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Previous studies on commensal Escherichia coli from healthy children in the Bolivian Chaco have shown remarkable resistance rates to the old antibiotics since the early 1990s, and the emergence of resistance to newer drugs (fluoroquinolones and expanded-spectrum cephalosporins) in the 2000s. Here we report the results of a new survey conducted in 2011 in the same setting. Rectal swabs were obtained from 482 healthy children (aged 6-72 months) from three urban areas of the Bolivian Chaco. Screening for antibiotic-resistant E. coli was performed by a direct plating method, as in the previous studies. The blaCTX-M genes were investigated by PCR/sequencing, and CTX-M-producing isolates were subjected to genotyping and detection of several plasmid-mediated quinolone resistance mechanisms. Results showed high rates of resistance to nalidixic acid (76%), ciprofloxacin (44%) and expanded-spectrum cephalosporins (12.4%), demonstrating a relentless increase of resistance to those drugs over the past two decades. CTX-M-type extended-spectrum beta-lactamases were found to be widespread (12%, 97% of extended-spectrum beta-lactamase producers). Compared with the previous studies, CTX-M-producing E. coli underwent a dramatic dissemination (120-fold increase since early 2000s) and a radical change of dominant CTX-M groups (CTX-M-1 and CTX-M-9 groups versus CTX-M-2 group). Most CTX-M producers were not susceptible to quinolones (91%), and 55% carried plasmid-mediated quinolone resistance genes (different combinations of aac(6')-Ib-cr, qnrB and qepA). This study shows the rapid and remarkable increasing trend for resistance to fluoroquinolones and expanded-spectrum cephalosporins in one of the poorest regions of Latin America, and underscores the need for urgent control strategies aimed at preserving the efficacy of those drugs in similar settings.

f During the last decade, a significant diffusion of CTX-M-type extended-spectrum ␤-lactamases (ESBLs) was observed in commensal Escherichia coli from healthy children in the Bolivian Chaco region, with initial dissemination of CTX-M-2, which was then replaced by CTX-M-15 and CTX-M-65. In this work, we demonstrate that the widespread dissemination of CTX-M-65 observed in this context was related to the polyclonal spreading of an IncI1 sequence type 71 (ST71) epidemic plasmid lineage. The structure of the epidemic plasmid population was characterized by complete sequencing of four representatives and PCR mapping of the remainder (n ‫؍‬ 16). Sequence analysis showed identical plasmid backbones (similar to that of the reference IncI1 plasmid, R64) and a multiresistance region (MRR), which underwent local microevolution. The MRR harbored genes responsible for resistance to ␤-lactams, aminoglycosides, florfenicol, and fosfomycin (with microevolution mainly consisting of deletion events of resistance modules). The bla CTX-M-65 module harbored by the IncI1 ST71 epidemic plasmid was apparently derived from IncN-type plasmids, likely via IS26-mediated mobilization. The plasmid could be transferred by conjugation to several different enterobacterial species (Escherichia coli, Cronobacter sakazakii, Enterobacter cloacae, Klebsiella oxytoca, Klebsiella pneumoniae, and Salmonella enterica) and was stably maintained without selective pressure in these species, with the exception of K. oxytoca and S. enterica. Fitness assays performed in E. coli recipients demonstrated that the presence of the epidemic plasmid was apparently not associated with a significant biological cost. C TX-M-type ␤-lactamases, first described in the late 1980s, have become the most common extended-spectrum ␤-lactamases (ESBLs) among Enterobacteriaceae worldwide (1, 2). Multiple sublineages (groups) of CTX-M-type ESBLs exist, and their dissemination has followed complex trajectories, depending on the spreading ability of the epidemic plasmids and bacterial clones with which bla CTX-M genes have become associated (1). Paradigmatic examples are the dissemination of bla CTX-M-15 , mediated by IncFII and IncX plasmids associated with the Escherichia coli sequence type 131 (ST131) clone (3, 4); the dissemination of bla CTX-M-14 in Spain and the United Kingdom, mediated by IncK plasmids (5-8); and the dissemination of bla CTX-M-65 in China, mediated by F33:A-:B-plasmids (9).In South America, members of the CTX-M-2 group emerged first and underwent wide dissemination during the 1990s (1, 10-12). Subsequently, members of the CTX-M-1 group (mostly CTX-M-15) and of the CTX-M-9 group have also disseminated in the area, outnumbering and replacing CTX-M-2 (13-18).A similar epidemiological evolution was also observed in the Bolivian Chaco region, where we have been monitoring the evolution of antibiotic resistance among commensal E. coli isolates during the past 2 decades (from 1992 to 2011) (19)(20)(21). In that area, CTX-M-type ESBLs were first detected in 2002 and ...

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The tetra-branched peptide M33 (Pini et al. in FASEB J 24:1015-1022, 2010) is under evaluation in animal models for its activity as antimicrobial agent in lung infections and sepsis. The preclinical development of a new drug requires medium-scale manufacture for tests of efficacy, biodistribution, pharmacokinetics and toxicity. In order to produce the most suitable peptide form for these purposes, we evaluated the behaviour of the peptide M33 obtained with different counter-ions. We compared activity and toxicity in vitro and in vivo of the peptide M33 produced as trifluoroacetate salt (TFacetate) and as acetate salt. The two forms did not differ substantially in terms of efficacy in vitro or in vivo but showed different toxicities for human cells and in animals. M33-TFacetate proved to be 5-30% more toxic than M33-acetate for cells derived from normal bronchi and cells carrying ΔF508 mutation in the CFTR gene, the most frequent variant in cystic fibrosis. M33-TFacetate produced manifest signs of in vivo toxicity immediately after administration, whereas M33-acetate only generated mild signs, which disappeared within a few hours. The peptide M33-acetate proved more suitable for the development of a new drug, and was therefore chosen for further characterization.