Comparison of hsp60 gene sequencing and MALDI-TOF MS for species identification of Enterobacter cloacae complex and the characteristics of resistance mechanisms and clinical features for the third-generation cephalosporin resistant Enterobacter cloacae complex

• Main Authors: Po-Hao Huang, 黃柏豪
• Other Authors: Chung-Yu Chang
• Format: Others
• Language: zh-TW
• Published: 2016
• Online Access: http://ndltd.ncl.edu.tw/handle/52692600509999858675

碩士 === 高雄醫學大學 === 醫學研究所碩士班 === 104 === Enterobacter cloacae complex is ubiquitous in different environments and has been increasingly isolated as nosocomial pathogens. Different species of E. cloacae complex may possess distinct infectious potentials and different pathogenicity towards humans, resulting in different clinical outcomes. However, conventional phenotypic identification of E. cloacae complex was difficult and unreliable. Therefore, relevant studies were lacking. This study aimed to identify species of E. cloacae complex by hsp60 sequencing and MALDI-TOF MS and to compare their application on species identification of E. cloacae complex. Furthermore, the prevalence of multidrug resistance among isolates of the E. cloacae complex in human infections is rising. This study investigated the prevalence of resistance to antimicrobial agents in E. cloacae complex. The β-lactamase genes, class 1 integrons and gene cassettes were characterized by PCR and sequencing. Clinical features of E. cloacae complex infection were also elucidated in this study. One hundred and eighty four isolates of E. cloacae complex were collected consecutively from December 2013 to June 2014 from Kaohsiung Medical University Hospital. hsp60 gene sequencing was performed by amplifying and sequencing a fragment of the hsp60 gene. 95.7% (176/184) of the isolates were assigned to their respective species, subspecies, or genetic clusters by hsp60 gene sequencing. The four most frequently identified species and subspecies were E. hormaechei subsp. steigerwaltii (55/184, 29.9%), E. hormaechei subsp. ohrae (37/184, 20.1%), E. cloacae subsp. cloacae (22/184, 12%) and E. kobei (19/184, 10.3%). MALDI-TOF identified the majority of the isolates as E. cloacae (110/184, 59.8%), followed by E. asburiae (44/184, 23.9%), E. cloacae subsp. cloacae (22/184, 12%), E. kobei (6/184, 3.3%), and E. cloacae subsp. dissolvens (2/184, 1.1%). Comparing hsp60 sequencing with MALDI-TOF, the identification for E. cloacae subsp. cloacae and E. cloacae subsp. dissolvens was the same between these two methods. The results of MALDI-TOF had 22.8% coincidence with those obtained by hsp60 sequencing when taking the hsp60 sequencing as the standard. Sixty four (34.8%) of the 184 isolates were nonsusceptible to one of the third-generation cephalosporins, 51 isolates (27.7%) to ceftazidime and 63 isolates (34.2%) to ceftriaxone. Seven kinds of β-lactamase genes were detected, including SHV-12, CTX-M-15, DHA-1, ACT-1/MIR-1, TEM-1, OXA-1 and IMP-8. Among the 64 third-generation cephalosporin-nonsusceptible isolates, 32 (32/64, 50%) had at least one kind of ESBL, AmpC β-lactamase or carbapenemase genes. Forty-five isolates (45/184, 24.5%) carried the class 1 integron gene intI1. The antibiotic resistant gene cassettes included those encoding resistance to trimethoprim (dfrA7, 12, 15, 27), gentamicin (aadB), streptomycin (aadA1, 2), erythromycin (ereA2), rifampin (arr3), aminoglyco¬side-3’-N-acetyltransferase aac3, and aminoglyco¬side-6’-N-acetyltransferase aac(6&apos;&apos;)-Ib-cr and aac(6&apos;&apos;)-IIc. The non-susceptibilities for seven antibiotics were significantly associated with the presence of class 1 integrons (p < 0.001). These antibiotics were ceftazidime, ceftriaxone, gentamicin, levofloxacin, trimethoprim-sulfamethoxazole, tigecycline and piperacillin/tazobactam. The PFGE analysis revealed that 34 isolates belonged to 13 pulsotypes (A~M). Isolates in the same pulsotype belonged to the same hsp60-based genetic cluster except for pulsotypes B, H and L. The clinical features of E. cloacae complex infection demonstrated that the rates of the third-generation cephalosporin-nonsusceptible isolates were significantly higher in dialysis patients and in 30-day and 100-day mortality. The relevant factors for infection of the third-generation cephalosporin-nonsusceptible isolates including age > 65years old (p = 0.031), inpatient (p = 0.006), renal disease (p = 0.002), dialysis (p = 0.024), catheter usage (p = 0.020) and stay in ICU (p = 0.001). The outcomes revealed the rates of 30-day and 100-day mortality were significantly higher in patients infected with third-generation cephalosporin-nonsusceptible isolates than those in patients infected with third-generation cephalosporin-susceptible isolates. Comparing cluster XI with clusters VI and VIII, cluster XI caused community infections more usually than clusters VI and VIII (cluster XI vs. VI, p = 0.006; cluster XI vs. VIII, p = 0.034). The 30-day and 100-day mortality of cluster XI -infected patients was significantly higher than that of clusters II and VIII-infected patients. Moreover, multivariate analysis showed that stay in ICU was significantly associated with higher patient mortality. In conclusion, hsp60 sequencing can identify species, subspecies and genetic clusters of E. cloacae complex efficiently. MALDI-TOF MS can not identify E. hormaechei and its subspecies. Therefore, hsp60 sequencing is superior to MALDI-TOF MS for species identification of E. cloacae complex. Cluster XI infections usually occur in community. Stay in ICU was significantly associated with patient mortality with E. cloacae complex infections.