New Class 2 Integron In 2-4 Among IncI1-Positive Escherichia coli Isolates Carrying ESBL and PMAβ Genes from Food Animals in Portugal

Introduction

The worldwide rate of infections caused by third-generation cephalosporin-resistant Escherichia coli has increased alarmingly in the past decades. The production of extended-spectrum β-lactamases (ESBL) and plasmid-mediated AmpC β-lactamases (PMAβ) has become widespread among different reservoirs (de Been et al., 2014). This sort of dissemination process has mostly been attributed to mobile genetic elements (MGE) that may encode resistance to different antibiotics and promote their spread among different microorganisms and settings (de Been et al., 2014). In Portugal, the detection of mechanisms of resistance in food animals had already led to important findings (Jones-Dias et al., 2013; Clemente et al., 2015). However, many studies fail to investigate the association of specific MGE to the spread of clinically important antibiotic resistance genes. Moreover, knowledge about the design of MGE and their interaction with antibiotic resistance is vital to the understanding of the dynamics of antimicrobial resistance (Stokes et al., 2011).

The aim of the present study was to investigate the importance of ESBL and PMAβ within a group of E. coli from food animals, and to determine the extent of their association with MGE; in addition, we expect to contribute to the architectural knowledge of a new type of class 2 integron.

Materials and Methods

A collection of 179 E. coli isolates was gathered in a food safety laboratory covering four regions of Portugal (North, Center, Lisbon and Tagus Valley, and South). All E. coli isolates were recovered from routine diagnostic samples (macerated organs [n = 174] and fecal samples [n = 5]), during 1 year, identified by automated methods and sent to the NRL-AR/HAI at the National Institute of Health in Lisbon, without any prior selection criteria. The samples were recovered from three distinct animal origins, as follows: poultry (n = 164), pigs (n = 13), and rabbits (n = 2), and collected as reported elsewhere (Jones-Dias et al., 2013). Antimicrobial susceptibility of all isolates was performed by disk-diffusion method, as previously reported (Bonnet et al., 2013). Isolates nonsusceptible to third-generation cephalosporins were investigated for the presence of ESBL- and PMAβ-encoding genes, transferability of resistance, multilocus sequence typing (MLST), plasmid MLST, integrons, and genetic environment of interest genes, as reported elsewhere (Clemente et al., 2015). Isolates were considered multidrug-resistant (MDR) if they presented nonsusceptibility against three or more structurally unrelated antibiotics (Magiorakos et al., 2012).The content of the class 2 integron from isolate INSLA289 was also assessed through polymerase chain reaction mapping, using a targeting sequencing approach with primers described elsewhere (Ramírez et al., 2005; Sáenz et al., 2010). The new version of integron In2-4 was submitted to the European Nucleotide Archive with accession number LN827615.

Results and Discussion

Overall, among the 179 E. coli isolates recovered, we detected 15 isolates nonsusceptible to third-generation cephalosporins, all of them harboring either ESBL or PMAβ (Table 1). bla _CTX-M-1 (n = 8) was the most frequent ESBL-encoding gene, followed by bla _SHV-12 (n = 2) and bla _CTX-M-14 (n = 1); bla _CMY-2 (n = 5) was the only PMAβ identified. More than half of the isolates were also identified as MDR, as shown in Table 1. The presence of the same β-lactamases (TEM-1, CTX-M-1, CMY-2) in animals from different regions points to different sources of these β-lactamases, spread across three important regions of the country (Table 1). This is in agreement with the high genetic diversity noticed: one clonal complex (ST88 and ST23) and eight additional singletons (Table 1). Thus, the spread of ESBL and PMAβ in E. coli from food animals in the country strongly suggests horizontal transfer driven by MGE, rather than a clonal dissemination.

All the transconjugants carrying ESBL- or PMAβ-encoding genes harbored IncI1 (14/15), and three of them also carried IncFIB (3/13) plasmids (Table 1). However, E. coli isolates harbored plasmids belonging to different replicon types: IncI1 (n = 15), IncFIA (n = 1), IncFIB (n = 13), and IncFIC (n = 7) (Table 1). In fact, the mobilization of the detected bla genes was driven by five different IncI1 plasmids: ST26/IncI1-harboring bla _SHV-12, ST12/IncI1-harboring bla _CMY-2, ST3 and ST38/IncI1-harboring bla _CTX-M-1, and ST1/IncI1-harboring bla _CTX-M-14. bla _CTX-M-1 was associated with two different plasmids from clonal complex 3, suggesting the occurrence of intraplasmid evolution. ST12/IncI1 and ST3/IncI1 have been associated with the spread of CTX-M-1 and CMY-2 β-lactamases in animals, which represents a concern, because they might be easily disseminated to humans, water, and soil (Ben Sallem et al., 2014). Globally, although the isolates carried other plasmids, IncI1 plasmids seem to be responsible for this epidemic-like spread, which has already been cause for concern in other countries (de Been et al., 2014).

The characterization of the genetic context of ESBL- and PMAβ-encoding genes showed the presence of ISEcp1 upstream of the nine bla _CTX-M-1 and of the five bla _CMY-2 genes, while the two bla _SHV-12 genes were flanked upstream by IS26. The downstream genetic context of the genes consisted of orf477 in bla _CTX-M-1 genes (n = 8), IS903 in bla _SHV-12 and a blc-sugE structure in bla _CMY-2 (Table 1). The detection of those IS elements, despite the plasmid background or ST, indicates that they might play an important part in the capture, expression, and mobilization of bla _ESBL and bla _PMAβ genes (Stokes et al., 2011; de Been et al., 2014).

Furthermore, five isolates were positive for class 1 integrons, and the isolate INSLA289 for a class 2 integron; the array for the latter revealed a new arrangement (Table 1). The integron sequence resembled the classic In2-4, except within the recombination site where the array containing dfrA1, sat2, and aadA1y was preceded by a complete IS10 that resulted in the disruption of the original attI2 and reconstruction of a second attI2 immediately after the inserted IS10 (Fig. 1) (Ramírez et al., 2005, 2010). Consequently, integrating abilities of In2 remained intact, and the new construct benefits from an additional capability to move using the recently acquired IS10 transposase, which may have important consequences on resistance dissemination. Moreover, an amino acid alteration (H379Y) was detected within TnsD protein.

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FIG. 1.

Scheme of the In2-4 harbored by isolate INSRA289 (LN827615) where the attI2 recombination site (793-809bp and 2148-2193bp) is disrupted by the insertion of a complete IS10 (1329bp, at position 810-2138bp).

This study reports multiple animal sources of ST3/IncI1-harboring bla _CTX-M-1, and other ESBL/PMAβ-encoding genes associated with the spread of epidemic plasmids within and among different farms. These genes have been increasingly identified in humans and animals across Europe, highlighting the widespread distribution of those MGE along the food chain.