Chromosomal location of blaCTX-M genes in clinical isolates of Escherichia coli from Germany, The Netherlands and the UK

doi:10.1016/j.ijantimicag.2014.02.019

International Journal of Antimicrobial Agents

Volume 43, Issue 6, June 2014, Pages 553-557

https://doi.org/10.1016/j.ijantimicag.2014.02.019 Get rights and content

Abstract

This study aimed to detect and characterise clinical Escherichia coli isolates suspected of carrying chromosomally encoded CTX-M enzymes. Escherichia coli (n = 356) obtained in Germany, The Netherlands and the UK (2005–2009) and resistant to third-generation cephalosporins were analysed for the presence of ESBL-/AmpC-encoding genes within the European SAFEFOODERA-ESBL project. β-Lactamases and their association with IS26 and ISEcp1 were investigated by PCR. Isolates were typed by phylogenetic grouping, MLST and PFGE. Plasmids were visualised by S1 nuclease PFGE, and the location of bla_CTX-M genes was determined by Southern hybridisation of XbaI-, S1- and I-CeuI-digested DNA. ESBL enzymes could not be located on plasmids in 17/356 isolates (4.8%). These 17 isolates, from different countries and years, were ascribed to phylogenetic groups D (9), B2 (6) and B1 (2), and to seven sequence types, with ST38 being the most frequent (7 phylogroup D isolates). Eleven isolates produced CTX-M-15. bla_CTX-M-15 genes were associated with ISEcp1. The remaining isolates expressed the CTX-M group 9 β-lactamases CTX-M-14 (4), CTX-M-9 (1) and CTX-M-51 (1). bla_CTX-M probes hybridised with I-CeuI- and/or XbaI-digested DNA, but not with S1-digested DNA, corroborating their chromosomal location. To summarise, only 4.8% of a large collection of ESBL-producing E. coli isolates harboured chromosomal bla_CTX-M genes. These isolates were of human origin and belonged predominantly to ST38 and ST131, which possibly indicates the role of these sequence types in this phenomenon. However, heterogeneity among isolates was found, suggesting that their spread is not only due to the dispersion of successful E. coli clones.

Introduction

Production of β-lactamases is the main mechanism responsible for resistance to β-lactams in Enterobacteriaceae. Extended-spectrum β-lactamases (ESBLs) are able to hydrolyse third- and fourth-generation cephalosporins and monobactams, limiting therapeutic options in serious infections caused by Enterobacteriaceae. Over the last decade, the number of ESBL-producing bacteria has increased in many different genera of Enterobacteriaceae and represents a public health threat [1].

CTX-M-type ESBLs are a complex and heterogeneous family of enzymes and may be subdivided into five major groups (CTX-M-1, CTX-M-2, CTX-M-8, CTX-M-9 and CTX-M-25 groups) [2]. These enzymes have spread globally and are the most common ESBLs detected in Enterobacteriaceae, not only in hospitals but also in the community, changing the epidemiology of ESBLs. Among the different CTX-M enzymes, CTX-M-15 (belonging to CTX-M group 1) and CTX-M-14 (belonging to CTX-M group 9) are of high relevance because of their ubiquity, being detected not only in humans and animals but also in environmental samples in many different countries [1], [2]. The successful spread, diversification and maintenance of CTX-M enzymes is due to a combination of different factors: their association with transposable elements and integrons; the capture of these structures by conjugative plasmids; and their transfer to and maintenance in successful bacterial clones [2]. The latter are typified by Escherichia coli clone O25b-ST131, which is a pandemic, multiresistant and uropathogenic lineage frequently associated with the expression of CTX-M-15 and that has contributed significantly to the worldwide spread of this ESBL [3]. This clone habitually harbours the bla_CTX-M-15 gene located on plasmids belonging to the IncF family, typically IncFII or multireplicon FII, FIA and FIB [3].

Despite being predominantly plasmid-mediated enzymes, chromosomally located bla_CTX-M genes have also been described [4], [5], [6]. In this case, the chromosomal location does not enhance the spread of the gene but does assist its stabilisation and maintenance in the bacterium. The objective of this study was to seek and characterise clinical E. coli isolates suspected of expressing chromosomally encoded CTX-M enzymes from Germany, The Netherlands and the UK.

Section snippets

Bacterial isolates and detection of β-lactamase-encoding genes

Within the European SAFEFOODERA-ESBL project (EU ERA-Net, Ref. 08176), a total of 629 E. coli isolates were selected from the strain collections of the Animal Health and Veterinary Laboratories Agency (AHVLA, UK), Public Health England (PHE, UK), Central Veterinary Institute (CVI, The Netherlands), Friedrich-Loeffler-Institut (FLI, Germany) and Federal Institute for Risk Assessment (BfR, Germany). All isolates were cefotaxime-non-susceptible [minimum inhibitory concentrations above the

Results and discussion

Attempts to transform ESBL genes into a recipient strain failed repeatedly for only 17 (4.8%) of the 356 E. coli isolates and these were selected to investigate chromosomally encoded ESBLs. Of the 17 isolates, 11 produced CTX-M-15 enzyme, usually in addition to narrow-spectrum β-lactamases such as TEM-1 (5 isolates), OXA-1 (2) or both (2) (Table 1). The remaining six isolates expressed CTX-M group 9 ESBLs; four produced CTX-M-14 (with two co-producing TEM-1), whilst CTX-M-9 and CTX-M-51 ESBLs

Acknowledgments

The authors thank S. Schmoger, B. Baumann, W. Barownick and the NRL-Salm staff for their helpful assistance.

Funding: The Federal Institute for Risk Assessment (BfR) [BfR-45-004; BfR-46-001] and the EU-SAFEFOODERA project EU ERA-Net, Ref. 08176, entitled ‘The role of commensal microflora of animals in the transmission of extended spectrum β-lactamases’. During the experimental execution of this work, IR was a postdoctoral student at the BfR (Berlin, Germany), with a grant from the Fundación

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The One Health approach of antimicrobial resistance highlighted the role of the aquatic environment as a reservoir and dissemination source of resistance genes and resistant bacteria, especially due to anthropogenic activities. Resistance to extended-spectrum cephalosporins (ESC) conferred by extended-spectrum beta-lactamases (ESBLs) in E. coli has been proposed as the major marker of the AMR burden in cross-sectoral approaches. In this study, we investigated wastewater, surface water and seawater that are subjected to official water quality monitoring in Monastir, Tunisia. While all but one sample were declared compliant according to the official tests, ESC-resistant bacteria were detected in 31 (19.1 %) samples. Thirty-nine isolates, coming from urban, industrial and surface water in Monastir, were collected and characterized using antibiograms and whole-genome sequencing. These isolates were identified as 27 Escherichia coli (69.3 %) belonging to 13 STs, 10 Klebsiella pneumoniae (25.6 %) belonging to six STs, and two Citrobacter freundii (5.1 %). We observed the persistence and dissemination of clones over time and in different sampling sites, and no typically human-associated pathogens could be identified apart from one ST131. All isolates presented a bla_CTX-M gene – bla_CTX-M-15 (n = 22) and bla_CTX-M-55 (n = 8) being the most frequent variants – which were identified on plasmids (n = 20) or on the chromosome (n = 19). In conclusion, we observed ESC resistance in rather ubiquitous bacteria that are capable of surviving in the water environment. This suggests that including the total coliform count and the ESBL count as determined by bacterial growth on selective plates in the official monitoring would greatly improve water quality control in Tunisia.
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Whole genome sequencing (WGS) of extended-spectrum β-lactamase-producing Escherichia coli (ESBL-E. coli) in developing countries is lacking. Here we describe the population structure and molecular characteristics of ESBL-E. coli faecal isolates in rural Southern Niger.
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Most isolates belonged to the commensalism-adapted phylogroup A (83.5%), with high clonal diversity. The bla_CTX-M-15 gene was the major ESBL determinant (98.1%), chromosome-integrated in approximately 50% of cases, in multiple integration sites. When plasmid-borne, bla_CTX-M-15 was found in IncF (57.4%) and IncY plasmids (26.2%). Closely related plasmids were found in different genetic backgrounds. Genomic environment analysis of bla_CTX-M-15 in closely related strains argued for mobilisation between plasmids or from plasmid to chromosome.
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Proper use of antimicrobials should be combined with other farm management strategies for the effective control of cephalosporin-resistant E. coli isolates in commercial layer farms.
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Citation Excerpt :
ESBL genes are often found on plasmids, which facilitates their horizontal spread (Castanheira et al., 2021). However, some previous studies also detected ESBL genes on chromosomes and suggested that integration of ESBL genes into chromosomes could contribute to the stabilization and maintenance of ESBL genes in the absence of antibiotic selection pressure (Hirai et al., 2013; Nair et al., 2021; Rodriguez et al., 2014). Thus, information on the location (chromosome or plasmid) of ESBL genes is important to predict the mobility and stability of these genes.
Occurrence of extended-spectrum β-lactamase (ESBL)-producing Escherichia coli (ESBLEC) in environmental waters is of great concern. However, unlike clinical ESBLEC, their genetic characteristics, in particular the genetic contexts of ESBL genes, are not well understood. In this study, we sequenced and analyzed the genomes of CTX-M-producing E. coli isolates recovered from river water to fully characterize the genetic contexts of bla_CTX-M genes. Among the 14 isolates with completed genomes, bla_CTX-M genes were detected on the chromosome in nine isolates. All but one chromosomal bla_CTX-M genes were associated with ISEcp1 and were carried on different transposition units ranging in size from 2,855 bp to 11,093 bp; the exception, bla_CTX-M-2, was associated with ISCR1. The remaining five isolates carried bla_CTX-M genes on epidemic IncI1 plasmids of different sequence types (STs) (ST3, ST16, ST113, and ST167) (n = 4) or on an IncB/O/K/Z plasmid (n = 1). This study revealed that environmental E. coli carry bla_CTX-M genes in diverse genetic contexts. Apparent high prevalence of chromosomal bla_CTX-M potentially indicates that some E. coli can stably maintain bla_CTX-M genes in environmental waters, though further studies are needed to confirm this.
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¹: This consortium also includes: Martin J. Woodward and Nick Coldham, Animal Health and Veterinary Laboratories Agency (AHVLA), UK; Kristina Kadlec and Stefan Schwarz, Friedrich-Loeffler-Institut (FLI), Germany; John Threlfall, Neil Woodford and John Wain, Public Health England (PHE), UK; and Cindy Dierikx, Central Veterinary Institute (CVI), The Netherlands.

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International Journal of Antimicrobial Agents

Abstract

Introduction

Section snippets

Bacterial isolates and detection of β-lactamase-encoding genes

Results and discussion

Acknowledgments

References (15)

Diversity of CTX-M β-lactamases and their promoter regions from Enterobacteriaceae isolated in three Parisian hospitals

FEMS Microbiol Lett

Plasmids in Gram negatives: molecular typing of resistance plasmids

Int J Med Microbiol

Scientific opinion on the public health risks of bacterial strains producing extended-spectrum β-lactamases and/or AmpC β-lactamases in food and food-producing animals

EFSA J

CTX-M enzymes: origin and diffusion

Front Microbiol

Escherichia coli O25b-ST131: a pandemic, multiresistant, community-associated strain

J Antimicrob Chemother

Dissemination of clonally related Escherichia coli strains expressing extended-spectrum β-lactamase CTX-M-15

Emerg Infect Dis

Characterization of IncF plasmids carrying the bla_CTX-M-14 gene in clinical isolates of Escherichia coli from Korea

J Antimicrob Chemother

Cited by (72)

Occurrence and persistence of multidrug-resistant Enterobacterales isolated from urban, industrial and surface water in Monastir, Tunisia

Faecal carriage of extended-spectrum β-lactamase-producing Escherichia coli in a remote region of Niger

Clonal and plasmid-mediated flow of ESBL/AmpC genes in Escherichia coli in a commercial laying hen farm

Chromosomal integration of bla<inf>CTX-M</inf> genes in diverse Escherichia coli isolates recovered from river water in Japan

Dynamics of extended-spectrum cephalosporin-resistant Escherichia coli in pig farms: A longitudinal study

Molecular characterization of a carbapenem-resistant Enterobacter hormaechei ssp. xiangfangensis co-harbouring bla<inf>NDM-1</inf> and a chromosomally encoded phage-linked bla<inf>CTX-M-15</inf> genes

Short CommunicationChromosomal location of blaCTX-M genes in clinical isolates of Escherichia coli from Germany, The Netherlands and the UK

Abstract

Introduction

Section snippets

Bacterial isolates and detection of β-lactamase-encoding genes

Results and discussion

Acknowledgments

FEMS Microbiol Lett

Int J Med Microbiol

Scientific opinion on the public health risks of bacterial strains producing extended-spectrum β-lactamases and/or AmpC β-lactamases in food and food-producing animals

EFSA J

CTX-M enzymes: origin and diffusion

Front Microbiol

Escherichia coli O25b-ST131: a pandemic, multiresistant, community-associated strain

J Antimicrob Chemother

Dissemination of clonally related Escherichia coli strains expressing extended-spectrum β-lactamase CTX-M-15

Emerg Infect Dis

Characterization of IncF plasmids carrying the blaCTX-M-14 gene in clinical isolates of Escherichia coli from Korea

J Antimicrob Chemother

Short Communication
Chromosomal location of bla_CTX-M genes in clinical isolates of Escherichia coli from Germany, The Netherlands and the UK

Characterization of IncF plasmids carrying the bla_CTX-M-14 gene in clinical isolates of Escherichia coli from Korea