A One Health approach based on genomics for enhancing the Salmonella enterica surveillance in Colombia.

Objectives: This study aimed to provide evidence of the domestic benefits of introducing an integrative genomic analysis from the One Health approach in the national surveillance of Salmonella enterica between 1997-2017 in Colombia. Methods: Data on Salmonella from clinical laboratory-based surveillance between 1997-2017 and from a national cross-sectional study at chicken retail stores in Colombia were compared using a phenotypic, molecular, and genomic approaches. Additional analysis by serovar using single nucleotide polymorphism was developed to increase the resolution of the relatedness between the interfaces. Results: Locally, the diversity and pathogenic factors of the prevalent S. enterica serovars associated with foodborne disease in Colombia were described using laboratory, pulse field gel electrophoresis, and whole genome sequencing data. For example, the resolution of pulse field gel electrophoresis allowed the description of two main foodborne clusters of Salmonella Enteritidis isolates, which were expanded to eight foodborne clades using whole genome sequencing. Likewise, virulence factors and antimicrobial resistance determinants, and mobile genetic elements that converged in the foodborne clades should be considered a public health concern in Colombia. All results by serovar were compiled in an interactive easy to share report. Conclusion: Whole genome sequencing is a technology that provides a precise assessment of emerging foodborne risks such as the Salmonella foodborne clades, but it requires an integrative and continued collaboration between the stakeholders across the One Health sectors to promote appropriated actions and policies in public health.

Genomic epidemiology of Staphylococcus aureus isolated from bloodstream infections in South America during 2019 supports regional surveillance.

Staphylococcus aureus remains one of the leading causes of infections worldwide and a common cause of bacteraemia. However, studies documenting the epidemiology of S. aureus in South America using genomics are scarce. We hereby report on the largest genomic epidemiology study to date of both methicillin-resistant S. aureus (MRSA) and methicillin-susceptible S. aureus (MSSA) in South America, conducted by the StaphNET-SA network. We characterised 404 genomes recovered from a prospective observational study of S. aureus bacteraemia in 58 hospitals from Argentina, Bolivia, Brazil, Paraguay and Uruguay between April and October 2019. We show that a minority of S. aureus isolates are phenotypically multi-drug resistant (5.2%), but more than a quarter are resistant to macrolide-lincosamide-streptogramin B (MLSb). MSSA were more genetically diverse than MRSA. Lower rates of associated antimicrobial resistance in community-associated(CA)-MRSA versus hospital-associated (HA)-MRSA were found in association with three S. aureus genotypes dominating the MRSA population: CC30-MRSA-IVc-t019-lukS/F-PV+, CC5-MRSA-IV-t002-lukS/F-PV- and CC8-MRSA-IVc-t008-lukS/F-PV+-COMER+. These are historically from a CA origin, carry on average fewer antimicrobial resistance determinants, and often lack key virulence genes. Surprisingly, CC398-MSSA-t1451-lukS/F-PV- related to the CC398 human-associated lineage is widely disseminated throughout the region, and is described here for the first time as the most prevalent MSSA lineage in South America. Moreover, CC398 strains carrying ermT (largely responsible for the MLSb resistance rates of MSSA strains: inducible iMLSb phenotype) and sh_fabI (related to triclosan resistance) were recovered from both CA and HA origin. The frequency of MRSA and MSSA lineages differed between countries but the most prevalent S. aureus genotypes are high-risk clones widely distributed in the South American region without a clear country-specific phylogeographical structure. Therefore, our findings underline the need for continuous genomic surveillance by regional networks such as StaphNET-SA. This article contains data hosted by Microreact.

Complexity of Genomic Epidemiology of Carbapenem-Resistant Klebsiella pneumoniae Isolates in Colombia Urges the Reinforcement of Whole Genome Sequencing-Based Surveillance Programs.

BACKGROUND: Carbapenem-resistant Klebsiella pneumoniae (CRKP) is an emerging public health problem. This study explores the specifics of CRKP epidemiology in Colombia based on whole genome sequencing (WGS) of the National Reference Laboratory at Instituto Nacional de Salud (INS)'s 2013-2017 sample collection. METHODS: A total of 425 CRKP isolates from 21 departments were analyzed by HiSeq-X10®Illumina high-throughput sequencing. Bioinformatic analysis was performed, primarily using the pipelines developed collaboratively by the National Institute for Health Research Global Health Research Unit (GHRU) on Genomic Surveillance of Antimicrobial Resistance (AMR), and AGROSAVIA. RESULTS: Of the 425 CRKP isolates, 91.5% were carbapenemase-producing strains. The data support a recent expansion and the endemicity of CRKP in Colombia with the circulation of 7 high-risk clones, the most frequent being CG258 (48.39% of isolates). We identified genes encoding carbapenemases blaKPC-3, blaKPC-2, blaNDM-1, blaNDM-9, blaVIM-2, blaVIM-4, and blaVIM-24, and various mobile genetic elements (MGE). The virulence of CRKP isolates was low, but colibactin (clb3) was present in 25.2% of isolates, and a hypervirulent CRKP clone (CG380) was reported for the first time in Colombia. ST258, ST512, and ST4851 were characterized by low levels of diversity in the core genome (ANI > 99.9%). CONCLUSIONS: The study outlines complex CRKP epidemiology in Colombia. CG258 expanded clonally and carries specific carbapenemases in specific MGEs, while the other high-risk clones (CG147, CG307, and CG152) present a more diverse complement of carbapenemases. The specifics of the Colombian situation stress the importance of WGS-based surveillance to monitor evolutionary trends of sequence types (STs), MGE, and resistance and virulence genes.

Genomic Epidemiology of CC30 Methicillin-Resistant Staphylococcus aureus Strains from Argentina Reveals Four Major Clades with Distinctive Genetic Features.

Staphylococcus aureus clonal complex 30 (CC30) has given rise to epidemics worldwide and is one of the most prevalent lineages in Argentina, represented by sequence type 30 methicillin-resistant S. aureus SCCmec type IV (ST30-MRSA-IV). ST30-MRSA-IV has displaced previous prevalent clones in the country and demonstrated increased virulence. Despite the burden of infections caused by ST30-MRSA-IV both in hospitals and in communities in Argentina, no detailed genome-based characterization of this clone is available to date. In this study, we used whole-genome sequencing (WGS) to evaluate the genetic diversity, population structure, and genomic characteristics of 190 CC30-MRSA strains circulating in Argentina between 2004 and 2015. Phylogenetic analysis revealed the existence of 4 major clades: ARG-1 (CC30-MRSA-IVc-spa t012), ARG-2 (ST30-MRSA-IVc-spa t021 related), ARG-3 (ST30-MRSA-IVh/j-spa t021 and related), and ARG-4 (CC30-MRSA-IVc-spa t019 and related). The clades were characterized by different distributions of antimicrobial resistance determinants, virulence genes, and mobile genetic elements (MGEs). While ARG-1 and ARG-4 were related to global epidemic MRSA-16 (EMRSA-16) and South West Pacific (SWP) clones, respectively, ARG-3 was phylogenetically distinct from previously defined CC30 epidemic clones. ARG-4, the most prevalent and geographically disseminated in the collection (N = 164), was characterized by specific MGEs and chromosomal mutations that might have contributed to its virulence and success. To our knowledge, this is the first genomic epidemiology study of CC30-MRSA in Argentina, which will serve as baseline genomic data going forward to inform public health measures for infection prevention and control.IMPORTANCE The rise in prevalence of community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) is of public health concern. In Argentina, several studies documented a shift in the epidemiology of CA-MRSA since 2009, with clonal complex 30 (CC30) and, in particular, sequence type 30 MRSA SCCmec type IV (ST30-MRSA-IV) replacing other clones both in communities and in hospitals and possibly displaying increased virulence. By sequencing the whole genomes of 190 CC30 MRSA isolates recovered from Argentina between 2005 and 2015, we showed that they represented a diverse population composed of 4 major clades. The predominant clade evolved from the South West Pacific clone but has acquired a distinct repertoire of mobile genetic elements, virulence genes, and chromosomal mutations that might play a role in its success. Our work is the first extensive genomic study of CC30 S. aureus in Argentina and will contribute not only to the development of genomic surveillance in the region but also to our understanding of the global epidemiology of this pathogen.

Mucor rouxii Rho1 protein; characterization and possible role in polarized growth.

We have previously shown that protein kinase A of the medically important zygomycete Mucor rouxii participates in fungal morphology through cytoskeletal organization. As a first step towards finding the link between protein kinase A and cytoskeletal organization we here demonstrate the cloning of the Rho1 gene and the characterization of its protein product. The RHO1 protein primary sequence shows 70-85% identity with fungal RHO1 or mammalian RhoA. Two protein kinase A phosphorylation sequences in adequate context are predicted, Ser73 and Ser135. The peptide IRRNSQKFV, containing Ser135 proved to be a good substrate for M. rouxii protein kinase A catalytic subunit. The over-expressed Rho1 fully complements a Saccharomyces cerevisiae null mutant. The endogenous protein was identified by western blot against a developed antibody and by ADP-ribosylation. Localization in germlings was visualized by immunofluorescence; the protein was localized in patches in the mother cell surface and excluded from the germ tube. Measurement of Rho1 expression during germination indicates that Rho1, at both the mRNA and protein levels, correlates with differentiation and not with growth. Rho1 has been shown to be the regulatory protein of the beta-1,3-glucan synthase complex in fungi in which beta-1,3-glucans are major components of the cell wall. Even though glucans have not been detected in zygomycetes, caspofungin, an echinochandin known to be an inhibitor of beta-1,3-glucan synthase complex, is shown here to have a negative effect on growth and to produce an alteration on morphology when added to M. rouxii growth culture medium. This result has an important impact on the possible participation of beta-1,3-glucans on the regulation of morphology of zygomycetes.

RGD-containing peptides and cyclic AMP have antagonistic roles in the morphology of Mucor rouxii.

The tripeptide Arg-Gly-Asp (RGD) (1 mM) as well as the polymer ProNectin F (20 nM) added to culture medium of the fungus Mucor rouxii (defined medium) produced a delay in the switch from isodiametric growth to tip growth; at the time of germination the mother cell had a 4.6 times larger volume with 3.6 times more germ tubes per cell than control germinating sporangiospores. Disruption of the actin network with 2 micro g of cytochalasin A per ml blocked the switch to tip growth; the effect was analogous to the one of 150 micro M dibutyryl-cyclic AMP (cAMP), which we previously described to promote isodiametric growth via protein kinase A. 150 micro M dibutyryl-cAMP antagonises partially the effect of 1 mM RGD; the cells still emit several germ tubes per mother cell but their number is smaller and the volume of the cell at germ tube emission is larger than with RGD alone. At higher concentrations the dibutyryl-cAMP overrides completely the effect of RGD. Our results suggest that M. rouxii has an RGD recognition system and demonstrate that RGD-containing peptides have a profound effect on the isotropic stage of growth and on the establishment of cell polarity and that cAMP analogues can override this effect.