Basic characteristics of the CREC isolates
A total of 29 consecutive nonduplicate CREC isolates were identified during 2010-2019 (Table 1), which originated from different anatomical sites: urine (n=8, 27.6%), blood (n=6, 20.7%), drainage (n=5, 17.2%), sputum (n=5, 17.2%), puncture fluid (n=1, 3.4%), catheter (n=1, 3.4%), tissue (n=1, 3.4%), secretion (n=1, 3.4%), and semen (n=1, 3.4%). The majority of patients were in the intensive care unit (n=9, 31.0%), followed by hepatobiliary surgical ward (n=3, 10.3%), urinary surgery ward (n=3, 10.3%), respiratory ward (n=3, 10.3%), cardiac surgery ward (n=2, 6.9%), surgical clinic (n=2, 6.9%), orthopedic ward (n=1, 3.4%), hematology ward (n=1, 3.4%), neurosurgery ward (n=1, 3.4%), neonatal ward (n=1, 3.4%), infection ward (n=1, 3.4%), otolaryngology clinic (n=1, 3.4%), and emergency center (n=1, 3.4%).
The first isolate of CREC dates back to 2012 in the hematology ward. During the 10-year period, the CREC detection rate increased notably from 0.00% in 2010 to 6.48% in 2019, with a peak of 8.11% in 2018 (Fig. 1). This rapid increase deserves further molecular epidemiology research.
Clinical outcomes of CREC infections
The overall 30-day crude mortality of CREC infection/colonization was 13.8% (4/29). The specimen types of the four patients were as follows: drainage (n=2), catheter (n=1), sputum (n=1). None of the patients with bloodstream infections died. Furthermore, all four patients were accompanied with other infections: two patients with serious abdominal infection, one patient with cytomegalovirus pneumonia, and one patient with Acinetobacter baumannii bloodstream infection. This indicated that CREC may be not the main reason of death.
Antimicrobial susceptibility testing
The antimicrobial susceptibility testing was summarized in Table 2, which showed that 100.0% (18/18) isolates and 72.2% (13/18) isolates were resistant to imipenem and meropenem respectively. Among them, nine isolates showed extreme resistance (MIC ≥16) to both drugs. In addition, these isolates showed high resistance to ceftriaxone (94.4%), ceftazidime (94.4%), piperacillin/tazobactam (77.8%), cefepime (72.2%), ciprofloxacin (66.7%), and levofloxacin (61.1%). Moreover, 55.6%, 55.6%, 44.4% and 44.4% strains were resistant to gentamicin, sulfamethoxazole-trimethoprim, nitrofurantoin and tobramycin respectively. In contrast, 100%, 100% and 22.2% were susceptible to tigecycline, polymyxin B and amikacin.
Carbapenemase producers were detected in 94.4% (17/18) of the isolates (Table 3). Overall, 94.4% (17/18) of the CREC isolates harbored carbapenemase-encoding genes. Among them, four types of carbapenemases were detected in these isolates: blaNDM-1 (n=9, 50.0%), blaNDM-5 (n=7, 38.9%), blaIMP-4 (n=2, 11.1%)，and blaKPC-2 (n=1, 5.6%). Of note, co-occurrence of blaNDM-1 and blaIMP-4 was identified in two isolates (CMU10 and CMU29). For the β-lactamase genes, eight isolates had TEM-1, three isolates had CTX-M-15, three isolates had CTX-M-3, two isolates had SHV-12, and one isolate had CTX-M-14, indicating that TEM-1 was the prevalent β-lactamase genotype.
It revealed 11 sequence types among the 18 CREC isolates, with ST93 was the predominant epidemic type (n=6, 33.3%), followed by ST171 (n=3, 16.7%) and ST145 (n=2, 11.1%). The other types contained one isolate for each: ST13, ST66, ST114, ST528, ST1120 (n=1, 5.6%).
Clonal relatedness analysis
To analyze the phylogenetic relationships between these 18 isolates, a maximum likelihood tree (Fig. 2A) was constructed with the concatenated sequences (6090 bp) of the seven loci MLST genes and 16s rRNA, which formed two separate clades. Clade A was sub-divided into four subclades, clade A1, A2, A3, A4. Clade A1 were E. hormaechei subsp. steigerwaltii, including ST93 and ST 1120; Clade A2 were E. hormaechei subsp. oharae, including ST66, ST114, and ST171; Clade A3 were E. kobei typed as ST145, Clade A4 were E. hormaechei subsp. hormaechei typed as ST528. Whereas clade B had two sequence types and were identified as E. ludwigii and E. asburiae respectively. In short, phylogenetic analysis of 18 CREC isolates showed genetic diversity with E. hormaechei as the predominant species.
Furthermore, we compared the antimicrobial resistance patterns of E. hormaechei subsp. steigerwaltii (clade A1) and E. hormaechei subsp. oharae (clade A2). As shown in Figure 2B, clade A1 were characterized with higher gentamicin resistance rate relative to clade A2 (p<0.05). However, due to the small sample size, further confirmation was needed.