Dynamics of the MRSA Population in a Chilean Hospital: a Phylogenomic Analysis (2000-2016).

The global dissemination of methicillin-resistant Staphylococcus aureus (MRSA) is associated with the emergence and establishment of clones in specific geographic areas. The Chilean-Cordobes clone (ChC) (ST5-SCCmecI) has been the predominant MRSA clone in Chile since its first description in 1998, despite the report of other emerging MRSA clones in recent years. Here, we characterize the evolutionary history of MRSA from 2000 to 2016 in a Chilean tertiary health care center using phylogenomic analyses. We sequenced 469 MRSA isolates collected between 2000 and 2016. We evaluated the temporal trends of the circulating clones and performed a phylogenomic reconstruction to characterize the clonal dynamics. We found a significant increase in the diversity and richness of sequence types (STs; Spearman r = 0.8748, P < 0.0001) with a Shannon diversity index increasing from 0.221 in the year 2000 to 1.33 in 2016, and an effective diversity (Hill number; q = 2) increasing from 1.12 to 2.71. The temporal trend analysis revealed that in the period 2000 to 2003 most of the isolates (94.2%; n = 98) belonged to the ChC clone. However, since then, the frequency of the ChC clone has decreased over time, accounting for 52% of the collection in the 2013 to 2016 period. This decline was accompanied by the rise of two emerging MRSA lineages, ST105-SCCmecII and ST72-SCCmecVI. In conclusion, the ChC clone remains the most frequent MRSA lineage, but this lineage is gradually being replaced by several emerging clones, the most important of which is clone ST105-SCCmecII. To the best of our knowledge, this is the largest study of MRSA clonal dynamics performed in South America. IMPORTANCE Methicillin-resistant Staphylococcus aureus (MRSA) is a major public health pathogen that disseminates through the emergence of successful dominant clones in specific geographic regions. Knowledge of the dissemination and molecular epidemiology of MRSA in Latin America is scarce and is largely based on small studies or more limited typing techniques that lack the resolution to represent an accurate description of the genomic landscape. We used whole-genome sequencing to study 469 MRSA isolates collected between 2000 and 2016 in Chile providing the largest and most detailed study of clonal dynamics of MRSA in South America to date. We found a significant increase in the diversity of MRSA clones circulating over the 17-year study period. Additionally, we describe the emergence of two novel clones (ST105-SCCmecII and ST72-SCCmecVI), which have been gradually increasing in frequency over time. Our results drastically improve our understanding of the dissemination and update our knowledge about MRSA in Latin America.

Heavy Metal Pollution From a Major Earthquake and Tsunami in Chile Is Associated With Geographic Divergence of Clinical Isolates of Methicillin-Resistant Staphylococcus aureus in Latin America.

Methicillin-resistant Staphylococcus aureus (MRSA) is a priority pathogen listed by the World Health Organization. The global spread of MRSA is characterized by successive waves of epidemic clones that predominate in specific geographical regions. The acquisition of genes encoding resistance to heavy-metals is thought to be a key feature in the divergence and geographical spread of MRSA. Increasing evidence suggests that extreme natural events, such as earthquakes and tsunamis, could release heavy-metals into the environment. However, the impact of environmental exposition to heavy-metals on the divergence and spread of MRSA clones has been insufficiently explored. We assess the association between a major earthquake and tsunami in an industrialized port in southern Chile and MRSA clone divergence in Latin America. We performed a phylogenomic reconstruction of 113 MRSA clinical isolates from seven Latin American healthcare centers, including 25 isolates collected in a geographic area affected by an earthquake and tsunami that led to high levels of heavy-metal environmental contamination. We found a divergence event strongly associated with the presence of a plasmid harboring heavy-metal resistance genes in the isolates obtained in the area where the earthquake and tsunami occurred. Moreover, clinical isolates carrying this plasmid showed increased tolerance to mercury, arsenic, and cadmium. We also observed a physiological burden in the plasmid-carrying isolates in absence of heavy-metals. Our results are the first evidence that suggests that heavy-metal contamination, in the aftermath of an environmental disaster, appears to be a key evolutionary event for the spread and dissemination of MRSA in Latin America.

Evaluation of the Persistence and Characterization of Listeria monocytogenes in Foodservice Operations.

Listeria monocytogenes is a major foodborne pathogen that can contaminate food products and colonize food-producing facilities. Foodservice operations (FSOp) are frequently responsible for foodborne outbreaks due to food safety practices failures. We investigated the presence of and characterized L. monocytogenes from two FSOp (cafeterias) distributing ready-to-eat meals and verified FSOp's compliance with good manufacturing practices (GMP). Two facilities (FSOp-A and FSOp-B) were visited three times each over 5 months. We sampled foods, ingredients, and surfaces for microbiological analysis, and L. monocytogenes isolates were characterized by phylogenetic analyses and phenotypic characteristics. GMP audits were performed in the first and third visits. A ready-to-eat salad (FSOp-A) and a frozen ingredient (FSOp-B) were contaminated with L. monocytogenes, which was also detected on Zone 3 surfaces (floor, drains, and a boot cover). The phylogenetic analysis demonstrated that FSOp-B had persistent L. monocytogenes strains, but environmental isolates were not closely related to food or ingredient isolates. GMP audits showed that both operations worked under "fair" conditions, and "facilities and equipment" was the section with the least compliances. The presence of L. monocytogenes in the environment and GMP failures could promote food contamination with this pathogen, presenting a risk to consumers.

Antilisterial Effect and Influence on Listeria monocytogenes Gene Expression of Enterocin or Enterococcus faecalis in Sliced Dry-Cured Ham Stored at 7°C.

In this study, we focused on the effect of an enterocin or an Enterococcus faecalis strain added onto sliced dry-cured ham that was artificially inoculated with Listeria monocytogenes and stored at 7°C. The population of L. monocytogenes and the expression of five genes were monitored throughout the storage period. A persistent and a nonpersistent strain were tested, and both were influenced by the presence of the enterocin; both populations were reduced by more than 2 Log CFU/g after 14 days compared with the control, noninoculated ham. The presence of E. faecalis, a bacteriocin-producing lactic acid bacterium, had a less pronounced effect on the viable counts for both strains. Concerning gene expression, a common trend observed for both strains in the presence of enterocin was the down-regulation of genes tested after 30 min of storage at 7°C. For the remainder of the storage period, the expression fluctuated but was mostly reduced. Similarly, the presence of E. faecalis led to an overall down-regulation of genes. The effect on gene expression of both enterocin and E. faecalis was more pronounced on the nonpersistent L. monocytogenes strain. Although the potential of a bacteriocin and a bacteriocin-producing microorganism to control L. monocytogenes was confirmed, this study highlights that gene expression may be influenced and needs to be evaluated when considering such biopreservation interventions.

The Combined Effect of Cold and Copper Stresses on the Proliferation and Transcriptional Response of Listeria monocytogenes.

Listeria monocytogenes is a foodborne pathogen that can cause severe disease in susceptible humans. This microorganism has the ability to adapt to hostile environmental conditions such as the low temperatures used by the food industry for controlling microorganisms. Bacteria are able to adjust their transcriptional response to adapt to stressful conditions in order to maintain cell homeostasis. Understanding the transcriptional response of L. monocytogenes to stressing conditions could be relevant to develop new strategies to control the pathogen. A possible alternative for controlling microorganisms in the food industry could be to use copper as an antimicrobial agent. The present study characterized three L. monocytogenes strains (List2-2, Apa13-2, and Al152-2A) adapted to low temperature and challenged with different copper concentrations. Similar MIC-Cu values were observed among studied strains, but growth kinetic parameters revealed that strain List2-2 was the least affected by the presence of copper at 8°C. This strain was selected for a global transcriptional response study after a 1 h exposition to 0.5 mM of CuSO4 × 5H2O at 8 and 37°C. The results showed that L. monocytogenes apparently decreases its metabolism in response to copper, and this reduction is greater at 8°C than at 37°C. The most affected metabolic pathways were carbohydrates, lipids and nucleotides synthesis. Finally, 15 genes were selected to evaluate the conservation of the transcriptional response in the other two strains. Results indicated that only genes related to copper homeostasis showed a high degree of conservation between the strains studied, suggesting that a low number of genes is implicated in the response to copper stress in L. monocytogenes. These results contribute to the understanding of the molecular mechanisms used by bacteria to overcome a combination of stresses. This study concluded that the application of copper in low concentrations in cold environments may help to control foodborne pathogens as L. monocytogenes in the industry.

Synergistic effect of copper and low temperature over Listeria monocytogenes.

The capacity to grow at low temperatures has allowed Listeria monocytogenes to become one of the primary food pathogens to date, representing a major public health problem worldwide. Several works have described the homeostatic response of L. monocytogenes under different copper (Cu) treatments growing at mild temperature (30 °C). The aims of this report were to evaluate if changes in the external concentration of Cu affected viability and Cu homeostasis of L. monocytogenes growing at low temperature. Ours results showed that L. monocytogenes growing at 8 °C had a reduced viability relative to 30 °C when exposed to Cu treatments. This decrease was correlated with an increase in the internal concentration of Cu, probably linked to the transcriptional down-regulation of mechanisms involved in Cu homeostasis. This combined effect of Cu and low temperature showed a synergistic impact over the viability and homeostasis of L. monocytogenes, where low temperature exacerbated the toxic effect of Cu. These results can be useful in terms of the use of Cu as an antibacterial agent.

Potential of Chilean native corn (Zea mays L.) accessions as natural sources of phenolic antioxidants and in vitro bioactivity for hyperglycemia and hypertension management.

Thirty-three Chilean corn accessions were screened for the first time regarding their phenolic profiles, total phenolic contents (TPC), antioxidant capacity (DPPH and ABTS), and in vitro inhibition against key enzymes relevant for hyperglycemia (α-amylase and α-glucosidase) and hypertension (angiotensin I-converting enzyme, ACE-I) in both free and cell wall-bound fractions. TPC varied from 132.2 to 262.5 mg of gallic acid equivalents/100g dry weight (DW), and around 88% of TPC and antioxidant capacity were found in the bound form. Vanillin, vanillic, protocatechuic, ferulic, and p-coumaric acids were detected by HPLC in free fractions, whereas ferulic and p-coumaric acids were found in the bound form. Pisankalla accession (red kernel) had the highest ferulic acid content (269.5 mg/100g DW). No α-amylase and ACE-I inhibition were found; however, all free fractions inhibited α-glucosidase (10.8-72.5%). Principal component analysis revealed that darker samples (free fraction) showed higher TPC and antioxidant capacity, while α-glucosidase inhibition was related to yellow-colored samples.