KENTUCKY

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The Project #KENTUCKY

Start:  January 2020
Duration:  3 Years
Domain: Antimicrobial Resistance, Foodborne Zoonoses
Members: Sciensano- Belgium, INRA- France
Contact: Dr Pieter-Jan Ceyssens (Sciensano)

KENTUCKY: Exploring the evolutionary success of the antibiotic resistant Salmonella Kentucky ST198

Salmonella enterica serovar Kentucky (S. Kentucky) is a common causative agent of gastroenteritis in humans. It is one of most notorious Salmonella serotypes, as it is strongly associated with antimicrobial resistance (AMR).  Ciprofloxacin-resistant S. Kentucky (CIPR S. Kentucky) belongs to a single sequence type (ST198), which acquired of a variant of the Salmonella genomic island 1 (SGI1) conferring resistance to first-line antimicrobials (b-lactams, aminoglycosides, sulphonamides, tetracyclines). The MDR clone has since then also accumulated various substitution mutations in the quinolone resistance determining regions (QRDR) of DNA gyrase (gyrA) and DNA topoisomerase (parC), such that most strains carry three QRDR mutations which together confer full resistance to ciprofloxacin as well. Phylogeographic analysis indicates this clone first emerged in Egypt ca. 1989, before disseminating into Northern, Southern and Western Africa, and then further to the Middle East, Asia and Europe.

The main reservoir of S. Kentucky is poultry, and domestic poultry has played an important role in its global spread (most recently in South Asia and Europe). Ciprofloxacin resistance is rare in Salmonella and is hypothesised to be linked to strong selective pressure exerted by fluoroquinolone use in poultry.

In addition to CIPR, S. Kentucky is able to gain additional antibiotic resistance determinants through the acquisition of locally circulating plasmid-borne ESBL, AmpC and/or carbapenemase genes. Additionally, the geographic distribution of ciprofloxacin-resistant (CIPR) S. Kentucky ST198 overlaps with other highly drug resistant Enterobacteriaceae carrying plasmid-borne ESBL, AmpC and/or carbapenemase genes, leading to predictions that highly-drug resistant Kentucky ST198 strains are likely to become more frequent in the near future due to novel plasmid acquisitions.

Most recently, the situation has worsened, as ECDC launched an Urgent Inquiry (UI-464) on a CIPR S. Kentucky ST198 strain carrying a chromosomally integrated blaCTX-M-14b gene encoding for cephalosporin resistance. The insertion event was traced back to Malta, but the strain has already spread to Belgium, UK, The Netherlands and five other EU countries. To date, this clone is only reported in humans, as opposed to (for example) the CipS S. Kentucky ST152 clone widely found in poultry in the USA but rarely reported in humans.

The KENTUCKY project investigates

  • What explains the evolutionary success of the multidrug resistant S. Kentucky ST198 clone?
  • What is the mechanisms of the integration (and potential further transfer) of the ESBL gene in its chromosome?
  • Are there genetic determinants of different human-animal host ranges in epidemic S. Kentucky ST198 and ST152?

Project Outputs

1. Construction of a reporter plasmid based on  P1, pMT  parS/ ParB partition system

IncHI plasmids are important spreaders of antibiotic resistance genes, ca. 40% of colistin resistance-carrying plasmids in Europe, belong to the IncHI group. Due to its clinical significance, the S. Kentucky resident-IncHI- mega-plasmid carrying the ISECP1-blaCTX-M-14B transposition unit was chosen as a focal point for our fluorescent tagging strategy. Yet, wet-lab experiments backed with in silico analysis indicated that the plasmid is non-conjugable, most probably due to truncation in the transfer region. Thus, we reasoned to shift to the prototype IncHI1 plasmid (R27), a self-transmissible plasmid with fully characterized conjugation machinery (Craig et al.,2000) that is capable of transfer between members of the Enterobacteriaceae. pR27 displays a conjugation efficiency of ca. 10-5 at 25°C and ca. 10-7 at 37°C. Since conjugation of the wild-type R27 plasmid is only a one in a 10-5 event, genetic engineering approaches were instigated to increase the conjugation efficiency of pR27 to maximize the likelihood of observing the conjugation in Real-time.  we opted for knocking out the conjugation repressor HtdA which is involved in the repression of four tra operons and has a pivotal role in the growth phase dependency of R27 conjugation. Replacing HtdA by pMT parS , increased the conjugation efficiency from ca. 10-5 ( pR27 wild type) to ca. one in 100. Next, We labeled the transposition unit ISECP1-blaCTX-M-14B with P1-parS and cloned it into pR27 yielding double parS labeled pR27 that can be tracked in Real-time using a fluorescent microscope.

2. Construction of the fluorescent recipient strain to visualize the transfer of pR27 in real-time.

To visualize the double parS tagged R27 plasmid, a recipient strain which encodes orthogonal ParB proteins ( Nielsen et al., 2006) fused to fluorescent reporters was assembled. To validate both the reporter plasmid and the recipient strain, pR27 plasmid labelled with pMTparS and P1parS was conjugated to the recipient strain and the resulting transconjugants were tracked in real-time with fluorescence microscopy. This results in overlapping foci between the YGFP (the plasmid backbone) and CFP (the transposon) channel as can be seen in the below figure.

Project Assets

Albasiony, A., Ceyssens, PJ., Aertsen, A. (2022). Exploring the evolutionary success of the antibiotic-resistant Salmonella Kentucky ST198. Oral presentation at International Symposium Salmonella and Salmonellosis (I3S2022), Saint-Malo, France. 20-22th June 2022.

Albasiony, A., Ceyssens, PJ., Aertsen, A. (2022). Exploring the evolutionary success of the antibiotic-resistant Salmonella Kentucky ST198. Poster presentation at One Health EJP Annual Scientific Meeting , Orvieto, Italy. 11-13th April 2022. DOI: https://doi.org/10.5281/zenodo.7594718

Albasiony, A., Ceyssens, PJ., Aertsen, A. (2021) Exploring the ISECP-1 mediated chromosomal integration of blaCTX-M-14 in Salmonella Kentucky. Poster presentation at One Health EJP Annual Scientific Meeting, Copenhagen, Denmark. 9-11th June 2021.

More information coming soon!

Alaa Albasiony OHEJP profile photo

Alaa Albasiony

About me:
I am a self-motivated pharmacist and bioengineer. I possess over 9 years of experience within the pharmaceutical industry and clinical settings. I am genuinely interested in synthetic biology and our attempts to create semi-synthetic, and maybe “non-ACGT” based life. I hold a master’s degree in molecular biology through which I have developed a special interest in the research focusing on re-programming the bacterial cells to tackle the limitations in the current therapeutic approaches. My motivation is to develop and optimise bacterial delivery systems that can harbour the coding sequence of a therapeutic cargo for in vivo manufacturing and delivery.

What motivated me to do a PhD:
I am passionate about engineering and re-programming bacteria to be utilised in clinical settings. Yet, the concept of a bacterial delivery system suffers a major challenge which is the ability of bacteria to exchange genetic data through a process termed horizontal gene transfer. During the Ph.D. project, we aim to elucidate the molecular basis of horizontal gene transfer and conceptualise the dynamics of mobile genetic elements in prokaryotes. We attempt to generate mechanistic insights that could help to develop models and/or intervention strategies to recede the pathogenesis of <em>Salmonella Kentucky</em> ST198 clone that is extremely resistant to several clinically significant antibiotics.

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