Virulence potential of a multidrug-resistant Escherichia coli belonging to the emerging clonal group ST101-B1 isolated from bloodstream infection

Escherichia coli EC121 is a multidrug-resistant (MDR) strain isolated from bloodstream infection of an inpatient with persistent gastroenteritis and Zone T lymphoma that died due to septic shock. Despite causing an extraintestinal infection, strain EC121 harbors very few known virulence factors associated with extraintestinal pathogenic E. coli (ExPEC). Furthermore, this strain was assigned to phylogenetic group B1, which is usually related to commensals or diarrheagenic E. coli (DEC) strains, being rarely linked to extraintestinal infections. The question rises if E. coli EC121 is opportunistic or does have a true virulence potential. To address this question the genome of strain EC121 was sequenced, and an in vitro characterization of some pathogenic-associated properties was performed. The data retrieved from genome analyses showed that E. coli strain EC121 belongs to the O154:H25 serotype, and to the sequence type 101-B1 (ST101-B1), which has been epidemiologically linked to extraintestinal infections and antimicrobial resistance spread as well. Moreover, it is shown to be closely related to Shiga-toxin producing E. coli (STEC). Besides, strain EC121 is an MDR strain harboring 14 antimicrobial resistance genes, including blaCTX-M-2, and more than 50 complete virulence genetic clusters, which are reported to be associated either with DEC or ExPEC, or both. Strain EC121 also displays the capacity to adhere to and invade HeLa cells, intestinal Caco-2 cells and bladder T24 cells, as well as the ability to form biofilms on abiotic surfaces, and survive the bactericidal serum complement activity. Altogether, the genetic and phenotypic traits presented by E. coli EC121 may be unveiling a pathogen powered by its multi-drug resistance characteristic. The recognition of such MDR pathogens makes it essential to carry out studies providing accurate information about their virulence potential. Such studies will help in the development of alternative therapies of infection management and spread control of MDR strains. Author summary Extraintestinal pathogenic Escherichia coli are mostly associated with phylogenetic group B2 and the majority of the studies regarding extraintestinal infection focus on the most virulent strains, which might also possess multidrug-resistant phenotype, like the ST131 strains. On the other hand, strains that belong to phylogroup B1 and are isolated from extraintestinal infections are almost neglected, being considered as merely opportunist pathogens, and the majority of studies reporting strains from this phylogroup focus on antimicrobial resistance. We concentrated our research on one multidrug-resistant strain isolated from bloodstream infection that belongs to phylogenetic group B1 to enlarge the knowledge about the virulence of this kind of strain. We demonstrated that strain EC121 has full potential to be considered a pathogenic strain, because it adheres to and invades intestinal and bladder human cells and survives in human serum; moreover, our data highlight some important EC121 features, which are typical of ST101 strains, like its involvement in the spread of antimicrobial resistance genes, its relationship with extraintestinal infections from diverse sources, and its close relatedness to Shiga toxin-producing E. coli. All these data are important for the development of global actions concerning the spread of antimicrobial resistance, as well as to elucidate the virulence potential of strains that at first glance may be considered as a mere opportunist.

The spread of MDR pathogens is a major Public Health concern that needs to be 1 0 2 adequately addressed towards efficient control. Based on that, the World Health factors as targets can be problematic as they can adversely affect the gut microbiota. The E. coli strain EC121 was isolated from the blood of a patient diagnosed with 1 2 9 T-zone lymphoma and persistent infectious gastroenteritis, who had been hospitalized in to septic shock two days after the isolation of the EC121. The EC121 strain was kept showed that EC121 strain belonged to phylogroup B1, presented an MDR phenotype by 1 3 5 routine susceptibility testing, and harbored few known virulence genes (fim, hra, cvaC, 1 3 6 ompA, ompT, sitA, iroN). Furthermore, it was not considered a pathogenic strain afaBC, iuc/iut, and kpsMTII) [25]. Total DNA extraction, whole-genome sequencing (WGS), and genome assembly. tRNA, and 13 rRNA. One CRISPR locus was identified as type 1-IE and presented two 3 7 9 arrays and 30 CRISPR-repeat regions ( Figure 3A). Among the CDSs annotated, 702 belonging to systems involved in response to stress, virulence, and defense (Table 1 and   3  8  3 Figures 3B-C).

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Analysis using the MacSyFinder tool identified 10 type V secretion system 3 8 5 proteins, nine of which were from type T5aSS and one from T5cSS (EhaG) ( Table S2).

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All genes that were reported by the PATRIC virulence factor database were 3 9 0 manually curated to provide information about their full sequence. As shown in Tables   3  9  1 1 8 cells, iron acquisition and immune evasion were also detected in the EC121 genome.

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Furthermore, other clusters associated with Urinary Tract Infections (UTI) were 4 0 2 detected (Tables 2 and S3). of different phages, ranging from 6 to 31 kb (  (Table 3 and Table S4). However, no toxin-encoding genes were identified in strain findings, as can be seen in Table 4. The EC121 strain harbors multiple plasmids. Four bands were detected by agarose 4 3 0 gel electrophoresis, suggesting that EC121 harbor multiple plasmids ( Figure S2). Although the EC121 genome is still in draft, we analyzed the replicon containing  roggenkampii strain R11 ( Figure S4).

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The other replicons were segregated into four different contigs, with IncFIB and IncFII  The IncFIB replicon's contig also contained the virulence genes iroN, iss, and traT as to the pAPEC plasmid that carries virulence and resistance genes simultaneously. Although the IncFII replicon was identified into a different contig, the data indicated 4 5 1 that both the IncFII and IncFIB replicons represent segments of the same plasmid.

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Further studies are required to unravel this plasmid genetic composition. and IncHI2), as well as the hlyF, sitA, and iroN genes (Table 5) IncHI2A/IncHI2 plasmid.

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One transconjugant strain of each type was submitted to the microdilution assay to susceptibility to all beta-lactams ( indicating that they were all conjugative or mobilizable plasmids. pathogenic bacteria must be able to survive the serum bactericidal activity. To identify 4 9 0 such a feature in EC121, we determined the lowest serum-resistant bacterial inoculum 4 9 1 using a pool of normal human sera (NHS). The lowest inoculum of EC121 strain that could potentially transfer plasmids to other bacteria, even from distinct genera. Such 6 0 0 cross genera plasmid transfer could be easily identified in the plasmids reported in the 6 0 1 present study; IncM1 plasmid, for example, is closely related to plasmids found in 6 0 2 Klebsiella spp. and Enterobacter spp., while IncHI2/HI2A is related to Salmonella spp. and Yersinia spp. plasmids. Together, these findings reinforce the high risks associated 6 0 4 with strains belonging to the ST101 complex due to their ability to colonize humans and 6 0 5 animals' gut, to easily disseminate via retail food and water, being able to acquire and 6 0 6 spread antimicrobial resistance-encoding genes. The genomic analysis of the EC121 strain showed a high number of virulence 6 1 3 genes, demonstrating that it presents all the traits necessary to be considered as an phenotype that ensures its pathogenicity. However, even considering the completeness 6 1 8 of each sequence and each operon, which was manually checked, the presence of 6 1 9 virulence genes "per se" does not guarantee that all of them are expressed. Therefore, to 6 2 0 evaluate the expression of such traits, distinct phenotypic assays were performed and 6 2 1 confirmed the virulence genetic background of EC121. To test the bacterial ability to resist the serum complement activity that could be performed, in which one particle can traverse all circulatory system at least twice, so a 6 2 5 pathogen that resists complement's activity during this period, even with a small The ability to colonize and attach to surfaces is also an important trait for any 6 3 6 pathogenic bacteria; in this way, the assays carried out showed not only that EC121 6 3 7 strain was able to adhere to and invade different cell lineages, including bladder T24 6 3 8 cells but that it could also attach and produce biofilm on abiotic surfaces. Peirano et al. [90] showed that ExPEC negative ST101 MDR strains isolated from extraintestinal 6 4 0 infections could interact with HEp-2 and Caco-2 cells more efficiently than strains 6 4 1 the immune system and confer protection against antimicrobials agents. However, further studies are required to prove this hypothesis.
6 5 1 Ten virulence encoding-genes involved with biofilm production were identified 6 5 2 in the EC121 genome (Table S3). The capacity to produce biofilm might confer many 6 5 3 advantages to bacteria, like protection against the immunological system and 6 5 4 antibiotics, assisting its persistence and spread in the environment. However, biofilm 6 5 5 production depends on many factors, like temperature and presence of specific nutrients 6 5 6 in the media or environment [92][93][94]. Moreover, the ability to produce biofilm has been 6 5 7 reported to vary among ExPEC strains and that strains with this capacity are considered 6 5 8 to be more pathogenic [95].

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Interestingly, many of the EC121 virulence factors detected in the draft genome and food were found to carry stx1. Interestingly, most Stx-converting phages remains 6 7 8 found in EC121 were similar to those commonly related to Stx1a production, corroborating with the results presented here. The genome of three non-STEC strains 6 8 0 from diarrheic patients was found in GenBank, one of which was devoid of DEC       Comparative study on the high pressure inactivation behavior of the Shiga toxin- producing Escherichia coli O104:H4 and O157:H7 outbreak strains and a non- 40. Abby SS, Cury J, Guglielmini J, Néron B, Touchon M, Rocha EPC.  Wooten RM, editor. PLoS One. 2015;10: e0121271. Communities: A Potential Etiology for Chronic Lower Urinary Tract Symptoms. Phylogenetic and molecular analysis of food-borne shiga toxin-producing