Prevalence and Molecular Characterization of Vancomycin Variable Enterococcus faecium Isolated From Clinical Specimens
2024; 44(5): 450-454
Ann Lab Med 2022; 42(2): 203-212
Published online March 1, 2022 https://doi.org/10.3343/alm.2022.42.2.203
Copyright © Korean Society for Laboratory Medicine.
Bongyoung Kim , M.D., Ph.D.1, Jin-Hong Kim , M.D.2, and Yangsoon Lee, M.D., Ph.D.2
Departments of 1Internal Medicine and 2Laboratory Medicine, Hanyang University College of Medicine, Seoul, Korea
Correspondence to: Yangsoon Lee, M.D., Ph.D.
Department of Laboratory Medicine, Hanyang University Seoul Hospital, Hanyang University College of Medicine, 222-1 Wangsimni-ro, Seongdong-gu, Seoul 04763, Korea
Tel: +82-2-2290-8973
Fax: +82-2-2290-9193
E-mail: yangsoon@hanyang.ac.kr
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background: Extraintestinal pathogenic Escherichia coli (ExPEC) causes various infections, including urinary tract infection (UTI), sepsis, and neonatal meningitis. ExPEC strains have virulence factors (VFs) that facilitate infection by allowing bacterial cells to migrate into and multiply within the host. We compared the microbiological characteristics of ExPEC isolates from blood and urine specimens from UTI patients.
Methods: We conducted a single-center, prospective study in an 855-bed tertiary-care hospital in Korea. We consecutively recruited 80 hospitalized UTI patients with E. coli isolates, which were isolated from blood and/or urine, and urine alone between March 2019 and May 2020. We evaluated the 80 E. coli isolates for the presence of bacterial genes encoding the sequence types (STs), antimicrobial resistance, and VFs using whole-genome sequencing (WGS).
Results: We found no significant differences in STs, antimicrobial resistance patterns, or VFs between isolates from blood and urine specimens. ST131, a pandemic multidrug-resistant clone present in both blood and urine, was the most frequent ST (N=19/80, 24%), and ST131 isolates carried more virulence genes, especially, tsh and espC, than non-ST131 isolates. The virulence scores of the ST131 group and the ST69, ST95, and ST1193 groups differed significantly (P<0.05).
Conclusions: We found no STs and VFs associated with bacteremia in WGS data of E. coli isolates from UTI patients. ST131 was the most frequent ST among UTI causing isolates and carried more VF genes than non-ST131 isolates.
Keywords: Escherichia coli, ST131, Urinary tract infection, Whole-genome sequencing, tsh, Virulence factors
Extraintestinal pathogenic
Among multidrug-resistant ExPEC strains, the most frequent sequence type (ST) is ST131, which is globally disseminated and resistant to multiple antibiotics [3, 4]. Recently, ST1193, which is resistant to fluoroquinolones, has spread rapidly [4]. ExPEC strains have virulence factors (VFs), including adhesion molecules, iron acquisition systems, invasion proteins, and toxins, which facilitate infection by allowing bacterial cells to migrate into and multiply within the host. Several ExPEC VF genes, including
ExPEC strains involved in UTIs are believed to express a diverse repertoire of VFs to colonize and infect the urinary tract in an ascending manner [1]. However, the role of VFs in the pathogenesis and clinical outcomes of
We conducted a prospective study in the 855-bed Hanyang University Hospital, Seoul, Korea. We consecutively recruited hospitalized UTI patients with
Clinical factors possibly influencing the culture results of blood and urine specimens
Clinical factor (%) | Blood (N=40) | Urine (N=40) | |
---|---|---|---|
Age, median (IQR) | 72.5 (59–81) | 70 (54.5–78.5) | 0.473 |
Male sex, N (%) | 4 (10.0) | 2 (5.0) | 0.675 |
Time lag between fever or UTI symptom onset and bacterial culture sampling, days, median (IQR) | 2 (1–3.75) | 1 (0–4) | 0.382 |
History of antibiotic use within three days prior to bacterial culture sampling | 8/39 (20.5) | 7 (17.5) | 0.733 |
Antimicrobial use concordant with the antimicrobial susceptibility of the causative organisms | 3/39 (7.7) | 1 (2.5) | 0.359 |
Existence of any underlying urinary tract abnormalities | 18 (45.0) | 17 (42.5) | 0.822 |
Benign prostatic hypertrophy among male patients | 1/4 (25.0) | 2/2 (100) | 0.400 |
Neurogenic bladder | 1 (2.5) | 2 (5.0) | 1.000 |
Urolithiasis | 12 (30.0) | 5 (12.5) | 0.056 |
Urinary retention | 0 (0) | 1 (2.5) | 1.000 |
Vesicoureteral reflux | 0 (0) | 0 (0) | - |
Bladder diverticulum | 0 (0) | 1 (2.5) | 1.000 |
Prolapse of vagina among female patients | 0 (0) | 0 (0) | - |
Intubated urinary tract at the time of culture sampling | 5 (12.5) | 9 (22.5) | 0.239 |
Intermittent catheterization | 0 (0) | 2 (5.0) | 0.494 |
History of urinary catheterization during one month prior to inclusion | 4 (10.0) | 3 (7.5) | 1.000 |
Polycystic kidney | 0 (0) | 0 (0) | - |
Kidney tumor | 0 (0) | 1 (2.5) | 1.000 |
Urinary tract surgery during three months prior to inclusion | 2 (5.0) | 1 (2.5) | 1.000 |
Surgically reconstructed bladder | 0 (0) | 0 (0) | - |
Abbreviations: IQR, interquartile range; UTI, urinary tract infection.
Blood culture bottles were incubated in the BACT/ALERT VIRTUO System (bioMérieux, Marcy l’Étoile, France). Urine specimens were inoculated on blood agar and MacConkey agar plates, and the plates were incubated at 35°C for 24 hours. Bacterial species were identified by matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometry using the MALDI Biotyper system and related software (version 2.3, Bruker Daltonics, Bremen, Germany). Antimicrobial susceptibility was tested using the MicroScan Walkaway system (Beckman Coulter, Brea, CA, USA), and the results were interpreted according to the Clinical and Laboratory Standards Institute (CLSI) guidelines, with intermediate isolates designated as resistant [12]. All isolates were maintained in 20% skim milk at –80°C.
Bacterial isolates cultured on MacConkey agar plates were sent to Macrogen (Seoul, Korea) for WGS that was performed using the Illumina system (Illumina, Inc., San Diego, CA, USA).
The chi-square test (Fisher’s exact test) was used to compare clinical factors, ST, antimicrobial resistance rates between blood and urine groups, and the Kruskal–Wallis test was used to compare virulence scores according to ST. Data was analyzed using SPSS 26 (SAS Institute Inc., Cary, NC, USA), and
The MLST results revealed a diverse population comprising 18 STs (Table 2). The most frequent STs were ST131 (N=19, 24%), ST95 (N=16, 20%), ST69 (N=11, 14%), and ST1193 (N=9, 11%). We detected five ST73 isolates and two ST127 isolates. There were no significant differences in ST frequencies between the blood and urine specimens. ST12 was more frequently found in urine specimens than in blood specimens (
Number of isolates (%) according to MLST in blood and urine specimens
ST- | Blood (N=40) | Urine (N=40) | |
---|---|---|---|
ST131 | 8 (20) | 11 (28) | 0.431 |
ST131- | 6 (15) | 8 (0.556) | 0.556 |
ST131- | 2 (5) | 2 (1.000) | 1.000 |
ST131- | 0 (0) | 1 (0.314) | 0.314 |
ST95 | 9 (23) | 7 (18) | 0.576 |
ST95- | 6 (15) | 3 (0.288) | 0.288 |
ST95- | 1 (3) | 1 (1.000) | 1.000 |
ST95- | 2 (5) | 2 (1.000) | 1.000 |
ST95- | 0 (0) | 1 (0.314) | 0.314 |
ST69- | 8 (20) | 3 (8) | 0.105 |
ST1193 | 4 (10) | 5 (13) | 0.723 |
ST1193- | 3 (8) | 4 (0.692) | 0.692 |
ST1193- | 1 (3) | 1 (1.000) | 1.000 |
ST73 | 3 (8) | 2 (5) | 0.644 |
ST12 | 0 (0) | 4 (10) | 0.040 |
ST38 | 1 (3) | 2 (5) | 0.556 |
ST127 | 0 (0) | 2 (5) | 0.152 |
ST357 | 1 (3) | 1 (3) | 1.000 |
Others* | 6 (15) | 3 (8) | 0.288 |
*Nine isolates belonging to ST10, ST14, ST70, ST372, ST394, ST421, ST457, ST998, and ST2613.
Abbreviations: ST, sequence type; MLST, multilocus sequence type.
The ampicillin and ampicillin/sulbactam resistance rates were 72.5% and 40.0%, respectively, for blood isolates and 75.0% and 45.0%, respectively, for urine isolates. In addition, 20.0% of blood isolates and 27.5% of urine isolates were extended spectrum β-lactamase (ESBL) producers. We found no significant differences in antimicrobial resistance rates between blood and urine isolates (Table 3). However, ST131 and ST1193 isolates showed higher resistance rates than the other isolates. The cefotaxime, ceftazidime, aztreonam, and cefepime resistance rates of ST131 isolates (78.9%) were higher than those of non-ST131 isolates (0%–12.0%). Among the 19 ST131 isolates, 15 carried ESBL-associated genes including CTX-M-15, CTX-M-27, CTX-M-55, and CTX-M-14. CTX-M-14 genes were detected in ST95, ST38, and ST457, and the
Antimicrobial resistance rates (%) according to ST and specimen type
Antimicrobial agent | ST (N of isolates) | Specimen type (N isolates) | |||||
---|---|---|---|---|---|---|---|
ST131 (19) | ST95 (16) | ST69 (11) | ST1193 (9) | Others (25) | Blood (40) | Urine (40) | |
Ampicillin | 89.5 | 31.3 | 81.8 | 88.9 | 80.0 | 72.5 | 75.0 |
Piperacillin | 84.2 | 31.3 | 81.8 | 88.9 | 72.0 | 72.5 | 67.5 |
Cefoxitin | 0 | 0 | 0 | 11.1 | 4.0 | 0 | 5.0 |
Ceftazidime | 78.9 | 6.3 | 0 | 0.0 | 12.0 | 20.0 | 27.5 |
Cefotaxime | 78.9 | 6.3 | 0 | 11.1 | 12.0 | 20.0 | 30.0 |
Cefuroxime | 78.9 | 6.3 | 9.1 | 11.1 | 12.0 | 22.5 | 30.0 |
Aztreonam | 78.9 | 6.3 | 0 | 11.1 | 12.0 | 22.5 | 27.5 |
Cefepime | 78.9 | 6.3 | 0.0 | 0.0 | 12.0 | 20.0 | 27.5 |
Ampicillin/sulbactam | 31.6 | 12.5 | 63.6 | 55.6 | 56.0 | 40.0 | 45.0 |
Amoxicillin/clavulanate | 26.3 | 0.0 | 9.1 | 11.1 | 12.0 | 2.5 | 22.5 |
Piperacillin/tazobactam | 5.3 | 0 | 9.1 | 0 | - | 2.5 | 2.5 |
Doripenem | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
Ertapenem | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
Meropenem | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
Imipenem | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
Amikacin | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
Gentamicin | 47.4 | 0 | 27.3 | 22.2 | 16.0 | 25.0 | 20.0 |
Tobramycin | 52.6 | 0.0 | 27.3 | 22.2 | 8.0 | 22.5 | 20.0 |
Trimethoprim/sulfamethoxazole | 52.6 | 12.5 | 45.5 | 33.3 | 40.0 | 42.5 | 32.5 |
Ciprofloxacin | 73.7 | 6.3 | 9.1 | 100.0 | 8.0 | 30.0 | 37.5 |
Levofloxacin | 73.7 | 0 | 0 | 88.9 | 8.0 | 22.5 | 37.5 |
Chloramphenicol | 0 | 6.3 | 0 | 0.0 | 8.0 | 2.5 | 5.0 |
Fosfomycin | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
Tetracycline | 57.9 | 12.5 | 18.2 | 33.3 | 32.0 | 30.0 | 35.0 |
Minocycline | 0 | 0 | 0 | 11.1 | 16.0 | 5.0 | 7.5 |
Tigecycline | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
Colistin | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
ESBL positive | 78.9 | 6.3 | 0 | 0 | 12.0 | 20.0 | 27.5 |
Resistance rates >50% are indicated in bold.
Abbreviations: ST, sequence type; ESBL, extended-spectrum β-lactamase.
Antimicrobial resistance mechanisms of ESBL-producing and fluoroquinolone-resistant isolates according to ST-
ST- | Antimicrobial resistance mechanism | N isolates | |||
---|---|---|---|---|---|
ESBL gene | QRDR variation in | ||||
ST131- | CTX-M-15 | S83L, D87N | S80I, E84V | I529L | 8 |
CTX-M-14, 27 | S83L, D87N | S80I, E84V | I529L | 2 | |
CTX-M-55 | S83L, D87N | S80I, E84V | I529L | 2 | |
- | S83L, D87N | S80I, E84V | I529L | 1 | |
ST131- | CTX-M-27 | S83L | - | I529L | 2 |
ST131- | CTX-M-27 | S83L, D87N | S80I, E84V | I529L | 1 |
ST95- | CTX-M-14 | - | - | - | 1 |
ST95- | - | S83L, D87Y | S80R | - | 1 |
ST38- | CTX-M-14 | S83L, D87Y | S80I | S458A | 1 |
ST38- | - | S83L, D87N | S80I, E84V | - | 1 |
ST457- | CTX-M-14 | - | - | - | 1 |
ST12- | DHA-1 | - | - | - | 1 |
ST1193- | - | S83L, D87N | S80I | L416F | 5 |
- | S83L, D87N | - | - | 2 | |
ST1193- | - | S83L, D87N | S80I | L416F | 1 |
- | S83L, D87N | - | - | 1 | |
ST69- | - | S83L, D87N | S80I | - | 1 |
Abbreviations: ESBL, extended-spectrum β-lactamase; QRDR, quinolone resistance-determining region; -, not detected; ST: sequence type.
Reference strains were uropathogenic
Proportions (%) of VFs according to ST and specimen type
VF | VF gene | ST (N isolates) | Specimen type (N isolates) | Total (80) | |||||
---|---|---|---|---|---|---|---|---|---|
ST131 (19) | ST95 (16) | ST69 (11) | ST1193 (9) | Others (25) | Blood (40) | Urine (40) | |||
Adherence | |||||||||
100 | 100 | 100 | 100 | 100 | 100 | 100 | 100 | ||
Hemorrhagic | 100 | 100 | 100 | 100 | 100 | 100 | 100 | 100 | |
EaeH | 100 | 100 | 100 | 100 | 100 | 100 | 100 | 100 | |
Type I fimbriae | 100 | 100 | 91 | 100 | 100 | 100 | 100 | 100 | |
P fimbriae | 95 | 94 | 64 | 100 | 80 | 85 | 88 | 86 | |
F1C fimbriae | 0 | 0 | 0 | 0 | 52 | 13 | 20 | 16 | |
Afimbrial adhesin AFA-I | 16 | 6 | 9 | 0 | 32 | 18 | 15 | 16 | |
S fimbriae | 0 | 0 | 0 | 0 | 40 | 10 | 15 | 13 | |
AAF/II fimbriae | 21 | 0 | 9 | 0 | 8 | 15 | 3 | 9 | |
0 | 0 | 0 | 0 | 24 | 8 | 8 | 8 | ||
CFA/I fimbriae | 0 | 0 | 0 | 0 | 16 | 5 | 5 | 5 | |
AAF/III fimbriae | 0 | 0 | 0 | 0 | 4 | 0 | 3 | 1 | |
K88 fimbriae | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | |
Curli fibers | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | |
EtpA | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | |
Dispersin | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | |
Intimin | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | |
Porcine attaching-effacing associated protein | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | |
ToxB | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | |
Autotransporters | |||||||||
UpaG adhesin, trimeric AT | 95 | 100 | 100 | 100 | 88 | 95 | 95 | 95 | |
EhaB, AIDA-I type | 89 | 88 | 100 | 100 | 88 | 93 | 90 | 91 | |
Antigen 43, AIDA-I type | 89 | 69 | 45 | 100 | 60 | 70 | 73 | 71 | |
Temperature-sensitive hemagglutinin | 100 | 6 | 0 | 100 | 32 | 40 | 53 | 46 | |
Sat | 79 | 0 | 55 | 89 | 32 | 55 | 38 | 46 | |
Vacuolating autotransporter gene | 0 | 81 | 9 | 44 | 68 | 45 | 43 | 44 | |
Cah, AIDA-I type | 63 | 38 | 18 | 0 | 44 | 35 | 43 | 39 | |
EspC, SPATE | 100 | 6 | 0 | 0 | 16 | 25 | 35 | 30 | |
Enteroaggregative immunoglobulin repeat protein | 0 | 0 | 100 | 0 | 24 | 28 | 15 | 21 | |
Contact-dependent growth inhibition system | 11 | 0 | 0 | 0 | 48 | 10 | 25 | 18 | |
UpaH, AIDA-I type | 0 | 0 | 9 | 33 | 12 | 10 | 8 | 9 | |
Pic | 0 | 0 | 0 | 0 | 24 | 13 | 3 | 8 | |
AIDA-I | 11 | 0 | 0 | 0 | 0 | 5 | 0 | 3 | |
EhaA, AIDA-I type | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | |
EspI, SPATE | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | |
Pet, SPATE | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | |
AIDA-I type | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | |
AatA, AIDA-I type | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | |
EspP | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | |
Invasion | |||||||||
Invasion of brain endothelial cells | 0 | 0 | 0 | 0 | 16 | 5 | 5 | 5 | |
Tia/Hek | 53 | 88 | 18 | 0 | 68 | 53 | 55 | 54 | |
Iron uptake | |||||||||
Iron/manganese transport | 100 | 100 | 100 | 100 | 92 | 98 | 98 | 98 | |
Heme uptake | 100 | 100 | 91 | 100 | 96 | 98 | 98 | 98 | |
Yersiniabactin siderophore | 100 | 100 | 91 | 100 | 100 | 98 | 100 | 99 | |
Aerobactin siderophore | 74 | 44 | 73 | 89 | 52 | 73 | 53 | 63 | |
Iron-regulated element | 5 | 88 | 18 | 0 | 44 | 40 | 30 | 35 | |
Salmochelin siderophore | 5 | 13 | 27 | 0 | 68 | 28 | 30 | 29 | |
Toxins | |||||||||
Hemolysin/cytolysin A | 100 | 100 | 100 | 100 | 100 | 100 | 100 | 100 | |
Colicin-like Usp | 100 | 100 | 0 | 100 | 72 | 73 | 83 | 78 | |
Enterotoxin SenB/TieB | 79 | 38 | 45 | 56 | 52 | 53 | 58 | 55 | |
Alpha-hemolysin | 53 | 0 | 27 | 0 | 60 | 28 | 43 | 35 | |
Cytotoxic necrotizing factor 1 | 53 | 0 | 0 | 0 | 48 | 20 | 35 | 28 | |
Cytolethal distending toxin | 0 | 0 | 0 | 0 | 4 | 0 | 3 | 1 | |
Shiga-like toxin | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | |
Heat-labile enterotoxin | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | |
Heat-stable enterotoxin 1 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | |
Enterotoxin 1 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
Abbreviations: ST, sequence type; VF, virulence factor; CFA, colonization factor antigen; AAF, aggregative adhesion fimbria; SPATE, serine protease autotransporters of
In ST131 and ST1193 isolates, yersiniabactin and aerobactin siderophores were more frequent than salmochelin siderophores. In contrast, salmochelin siderophores were more frequent (68%) in other isolates than ST131 (5%) and ST1193 (0%) isolates. Several toxin genes were detected in UTI-associated isolates. The hlyE gene was detected in all isolates. The
The median virulence score for all 80 isolates was 15 (range, 0–21). Virulence scores differed significantly among ST groups (Fig. 1). ST12 and ST73 isolates had high virulence scores, ranging from 17–20.
We hypothesized that we could define specific VFs of
We classified isolates according to ST-
Adhesins, including fimbriae and afimbrial adhesins, play a significant role in host cell colonization [7, 15]. We detected type I fimbriae,
Some of the most frequently detected toxin genes in ExPEC are
ST131 isolates carried more VF genes, including
ST131 isolates reportedly had a higher virulence potential than other
Our study had some limitations, mainly related to the relatively small specimen size. Because of the limited number of
In conclusion,
None.
Lee Y and Kim B designed the study; Kim J collected and identified clinical isolates and performed molecular studies; Lee Y, Kim B, and Kim J analyzed the data; Lee Y and Kim B wrote, edited, and reviewed the manuscript. All authors revised and accepted the final version of the manuscript.
No potential conflicts of interest relevant to this article were reported.
This work was supported by a National Research Foundation of Korea (NRF) grant funded by the Korean government (MSIT) (2019R1F1A1061400).