Emergence of phylogenetically diverse and fluoroquinolone resistant Salmonella Enteritidis as a cause of invasive nontyphoidal Salmonella disease in Ghana

Abstract

Background: Salmonella enterica serovar Enteritidis is a cause of both poultry- and egg-associated enterocolitis globally and bloodstream-invasive nontyphoidal Salmonella (iNTS) disease in sub-Saharan Africa (sSA). Distinct, multi-drug resistant genotypes associated with iNTS disease in sSA have recently been described, often requiring treatment with fluoroquinolone antibiotics. In industrialised countries, antimicrobial use in poultry production has led to frequent fluoroquinolone resistance amongst globally prevalent enterocolitis-associated lineages.

Methodology/principal findings: Twenty seven S. Enteritidis isolates from patients with iNTS disease and two poultry isolates, collected between 2007 and 2015 in the Ashanti region of Ghana, were whole-genome sequenced. These isolates, notable for a high rate of diminished ciprofloxacin susceptibility (DCS), were placed in the phyletic context of 1,067 sequences from the Public Health England (PHE) S. Enteritidis genome database to understand whether DCS was associated with African or globally-circulating clades of S. Enteritidis. Analysis showed four of the major S. Enteritidis clades were represented, two global and two African. All thirteen DCS isolates, containing a single gyrA mutation at codon 87, belonged to a global PT4-like clade responsible for epidemics of poultry-associated enterocolitis. Apart from two DCS isolates, which clustered with PHE isolates associated with travel to Spain and Brazil, the remaining DCS isolates, including one poultry isolate, belonged to two monophyletic clusters in which gyrA 87 mutations appear to have developed within the region.

Conclusions/significance: Extensive phylogenetic diversity is evident amongst iNTS disease-associated S. Enteritidis in Ghana. Antimicrobial resistance profiles differed by clade, highlighting the challenges of devising empirical sepsis guidelines. The detection of fluoroquinolone resistance in phyletically-related poultry and human isolates is of major concern and surveillance and control measures within the region's burgeoning poultry industry are required to protect a human population at high risk of iNTS disease.

Fig 1. Midpoint-rooted maximum likelihood phylogeny of S. Enteritidis based on 1067 Public Health England surveillance isolates and the 29 Ghanaian isolates.

The majority of the Ghanaian isolates fall into four major clades (a global epidemic clade, a second global clade containing a lineage associated with North American poultry, a Central/ East African clade and a West African clade). Within the global epidemic clade the Ghanaian isolates cluster into sub-clades. Numbers of Ghanaian isolates in each clade or sub-clade are noted in brackets (n =). An asterisk represents clades containing the poultry isolates (*). All clades are supported by bifurcating nodes with 100% bootstrap support. The scale bar shows nucleotide substitutions per site.

Fig 2. Zoomed-in maximum likelihood tree showing the largest monophyletic cluster of Ghanaian isolates within the global epidemic clade (Ghana clade 1).

The ten Ghanaian isolates within this clade are highlighted in red (nine human bloodsteam isolates and one poultry isolate, SRR7072859). Each isolate in the tree is represented by its accession number followed by the country of origin. In the case of PHE isolates, second countries listed after the country of origin represent reported travel destinations. A heatmap showing the resistance genes present in each isolate (the gyrA 87:D-G mutation is shown as a red box) is displayed on the right. The scale bar shows nucleotide substitutions per site.

Fig 3. Zoomed-in maximum likelihood tree showing the smaller monophyletic cluster of Ghanaian isolates within the global epidemic clade (Ghana clade 2).

The three Ghanaian human bloodstream isolates within this clade are highlighted in red. Each isolate in the tree is represented by its accession number followed by the country of origin. In the case of PHE isolates, second countries listed after the country of origin represent reported travel destinations. A heatmap showing the resistance genes present in each isolate (the gyrA 87:D-N mutation is shown as a pink box) is displayed on the right. The scale bar shows nucleotide substitutions per site.

Fig 4. Zoomed-in maximum likelihood tree showing the Spanish travel-associated sub-clade of the global epidemic clade.

One Ghanaian human bloodstream isolate was related to this sub-clade and is highlighted in red. Each isolate in the tree is represented by its accession number followed by the country of origin. In the case of PHE isolates, second countries listed after the country of origin represent reported travel destinations. A heatmap showing the resistance genes present in each isolate is displayed on the right. The gyrA 87:D-Y mutation (shown as a blue box) is present in every isolate of this sub-clade. The scale bar shows nucleotide substitutions per site.