MDR Escherichia coli carrying CTX-M-24 (IncF[F-:A1:B32]) and KPC-2 (IncX3/IncU) plasmids isolated from community-acquired urinary trainfection in Brazil

ABSTRACT

Acquired antibiotic resistance in bacteria has become an important worldwide challenge. Currently, several bacteria, including Escherichia coli, have multidrug resistance profiles. Genes such as bla CTX-M-24 and bla KPC-2 (carbapenemase) are widespread. This research letter reports about a genomic surveillance study where multidrug-resistant E. coli containing CTX-M-24(IncF [F-:A1:B32]) and KPC-2(IncX3/IncU) plasmids were obtained from community- acquired urinary tract infection in Brazil.

Keywords:
Escherichia coli ; Urinary tract infection; CTX-M-24; KPC-2

Acquired antibiotic resistance in bacteria has become an important worldwide challenge.¹ Currently, several bacteria, including Escherichia coli, have multidrug resistance profiles.² Genes such as bla_CTX-M-24 and bla_TEM-1B that encodes extended-spectrum beta-lactamase (ESBL) are widespread in E. coli strains.², ³ These genes can suppress the action of cephalosporin antibiotics.⁴ Besides, the presence of bla_KPC-2 (carbapenemase) gene in the bacterial genome may confer a carbapenem resistance profile.⁵, ⁶ During a genomic surveillance study, a multidrug-resistant Escherichia coli containing CTX-M-24 (IncF [F-:A1:B32]) and KPC-2 (IncX3/IncU) plasmids was obtained from community-acquired urinary tract infection in Brazil. This microorganism was isolated in 2016 from a urine sample from a community 79-year old female patient, without previous hospitalization in the last year, living in the south region (Londrina, state of Parana, Brazil) and it was associated with ST354 strain after sequencing type analysis.

For genome sequencing, total DNA was extracted using a PureLink™ Quick Gel Extraction Kit (Life Technologies, CA). Libraries were prepared with a NexteraXT library prep kit (Illumina Inc., San Diego, CA). The samples were sequenced via Illumina NextSeq 550 platform (Illumina Inc., San Diego, CA), using 2 × 150-bppaired-end reads. Reads were de novo assembled using Unicyler v0.4.0 software (7). Read with a PHRED quality score below 20 were discarded, and adapters were trimmed using TrimGalore v0.6.5 (https://github.com/FelixKrueger/TrimGalore). The E. coli ST354 genome was annotated using the Prokaryotic Genome Annotation Pipeline v.3.2 (PGAP/NCBI). Multilocus sequence type (MLST), antimicrobial resistance (AMR) genes, virulence factors and plasmid replicons were predicted using the MLST v2.0, pMLSTv2.0, ResFinderv4.1, FimTyperv1.0, VirulenceFinderv2.0 and PlasmidFinder v2.1 (https://cge.cbs.dtu.dk/services/).⁷, ⁸

The E. coli ST354 strain exhibited as MDR profile by antimicrobial susceptibility testing (Table 1) according to the Clinical and Laboratory Standards Institute guidelines (CLSI, 2020)⁹ and by minimum inhibitory concentration (MIC) for colistin using automated Vitek^® 2 system.

Table 1
Antimicrobial susceptibility profile of E. coli ST354, containing CTX-M-24 and KPC-2 enzymes, obtained from community-acquired urinary tract infection in Brazil.

Antimicrobials	Susceptibility profile
	Sensitive (S)	Resistant (R)
Ampicillin (AMP)	-	R
Amoxicillin/clavulanate (AMC)	-	R
Trimethoprim-sulfamethoxazole (STX)	S	-
Piperacillin-tazobactam (TZP)	-	R
Cefalexin (CFX)	-	R
Cefuroxime (CXM)	-	R
Ceftriaxone (CRO)	-	R
Cefepime (FEP)	-	R
Meropenem (MEM)	-	R
Ertapenem (ERP)	-	R
Amikacin (AK)	S	-
Gentamicin (CN)	S	-
Ciprofloxacin (CIP)	-	R
Norfloxacin (NOR)	-	R
Nitrofurantoin (F)	S	-
Nalidixic acid (NA)	-	R

E. coli ST354 is associated with zoonosis and human infections (10). Generally, this strain causes extra-intestinal infections in humans and other animals. Besides, ST354 strain has a resistant profile to fluoroquinolone.¹⁰ The virulome of this isolate from Brazil showed a vast repertoire of virulence consisting of eilA (Salmonella HilA homologue), ipfA (long polar fimbriae), air (enteroaggregative immunoglobulin repeat protein), iss (increased serum survival) and gad (glutamate decarboxylase alphagenes). The presence of these virulence factors provides information regarding the high pathogenicity profile of E. coli ST354 lineage isolated from community-acquired urinary tract infection.

Concerning the resistome of E. coli ST354, it contains resistance factors to beta-lactam antibiotics. These factors are TEM-1B, CTX-M-24 and KPC-2. The blaCTX-M-24 gene is present in the IncF (IncF [F-:A1:B32]) plasmid while blaKPC-2 was identified within the IncX3/IncU replicons. Additionally, this strain carries the tet(B) gene, which confers resistance to tetracycline. Finally, many point mutations in the parC (Ser80Ile, Glu84Gly, Ser57Thr), parE (Ile355Thr, Leu416Phe) and gyrA (Ser83Leu, Asp87Asn) genes have been identified and are associated with resistance to fluoroquinolones (FQ) drugs. GyrA and ParC proteins are FQ targets. Point alterations in their genes can lead to FQ resistance.¹¹ The most frequent substitutions are Ser83Leu and Asp87Asn for gyrA and Ser80-Ile for parC gene.¹¹, ¹² The presence of resistance factors in replicons and chromosomal DNA reinforces the strong, resistant profile of E. coli ST354 strain making it become a potent MDR microorganism.

Many incompatibility group (Inc) plasmids are involved with resistance to several drugs in E. coli lineages. These replicons carry various combinations of resistance genes, and they can be transferred by conjugation.¹³ IncF and IncX are prevalent plasmids type in E. coli. IncX4, for example, is frequently identified as a carrier of resistance genes related to FQ and β-lactam antibiotics resistance.¹⁴, ¹⁵ Moreover, IncX4 has a high frequency of self-transfer (10⁻¹-10⁻⁴).¹³ In this study, the IncF [F-:A1:B32] plasmid harbors the blaCTX-M-24 gene and IncX3/IncU carry the blaKPC-2 gene.

These findings suggest that the presence of blaCTX-M-24, blaTEM-1B and blaKPC-2 genes in E. coli ST354 could be related to the multidrug resistance profile obtained in the antimicrobial susceptibility test. This strain was mainly resistant to beta-lactam antibiotics such as cephalosporins. Moreover, point mutations in parC, parE and gyrA genes observed in E. coli ST354 could influence the antimicrobial resistance profile to FQ antibiotics observed in this study. In addition to this resistant behavior, this strain contains several virulent factors such as air, gad, eilA, iphA and iss, which shows its high pathogenic genome content.

Our data could help understand the genetic basis of high pathogenicity of the MDR E. coli ST354 isolated from community-acquired urinary tract infection in Brazil. The presence of blaCTX-M-24, blaTEM-1B and blaKPC-2 genes, as well as incompatibility plasmids such as IncF [F-:A1:B32] and point mutations in the parC, parE and gyrA chromosomal genes, may help increase the spectrum of antimicrobial resistance in this microorganism and contribute to its pathogenicity.

Acknowledgment

The authors would like to thank the financial support of the Bill and Melinda Gates Foundation’s Grand Challenges Explorations Brazil - New Approaches to characterize the global burden of antimicrobial resistance (OPP1193112), the Research Support Facilities Center of the State University of São Paulo (CEFAPUSP), Institute of Biomedical Sciences of State University of São Paulo (ICB-USP) and Master’s program Clinical and Laboratory Pathophysiology of the State University of Londrina.

• Edited by Dr. L Goldani
• Ethical approval
  The study was approved by the Ethics and Research Committee of the State University of Londrina CAAE 56869816.0.000.5231.
• Data availability
  Draft whole-genome assembly was deposited in DDBJ/ENA/GenBank under the SRA accession numberSRR10310377.

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Publication Dates

• Publication in this collection
  23 Jan 2023
• Date of issue
  2022

History

• Received
  21 May 2022
• Accepted
  17 Sept 2022
• Published
  11 Oct 2022