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With the increasing use of antibiotics, extended-spectrum β-lactamase (ESBL) production has become a prominent survival strategy in Escherichia coli, a common pathogen in urinary tract infections (UTIs). Among ESBLs, the CTX-M family has spread rapidly since the late 1990s and become predominant worldwide [1, 2]. In Korea, as in other parts of the world, CTX-M-15 is the most frequent genotype in clinical isolates [3, 4].
CTX-M enzymes exhibit strong hydrolytic activity toward cephalosporins, particularly cefotaxime, and their genes are located on plasmids, enabling horizontal gene transfer [5]. The CTX-M family comprises various subtypes, most of which originate from genetic mutations. New subtype can arise even from single amino-acid substitutions, which may affect enzyme substrate specificity or structural stability [6–8].
Since its discovery in 2007, CTX-M-55, which differs from CTX-M-15 by a single amino-acid substitution (Ala77Val), has been increasingly reported, particularly in China [6, 7, 9]. However, its molecular epidemiology and clinical implications in Korea remain insufficiently characterized, especially in the context of UTIs. Given that even a single point mutation in the CTX-M gene can alter antibiotic resistance patterns, influencing antibiotic options in UTI patients, careful attention should be paid to this emerging subtype.
We identified E. coli with CTX-M-55 genotype among isolates initially presumed to harbor CTX-M-15 and compared their microbiological features, including antimicrobial susceptibility profiles and the co-existence of other virulence factors and antibiotic resistance genes.
E. coli isolates confirmed as the etiological agent of community-acquired acute pyelonephritis were obtained from blood and urine cultures between July 2019 and December 2021 at Hanyang University Seoul Hospital, an 860-bed tertiary-care hospital, in Korea. The study protocol was approved by the Institutional Review Board of the Hanyang University Hospital, Seoul, Korea (IRB No: 2025-07-050), and the requirement for written informed consent was waived because of the retrospective design.
Only the first isolate from each patient was included in the study. E. coli was identified as the etiological agent if it was detected in blood cultures or isolated from urine cultures at a concentration of ≥105 colony-forming units/mL. When blood and urine isolates differed, blood culture isolates were prioritized for analysis.
Isolates were screened for ESBL production according to the CLSI guidelines [10]. Isolates were subjected to disk diffusion tests with ceftazidime, aztreonam, and cefotaxime (30 µg), and those producing inhibition zones with diameters of ≤22 mm for ceftazidime, ≤27 mm for aztreonam, and ≤27 mm for cefotaxime were classified as probable ESBL producers. ESBL production was confirmed using the double-disk synergy test (DDST) [10].
For confirmed ESBL producers, minimum inhibitory concentrations (MICs) were determined to evaluate antibiotic susceptibility. MICs of amoxicillin/clavulanate, aztreonam, cefotaxime, and ceftazidime were measured using a semi-automated system (bioMérieux Vitek, Hazelwood, MO, USA, or MicroScan Dade Behring, West Sacramento, CA, USA). Additionally, MICs of nine additional antibiotics, including cefodizime, cefepime, piperacillin/tazobactam, imipenem, ciprofloxacin, amikacin, fosfomycin, trimethoprim/sulfamethoxazole (TMP/SMX), and nitrofurantoin, were determined using the agar dilution method or broth microdilution method. Susceptibility was interpreted according to the CLSI breakpoints [10].
ESBL-producing isolates were genotypically analyzed. ESBL genes (blaCTX-M, blaSHV, and blaTEM), plasmid-mediated AmpC β-lactamase (blaCMY, blaDHA, blaACT), and plasmid-mediated quinolone resistance-encoding genes (qnr and aac(6′)-lb-cr) were detected using PCR with specific primers [11, 12]. Isolates positive for blaCTX-M-15 were further subjected to Sanger sequencing and mutation analysis via Basic Local Alignment Search Tool (BLAST) searches against reference sequences (GenBank accession No.: JX294480), multiplex PCR-based screening for 11 virulence factors (fimH, papA, papEF, sfa/foc, ompT, hlyA, sat, fyu, iutA, kpsMTII, and usp) [13, 14], phylogenetic classification (A, B1, B2, or D) via multiplex PCR targeting chuA, yjaA, arpA, and TspE4.C2 [15], and multilocus sequence type analysis using PCR products of seven housekeeping genes (adk, fumC, gyrB, icd, mdh, purA, and recA) [16]. Data were statistically analyzed using the chi-square or Fisher’s exact test for categorical variables, and all analyses were conducted using the R version 4.3.2 (R Core Team, R Foundation for Statistical Computing, Vienna, Austria).
In total, 247 isolates were collected as causative pathogens of acute pyelonephritis, and DDST was performed on 179 isolates. ESBL production was experimentally confirmed for 99 isolates. For 38 of these isolates initially identified to possess CTX-M-15, gene sequencing revealed that eight isolates harbored the A77V substitution, conferring them the CTX-M-55 genotype.
Table 1 summarizes the characteristics of the eight CTX-M-55 isolates. Most isolates (6/8) belonged to phylogenetic group B2; the remaining two belonged to group D. Sequence types (STs) were diverse (ST131, ST14, ST3185, ST4456, ST1193, and ST94). Demographic and clinical data of the patients are summarized in Supplemental Data Table S1. Six patients were female, with a median age of 63.5 yrs. Three patients had a history of UTIs, and two required intensive care unit admission; nevertheless, all eight patients survived.
Antimicrobial susceptibility patterns are detailed in Table 2. None of the observed differences reached statistical significance. As for beta-lactam antibiotics, all CTX-M-55 isolates were resistant to aztreonam and cefotaxime, with MICs exceeding 16 and 32 µg/mL, respectively. In comparison, CTX-M-15 isolates had a broader MIC range and lower resistance rates (100% vs. 66.7%, P=0.082). CTX-M-55 isolates were also more resistant to ceftazidime compared with CTX-M-15 isolates (87.5% vs. 56.7%, P=0.216), with the MICs concentrated in the higher section (MIC range, 4 µg/mL to >16 µg/mL; MIC50, >16 µg/mL) compared with CTX-M-15 isolates (MIC range, ≤1 µg/mL to >16 µg/mL; MIC50, 8 µg/mL). Cefepime resistance was higher in CTX-M-55 isolates than in CTX-M-15 isolates, in terms of resistance rate (62.5% vs. 36.7%, P=0.243) and MIC50 (32 µg/mL vs. 1 µg/mL). For other beta-lactam antibiotics, CTX-M-55 isolates showed lower resistance rates compared with CTX-M-15 isolates. For non-beta-lactam antibiotics, resistance rates to amikacin and nitrofurantoin were higher in CTX-M-55 than in CTX-M-15, whereas resistance rates to fosfomycin and TMP/SMX were lower, albeit not significantly.
Molecular characteristics are summarized in Table 3. Most CTX-M-55 (87.5%) and CTX-M-15 (83.3%) isolates belonged to phylogenetic group B2, whereas the proportion of ST131 was lower in CTX-M-55 (25%) than in CTX-M-15 (53.4%) isolates, and the ST distribution was more diverse among CTX-M-55 isolates. TEM-type β-lactamase gene was the most prevalent β-lactamase in both groups (62.5% vs. 46.7%, P=0.693). All CTX-M-55 isolates (100%) and 86.7% of CTX-M-15 isolates harbored at least one PABL gene, and PMQR genes were detected in 87.5% of CTX-M-55 and 80% of CTX-M-15 isolates (P=1.000). Resistance gene and virulence factor molecular profiles differed between the two groups, although not significantly.
Among uropathogenic E. coli isolates initially identified as harboring CTX-M-15 based on PCR, 21.1% (8/38) (95% confidence interval, 0.096–0.373) were confirmed to harbor CTX-M-55. In China, CTX-M-55 has been frequently reported since its first description in 2007 and, in some settings, even surpasses CTX-M-15 in prevalence [9]. In contrast, reports in Korea are limited and mostly involve isolates from environmental or livestock sources [17]. Our findings suggest that CTX-M-55 isolates may be more widespread than anticipated in Korea and even be involved in UTIs.
The A77V mutation, which distinguishes CTX-M-55 from CTX-M-15, stabilizes the structural instability induced by the D240G mutation, which enhances ceftazidime resistance [6, 7]. We found that CTX-M-55-harboring isolates tended to exhibit increased resistance rates to most cephalosporins and demonstrated elevated MIC50 values for ceftazidime and cefepime. Consistent with previous findings [18], most of these isolates also harbored TEM and PMQR genes, raising concerns for multidrug resistance and therapeutic challenges. However, resistance rates of CTX-M-55 producers to β-lactam/β-lactamase inhibitors and imipenem were lower than those of CTX-M-15 producers, suggesting that, as for CTX-M-15, carbapenems or β-lactam/β-lactamase inhibitor combinations may be preferable for managing infections caused by these strains.
Regarding phylogenetic groups, most CTX-M-55-harboring isolates belonged to group B2, which is typically associated with extraintestinal pathogenic E. coli [19]. However, we found CTX-M-55 genotype across diverse STs. ST131, the clone commonly associated with CTX-M-producing E. coli, was less prevalent in CTX-M-55 than in CTX-M-15 [19]. While Zeng et al. have suggested that, unlike other CTX-M subtypes, ST1193 may be a potential epidemic clone driving the dissemination of CTX-M-55 [18], our findings reveal a broader clonal diversity, likely reflecting plasmid-mediated horizontal gene transfer.
This study had several limitations. First, it was a single-center study with a limited sample size, limiting the generalizability of our findings. Furthermore, because of laboratory constraints, broth microdilution tests could not be performed for all antibiotics. The lowest detectable MICs vary among antibiotics, which may have affected precise resistance profiling. Finally, we could not thoroughly delineate the clinical implications of CTX-M-55 in UTIs, and further studies are required. Nonetheless, our study highlights the presence of CTX-M-55 in uropathogenic E. coli in Korea. Our findings indicate that CTX-M-55 may have been underrecognized in clinical settings and should be considered in the management of UTIs.
In conclusion, CTX-M-55 was identified in uropathogenic E. coli isolates from Korea. CTX-M-55-harboring isolates exhibited enhanced MIC50 values for certain cephalosporins, reflecting the enhanced structural stability of the enzyme. Further studies are required to clarify the clinical and epidemiological significance of CTX-M-55.
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