Determining the Virulence Properties of Escherichia coli ST131 Containing Bacteriocin-Encoding Plasmids Using Short- and Long-Read Sequencing and Comparing Them with Those of Other E. coli Lineages

microorganisms Article Determining the Virulence Properties of Escherichia coli ST131 Containing Bacteriocin-Encoding Plasmids Using Short- and Long-Read Sequencing and Comparing Them with Those of Other E. coli Lineages Ana Carolina da Cruz Campos 1,2 , Francis M. Cavallo 2 , Nathália L. Andrade 1 , Jan Maarten van Dijl 2 , Natacha Couto 2 , Jan Zrimec 3 , Jerome R. Lo Ten Foe 2 , Ana C. P. Rosa 1 , Paulo V. Damasco 4 , Alex W. Friedrich 2 , Monika A. Chlebowicz-Flissikowska 2 and John W. A. Rossen 2, * 1 2 3 4 * Departamento de Microbiologia, Imunologia e Parasitologia, Universidade do Estado do Rio de Janeiro, Rio de Janeiro 20550-170, Brazil; (A.C.d.C.C.); (N.L.A.); (A.C.P.R.) Department of Medical Microbiology and Infection Prevention, University Medical Center Groningen, University of Groningen, Hanzeplein 1, 9713GZ Groningen, The Netherlands; (F.M.C.); (J.M.v.D.); (N.C.); (J.R.L.T.F.); (A.W.F.); (M.A.C.-F.) Department of biology and Biological Engineering, Chalmers University of Technology, Chalmersplatsen 4, 412 96 Göteborg, Sweden; Departamento de Doenças Infecciosas e Parasitárias, Universidade Federal do Estado do Rio de Janeiro, Rua Voluntário da Patria, 21, Rio de Janeiro 941-901107, Brazil; Correspondence: ; Tel.: +31-50-361-3480; Fax: +31-50-361-9105 Received: 19 September 2019; Accepted: 1 November 2019; Published: 6 November 2019



 Abstract: Escherichia coli ST131 is a clinical challenge due to its multidrug resistant profile and successful global spread. They are often associated with complicated infections, particularly urinary tract infections (UTIs). Bacteriocins play an important role to outcompete other microorganisms present in the human gut. Here, we characterized bacteriocin-encoding plasmids found in ST131 isolates of patients suffering from a UTI using both short- and long-read sequencing. Colicins Ia, Ib and E1, and microcin V, were identified among plasmids that also contained resistance and virulence genes. To investigate if the potential transmission range of the colicin E1 plasmid is influenced by the presence of a resistance gene, we constructed a strain containing a plasmid which had both the colicin E1 and blaCMY-2 genes. No difference in transmission range was found between transformant and wild-type strains. However, a statistically significantly difference was found in adhesion and invasion ability. Bacteriocin-producing isolates from both ST131 and non-ST131 lineages were able to inhibit the growth of other E. coli isolates, including other ST131. In summary, plasmids harboring bacteriocins give additional advantages for highly virulent and resistant ST131 isolates, improving the ability of these isolates to compete with other microbiota for a niche and thereby increasing the risk of infection. Keywords: ST131; E. coli; bacteriocins; plasmids; AmpC-beta-lactamase; UTIs; virulence 1. Introduction Pathogenic Extraintestinal Escherichia coli (ExPEC), including the uropathogenic E. coli (UPEC) pathotype, is often associated with urinary tract infections (UTIs) [1]. They carry a high number of virulence factors such as adhesins, fimbriae, hemolysins, aerobactin and others that allow these Microorganisms 2019, 7, 534; doi:10.3390/microorganisms7110534 www.mdpi.com/journal/microorganisms Microorganisms 2019, 7, 534 2 of 15 bacteria to live in the human gut but also, to cause infections at other sites [2–4]. One of the survival strategies of E. coli is the production of bacteriocins, a group of antibacterial peptides often encoded by genes located on plasmids and able to kill normally closely related surrounding bacteria [5]. Although not required for growth, they help to outcompete other microorganisms (bacteria and fungi) for the limiting nutrients in the environment [6,7]. Colicins and microcins are the types of bacteriocins most often found in pathogenic and in approximately 30% of commensal E. coli [8–10]. Colicin genes are mostly located in operons, also containing the colicin immunity gene, important for neutralizing its toxic effect on the producer strain, and the lysis gene required for colicin release. The operon is activated by the SOS system [11]. In contrast, microcins are not inducible by the SOS system and are not toxic to producer strains. Colicins have different ways of action. They can act by forming a pore in the bacterial membrane, digesting bacterial DNA by their nuclease activity, or by interfering with cell wall synthesis [5,12]. The presence of multiple bacteriocins in E. coli isolates is common and increases their urovirulence [13] and the development of bacteremia of urinary tract origin [9]. Within the E. coli population, ST131 is one of the most successful lineages frequently causing UTIs and bloodstream infections (BSIs). Their success is partially associated with the presence of fluoroquinolone resistance (FQR), β-lactamases responsible for their ESBL phenotype and specific virulence genes [14,15]. Among a diversity of beta-lactamases genes, blaCMY-2 is frequently identified. It encodes for an AmpC type of β-lactamase that confers resistance to all β-lactam antibiotics except the fourth-generation cephalosporins and carbapenems [16]. ST131 E. coli, frequently belonging to phylogenetic group B2, are associated with a high virulence profile [17]. Due to this combination of a high resistance and virulence profile, infections caused by the ExPEC ST131 are a serious threat for patients. In addition, other successful and worldwide spread lineages, such as ST405 and ST648, have been associated with antibiotic resistance [18]. As mentioned, the production of bacteriocins, such as microcin, by ExPEC is associated with a more virulent profile of the bacterium [2]. Whole genome sequencing (WGS) has been used to reveal the epidemiology and evolution of the ST131 lineage [19,20] and the role of mobile genetic elements (MGE) herein [21]. MGEs, particularly plasmids, can be easily exchanged between isolates creating sub-lineage variants that become even more resistant and virulent [22]. As mentioned, in E. coli, several bacteriocins are encoded by genes located on plasmids and the presence of resistance genes and specific bacteriocins on single plasmids could potentially contribute to the success of ST131 E. coli. However, to the best of our knowledge, there are no studies that address the importance of plasmids encoding both resistance and bacteriocin genes for the successful dissemination and virulence potential of this high-risk clones and only a few studies have investigated the importance of bacteriocins with respect to the virulence of these successful lineages. Therefore, our study aimed to investigate the bacteriocin in vivo activity in E. coli isolates, focusing on successful lineages, particularly ST131, and to characterize resistance genes and bacteriocin-encoding plasmids of ST131 E. coli isolated from clinical urine samples in an attempt to reveal their role in the ba (...truncated)