Zhang Zhao, Zhai Yao, Guo Yatao, Li Daixi, Wang Zhanwei, Wang Jing, Chen Yusheng, Wang Qi, Gao Zhancheng
1 Department of Respiratory and Critical Care Medicine, Peking University People's Hospital , Beijing, China .
2 Laboratory Medicine, Peking University People's Hospital , Beijing, China .
Microb Drug Resist. 2018 Jul/Aug;24(6):799-806. doi: 10.1089/mdr.2017.0018. Epub 2017 Nov 1.
The current investigation explores whether extended-spectrum β-lactamase (ESBL) genes exist in clinical non-ESBL-producing Klebsiella pneumoniae isolates.
A total of 202 clinical isolates with non-ESBL-producing K. pneumoniae were collected from southern and middle of China. Thirteen β-lactamase genes (bla, , , , , , , , , , , , ) were screened by PCR and their identity confirmed by sequencing of PCR products. The ESBL-producing phenotype of the isolates that carried ESBL genes was tested and confirmed in 9 of the 18 isolates by a double-disc synergy test. The sequences upstream of ESBL genes of isolates with ESBL-producing genotype (+)/phenotype (-) were also subjected to PCR and sequencing. The ESBL genes and their upstream regions were cloned into Escherichia coli DH5α for functional evaluation.
A total of 8.9% (18/202) isolates carried ESBL genes. All of them harbored only one ESBL gene, including 33.3% (6/18) bla and 66.7% (12/18) bla. Among the isolates carrying ESBL genes, nine isolates were confirmed as ESBL phenotype (-). The ESBL genotype (+)/phenotype (-) isolates had bla (66.7%, 6/9) and bla (33.3%, 3/9). The upstream gene sequences, including promoters of these unexpressed ESBL genes, were intact without any mutations or spacers and effective among eight strains. The ISEcp1 element in the upstream region was not found in one isolate carrying an unexpressed bla gene.
Clinical non-ESBL-producing K. pneumoniae isolates could carry ESBL genes with intact promoter, but without the correlated phenotype. Specific silencing mechanisms may play an important role in regulating ESBL gene expression. This kind of isolates has the potential to transfer their ESBL genes to other bacteria with effective promoters, resulting in ESBL phenotype.
本研究探讨临床非产超广谱β-内酰胺酶（ESBL）的肺炎克雷伯菌分离株中是否存在ESBL基因。
从中国南部和中部地区收集了202株非产ESBL的肺炎克雷伯菌临床分离株。通过PCR筛选13种β-内酰胺酶基因（bla、、、、、、、、、、、、），并通过PCR产物测序确认其同一性。对携带ESBL基因的分离株进行产ESBL表型检测，18株中有9株通过双纸片协同试验得到确认。对产ESBL基因型（+）/表型（-）的分离株的ESBL基因上游序列也进行了PCR和测序。将ESBL基因及其上游区域克隆到大肠杆菌DH5α中进行功能评估。
共有8.9%（18/202）的分离株携带ESBL基因。它们均仅携带一种ESBL基因，其中bla占33.3%（6/18），bla占66.7%（12/18）。在携带ESBL基因的分离株中，9株被确认为ESBL表型（-）。ESBL基因型（+）/表型（-）的分离株中bla占66.7%（6/9），bla占33.3%（3/9）。这些未表达的ESBL基因的上游基因序列，包括启动子，均完整无任何突变或间隔序列，且在8株菌株中有效。在一株携带未表达bla基因的分离株中未发现上游区域的ISEcp1元件。
临床非产ESBL的肺炎克雷伯菌分离株可携带启动子完整但无相关表型的ESBL基因。特定的沉默机制可能在调节ESBL基因表达中起重要作用。这类分离株有可能将其ESBL基因转移至其他具有有效启动子的细菌，从而导致ESBL表型。
