Healthcare-associated infections caused by chlorhexidine-tolerant Serratia marcescens carrying a promiscuous IncHI2 multi-drug resistance plasmid in a veterinary hospital

The bacterium Serratia marcescens can cause opportunistic infections in humans and in animals. In veterinary settings, the diversity, reservoirs and modes of transmission of this pathogen are poorly understood. The phenotypes and genotypes of Serratia spp. isolated from dogs, cats, horses, a bird and a rabbit examined at an Australian veterinary hospital between 2008 and 2019 were characterised. The isolates were identified as S. marcescens (n = 15) or S. ureilytica (n = 3) and were placed into four distinct phylogenetic groups. Nine quasi-clonal isolates associated with post-surgical complications in different patients displayed high levels of resistance to the antimicrobials fluoroquinolones, cephalosporins, aminoglycosides, and to the disinfectant chlorhexidine. A Serratia sp. with a similar resistance profile was also isolated from chlorhexidine solutions used across the Hospital, suggesting that these infections had a nosocomial origin. A genomic island encoding a homolog of the Pseudomonas MexCD-OprJ biocide efflux system was detected in the chlorhexidine-tolerant Serratia. The nine multi-drug resistant Serratia isolates also possessed a Ser-83-Ile mutation in GyrA conferring fluoroquinolone resistance, and carried a large IncHI2 conjugative plasmid encoding antimicrobial and heavy metal resistances. This replicon was highly similar to a plasmid previously detected in a strain of Enterobacter hormaechei recovered from the Hospital environment. IncHI2 plasmids are commonly found in Enterobacteriaceae, but are rarely present in Serratia spp., suggesting that this plasmid was acquired from another organism. A chlorhexidine-tolerant Serratia isolate which lacked the IncHI2 plasmid was used in mating experiments to demonstrate the transfer of multi-drug resistance from a E. hormaechei donor. This study illustrates the importance of environmental surveillance of biocide-resistance in veterinary hospitals.</jats:p

Genes present in the chlorhexidine-tolerant strains CM2015_854 and CM2017_569 but absent from the chlorhexidine-susceptible strains.

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Sequenced humans isolates of <i>Serratia</i> that are predicted to carry a IncHI2/IncHI2A plasmid, illustrating the rarity of the incompatibility group within this genus.

Sequenced humans isolates of Serratia that are predicted to carry a IncHI2/IncHI2A plasmid, illustrating the rarity of the incompatibility group within this genus.</p

Heatmap of all-against-all ANI values for 133 complete <i>Serratia</i> spp. genomes and 18 veterinary isolates, showing distinct intra-specific subgroups within <i>S</i>. <i>marcescens sensu lato</i>.

The top dendrogram was generated from a distance matrix calculated from the ANI values using the “dist” function (euclidean method) followed by hierarchical clustering using the “hclust” function (complete method) in R. The ANI values are represented by a scale of grey. The darkest shade represents ANI values >97.5%, which is used as the lowest limit to define groups A-F within the S. marcescens sensu lato group. The strain names and ANI groups are indicated on the right side of the map and their corresponding accession numbers are listed in the S1 Table. The GenBank files headers were used to extract the reported taxonomic classification (top sidebar) and the host or origin (left sidebar) of each strain, and this information was color-coded accordingly. The 18 animal isolates from the U-vet are indicated by a pink square on the right side of the map. The number of ARGs per genome was calculated from individual ABRicate reports and color-coded on the far-left sidebar.</p

Comparison of MICs values (μg/mL) of donor strain <i>E</i>. <i>hormachei</i> CM2018_216, recipient strain <i>S</i>. <i>marcescens</i> CM2017_569 and 2 <i>Serratia</i> transconjugants T4 and T6, demonstrating the occurrence of transfer of resistance <i>in vitro</i>.

Comparison of MICs values (μg/mL) of donor strain E. hormachei CM2018_216, recipient strain S. marcescens CM2017_569 and 2 Serratia transconjugants T4 and T6, demonstrating the occurrence of transfer of resistance in vitro.</p

Phylogenetic diversity and plasmid distribution in <i>S</i>. <i>marcescens</i> genomes.

A maximum likelihood tree was generated from concatenated multiple alignments with MEGA using the General Time Reversible model with discrete Gamma distribution and Invariable sites (GTR+G+I), selected by the lowest Bayesian Information Criterion (BIC) score. Top: entire tree derived from the analysis of 671 complete and partial genomes from NCBI. Bottom: close-up showing the position of 9 MDR isolates in the tree. The presence and incompatibility groups of plasmids were predicted by ABRicate with the database plasmidfinder. The presence of the ARGs mcr-9.1 and blaSHV-12 was predicted by ABRicate with the database resfinder. The unrooted tree was decorated with iTOL. (TIFF)</p

Clinical history and key phenotypic characters of <i>Serratia</i> spp. isolates.

Clinical history and key phenotypic characters of Serratia spp. isolates.</p

Predicted ARGs in fully assembled genomes of representative <i>Serratia</i> spp. isolates, showing the high number of predicted resistance markers carried by a IncHI2 plasmid in strain CM2015_854.

Predicted ARGs in fully assembled genomes of representative Serratia spp. isolates, showing the high number of predicted resistance markers carried by a IncHI2 plasmid in strain CM2015_854.</p

Chlorhexidine Minimum Inhibitory Concentrations and Minimum Bactericidal Concentrations for <i>Serratia</i> spp. isolates, showing a higher resistance to this biocide within the MDR strains compared to the other strains.

Chlorhexidine Minimum Inhibitory Concentrations and Minimum Bactericidal Concentrations for Serratia spp. isolates, showing a higher resistance to this biocide within the MDR strains compared to the other strains.</p

Minimum inhibitory concentration of <i>Serratia</i> spp. isolates from the U-Vet.

Minimum inhibitory concentration of Serratia spp. isolates from the U-Vet.</p