The present work describes the abrupt emergence of Klebsiella pneumoniae carbapenemase (KPC) and characterizes the first 79 KPC-producing enterobacteria from Argentina (isolated from 2006 to 2010). The emergence of bla(KPC-2) was characterized by two patterns of dispersion: the first was the sporadic occurrence in diverse enterobacteria from distant geographical regions, harbouring plasmids of different incompatibility groups and bla(KPC-2) in an unusual genetic environment flanked by ISKpn8-Δbla(TEM-1) and ISKpn6-like. bla(KPC-2) was associated with IncL/M transferable plasmids; the second was the abrupt clonal spread of K. pneumoniae ST258 harbouring bla(KPC-2) in Tn4401a.

Transferable quinolone resistance has not previously been reported in Argentina. Here we describe three complex class 1 integrons harboring the novel allele qnrB10 in a unique region downstream of orf513, one of them also containing aac(6)-Ib-cr within the variable region of integrons. The three arrays differed from bla CTX-M-2 -bearing integrons, which are broadly distributed in Argentina.

A number of genes encoding bacterial glycosyltransferases have been sequenced during the last few years, but their low sequence similarity has prevented a straightforward grouping of these enzymes into families. The sequences of several bacterial alpha-mannosyltransferases have been compared using current alignment algorithms as well as hydrophobic cluster analysis (HCA). These sequences show a similarity which is significant but too low to be reliably aligned using automatic alignment methods. However, a region spanning approx. 270 residues in these proteins could be aligned by HCA, and several invariant amino acid residues were identified. These features were also found in several other glycosyltransferases, as well as in proteins of unknown function present in sequence databases. This similarity most probably reflects the existence of a family of proteins with conserved structural and mechanistic features. It is argued that the present IUBMB classification of glycosyltransferases could be complemented by a classification of these enzymes based on sequence similarities analogous to that which we proposed for glycosyl hydrolases [Henrissat, B. (1991) Biochem. J. 280, 309-316].

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We have characterized nine mcr-1-harboring plasmids from clinical Escherichia coli isolates previously described in Argentina and Canada. Three of these plasmids carried a mcr-1-variant called here mcr-1.5. All these E. coli isolates were not clonally related and were recovered in different years and locations. However, their mcr-1-harboring plasmids showed high identity among them and to others characterized in other countries, which strongly suggests that this plasmid-type is playing an important role in spreading this mechanism of resistance to polymyxins.

cWe studied a collection of 105 clinical enterobacteria with unusual phenotypes of quinolone susceptibility to analyze the occurrence of plasmid-mediated quinolone resistance (PMQR) and oqx genes and their implications for quinolone susceptibility. The oqxA and oqxB genes were found in 31/34 (91%) Klebsiella pneumoniae and 1/3 Klebsiella oxytoca isolates. However, the oqxAand oqxB-harboring isolates lacking other known quinolone resistance determinants showed wide ranges of susceptibility to nalidixic acid and ciprofloxacin. Sixty of the 105 isolates (57%) harbored at least one PMQR gene [qnrB19, qnrB10, qnrB2, qnrB1, qnrS1, or aac(6=)-Ib-cr)], belong to 8 enterobacterial species, and were disseminated throughout the country, and most of them were categorized as susceptible by the current clinical quinolone susceptibility breakpoints. We developed a disk diffusionbased method to improve the phenotypic detection of aac(6=)-Ib-cr. The most common PMQR genes in our collection [qnrB19, qnrB10, and aac(6=)-Ib-cr] were differentially distributed among enterobacterial species, and two different epidemiological settings were evident. First, the species associated with community-acquired infections (Salmonella spp. and Escherichia coli) mainly harbored qnrB19 (a unique PMQR gene) located in small ColE1-type plasmids that might constitute its natural reservoirs. qnrB19 was not associated with an extended-spectrum ␤-lactamase phenotype. Second, the species associated with hospital-acquired infections (Enterobacter spp., Klebsiella spp., and Serratia marcescens) mainly harbored qnrB10 in ISCR1-containing class 1 integrons that may also have aac(6=)-Ib-cr as a cassette within the variable region. These two PMQR genes were strongly associated with an extended-spectrum ␤-lactamase phenotype. Therefore, this differential distribution of PMQR genes is strongly influenced by their linkage or lack of linkage to integrons.

Since 1992 there have been seven major outbreaks of cholera in Argentina. Susceptibility analysis of 1,947 isolates (40% of reported cases) of Vibrio cholerae O1 biotype El Tor suggested the presence of extendedspectrum ␤-lactamases (ESBLs) in 28 isolates. Because of their different susceptibility profiles, V. cholerae isolates M1502, M1516, M1573, and M3030 (all of which are of the Ogawa serotype) were selected for the present study. By susceptibility analysis, isoelectric focusing, and PCR-based restriction fragment length polymorphism analysis, CTX-M-type enzymes were identified in three isolates, whereas a PER-2-type enzyme, in addition to a TEM-1-like enzyme, was identified in the other isolate. The presence of these ESBLs in V. cholerae isolates resulted in MICs well below those commonly observed for members of the family Enterobacteriaceae. Genes that encode both ESBLs were transferred to Escherichia coli by conjugation, together with all determinants of resistance to non-␤-lactam antibiotics (gentamicin, kanamycin, and sulfamethoxazole for all isolates; amikacin and streptomycin for three isolates; trimethoprim, tetracycline, and chloramphenicol for two isolates). Plasmid profile analysis and Southern blotting revealed the presence of single plasmids of about 150 kb in the four V. cholerae isolates and their respective transconjugants and revealed that the plasmids harbored genes encoding CTX-M-type or PER-2-type ESBLs. These results strongly suggest the broad spread of these ESBLs among genera belong to families other than the Enterobacteriaceae.

Plasmids pPAB19-1, pPAB19-2, pPAB19-3, and pPAB19-4, isolated from Salmonella and Escherichia coli clinical strains from hospitals in Argentina, were completely sequenced. These plasmids include the qnrB19 gene and are 2,699, 3,082, 2,989, and 2,702 nucleotides long, respectively, and they share extensive homology among themselves and with other previously described small qnrB19-harboring plasmids. The genetic environment of qnrB19 in all four plasmids is identical to that in these other plasmids and in transposons such as Tn2012, Tn5387, and Tn5387-like. Nucleotide sequence comparisons among these and previously described plasmids showed a variable region characterized by being flanked by an oriT locus and a Xer recombination site. We propose that this arrangement could play a role in the evolution of plasmids and present a model for DNA swapping between plasmid molecules mediated by site-specific recombination events at oriT and a Xer target site. Qnrs are pentapeptide repeat proteins that mediate resistance to quinolones by protecting type II DNA topoisomerases (14, 28). They are known since 1998 when the first qnr gene was found in the multiresistance plasmid pMG252 harbored by a Klebsiella pneumoniae strain isolated from the urine of a patient at the University of Alabama (20). Since then five qnr families (qnrA, qnrB, qnrC, qnrD, and qnrS) have been found, usually hosted in large plasmids (31). The first qnrB gene (qnrB1) was identified in a plasmid from a K. pneumoniae strain isolated in South India (16), and 38 members of the family quickly followed (http://www.lahey .org/qnrStudies/) (13). The qnrB19 gene has been found in several genera of Enterobacteriaceae isolated from humans (healthy people and clinical isolates), animals, and food of animal origin in numerous geographical regions (6,9,12,17,21,22,27). An interesting characteristic of the qnrB19 allele is that it has been found within large plasmids, associated to ISEcp1C-based transposons (6,9,27), and in small plasmids (ϳ3 kbp) lacking ISEcp1C or any other insertion sequence (12,17,22) (Table 1). However, in spite of being located in such dissimilar elements, the qnrB19 genes share a conserved genetic environment (22).We have recently analyzed a collection of clinical enterobacterial isolates with decreased quinolone susceptibility, and we found four small plasmids harboring qnrB19 (2). We describe here their molecular features and characterize their relationships with other qnrB19-harboring genetic platforms. Furthermore, we propose possible pathways of evolution of the qnrB19 environment as well as a site-specific recombination-based model for DNA modifications at a variable region found in these plasmids. MATERIALS AND METHODSBacterial strains and plasmids. The plasmids pPAB19-1, pPAB19-2, pPAB19-3, and pPAB19-4 analyzed in the present study were isolated from Salmonella enterica serovar Infantis M7849, Escherichia coli M9996, E. coli M9888, and Salmonella sp. strain M9397, respectively (Table 1). Salmonella Infantis M7849 was isolated at the ...