Welcome to the IUMS 2022 Interactive Program

Abstract Body

Bacteria that have acquired resistance to last-resort antibiotics are a major threat to modern healthcare. Here, I will discuss recent work on the evolutionary trajectories that lead to resistance to the antibiotic colistin in the Gram-negative bacteria Escherichia coli and Klebsiella pneumoniae and to the antibiotic vancomycin in the Gram-positive bacterium Enterococcus faecium.

Colistin resistance can emerge through the acquistion of mobilisabile colistin resistance (mcr) genes, but also, and perhaps more commonly, through the accumulation of chromosomal mutations. We show that in Enterobacteriaceae strains isolated from clinical sites, mutations in genes encoding two-component systems are commonly associated with colistin resistance. In all colistin-resistant strains, the lipid A moieties of LPS were modified, through hydroxylation, palmitoylation, and/or the addition of 4-amino-4-deoxy-L-arabinose (by ArnT) or phosphoethanolamine. These mutations, and their associated modifications of LPS, can have variable impacts on fitness and virulence characteristics.

Vancomycin resistance in Enterococcus faecium is mediated through the acquisition of a gene cluster that replaces the terminal amino acids of peptidoglycan precursors from D-Ala-D-Ala to D-Ala-D-lactate or, less commonly, to D-Ala-D-Serine. At least 9 vancomycin resistance gene clusters have been identified, but the vanA-type is globally the most common cause of vancomycin resistance in E. faecium. In a recent genomic study, we studied the genomic epidemiology of the vancomycin-resistant E. faecium strains in a local hospital. We found strains that had the vanA-type vancomycin resistance genes but were phenotypically susceptible. However, these strains quickly reverted, due to plasmid rearrangements and integration in the chromosome, to a resistant phenotype upon growth in the presence of low levels of vancomycin.

Our works shows the remarkable variety of mechanisms by which opportunistic pathogens can acquire resistance to important antibiotics and highlights the need for genome-based diagnostics to rapidly identify resistance mechanisms.

Background and Aims

Streptococcus pneumoniae inhabits the normal nasopharyngeal flora of human infants, though often causes life-threatening bacterial infections, including pneumonia, bacteremia, and meningitis. However, pneumococcal factors that determine host pathophysiology remain largely unknown. In this study, pneumococcal genetic factors related to clinical symptoms were explored using a bacterial genome-wide association approach.

Methods

A total of 6037 whole genome sequences of S. pneumoniae from 13 countries registered in the NCBI database were obtained, in addition to 58 sequences that we previously determined. Using Pyseer, v.1.3.4, a genome-wide association study (GWAS) of genetic variants including single nucleotide polymorphisms (SNPs) in core genes was conducted. To elucidate the influences of SNPs on protein functions, protein structures were predicted with use of AlphaFold2.

Results

The GWAS results revealed 329 SNPs that were significantly related to clinical symptoms. Furthermore, 32 significant SNPs were detected in the aroE gene, which encodes shikimate dehydrogenase and has been reported to have effects on expressions of other virulence factors. Protein structure prediction obtained with AlphaFold2 suggested that an amino acid substitution in AroE is located in a domain that interacts with nicotinamide adenine dinucleotide phosphate.

Conclusions

The present GWAS identified SNPs significantly related to clinical symptoms, of which approximately 10% were found in the aroE gene. These results indicate that AroE mutations may have important effects on metabolic processes and pneumococcal pathogenesis.

Background and Aims

Chronic wounds can readily become infected by microorganisms due to failure of mechanical and physiological first line innate immune responses. We report the characterization of host adaptation of three Escherichia coli ST131 genomes that occurred during a 10-year chronic wound infection after a foot fracture.

Methods

The three E. coli strains were characterized by various microbiological and genomic approaches as assessment of antimicrobial resistance, growth in different media, biofilm formation and genome sequencing by PacBio RSII. Phylogenetic analyses and genome alterations such as single nucleotide polymorphisms, deletion and rearrangements that led to pseudogenes and chromosomal inversions were documented. Relevant selected metabolic and physiological pathways were analyzed for integrity.

Results

The three E. coli ST131 strains showed a heavily host-adapted genome with high number of pseudogenes and a large chromosomal inversion compared to ST131 reference strains. Furthermore, two of three E. coli ST131 isolates were small colony variants with its genetic basis in multiple genome alterations including pseudogenes and deletions in the pathway for heme biosynthesis. Pseudogene analysis indicated three ST131 strains to be mutator strains. Enhanced capability of biofilm formation of the ST131 isolates was indicated by the agar plate assay.

Conclusions

ST131 clone members are ubiquitously found in patients and the environment. ST131 strains have perhaps already been acquired from the environment upon foot fracture and persisted in wounds showing outmost genome plasticity and adaptability which might causing the chronic infection. Although co-infection with E. faecalis might have supported chronicity, these findings indicate that in individuals with underlying metabolic diseases wound infection by ST131 E. coli isolates can be a health risk.

Background and Aims

Salmonella enterica serovar Typhimurium is mainly associated with enteric self-limiting disease. Its virulence mechanism has been studied using reference strains belonging to the worldwide prevalent ST19 genotype. However, the emergence of strains from different genotypes associated with multidrug resistance and non-typhoidal invasive infections has increased.

In Mexico, during an epidemiological surveillance program, Typhimurium strains, mostly belonging to the ST19 and ST213 genotypes, were isolated; the latter was the most abundant and was linked to five cases of systemic infection. ST213 strains possess distinctive characteristics such as a broad profile of antibiotic resistance encoded by large plasmids and lack the pSTV virulence plasmid. They have been mainly identified across North America and represent a public health risk.

This study aimed to phenotypically characterize ten representative ST213 strains isolated in Mexico compared to a prototype ST19 strain.

Methods

Biofilm formation was assessed by crystal violet staining and RDAR morphotype. Their ability to invade and survive/replicate in epithelial cell lines and macrophages was assessed by aminoglycoside protection assays.

Results

ST213 strains displayed a stronger ability to form biofilms than the prototype ST19 strain but exhibited lower internalization capacity and no difference in intracellular replication.

Conclusions

Even though the reductionistic approach used for the study of the pathogenesis of Typhimurium led the field to great advance, the emergence of new strains stresses the need for integrative approaches to further our understanding of host-Typhimurium interactions, as the reference strains do not always represent the genomic diversity present in clinical or food isolates.

Acknowledgements: DGAPA-PAPIIT-(IN215119/IN218322) and CONACyT-354699-ISF.