC-GGGCCGGATAAC). The purified whole PCR fragment ofthe bla SHV-111 gene containing only the coding region wascloned into the SmaI-digested plasmid vector pBAD33 using the TaKaRa Ligation kit (Takara Bio). This cloningvector has a chloramphenicol resistance gene and the P BADpromoter of the araBAD (arabinose) operon for geneexpression. The new recombinant plasmid was namedpSHV-111 and used to transform Escherichia coli TG1 forexpression of the cloned bla SHV-111 . E. coli TG1 expressingthe bla SHV-111 gene (TG1/pSHV-111) was phenotypicallycharacterized by the broth microdilution method. Theresults were interpreted according to CLSI [4] guidelines foran ESBL producer: an MIC value of at least one extended-spectrum cephalosporin (ceftazidime and ceftriaxone) oraztreonam of ?2 mg l ?1 . Analytical IEF was conducted todetect the isoelectric point (pI) and Southern blot analysiswas carried out for localization of this gene. Sequence analy-sis of the bla SHV-111 gene (GenBank accession number,AB372881) revealed five silent nucleotide substitutions (incomparison with bla SHV-1 , GenBank accession number,AF148850) of CfiT, AfiG, GfiA, CfiG and CfiG at posi-tions 357, 402, 705, 759 and 786, respectively. Interestingly,a codon change of CCC to TCC resulted in one amino acidsubstitution, serine instead of proline at the strictly con-served Ambler position 174, namely, P174S. This aminoacid substitution was not enough to widen its substrate pro-file (Table 1) to meet the criteria of CLSI [4] for ESBL pro-ducers. Analysis of the b-lactamase content by IEF showedthe presence of a single b-lactamase co-focusing with SHV-1 (pI 7.6). Southern hybridization analysis revealed chromo-somal localization of this gene (data not shown).
机译：ω-环是所有类Aβ-乳酰胺酶中的保守结构元素。该环中的氨基酸取代似乎改变了催化剂的构象柔韧性，导致B-内酰胺酶的演变。在扩展光谱B-乳酰胺酶（ESBLS）的emer斜率期间观察到的突变的逐步积累使得曝光抗生素抗生素抗生素潜在临床环境中的临床环境中发生的近阶的突变组合[1]。一个突变可以改变活跃的入学性学，另一个突变可以增强催化活性，并且有些突变是补偿性的。对B-乳酰胺酶的EVO-疏乳的目前的假设主要来自人临床研究和体外进化实验。然而，由Martínez等人的愿望。 [2]'对Evolu -tionary轨迹的准确预测，所有进化的步骤都需要产生给定表型，需要更好的衡量，并更完全了解人类和动物的所有选择压力，环境变化和进化的限制Consti-Tute对正确的自然途径的真实决定因素'。因此，了解新型氨基酸取代对Shv B-乳酰胺酶的表型Charac-Teristics的影响，使催化位于传统的B-内酰胺抗生素甚至是传统的B-内酰胺抗生素的重要性。鉴于B-内酰胺酶的演化中的非临床分离物的数据的稀缺性，我们表征了一种新的SHV B-Lactamase，SHV-111，Froma奶酪分离物（KP-DC6-2），其在称为期间鉴定传统的埃及Domiati奶酪ForeSBLS使用保守的引物[3]。通过PCCR使用引物对SHV-F2（CGGCCTTCACCAAGGATGTA）和SHV-R2（GTGCTGC-GGGCCGACAC）通过PCR溶解BLA SHV-111基因。使用Takara结扎试剂盒（Takara Bio）仅含有编码区的BLA SHV-111基因的纯化的整个PCR片段含有编码区域的编码区。该克隆仪具有氯霉素抗性基因和Arabad（阿拉伯糖）的P Badpromoter进行Geneexpression。新的重组质粒是NamedPSHV-111，用于转化克隆BLA SHV-111的大肠杆菌TG1表达。表达BLA SHV-111基因（TG1 / PSHV-111）的大肠杆菌TG1通过肉汤微稀释方法表现出了表型。根据Clsi解释了Theresults [4]指南澳大利亚州ESBL生产者：至少一种扩展谱头孢菌素（头孢他啶和头孢菌和头孢菌）oraztreonam的MIC值（ceftazidime和头孢菌蛋白）oraztreonam分析IEF进行了对该基因定位进行的等电点（PI）和Southern印迹分析。 BLA shv-111基因的序列分析（Genbank登录号，AB372881）揭示了五个沉默的核苷酸取代（与BLA Shv-1，Genbank登录号，AF148850）的CFIT，AFIG，GFIA，CFIG和CFIG在POSI - 分别为357,402,705,759和786。有趣的是，CCC对TCC的密码子改变导致一个氨基酸，丝氨酸代替脯氨酸在严格的倒置位置174，即P174s。这种氨基酸替换不足以扩展其基板Pro文件（表1）以满足ESBL Pro-DECERS的CLSI [4]的标准。 IEF的B-in酰胺酶含量分析显示出单一B-乳酰胺酶共聚焦的存在，具有SHV-1（PI 7.6）。 Southern杂交分析显示了该基因的Chromo-omal定位（数据未显示）。

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