The nature of bel-1 attC stabilizes its genetic environment and contributes to antibiotic resistance spreading

The rapid emergence of antibiotic resistance is a worldwide crisis, endangering the efficacy of antibiotic treatment. Multidrug resistance in Gram negatives is now recognized as an issue of worldwide interest. Those bacteria possess various resistance mechanisms compromising the efficacy of several classes of antibiotics such as beta-lactams.

In this study, we investigated the blaBEL-1 gene which encodes an extended-spectrum β-lactamase, BEL-1, that is present at the second position of the variable region of class 1 integrons identified in Pseudomonas aeruginosa. Class 1 integrons are genetic elements that can acquire and rearrange gene cassettes, including genes carrying antibiotic/disinfectant resistance genes, therefore participating in the evolution toward multidrug resistance. The bel-1 cassettes are associated with aacA4 and aadA5 gene cassettes, coding for an aminoglycoside-modifying enzyme, and also with the smr cassette, encoding resistance to antiseptics.

Schematic representation of integrons containing the blaBEL-1 gene

Fig. 1. Schematic representation of integrons containing the blaBEL-1 gene found in clinical strain P. aeruginosa 51170. Arrows, orientation of gene transcription; black and gray circles, attC and attI1 sites, respectively.

Integrons are bracketed by two segments at their 5’ and 3’ ends. The 5’ includes intI1, a gene encoding a site-specific recombinase of the DNA integrase family, with attI being the cassette integration site and the promoter Pc driving the expression of the cassettes. The gene cassettes are independent units each consisting of a gene bracketed by copies of a recombination site named attC. attC sites are involved in site-specific recombination catalyzed by the integrase IntI1 leading to cassette integration or excision.

We investigated here the putative mobility of the bel-1 gene cassette and the role of several antibiotic molecules in the putative induction of its mobility. Interestingly, we found after 10 days of P. aeruginosa culture with sub-inhibitory concentrations of antibiotics, that the bel-1 cassette remained at the second position in the integron, highlighting its stability in P. aeruginosa.

Therefore, the effect of IntI1 integrase overproduction was investigated. The cointegration frequencies depend on the recombination efficiency of the attC site available for recombination in the donor integron. We showed that the bel-1 attC site was likely inefficient for recombination with the attI1 site or an attC site in a receiving integron In3. The smr2 attC site was thus more efficient for recombination than the bel-1 attC site itself.

The excision/mobilization experiments showed that the attC site of the bel-1 gene cassette was inefficient and that this gene cassette was not mobilizable independently. This might likely be explained by the sequence of the bel-1 attC site itself, which does not correspond to the ones better recognized by the Int1 integrase. Here the extrahelical bases constituting the bel-1 attC bottom strand are distantly related to those well recognized by Int1. We also showed that the smr2 attC site is enhanced over the bel-1 attC site, as it is involved in almost all recombination events when both gene cassettes are present on a plasmid. smr2 always remained associated with the bel-1 gene cassette.

Overall, our work provides some insights into the organization of blaBEL-1-containing integrons. It is likely that those later evolved from a common ancestor carrying an early association between the bel-1 and smr2 gene cassettes. It is also possible that smr2 was responsible for bel-1 gene cassette recruitment and for the comobilization of bel-1–smr2 into class 1 integrons. Although blaBEL-1-containing integrons are subject to gene cassette rearrangements, we propose that the nature of bel-1 attC stabilizes its genetic environment, probably by impairing recombination events that could lead to its loss and thus maintaining antibiotic resistance.

Nacim Bouheraoua 1, Laurent Poirel 2-5, Patrice Nordmann 2-5
1Quinze-Vingts National Ophthalmology Hospital, Sorbonne Université, Paris, France
2Medical and Molecular Microbiology Unit, Department of Medicine, Faculty of Science, University of Fribourg, Fribourg, Switzerland
3INSERM European Unit (IAME, France), University of Fribourg, Fribourg, Switzerland
4Swiss National Reference Center for Emerging Antibiotic Resistance (NARA), University of Fribourg, Fribourg, Switzerland
5Institute for Microbiology, University of Lausanne and University Hospital Centre, Lausanne, Switzerland

Publication

Integrase-Mediated Recombination of the bel-1 Gene Cassette Encoding the Extended-Spectrum β-Lactamase BEL-1.
Bouheraoua N, Poirel L, Bourreau B, Bonnin R, Laroche L, Naas T, Nordmann P
Antimicrob Agents Chemother. 2018 Apr 26

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