A novel IS26 structure surrounds bla CTX-M genes in different plasmids from German clinical Escherichia coli isolates

Abstract

This report focuses on the molecular characterization of 22 extended-spectrum b-lactamaseproducing Escherichia coli isolates collected in a German university hospital during a period of 9 months in 2006. Relationship analysis of clinical isolates was done via PFGE, multilocus sequence typing, plasmid profiling and additionally PCR for bla ESBL detection and determination of phylogroups. After conjugal transfer, plasmid isolation and subsequent PCR for bla ESBL detection and determination of incompatibility groups were performed. Using one-primer walking, up to 3600 bp upstream and downstream of different bla CTX-M genes could be sequenced. b-Lactamases found were TEM-1 (n514), SHV-5 (n51) and a wide variety of CTX-M types (n521), i.e. CTX-M-15 (n512), CTX-M-1 (n54), CTX-M-14 (n52), CTX-M-9 (n51), CTX-M-3 (n51) and one new type, CTX-M-65 (n51). In 18 isolates, bla ESBL genes were located on conjugative plasmids of sizes between 40 and 180 kbp belonging to incompatibility groups FII (n59), N (n55) and I1 (n54). bla CTX-M was found to be associated with the common elements ISEcp1, IS26 and IS903-D, but with unusual spacer sequences for ISEcp1 in two isolates. These insertion sequences, connected to bla CTX-M as well as other genes, were located between two IS26 elements in a configuration that has not yet been described. The results reveal the emergence of bla ESBL , predominantly bla CTX-M , located on different plasmids harboured by genotypically different E. coli strains. The identical gene arrangement in the bla CTX-M neighbourhood in plasmids of different incompatibility groups indicates a main role of IS26 in distribution of mobile resistance elements between different plasmids. INTRODUCTIONResistance to extended-spectrum b-lactam antibiotics is mainly caused by extended-spectrum b-lactamases (ESBLs) such as bla TEM , bla SHV and bla CTX-M (Paterson & Bonomo, 2005). CTX-M-type b-lactamases seemed to be particularly successful in terms of spread. Since the first description in 1989, 86 variants have been found to date (www.lahey.org/ studies/). They are clustered in five subgroups (1, 2, 8, 9, 25) according to their amino acid homology (Tzouvelekis et al., 2000;Bonnet, 2004). Chromosomal genes of different Kluyvera species have been identified as a natural reservoir. A natural diversity of CTX-M types is also found among nosocomial isolates, which leads to the conclusion that the bla CTX-M genes have been acquired by different events (Rodriguez et al., 2004). A number of genetic mechanisms have apparently been involved in aquisition of CTX-M genes. Insertion sequences IS26, ISEcp1 and ISCR1 in association with class 1 integron structures, as well as phagerelated elements, seem to have played a prominent role in these processes (Arduino et al., 2002;Eckert et al., 2006;Oliver et al., 2005;Poirel et al., 2008). Moreover, ISEcp1 elements and remnants constitute an alternative promoter region (Karim et al., 2001) leading to increased, clinically relevant expression of the bla CTX-M gene, which is only weakly exp...

Analysis of bla CTX-M -Carrying Plasmids from Escherichia coli Isolates Collected in the BfT-GermVet Study

“…These results showed that the structural variability of the bla CTX-M-1 gene region resulted from interplasmid recombination events and the integration of insertion sequences. The gene bla CTX-M-1 in combination with the IS26-⌬ISEcp1-structure and the truncated mph(A)-mrx-mphR(A) gene cluster, which is followed by a second IS26 element, linked to IncN plasmids, was recently found in a German human clinical E. coli ST131 isolate, proposing a novel composite transposon with both IS26 elements being flanked by 8-bp direct repeats (5Ј-TTACCGGT-3Ј) (13). An identical sequence was located The recombination site between the mrx gene and orf477 is shown between the two maps, while the recombination site that played a role in the truncation of the mph(A) gene in pCTX168 is shown below the map of pCTX168.…”

“…A database search revealed that identical sequences downstream of the bla CTX-M-15 gene were present in a wide variety of bla CTX-M-15 -carrying plasmids from E. coli (3,13,15,32,37) and K. pneumoniae (33) but also Enterobacter cloacae (GenBank accession no. HM204572, 219230) and Serratia liquefaciens (GenBank accession no.…”

“…Molecular analyses. The ESBL genes were identified by previously described PCR assays (13) and initially confirmed by sequence analysis of the amplicons. Multilocus sequence typing (MLST) was performed for the bla CTX-M -positive E. coli isolates (36).…”

Characterization of Extended-Spectrum β-Lactamase Genes Found among Escherichia coli Isolates from Duck and Environmental Samples Obtained on a Duck Farm

“…Because most of the bla CTX-M-105 producers were clonally unrelated and the bla CTX-M-105 genes were located on different plasmids, we analyzed the genetic background surrounding it. bla CTX-M-105 was located in a Tn1721-like element on pHNDD81-1 and is associated with an ISEcp1-bla CTX-M -IS903D-iroN⌬ structure which has been seen in several cases associated with bla CTX-M-65 (7,33), bla CTX-M-24 (26), bla CTX-M-19 (24), and bla CTX-M-14 (17). ISEcp1 has been proved to be able to recognize part of the iroN gene sequence as its alternative IRR and then mobilize bla CTX-M , IS903, and the partial iroN gene (iroN⌬) into Tn1721.…”

Everyman's Guide to Bacterial Insertion Sequences

“…IS26 is encountered with increasing frequency in plasmids of clinical importance where it is involved in expression of antibiotic resistance genes and plasmid rearrangements (166)(167)(168)(169)(170)(171).…”