The contribution of integrons to the dissemination of extended-spectrum ␤-lactamases (ESBL) was analyzed on all ESBL-producing Escherichia coli isolates from 1988 to 2000 at Ramón y Cajal Hospital. We studied 133 E. coli pulsed-field gel electrophoresis types: (i) 52 ESBL-producing clinical strains (C-ESBL) (16 TEM, 9 SHV, 21 CTX-M-9, 1 CTX-M-14, and 5 CTX-M-10); (ii) 43 non-ESBL blood clinical strains (C-nESBL); and (iii) 38 non-ESBL fecal isolates from healthy volunteers (V-nESBL). Class 1 integrons were more common among C-ESBL (67%) than among C-nESBL (40%) or V-nESBL (26%) (P < 0.001) due to the high number of strains with bla CTX-M-9 , which is linked to an In6-like class 1 integron. Without this bias, class 1 integron occurrence would be similar in C-ESBL and C-nESBL groups (47% versus 40%). Occurrence of class 2 integrons was similar among clinical and community isolates (13 to 18%). No isolates contained class 3 integrons. The relatively low rate of class 1 integrons within transferable elements carrying bla TEM (23%) or bla SHV (33%) and the absence of class 2 integrons in all ESBL transconjugants mirror the assembly of translocative pieces containing bla TEM or bla SHV on local available transferable elements lacking integrons. The low diversity of class 1 integrons (seven types recovered in all groups) might indicate a wide dissemination of specific genetic elements in which they are located. In our environment, the spread of genetic elements encoding ESBL has no major impact on the dispersion of integrons, nor do integrons have a major impact on the spread of ESBL, except when bla ESBL genes are within an integron platform such as bla CTX-M-9 .

Extended-spectrum beta-lactamases (ESBL) constitute a key antibiotic-resistance mechanism affecting Gram-negative bacteria, and also an excellent model for studying evolution in real time. A shift in the epidemiology of ESBLs is being observed, which is characterized by the explosive diversification and increase in frequency of the CTX-M-type β-lactamases in different settings. This provides a unique opportunity for studying a protein evolutionary radiation by the sequential acquisition of specific mutations enhancing protein efficiency and fitness concomitantly. The existence of driver antibiotic molecules favoring protein divergence has been investigated by combining evolutionary analyses and experimental site-specific mutagenesis. Phylogenetic reconstruction with all the CTX-M variants described so far provided a hypothetical evolutionary scenario showing at least three diversification events. CTX-M-3 was likely the enzyme at the origin of the diversification in the CTX-M-1 cluster, which was coincident with positive selection acting on several amino acid positions. Sixty-three CTX-M-3 derivatives containing all combinations of mutations under positively selected positions were constructed, and their phenotypic efficiency was evaluated. The CTX-M-3 diversification process can only be explained in a complex selective landscape with at least two antibiotics (cefotaxime and ceftazidime), indicating the need to invoke mixtures of selective drivers in order to understand the final evolutionary outcome. Under this hypothesis, we found congruent results between the in silico and in vitro analyses of evolutionary trajectories. Three pathways driving the diversification of CTX-M-3 towards the most complex and efficient variants were identified. Whereas the P167S pathway has limited possibilities of further diversification, the D240G route shows a robust diversification network. In the third route, drift may have played a role in the early stages of CTX-M-3 evolution. Antimicrobial agents should not be considered only as selectors for efficient mechanisms of resistance but also as diversifying agents of the evolutionary trajectories. Different trajectories were identified using a combination of phylogenetic reconstructions and directed mutagenesis analyses, indicating that such an approach might be useful to fulfill the desirable goal of predicting evolutionary trajectories in antimicrobial resistance.

This study analyzes the diversity of In60, a class 1 integron bearing CR1 and containing bla CTX-M-9 , and its association with Tn402, Tn21, and classical conjugative plasmids among 45 CTX-M-9-producing clinical strains (41 Escherichia coli strains, 2 Klebsiella pneumoniae strains, 1 Salmonella enterica strain, and 1 Enterobacter cloacae strain). Forty-five patients in a Spanish tertiary care hospital were studied (1996 to 2003). The diversity of In60 and association of In60 with Tn402 or mercury resistance transposons were investigated by overlapping PCR assays and/or hybridization. Plasmid characterization included comparison of restriction fragment length polymorphism patterns and determination of incompatibility group by PCR-based replicon typing, sequencing, and hybridization. CTX-M-9 plasmids belonged to IncHI2 (n ‫؍‬ 26), IncP-1␣ (n ‫؍‬ 10), IncFI (n ‫؍‬ 4), and IncI (n ‫؍‬ 1) groups. Genetic platforms containing bla CTX-M-9 were classified in six types in relation to the In60 backbone and in eight subtypes in relation to Tn402 derivatives. They were associated with Tn21 sequences when located in IncP-1␣ or IncHI2 plasmids. Our study identified bla CTX-M-9 in a high diversity of CR1-bearing class 1 integrons linked to different Tn402 derivatives, often to Tn21, highlighting the role of recombination events in the evolution of antibiotic resistance plasmids. The presence of bla CTX-M-9 on broad-host-range IncP-1␣ plasmids might contribute to its dissemination to hosts that were not members of the family Enterobacteriaceae.The reasons driving the recent dramatic worldwide dissemination of CTX-M-producing microorganisms are far from understood. Chromosomal ␤-lactamase genes from different Kluyvera species are considered the ancestors of each of the five CTX-M groups described thus far (CTX-M-1, -2, -8, -9, and -