Characteristics of CR-EC:
In this study, a total of 10 non-duplicated CR-EC isolates were investigated. The detection rate of blood, urine and sputum was the same (30%). The samples were collected from 8 different clinical departments, with general internal medicine and surgical department accounting for a relatively high proportion (20%). The average age of infected individuals was 64 years old, the median was 66 years old. The general age of infected individuals was relatively high, 60% of them were over 60 years old, and most of them were male (80%). Please refer to Figure 1 for specific details. (Figure 1)
Results of antimicrobial susceptibility testing:
All experimental strains were classified as multidrug-resistant bacteria (MDR) (Magiorakos et al. 2012). (Figure 2) For carbapenems, the resistance rate of CR-EC to ertapenem was 100%, while the resistance rate of imipenem and meropenem was relatively reduced, both of which were 50%. Nevertheless, for traditional antibiotics such as cephalosporins, penicillins and β-lactam combination agents, the resistance rate of CR-EC can reach almost 100%. In addition, CR-EC also showed high resistance to quinolones (90%) and Chloramphenicol (100%) drugs.
Detection of resistance Genes:
blaNDM as the carbapenem resistance gene with the highest detection rate, was carried by 80%. After confirmation by Sanger sequence, the proportions of blaNDM-1, blaNDM-5, and blaNDM-13 were 25% (2/8), 62.5% (5/8), and 12.5% (1/8), respectively. Other common class A, B and D carbapenem genes were not detected. (Figure 3) In addition, the ESBL class gene blaCTX-M also has an extremely high detection rate (80%). Among them, blaCTX-M-14 and blaCTX-M-55 have the highest ratio, both accounting for 37.5%, blaCTX-M-3 (25%) and blaCTX-M-27 (12.5%) were also discovered. The genes related to colistin resistance: mcr-1 and mcr-9 were not detected.
Results of plasmid conjugation test:
Since CR-EC3809 and CR-EC3276 did not carry blaNDM, a total of 8 strains were tested for conjugation, and the conjugation success rate was 75% (6/8). All blaNDM-like was transferred from donor to recipient, while blaCTX-M—like was unable to transfer. The availability of blaNDM-like greatly increased the sensitivity of carbapenems, penicillins and β-lactam combination agents, and its MIC value was the same or slightly lower than that of donor. The specific results are detailed in Table 1.
Homology comparison:
PFGE analysis was conducted using the unweighted pair group method and the arithmetic mean (UPGMA) using a dice coefficient to judge the strain's affinity (Figure 3). The analysis showed two clusters, CR-EC3203 and CR-EC3264 have the same band, so they both belong to the cluster A, similarly, CR-EC3809 and CR-EC6301 belong to cluster B, it had a similar PFGE pattern that exceeded SAB (Similarity coefficient between bacteria) 0.8, thus indicating that these isolates shared a clonal relationship. It is worth noting that no matter how to adjust the experimental process of PFGE, CR-EC5129 and CR-EC6332 still could not get clear and complete bands. We believe that such results are related to the bacteria itself, such as: rapid degradation of nucleic acid, biochemical characteristics of strains, etc. All of the remaining samples have a similarity of < 80%, indicating that they belong to polyclonal and different origins.
In addition to the two novel ST types, ST410 was the most common type (30%, 3/10). While, the detection rates of ST648, ST88, ST1177, ST354, and ST602 are all 10%.
Results of WGS:
After quality control and assembly of all sequencing data, the GC content of all strains was in the range of 50-55%. In addition to blaNDM-like and blaCTX-M-like discovered by PCR, ESBLs (Extended Spectrum Beta-Lactamases): OXA-like, SHV-like and TEM-like; AmpC: CMY-like,ACT-like were also discovered. Besides common aminoglycoside, quinolone, and fosfomycin resistance genes, seven different antibiotic-resistance genes were also carried in CR-EC. A total of 9 plasmid replicators were detected. Among them, 5 strains carried IncFIB replicators. 3 strains carried IncN, 6 strains carried IncFIB, 2 strains carried IncX3 replicators, and only 1 strain carried IncFIA, IncFII, IncHI2, IncI1-1, IncX4 and IncFIC respectively. Please refer to Table 2 for specific carrying information.
The distribution of virulence factors in CR-EC is also not optimistic, with “Identity=100%” as the standard, a total of 6 functional proteins were predicted. According to the phylogenetic analysis method established by Clermont, Escherichia coli can be divided into four evolutionary groups (A, B1, B2, D) (Pitout et al. 2005). Among them, 2 strains belong to Group D, and 6 strains belong to Group A. Related to adhesion and invasion: fimH; related to outer membrane: traT; related to iron metabolism: iutA and sitA, the detection rates of them were 80%, 70%, 60% and 80%, respectively. The types of toxic factors contained in CR-EC are also relatively complex, as detailed in Table 3.