https://orcid.org/0000-0003-1108-0600
Figures
Abstract
Escherichia coli are one of the commonest bacteria causing bloodstream infection (BSI). The aim of the research was to identify the serotypes, MLST (Multi Locus Sequence Type), virulence genes, and antimicrobial resistance of E. coli isolated from bloodstream infection hospitalized patients in Cipto Mangunkusumo National Hospital Jakarta. We used whole genome sequencing methods rather than the conventional one, to characterized the serotypes, MLST (Multi Locus Sequence Type), virulence genes, and antimicrobial resistance (AMR) of E. coli. The composition of E. coli sequence types (ST) was as follows: ST131 (n = 5), ST38 (n = 3), ST405 (n = 3), ST69 (n = 3), and other STs (ST1057, ST127, ST167, ST3033, ST349, ST40, ST58, ST6630). Enteroaggregative E. coli (EAEC) and Extra-intestinal pathogenic E. coli (ExPEC) groups were found dominant in our samples. Twenty isolates carried virulence genes for host cells adherence and 15 for genes that encourage E. coli immune evasion by enhancing survival in serum. ESBL-genes were present in 17 E. coli isolates. Other AMR genes also encoded resistance against aminoglycosides, quinolones, chloramphenicol, macrolides and trimethoprim. The phylogeny analysis showed that phylogroup D is dominated and followed by phylogroup B2. The E. coli isolated from 22 patients in Cipto Mangunkusumo National Hospital Jakarta showed high diversity in serotypes, sequence types, virulence genes, and AMR genes. Based on this finding, routinely screening all bacterial isolates in health care facilities can improve clinical significance. By using Whole Genome Sequencing for laboratory-based surveillance can be a valuable early warning system for emerging pathogens and resistance mechanisms.
Citation: Paramita RI, Nelwan EJ, Fadilah F, Renesteen E, Puspandari N, Erlina L (2020) Genome-based characterization of Escherichia coli causing bloodstream infection through next-generation sequencing. PLoS ONE 15(12): e0244358. https://doi.org/10.1371/journal.pone.0244358
Editor: Iddya Karunasagar, Nitte University, INDIA
Received: August 21, 2020; Accepted: December 9, 2020; Published: December 23, 2020
Copyright: © 2020 Paramita et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All FASTA raw files are available from BioProject accession number PRJNA596854 and Sequence Read Archive accession numbers SRR10761126–SRR10761147.
Funding: This research was funded by Universitas Indonesia (Q1Q2 Grant; grant number NBK-0220/UN2.R3.1/HKP.05.00/2019).
Competing interests: The authors have declared that no competing interests exist.
Introduction
There is currently enlarge in the prevalence of infections worldwide due to multidrug-resistant (MDR). Due to their correlation with a high level of mortality and morbidity which are triggered by the insufficient of potent antibiotics, Gram-negative bacteria became a critical threat to global public health [1–3]. The production of Extended-spectrum beta-lactamase (ESBL) became the most significant determinant of AMR that spreads rapidly among Enterobacteriaceae [4, 5].
Escherichia coli naturally inhabits the human gastrointestinal tract (GIT) and classified as Gram-negative commensals bacterium. Two main classification of pathogenic E. coli—diarrheagenic E. coli (DEC) and extra-intestinal pathogenic E. coli (ExPEC)—are perceived, varying in their associated clinical syndromes and virulence genes. Extraintestinal pathogenic E. coli (ExPEC) can cause a diversity of infections, including sepsis, neonatal meningitis, and urinary tract infections (UTI). The urinary tract is the most commonly infected by these bacteria, which then, is a common source for bloodstream infections [6]. Through horizontal transfer and other mechanisms, commensal E. coli regularly obtains pathogenicity, virulence and multi-drug resistance characteristics of the pathogenic E. coli. Virulent E. coli strains then share pathogenic, virulence, and resistance genes with avirulent or less virulent strains, allowing the appearance of pathogenesis beyond their natural characters [7].
There are six well-recognized pathotypes of DEC such as enterotoxigenic E. coli [ETEC], enteropathogenic E. coli [EPEC], enteroaggregative E. coli [EAEC], Shiga toxin (Stx)-producing E. coli [STEC], enteroinvasive E. coli [EIEC], and diffusely adherent E. coli [DAEC], are identified as the leading etiological agents of childhood and travellers’ diarrhoea. EAEC has long been considered as an intestinal pathogen and thereof unlikely to cause disease in normal patients outside of the intestinal tract [8]. However, current studies have related EAEC strains with fatal haemolytic uremic syndrome outbreaks and urinary tract infection [8, 9].
Whole genome sequencing (WGS) as a sophisticated molecular diagnostics have shown the emergence of E. coli strains that cause fatal diarrhoea due to the combination of the virulence genes [10]. The virulence genes are important to understand the pathogenic properties of the causative organisms. The virulence gene is an agent that forms itself in or within its host by increasing its able to trigger disease effectively and efficiently. The virulence genes in certain E. coli strains was associated with serious outbreaks in Denmark between 2014–2015 [11]; in Italia between 2016–2017, where it caused 7.1% 30-day mortality rate [12]; in China in 1999, where it caused 177 deaths [13]; and in Germany in 2011, where it killed 54 people [14]. Molecular genomic studies of human infections of E. coli in Indonesia, especially in Jakarta are unusual and no report is available regarding specific subtypes causing infections in Jakarta. The purpose of the study was to characterize the serotypes, MLST (Multi Locus Sequence Type), virulence genes, and antimicrobial resistance of E. coli isolated from bloodstream infection patients in Cipto Mangunkusumo National Hospital Jakarta.
Methods
DNA isolation and whole genome sequencing
From blood samples from bloodstream infection patients the E. coli were isolated and then it cultured in Lactose Broth medium in the laboratory facilities at the Centre for Research and Development of Biomedical and Basic Health Technology, National Institute of Health Research and Development, Ministry of Health, Indonesia, between January–December 2018. The DNA was isolated from cultured E. coli using a Geneaid PrestoTM Mini gDNA Bacteria Kit. All of the isolation and whole genome sequencing methods of 22 samples of Escherichia coli were described in Nelwan, et al, 2020 [15]. This research was performed in strict conformity with the suggestions in the guide of Helsinki Declaration and the research protocol was approved by Health Research Ethics Committee, National Institute of Health Research and Development (HREC-NIHRD) with protocol approval number: LB.02.01/KE.014/2019.
Bioinformatics analysis
The paired-end reads from each Escherichia coli isolate were checked for the quality using FastQC 0.11.4 [16]. Before assembly, all reads with low quality were removed after quality control with Trimmomatic software [17]. Raw genome data have been submitted to the NCBI (https://www.ncbi.nlm.nih.gov/) and are available under BioProject accession number PRJNA596854 (https://www.ncbi.nlm.nih.gov/bioproject/PRJNA596854). According to this research aim, the analyses included: Multi-Locus Sequence Typing (MLST) determination using MLST 2.0.4 [18] (database version: 2.0.0 (2020-06-29)), serotyping using SeroTypeFinder 2.0.1 [19] (database version: 1.0.0 (2019-02-27)), virulence genes identification using VirulenceFinder 2.0 [20] (database version: 2020-05-29), and antimicrobial resistance (AMR) genes identification using ResFinder 3.2 [21] (database version: 2020-06-02). VirulenceFinder identified 29 virulence genes, of which 12 are ExPEC-associated, 16 are EAEC-associated, and 1 is EPEC-associated, irrespective of pathotype. For the detection standard parameters were set as follows: VirulenceFinder and SerotypeFinder, 85% sequence identity and 60% sequence coverage; ResFinder, 90% sequence identity and 60% sequence coverage; MLST, using the seven loci (gyrB, adk, icd, purA, recA, fumC, and mdh) scheme.
In this research, the 24 E. coli genomes were analyzed and compared with 26 diverse E. coli genomes (S1 Table) using PhaME pipelines [22] which applied a maximum likelihood phylogeny by RAxML v7.2.8, with the GTR model of nucleotide substitution, for model of rate heterogeneity we use the GAMMA, and 100 bootstrap replicates. The phylogeny was midpoint-rooted and diagramed using the interactive Tree of Life software (iTOL v.3).
Results
Multi-locus sequence typing and serotyping
From total of 22 E. coli isolates, 12 different STs were identified. Five (22,7%) E. coli samples are belonged to ST131 and all of this five had serotype O25:H4. Each of ST38, ST405, and ST69 were observed three (13,6%) out of 22 E. coli isolates. Of these three, two (66,7%) ST38 had serotype O86:H18, and another one had serotype unknown O-group but belonged to H30. All of the ST405 had serotype O102:H6 and the three of ST69 had different serotype, i.e O15:H6, O15:H18, O15:H1 (Table 1). Each of ST1057, ST127, ST167, ST3033, ST349, ST40, ST58, ST6630 were observed one (4,5%) out of 22 E. coli isolates. Strains with the same serotype and ST typically harboured the same set of virulence genes, such as O25:H4 (ST131) isolates, O102:H6 (ST405), and O86:H18 (ST38).
Distribution of virulence genes
Table 1 as well display the classification of virulence genes among the sequenced E. coli isolates. The virulence genes distribution in pathotypes species of E. coli were described in Table 2. A total of 20 (20,9%) E. coli isolates carried virulence genes for host cells adherence. These contained 11 (55%) for eilA, 10 (50%) for air, 7 (35%) for lpfA, 6 (30%) for iha, 3 (15%) for each nfaE and aap, 2 (10%) for each aggR and aatA, 1 (5%) for each aar, sfaS and eae. A total of 14 E. coli isolates had virulence genes that promote toxin production. The composition of virulence genes that promote toxin was 4 (28,6%) for cnf1, 6 (42,8%) for sat, 3 (21,4%) for vat, 5 (35,7%) for senB, 2 (14,3%) for each pic and astA, 1 (7,1%) for each ireA and tsh. A total of 5 E. coli isolates had virulence genes that produce iron acquisition systems (siderophores). Of these five, 5 (100%) for iroN and 1 (20%) for ireA. A total of 15 (68,2%) E. coli isolates had iss, a component that promotes E. coli immune evasion by increasing serum survival.
Isolates that harboured the virulence genes of EAEC were characteristically typical EAEC by genomic criteria. The EAEC showed highly heterogeneous with the existence of a substantial number of genes typically correlated with other E. coli pathotypes, i.e. extraintestinal pathogenic E. coli (ExPEC) and EPEC. ExPEC genes found among the EAEC strains included i) increased serum survival-encoding gene, iss (63.6%); ii) Secreted autotransporter toxin, sat (18.2%); iii) Plasmid encoded enterotoxin, senB (9.1%); iv) Temperature-sensitive hemagglutinin, tsh (9.1%), and Salmochelin receptor, iroN (18.2%). LpfA was the only virulence gene to differ in frequency between the phylogenetic groups, as it was found in all B1 isolates, phylogroup B2, phylogroup F, and phylogroup D.
Antimicrobial resistance
A total of 22 (100%) E. coli carried genes encoding macrolide resistance enzymes (Table 3). Those include 22 (100%) of mdf(A), 9 (40.9%) of mph(A), 1 (4.5%) for ere(B), 1 (4.5%) of erm(B), and 1 (4.5%) for Inu(F). A total of 19 (86.4%) E. coli isolates showed anti beta-lactam resistance genes. The proportions of genes encoding the beta-lactam enzymes are blaCTX-M-15 in 6 (27.3%) isolates, blaCTX-M-27 in 5 (22.7%) isolates, blaTEM-1B in 8 (36.4%) isolates, blaDHA-1 in 3 (13.6%) isolates, blaOXA-10 in 1 (4.5%) isolate, blaOXA-1 in 3 (13.6%) isolates, blaCTX-M-55 in 2 (9.1%) isolates, blaCARB-2 in 1 (4.5%) isolate and blaSHV-120 in 1 (4.5%) isolate. The overall proportion of dfrA genes encoding trimethoprim resistance enzymes was 12 (54.5%) isolates. The composition of dfrA17 was 5 (22.7%) isolates, followed by 3 (13.6%) for dfrA14, 2 (9.1%) for dfrA12 and 1 (4.5%) for each dfrA1, dfrA7, and dfrA16 genes. The compositions of genes encoding chloramphenicol resistance enzymes were 3 (13.6%) for catB3, 3 (13.6%) for catA1, 3 (13.6%) for floR and 1 (4.5%) for catA2. A total of 15 (63.6%) isolates carried genes against aminoglycoside. The gene aadA5 was detected in 4 (18.2%), aph(6)-Id in 8 (36.4%), aac(3)-IId in 5 (22.7%), aac(6')-Ib-cr in 3 (13.6%), aadA2 in 2 (9.1%), each aadA1 and aph(3”)-Ib in 2 (4.5%), aph(6)-Id in 1(4.5%), Aac(6”)-Iai in 1(4.5%), aac(3)-IIa in 3 (13.6%), and aadA2 in 2 (9.1%) E. coli isolates.
Ten of E. coli isolates were predicted to be resistant to ciprofloxacin (Table 4) due to amino acid substitutions in quinolone-resistance-determining-regions (QRDR) of parC gene (S80I, E84V) and/or QRDR of gyrA gene (S83L, D87N). Moreover, several SNPs resulting in amino acid substitutions were also defined in parE gene (I529L, S458A). From this result, E. coli isolates that show Ciprofloxacin isolates were ST131, ST405, and ST167.
Phylogenomic analysis of the E. coli isolates
In general, E. coli may be distributed into 7 phylogenetic categories as follows: A, B1, B2, C, D, E, and F [29]. Based on the phylogenetic tree of the E. coli isolates and 26 E. coli references as shown in Fig 1, eleven strains of the sequenced EAEC strains owned to the phylogroup D, and one strain of phylogroup F. Among phylogroup D, there were four subgroups, which showed in lineages 1–4. Strain ST349 was the only strain in lineage 1; lineage 2 strains included O102:H6 (ST405); lineage 3 strains included ST38; lineage 4 strains included ST69. Among phylogroup B2, there was also 4 lineage. In lineage 1, H31 isolate was included; O154:H5 (ST1057) isolate which is the only strain available, included in lineage 2; lineage 3 strains included four of O25:H4 (ST131); one of the O25:H4 (ST131) isolate was included in lineage 4 because it had different virulence genes than the other four (Fig 1).
Discussion
This research showed a huge variation of E. coli strains prevalent in Cipto Mangunkusumo hospital as assessed by Multi-Locus Sequence Typing (MLST) and Serotyping. Meanwhile, strains O25:H4-ST131 was found to predominate among other 11 ST: ST38, ST405, ST69, ST1057, ST127, ST167, ST3033, ST349, ST40, ST58, ST6630. Each of ST38, ST405, and ST69 was found in three isolates. But for each ST38 and ST69 have different serotypes within it. These results are identical to the results from other regions that described the distribution and prevalence of these endemic clones in health facilities [30–32]. The variation of clones discovered in the hospital might propose sporadic introductions of various strains to the hospital from the community. To investigate whether identical STs are clonal associated, SNP differences among isolates and phylogenetic assessment proposed the presence of multiple clones of E. coli in these settings.
Generally, the levels of antimicrobial resistance in E. coli isolates were inspected to be elevated. Based on assessment of resistance genes, 22 of the E. coli isolates carried genes encoding Macrolide, with mdf(A) being prevalent (100% appeared in 22 isolates) followed by mph(A), ere(B), erm(B), and Inu(F). Gene mdfA encodes a putative membrane protein (mdfA) of 410 amino acid residues which are the main facilitators of the transport protein superfamily. Cells that express mdfA from multicopy plasmids are considerably more resistant to various groups of zwitterionic lipophilic or cationic compounds such as benzalkonium, rhodamine, tetracycline, daunomycin, ethidium bromide, puromycin, rifampin, and tetraphenylphosphonium. Although, mdfA also provides resistance to chemically unrelated, clinically essential antibiotics such as fluoroquinolones, erythromycin, chloramphenicol, and aminoglycosides [33]. Gene mph(A) conferring resistance to macrolides including azithromycin and erythromycin also was discovered in a high quantity. The appearance of the mph(A) in E. coli isolates of the current research was 40.9%, and thus, higher than the 13% that was identified in E. coli from 5 countries from 4 continents by Nguyen et al [34]. and lower than the 50% that was found in E. coli from tertiary care hospital in Moshi, Tanzania by Sonda et al [7]. High risks to azithromycin and erythromycin can be one of the feasible reasons that allow the emergence of resistance to macrolides [35].
In this study, blaTEM-1B being predominant among other genes encoding beta-lactamase and followed by blaCTX-M-15 and blaCTX-M-27. In a tertiary hospital in Dar es Salaam, Manyahi et al [36] found blaCTX-M-15 as the most predominant gene (90.6%) and Sonda et al. [7] who found blaOXA-1 as the most predominant gene in a tertiary hospital in Moshi, Tanzania. In the previous research, O25:H4-ST131 was mentioned to be a main reason of MDR E. coli infections [7, 37, 38]. The presence of aac(6’)Ib-cr encodes low ciprofloxacin resistance by itself (as well as aminoglycoside resistance), and generally requires further mutations (e.g. in the gyrA or parC chromosomes) to provide high levels of resistance. Other research in the Korea [32], US [39], and Brazil [40] have confirmed identical results with this research of the co-carriage of aac(6’)Ib-cr, ESBL genes and the presence of ciprofloxacin resistance in E. coli ST131. This plausibly clarify the discovered relationship between transport of aac(6’)Ib-cr and CTX-M ESBL and ciprofloxacin resistance [41].
This current research also characterized virulence genes in all E. coli isolates. Based on our results, we found the specific virulence genes in ExPEC and EAEC groups. Adherence protein (iha) was found dominating in ExPEC adherence virulence genes. We also found several toxins which belong to ExPEC group, such as cnf1, ireA, sat, vat, senB, pic, and tsh. Another prevalent group of virulence gene were liable for E. coli immune evasion by increasing serum survival (iss). The iss gene recognized for its role in ExPEC for enhanced survival of bacteria in the serum. The iss gene is located on plasmid ColV, a huge virulence plasmid typical of avian pathogenic E. coli strains, which reveal that a replacement of plasmids and, as the result, replacement of those virulence genes is feasible between human and avian pathogenic E. coli strains [25].
EAEC-specific virulence genes present in our 12 isolates included aggregative adherence fimbria (AAF) variant I; AggR, a global regulator of EAEC virulence; AggR-activated regulator, aar; dispersin, required for proper dispersal of AAFs on the bacterial surface; and the aat transporter system, which mediates dispersin secretion [42]. The aggR gene was found in ST38 (H30) sample and both aggR and aar, was found in ST69 (O15:H18) sample. Strains harbouring the aggR regulon or its constituents have been termed typical EAEC. We also found two samples that harbouring EAEC toxin, i.e. astA, which both of them belong to ST38 (O86:H18). These virulence genes have been defined as suggestive of virulent serotypes and may be used as reliable markers for the detection of pathogenic E. coli [43].
Based on our results, the existence of a substantial number of genes characteristically correlated with another E. coli pathotypes, such extraintestinal pathogenic E. coli (ExPEC). ExPEC genes found among the EAEC strains included i) increased serum survival-encoding gene, iss (63.6%); ii) Secreted autotransporter toxin, sat (18.2%); iii) Plasmid encoded enterotoxin, senB (9.1%); iv) Temperature-sensitive hemagglutinin, tsh (9.1%), and Salmochelin receptor, iroN (18.2%). The similar result was also found in another study by Boisen et. al. 2020 [23], that showed ten ExPEC genes presented in EAEC strains. These finding showed that related to virulence genes, EAEC strains are highly heterogeneous.
Potential clinical implications for the results received are that caution must be taken when explaining and utilizing microbiology outcomes. A study from Havt et al [44], showed that malnutrition status of children aged 6–24 months was related to virulence genes of aatA and aar. This research underscore that E. coli should not be considered as non-pathogenic until the determinants of pathogenic and antimicrobial resistance have been verified to be missing. In addition, the WGS-based result suggested that there are nosocomial transmissions in the hospital, thus encouraging the formulation of pragmatic antimicrobial management, control initiatives and infection prevention.
We understand and acknowledge the limitations of this study. The assessment was carried out on a limited number of E. coli isolates, which may limit generalization of the results. With a limited number of the isolates analyzed, it is essential to figure out that another limitation that this work may suffer is the insufficiency of deeper statistical analysis to associate the isolates resistance and virulence results with patient characteristics such as room, age, ward, and gender. Also, the presence of genes that encode various resistance and virulence genes only shows the genes that are in the isolates.
Conclusions
The E. coli isolated from 22 patients in Cipto Mangunkusumo National Hospital Jakarta showed high diversity in serotypes, sequence types, virulence genes, and AMR genes. Based on this finding, routinely screening all bacterial isolates in health care facilities can improve clinical significance. By using Whole Genome Sequencing for laboratory-based surveillance can be a valuable early warning system for emerging pathogens and resistance mechanisms.
Supporting information
S1 Table. E. coli reference genomes included in the phylogenomic analysis.
https://doi.org/10.1371/journal.pone.0244358.s001
(XLSX)
References
- 1. Pitout JD, Laupland KB. Extended-spectrum β-lactamase-producing Enterobacteriaceae: an emerging public-health concern. The Lancet Infectious Diseases. 2008. pmid:18291338
- 2. Frieri M, Kumar K, Boutin A. Antibiotic resistance. Journal of Infection and Public Health. 2017. pmid:27616769
- 3. Kaye KS, Pogue JM. Infections Caused by Resistant Gram-Negative Bacteria: Epidemiology and Management. Pharmacotherapy. 2015; pmid:26497481
- 4. Poirel L, Bonnin RA, Nordmann P. Genetic support and diversity of acquired extended-spectrum β-lactamases in Gram-negative rods. Infection, Genetics and Evolution. 2012. pmid:22414916
- 5. Storberg V. ESBL-producing Enterobacteriaceae in Africa—a non-systematic literature review of research published 2008–2012. Infection Ecology and Epidemiology. 2014. pmid:24765249
- 6. Daga AP, Koga VL, Soncini JGM, De Matos CM, Perugini MRE, Pelisson M, et al. Escherichia coli Bloodstream Infections in Patients at a University Hospital: Virulence factors and clinical characteristics. Front Cell Infect Microbiol. 2019;9(JUN).
- 7. Sonda T, Kumburu H, van Zwetselaar M, Alifrangis M, Mmbaga BT, Aarestrup FM, et al. Whole genome sequencing reveals high clonal diversity of Escherichia coli isolated from patients in a tertiary care hospital in Moshi, Tanzania. Antimicrob Resist Infect Control. 2018;7(72):1–12.
- 8. Mandomando I, Vubil D, Boisen N, Quintó L, Ruiz J, Sigaúque B, et al. Escherichia coli st131 clones harbouring AGGR and AAF/V fimbriae causing bacteremia in Mozambican children: Emergence of new variant of FIMH27 subclone. PLoS Negl Trop Dis. 2020;14(5):1–21. pmid:32357189
- 9. Herzog K, Engeler Dusel J, Hugentobler M, Beutin L, Sägesser G, Stephan R, et al. Diarrheagenic enteroaggregative Escherichia coli causing urinary tract infection and bacteremia leading to sepsis. Infection. 2014 Apr 10;42(2):441–4. pmid:24323785
- 10. Olesen B, Scheutz F, Andersen RL, Menard M, Boisen N, Johnston B, et al. Enteroaggregative Escherichia coli O78:H10, the cause of an outbreak of urinary tract infection. J Clin Microbiol. 2012; pmid:22972830
- 11. Roer L, Hansen F, Thomsen MCF, Knudsen JD, Hansen DS, Wang M, et al. WGS-based surveillance of third-generation cephalosporin-resistant Escherichia coli from bloodstream infections in Denmark. J Antimicrob Chemother. 2017 Jul;72(7):1922–9. pmid:28369408
- 12. Trecarichi EM, Pagano L, Martino B, Candoni A, Di Blasi R, Nadali G, et al. Bloodstream infections caused by Klebsiella pneumoniae in onco-hematological patients: clinical impact of carbapenem resistance in a multicentre prospective survey. Am J Hematol. 2016 Nov;91(11):1076–81. pmid:27428072
- 13. Xiong Y, Wang P, Lan R, Ye C, Wang H, Ren J, et al. A novel escherichia coli O157:H7 clone causing a major hemolytic uremic syndrome outbreak in China. PLoS One. 2012;
- 14. Frank C, Werber D, Cramer JP, Askar M, Faber M, An Der Heiden M, et al. Epidemic profile of Shiga-toxin-producing Escherichia coli O104:H4 outbreak in Germany. N Engl J Med. 2011; pmid:21696328
- 15. Nelwan EJ, Puspandari N, Paramita RI, Erlina L, Renesteen E, Fadilah F. Whole genome sequencing data of Escherichia coli isolated from bloodstream infection patients in Cipto Mangunkusumo National Hospital, Jakarta, Indonesia. Data Br. 2020;
- 16. Andrews S. FastQC—A quality control tool for high throughput sequence data. http://www.bioinformatics.babraham.ac.uk/projects/fastqc/. Babraham Bioinformatics. 2010.
- 17. Bolger AM, Lohse M, Usadel B. Trimmomatic: A flexible trimmer for Illumina sequence data. Bioinformatics. 2014; pmid:24695404
- 18. Larsen M V., Cosentino S, Rasmussen S, Friis C, Hasman H, Marvig RL, et al. Multilocus sequence typing of total-genome-sequenced bacteria. J Clin Microbiol. 2012; pmid:22238442
- 19. Joensen KG, Tetzschner AMM, Iguchi A, Aarestrup FM, Scheutz F. Rapid and easy in silico serotyping of Escherichia coli isolates by use of whole-genome sequencing data. J Clin Microbiol. 2015; pmid:25972421
- 20. Joensen KG, Scheutz F, Lund O, Hasman H, Kaas RS, Nielsen EM, et al. Real-time whole-genome sequencing for routine typing, surveillance, and outbreak detection of verotoxigenic Escherichia coli. J Clin Microbiol. 2014;
- 21. Zankari E, Hasman H, Cosentino S, Vestergaard M, Rasmussen S, Lund O, et al. Identification of acquired antimicrobial resistance genes. J Antimicrob Chemother. 2012; pmid:22782487
- 22. Shakya M, Ahmed SA, Davenport KW, Flynn MC, Lo C-C, Chain PSG. Standardized phylogenetic and molecular evolutionary analysis applied to species across the microbial tree of life. Sci Rep. 2020 Dec 3;10(1):1723. pmid:32015354
- 23. Boisen N, Østerlund MT, Joensen KG, Santiago AE, Mandomando I, Cravioto A, et al. Redefining enteroaggregative Escherichia coli (EAEC): Genomic characterization of epidemiological EAEC strains. PLoS Negl Trop Dis. 2020;14(9):e0008613. pmid:32898134
- 24. Donnenberg MS, Tzipori S, McKee ML, O’Brien AD, Alroy J, Kaper JB. The role of the eae gene of enterohemorrhagic Escherichia coli in intimate attachment in vitro and in a porcine model. J Clin Invest. 1993 Sep 1;92(3):1418–24. pmid:8376595
- 25. Sarowska J, Futoma-Koloch B, Jama-Kmiecik A, Frej-Madrzak M, Ksiazczyk M, Bugla-Ploskonska G, et al. Virulence factors, prevalence and potential transmission of extraintestinal pathogenic Escherichia coli isolated from different sources: recent reports. Gut Pathog. 2019 Dec 21;11(1):10. pmid:30828388
- 26. Johnson JR, Delavari P, Kuskowski M, Stell AL. Phylogenetic Distribution of Extraintestinal Virulence‐Associated Traits in Escherichia coli. J Infect Dis [Internet]. 2001 Jan;183(1):78–88. Available from: https://academic.oup.com/jid/article-lookup/doi/10.1086/317656 pmid:11106538
- 27. Huang W-C, Liao Y-J, Hashimoto M, Chen K-F, Chu C, Hsu P-C, et al. cjrABC-senB hinders survival of extraintestinal pathogenic E. coli in the bloodstream through triggering complement-mediated killing. J Biomed Sci. 2020 Dec 6;27(1):86. pmid:32762693
- 28. Habouria H, Pokharel P, Maris S, Garénaux A, Bessaiah H, Houle S, et al. Three new serine-protease autotransporters of Enterobacteriaceae (SPATEs) from extra-intestinal pathogenic Escherichia coli and combined role of SPATEs for cytotoxicity and colonization of the mouse kidney. Virulence. 2019 Jan 1;10(1):568–87. pmid:31198092
- 29. Clermont O, Christenson JK, Denamur E, Gordon DM. The Clermont Escherichia coli phylo-typing method revisited: improvement of specificity and detection of new phylo-groups. Environ Microbiol Rep. 2013 Feb;5(1):58–65. pmid:23757131
- 30. Aibinu I, Odugbemi T, Koenig W, Ghebremedhin B. Sequence Type ST131 and ST10 Complex (ST617) predominant among CTX-M-15-producing Escherichia coli isolates from Nigeria. Clin Microbiol Infect. 2012; pmid:22192452
- 31. Oteo J, Diestra K, Juan C, Bautista V, Novais Â, Pérez-Vázquez M, et al. Extended-spectrum β-lactamase-producing Escherichia coli in Spain belong to a large variety of multilocus sequence typing types, including ST10 complex/A, ST23 complex/A and ST131/B2. Int J Antimicrob Agents. 2009; pmid:19464856
- 32. Cha MK, Kang CI, Kim SH, Cho SY, Ha YE, Wi YM, et al. Comparison of the microbiological characteristics and virulence factors of ST131 and non-ST131 clones among extended-spectrum β-lactamase-producing Escherichia coli causing bacteremia. Diagn Microbiol Infect Dis. 2016; pmid:26632660
- 33. Edgar R, Bibi E. MdfA, an Escherichia coli multidrug resistance protein with an extraordinarily broad spectrum of drug recognition. J Bacteriol. 1997; pmid:9079913
- 34. Nguyen MCP, Woerther PL, Bouvet M, Andremont A, Leclercq R, Canu A. Escherichia coli as reservoir for macrolide resistance genes. Emerg Infect Dis. 2009;
- 35. Noguchi N, Takada K, Katayama J, Emura A, Sasatsu M. Regulation of transcription of the mph(A) gene for macrolide 2’-phosphotransferase I in Escherichia coli: Characterization of the regulatory gene mphR(A). J Bacteriol. 2000;
- 36. Manyahi J, Moyo SJ, Tellevik MG, Ndugulile F, Urassa W, Blomberg B, et al. Detection of CTX-M-15 beta-lactamases in Enterobacteriaceae causing hospital- and community-acquired urinary tract infections as early as 2004, in Dar es Salaam, Tanzania. BMC Infect Dis. 2017;
- 37. Johnson JR, Johnston B, Clabots C, Kuskowski MA, Castanheira M. Escherichia coli Sequence Type ST131 as the Major Cause of Serious Multidrug‐Resistant E. coli Infections in the United States. Clin Infect Dis. 2010; pmid:20572763
- 38. Johnson JR, Porter S, Thuras P, Castanheira M. The pandemic H30 subclone of sequence type 131 (ST131) as the leading cause of multidrug-resistant Escherichia coli infections in the United States (2011–2012). Open Forum Infect Dis. 2017;
- 39. Peirano G, Costello M, Pitout JDD. Molecular characteristics of extended-spectrum β-lactamase-producing Escherichia coli from the Chicago area: High prevalence of ST131 producing CTX-M-15 in community hospitals. Int J Antimicrob Agents. 2010; pmid:20359869
- 40. Seki LM, Pereira PS, de Souza Conceição M, Souza MJ, Marques EA, Carballido JM, et al. Molecular epidemiology of CTX-M producing Enterobacteriaceae isolated from bloodstream infections in Rio de Janeiro, Brazil: Emergence of CTX-M-15. Brazilian J Infect Dis. 2013;
- 41. Valenza G, Nickel S, Pfeifer Y, Pietsch M, Voigtländer E, Lehner-Reindl V, et al. Prevalence and genetic diversity of extended-spectrum β-lactamase (ESBL)-producing Escherichia coli in nursing homes in Bavaria, Germany. Vet Microbiol. 2017; pmid:26494113
- 42. Boll EJ, Struve C, Boisen N, Olesen B, Stahlhut SG, Krogfelt KA. Role of Enteroaggregative Escherichia coli Virulence Factors in Uropathogenesis. Bäumler AJ, editor. Infect Immun. 2013 Apr;81(4):1164–71. pmid:23357383
- 43. Torres AG, Blanco M, Valenzuela P, Slater TM, Patel SD, Dahbi G, et al. Genes related to long polar fimbriae of pathogenic Escherichia coli strains as reliable markers to identify virulent isolates. J Clin Microbiol. 2009;
- 44. Havt A, Lima IF, Medeiros PH, Clementino MA, Santos AK, Amaral MS, et al. Prevalence and virulence gene profiling of enteroaggregative Escherichia coli in malnourished and nourished Brazilian children. Diagn Microbiol Infect Dis. 2017; pmid:28780245