Drug susceptibility and molecular epidemiology of Escherichia coli in bloodstream infections in Shanxi, China

Setting and study design

This retrospective study was conducted in a general teaching hospital affiliated to Shanxi Medical University (Shanxi Provincial People’s Hospital) in Taiyuan, the capital city of Shanxi Province in North China. Shanxi Provincial People’s Hospital is a comprehensive tertiary hospital, with 2,800 beds and a total of approximately 1.28 million patients annually. The hospital provides better medical services to the people of Shanxi Province, about 370,000 people every year.

The study was approved by the Ethical Committee of Shanxi Provincial People’s Hospital, Shanxi Medical University (Ethical Application Ref: 2020-7). Written informed consent was obtained from participants for our study.

Bacterial isolates

We performed a retrospective cross-sectional study of patients with E. coli BSIs between June 2019 and March 2020. Cases of E. coli BSIs were identified from the laboratory database in the Department of Clinical Laboratory, Shanxi Provincial People’s Hospital. Each patient was included only once, if multiple blood cultures from the same patient were positive, only the first episode was reviewed and recorded. All isolates were identified by matrix-assisted laser desorption ionization-time of flight mass spectrometer (bioMérieux, Marcy-l’Étoile, France) and stored in broth containing 30% glycerol at −80 °C until further experiments.

Antimicrobial susceptibility testing and detection of ESBLs and carbapenemases

Antimicrobial susceptibility testing was carried out by broth microdilution method according to Clinical and Laboratory Standard Institute (CLSI) recommendations (CLSI, 2019) for ampicillin, amoxicillin-clavulanate, piperacillin-tazobactam, cefazolin, cefoxitin, ceftriaxone, cefepime, cefotaxime, cefoperazone-sulbactam, aztreonam, ertapenem, imipenem, meropenem, amikacin, gentamicin, tobramycin, ciprofloxacin, levofloxacin, tigecycline and trimethoprim-sulfamethoxazole. The breakpoint of tigecycline was based on European Committee on Antimicrobial Susceptibility Testing (EUCAST) (EUCAST, 2019), other drugs were interpreted by CLSI and cefoperazone/sulbactam was referred to cefoperazone in CLSI (CLSI, 2019). E. coli ATCC 25922 and ATCC 35218 were used as quality control strains for antimicrobial susceptibility testing.

Broth dilution test (cefotaxime and cefotaxime-clavulanic acid, ceftazidime and ceftazidime-clavulanic acid) was used as confirmatory test for ESBLs producers following CLSI criteria (CLSI, 2019). Either cefotaxime or cefotadime combined with clavulanic acid, the MIC was decreased by threefold concentration, which can be considered as ESBLs positive. Klebsiella pneumoniae ATCC 700603 was used as positive control for ESBLs production. According to the susceptibility results, the ESBLs genes were further detected in the positive isolates.

Detection of resistance genes

Template DNA was extracted by boiling method as mentioned in previous article (Zhao et al., 2015). Polymerase chain reaction (PCR) were performed to screen for bla_TEM, bla_SHV, bla_{CTX-M(−1,−2,−8,−9,−25 group)}, bla_{V EB}, bla_GES, bla_{OXA(−1,−2,−10 group)}, and bla_PER genes, using reverse and forward primers pairs listed in Table 1, as previously described (Wang et al., 2016). Positive amplicons were sequenced using ABI3730xlDNAAnalyzer by Sangon Biotech (Shanghai, China) and the DNA sequences were compared in GenBank (http://blast.ncbi.nlm.nih.gov) to identify the allelic variant.

Phylogenetic group analysis and multilocus sequence typing

According to the well recognized phylogenetic grouping protocol proposed by Clermont, Bonacorsi & Bingen (2000), four major phylogenetic groups (A, B1, B2 and D) in the E. coli isolates were determined using the method of triple PCR based on three genetic markers, namely chuA, yjaA and TspE4.C2. chuA encodes outer membrane hemin receptor gene that involves in heme transport. yjaA encodes for gene responsible for cellular response to hydrogen peroxide and acid stress and TspE4.C2 DNA encodes for putative lipase esterase gene (Javed, Mirani & Pirzada, 2021). PCR was performed to determine the seven conserved housekeeping genes (adk, fumC, gyrB, icd, mdh, purA, and recA) of E. coli, and the primer pairs were listed in Table 2. The allelic profiles and STs were described by the combination of the seven alleles on line (http://mlst.warwick.ac.uk/mlst/dbs/Ecoli/).

Statistical analysis

The statistical analysis was performed using SPSS 25.0 (IBM, Armonk, NY, USA). When fitting a normal distribution, we presented continuous numerical variables as mean and standard deviation (SD). For categorical variables, results were expressed as the percentages of the groups from which they were derived. The χ² test was used to compare categorical variables. All tests were two-sided, and p < 0.05 was considered statistically significant.

Characteristics of total patient population

A total of 76 eligible E. coli isolates were enrolled during the study period. From the total of 76 E. coli causing BSIs they were isolates from females (42/76) and (34/76) from males. The age of patients ranged from 13 to 90 years and the mean age was 62.1 ± 14.9 years. The mean age did not differ significantly in ESBLs-producing isolates compared to non-ESBLs-producing E. coli.

Antimicrobial susceptibility tests

Antimicrobial susceptibility testing revealed that the top 6 E. coli resistant antibiotics were ampicillin (90.7%), ciprofloxacin (69.7%), cefazolin (65.7%), levofloxacin (63.1%), ceftriaxone and cefotaxime (56.5%). On the contrary, carbapenems, piperacillin-tazobactam, amikacin and tigecycline exhibited excellent activity against E.coli isolates in vitro with susceptibility up to 100.0%. Of the 76 isolates, 56.5% were confirmed as ESBLs-producing E. coli. Compared with ESBLs-producing E. coli, the non-ESBLs-producing E. coli showed higher susceptibility to cefazolin, ceftriaxone, cefepime, cefotaxime, aztreonam (P < 0.05) (Table 3). Despite the significant differences, both ESBLs-producing and non-ESBLs-producing E. coli showed lower susceptibility to ampicillin, ciprofloxacin, levofloxacin, trimethoprim-sulfamethoxazole and gentamicin. Out of total 76 E. coli isolates, 56 (73.6%) were multidrug resistant (MDR) (nonsusceptibility to ≥1 agent in ≥3 antimicrobial categories) isolates. The rates of MDR isolates were 97.6% and 42.4% in ESBL-producing and non-ESBL-producing E. coli, respectively.

Characterization of resistance genes

A total of 71 drug-resistant genes including 28 bla_CTX-M-14, 20 bla_TEM-1, 10 bla_CTX-M-15, 7 bla_OXA-1 and 6 bla_CTX-M-55 were identified among the 43 ESBLs-producing isolates, where they have one, two and three ESBLs genes in 19 isolates 20 and 4 isolates respectively. The highest detection rate was bla_CTX-M-14 (39.4%, 28/71), followed by bla_TEM-1 (28.2%, 20/71), bla_CTX-M-15 (14.1%, 10/71), bla_OXA-1 (9.8%, 7/71) and bla_CTX-M-55 (8.5%, 6/71) (Table 3). No bla_SHV, bla_{CTX-M (−2,−8,−25 group)}, bla_GES, bla_{V EB}, bla_{OXA (−2,−10 group)} or bla_PER genes were found.

Phylogenetic characterization

The phylogenetic lineages were determined in the 76 isolates: phylogenetic group D (42.2%) predominated, followed by group B2 (34.2%), group A (18.4%) and group B1 (5.2%). Among the 43 ESBLs-producers, phylogenetic group D (41.8%) predominated, followed by group B2 (37.3%), A (16.2%) and B1 (4.7%). On the other side, among the 33 non-ESBLs-producers, phylogenetic group D (42.4%) predominated, followed by group B2 (30.3%), A (21.2%) and B1 (6.1%). The percentage of phylo-groups identified were the same regardless of the ESBLs producing. Among the 40 bla_CTX-M positive strains, phylogenetic group B2 (40.0%) predominated, followed by group D (37.5%), group A (17.5%) and group B1 (5.0%) (Fig. 1).

Multilocus sequence typing

Among the 76 isolates, a total of 28 different sequence types (STs) were identified in this study (Table 4). The ST131 was the most frequently ST identified (19.7%, 15/76), followed by ST69 (15.7%, 12/76), ST38 (7.8%, 6/76), ST1193 (6.5%, 5/76), ST648 (5.2%, 4/76), ST73 (5.2%, 4/76), ST46 (3.9%, 3/76), ST405 (3.9%, 3/76) and other uncommon STs that were detected in one or two isolates. Among ESBLs-producing isolates, the most prevalent STs were ST131 (27.9%, 12/43), ST69 (9.3%, 4/43) and ST38 (9.3%, 4/43), while among non-ESBLs-producers, the predominant STs were ST69 (24.2%, 8/33), ST73 (12.1%, 4/33) and ST131 (9.0%, 3/33). As shown in Table 4, all ST131 assigned to phylogenetic group B2, harbored the TEM-1 (6/20), CTX-M-14 (11/28), CTX-M-15 (3/10), CTX-M-55 (1/6) and OXA-1 (3/7). All ST69 and ST38 assigned to phylogenetic group D, while, 3 (3/12) ST69 isolates and 4 (4/6) ST38 isolates were ESBL-producers.