IncR Plasmid Research Articles

Diversity of Plasmids Encoding Virulence and Resistance Functions in Salmonella enterica subsp. enterica Serovar Typhimurium Monophasic Variant 4,[5],12:i:- Strains Circulating in Europe

Plasmids encoding resistance and virulence properties in multidrug resistant (MDR) Salmonella enterica (S.) serovar Typhimurium monophasic variant 4,[5],12:i:- isolates recovered from pigs and humans (2006-2008) in Europe were characterised. The isolates were selected based on the detection by PCR-amplification of S. Typhimurium virulence plasmid pSLT genes and were analysed by multi-locus sequence typing (MLST). The resistance genes present in the isolates and the association of these genes with integrons, transposons and insertion sequences were characterised by PCR-sequencing, and their plasmid location was determined by alkaline lysis and by S1-nuclease pulsed-field gel electrophoresis (PFGE) Southern-blot hybridisation. Plasmids were further analysed by replicon typing, plasmid MLST and conjugation experiments. The 10 S. 4,[5],12,i:- selected isolates belonged to ST19. Each isolate carried a large plasmid in which MDR with pSLT-associated virulence genes were located. After analysis, eight different plasmids of three incompatibility groups (IncA/C, IncR and IncF) were detected. Two IncA/C plasmids represented novel variants within the plasmid family of the S. 4,[5],12:i:- Spanish clone, and carried an empty class 1 integron with a conventional qacEΔ1-sul1 3′ conserved segment or an In-sul3 type III with estX-psp-aadA2-cmlA1-aadA1-qacH variable region linked to tnpA440-sul3, part of Tn2, Tn21 and Tn1721 transposons, and ISCR2. Four newly described IncR plasmids contained the resistance genes within In-sul3 type I (dfrA12-orfF-aadA2-cmlA1-aadA1-qacH/tnpA440-sul3) and part of Tn10 [tet(B)]. Two pSLT-derivatives with FIIs-ST1+FIB-ST17 replicons carried cmlA1-[aadA1-aadA2]-sul3-dfrA12 and bla TEM-1 genes linked to an In-sul3 type I integron and to Tn2, respectively. In conclusion, three emerging European clones of S. 4,[5],12:i:- harboured MDR plasmids encoding additional virulence functions that could contribute significantly to their evolutionary success.

Klebsiella pneumoniae Sequence Type 11 from Companion Animals Bearing ArmA Methyltransferase, DHA-1 β-Lactamase, and QnrB4

Seven Klebsiella pneumoniae isolates from dogs and cats in Spain were found to be highly resistant to aminoglycosides, and ArmA methyltransferase was responsible for this phenotype. All isolates were typed by multilocus sequence typing (MLST) as ST11, a human epidemic clone reported worldwide and associated with, among others, OXA-48 and NDM carbapenemases. In the seven strains, armA was borne by an IncR plasmid, pB1025, of 50 kb. The isolates were found to coproduce DHA-1 and SHV-11 β-lactamases, as well as the QnrB4 resistance determinant. This first report of the ArmA methyltransferase in pets illustrates their importance as a reservoir for human multidrug-resistant K. pneumoniae.

Qnr, aac(6′)-Ib-cr and qepA genes in Escherichia coli and Klebsiella spp.: genetic environments and plasmid and chromosomal location

To characterize the location and genetic environments of qnr, aac(6')-Ib-cr and qepA genes related to quinolone resistance in 19 Escherichia coli, Klebsiella pneumoniae and Klebsiella oxytoca strains. Genetic environments of the indicated genes were studied by cloning, PCR mapping and sequencing. The location of these genes was analysed by S1-PFGE and PFGE-I-CeuI and hybridization with specific probes. Associated antibiotic resistance mechanisms and molecular typing of strains were also investigated. The studied strains carried the aac(6')-Ib-cr, qepA, qnrS1, qnrB6, qnrB4 and oqxAB genes, with aac(6')-Ib-cr being the most prevalent. E. coli strains belonged to sequence types (STs) ST648, ST131, ST224 and ST205, and K. pneumoniae strains to ST433, ST341, ST152, ST15 and ST431. Different genetic environments of quinolone resistance genes were observed, and some of them had not been previously detected and registered in GenBank. The aac(6')-Ib-cr gene was mainly located in class 1 integrons or associated with the Tn1721 transposon in E. coli and associated with the aac(3)-II gene in Klebsiella. All these structures contained mechanisms of gene acquisition and/or dissemination, such as IS26. The studied quinolone resistance genes were mostly detected in IncF and IncN plasmids in E. coli and in IncR plasmids in Klebsiella, but in some strains the chromosomal location of the aac(6')-Ib-cr gene was detected for the first time. The bla(CTX-M-15), bla(OXA-1), tet(A), aac(3)-II and aph(3')-Ia genes and class 1 integrons were found in most strains. The aac(6')-Ib-cr gene was detected for the first time in the chromosome, although a plasmidic location was the most frequently found, with differentiation of plasmids types in E. coli versus Klebsiella.

Investigation of diversity of plasmids carrying the blaTEM-52 gene

To investigate the diversity of plasmids that carry bla(TEM-52) genes among Escherichia coli and Salmonella enterica originating from animals, meat products and humans. A collection of 22 bla(TEM-52)-encoding plasmids was characterized by restriction fragment length polymorphism (RFLP), replicon typing (by PCR or replicon sequencing), susceptibility testing, assessment of plasmid ability to self-transfer by conjugation and typing of the genetic environment of the bla(TEM-52) gene. Detected IncI1 plasmids underwent further plasmid multilocus sequence typing. RFLP profiles demonstrated dissemination of bla(TEM-52) in Denmark (imported meat from Germany), France, Belgium and the Netherlands from 2000 to 2006 by mainly two different plasmids, one encoding bla(TEM-52b) (IncX1A, 45 kb) and the other bla(TEM-52c) (IncI1, 80 kb). In addition, bla(TEM-52b) was also found to be located on various other plasmids belonging to IncA/C and IncL/M, while bla(TEM-52c) was found on IncN-like as well as on IncR plasmids. In the majority of cases (n = 21) the bla(TEM-52) gene was located on a Tn3 transposon. Seven out of 10 bla(TEM-52) plasmids tested in conjugation experiments were shown to be capable of self-transfer to a plasmid-free E. coli recipient. The bla(TEM-52) gene found in humans could have been transmitted on transferable plasmids originating from animal sources. Some of the bla(TEM-52) plasmids carry replicons that differ from the classical ones. Two novel replicons were detected, IncX1A and IncN-like. Unlike its predecessor bla(TEM-1), the bla(TEM-52) gene was not detected on F-type replicons suggesting that this gene evolved on other types of plasmid scaffolds.