不同类型临床样本来源肺炎克雷伯菌耐药性和毒力分析

Analysis of drug resistance and virulence of Klebsiella pneumoniae from different clinical samples

目的分析不同类型临床样本来源肺炎克雷伯菌(KP)的耐药性、生物膜形成能力、毒力基因和耐药基因的携带特点,及其相互间的联系,为控制医院内感染提供参考。方法收集2021年11月—2022年9月新乡市3家教学医院分离自痰液、尿液(中段尿)和血液样本的148株KP。采用全自动微生物鉴定系统进行细菌鉴定和药物敏感性分析,采用聚合酶链反应(PCR)检测菌株荚膜血清型、毒力基因和耐药基因,采用拉丝试验检测菌株高黏液表型,采用结晶紫染色法检测菌株生物膜的形成情况。结果148株KP中,有55株(37.2%)分离自痰液样本,40株(27.0%)分离自血液样本,53株(35.8%)分离自尿液样本。尿液样本的强生物膜形成菌株分离率(54.3%)高于痰液样本(27.5%)和血液样本(29.7%),差异有统计学意义(P=0.023);耐药率(69.8%)高于分离自痰液样本的菌株(49.1%)(P=0.028)。携带率(85.8%)最高的耐药基因是blaSHV-1。分离自痰液样本的KP毒力基因iroN、rmpA检出率高于尿液样本(P=0.004)和血液样本(P=0.012);wabG、fimH和mrkD基因携带率均>93.2%。携带iroN、iucA、rmpA基因和K1、K2血清型的KP对抗菌药物较敏感,且易检出高黏液表型(P<0.05)。高黏液表型KP菌株同时携带4种及以上毒力基因的构成比高于非高黏液表型KP菌株(P<0.001),且对抗菌药物更敏感(P<0.05)。74.5%的痰液样本来源KP同时携带4种及以上毒力基因,高于血液样本来源(52.5%)和尿液样本来源(49.1%)的KP(P=0.016);形成生物膜的KP菌株wabG、mrkD、uge和fimH基因携带率更高(P<0.05)。结论尿液样本来源的KP耐药率较高,且易形成强生物膜。某些特定毒力基因普遍存在于KP临床分离株中。痰液样本来源的KP毒力基因构成复杂,iroN、rmpA基因与呼吸系统KP感染有一定关系。iroN、iucA、rmpA基因和K1、K2血清型与KP高黏液表型有关,且携带此类基因和血清型的KP菌株对抗菌药物更敏感。

Objective To analyze the characteristics of drug resistance,biofilm forming ability,virulence gene and drug resistance gene in Klebsiella pneumoniae(KP)from different clinical samples,and to provide a reference for controlling nosocomial infection.Methods Totally,148 KP isolates from sputum,midstream urine and blood samples were collected from 3 teaching hospitals in Xinxiang from November 2021 to September 2022.Bacterial identification and drug susceptibility test were performed by automatic microbial identification system.Polymerase chain reaction(PCR)was used to determine serotypes,virulence genes and drug resistance genes.Stringing test was used to determine high viscosity phenotype,and crystal violet staining was used to determine biofilm formation.Results Among 148 KP isolates,55(37.2%)isolates were isolated from sputum samples,40(27.0%)isolates were isolated from blood samples,and 53(35.8%)isolates were isolated from midstream urine samples.The isolation rate of strong biofilm forming isolates in midstream urine samples(54.3%)was higher than those in sputum samples(27.45%)and in blood samples(29.73%)(P=0.027),and the drug resistance rate(69.8%)was higher than that in sputum samples(49.1%)(P=0.028).The highest carrying rate of drug resistance gene was bla SHV-1(85.8%).The determination rates of KP virulence genes,iroN and rmpA,in sputum samples were higher than those in midstream urine samples(P=0.004)and blood samples(P=0.012).The carrying rates of wabG,fimH and mrkD genes were>93.2%.KP carrying iroN,iucA,rmpA genes and K1 and K2 serotypes were sensitive to antibiotics and easily determined high viscosity phenotype(P<0.05).The proportion of KP isolates with high viscosity phenotype was higher than that of KP isolates without high viscosity phenotype(P<0.001),and KP isolates with high viscosity were more sensitive to antibiotics(P<0.05).The 74.5%of sputum samples had KP with 4 or more virulence genes,which was higher than those of blood samples(52.5%)and midstream urine samples(49.1%)(P=0.016).The biofilm forming KP isolates carried more wabG,mrkD,uge and fimH genes(P<0.05).Conclusions The drug resistance rate of KP from midstream urine samples is high,which is easy to form strong biofilm.Some specific virulence genes are prevalent in KP.The composition of KP virulence genes from sputum samples is complex.The iroN and rmpA genes are associated with KP infection in respiratory system.The iroN,iucA,rmpA genes and K1,K2 serotypes are associated with high viscosity phenotypes,and KP isolates containing these genes and serotypes are more sensitive to antibiotics.