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.

Global epidemiology and clinical outcomes of carbapenem-resistant Pseudomonas aeruginosa and associated carbapenemases (POP): a prospective cohort study.

BACKGROUND: Carbapenem-resistant Pseudomonas aeruginosa (CRPA) is a global threat, but the distribution and clinical significance of carbapenemases are unclear. The aim of this study was to define characteristics and outcomes of CRPA infections and the global frequency and clinical impact of carbapenemases harboured by CRPA. METHODS: We conducted an observational, prospective cohort study of CRPA isolated from bloodstream, respiratory, urine, or wound cultures of patients at 44 hospitals (10 countries) between Dec 1, 2018, and Nov 30, 2019. Clinical data were abstracted from health records and CRPA isolates were whole-genome sequenced. The primary outcome was 30-day mortality from the day the index culture was collected. We compared outcomes of patients with CRPA infections by infection type and across geographic regions and performed an inverse probability weighted analysis to assess the association between carbapenemase production and 30-day mortality. FINDINGS: We enrolled 972 patients (USA n=527, China n=171, south and central America n=127, Middle East n=91, Australia and Singapore n=56), of whom 581 (60%) had CRPA infections. 30-day mortality differed by infection type (bloodstream 21 [30%] of 69, respiratory 69 [19%] of 358, wound nine [14%] of 66, urine six [7%] of 88; p=0·0012) and geographical region (Middle East 15 [29%] of 52, south and central America 20 [27%] of 73, USA 60 [19%] of 308, Australia and Singapore three [11%] of 28, China seven [6%] of 120; p=0·0002). Prevalence of carbapenemase genes among CRPA isolates also varied by region (south and central America 88 [69%] of 127, Australia and Singapore 32 [57%] of 56, China 54 [32%] of 171, Middle East 27 [30%] of 91, USA ten [2%] of 527; p<0·0001). KPC-2 (n=103 [49%]) and VIM-2 (n=75 [36%]) were the most common carbapenemases in 211 carbapenemase-producing isolates. After excluding USA patients, because few US isolates had carbapenemases, patients with carbapenemase-producing CRPA infections had higher 30-day mortality than those with non-carbapenemase-producing CRPA infections in both unadjusted (26 [22%] of 120 vs 19 [12%] of 153; difference 9%, 95% CI 3-16) and adjusted (difference 7%, 95% CI 1-14) analyses. INTERPRETATION: The emergence of different carbapenemases among CRPA isolates in different geographical regions and the increased mortality associated with carbapenemase-producing CRPA infections highlight the therapeutic challenges posed by these organisms. FUNDING: National Institutes of Health.

Multiomics characterization of methicillin-resistant Staphylococcus aureus (MRSA) isolates with heterogeneous intermediate resistance to vancomycin (hVISA) in Latin America.

BACKGROUND: Heterogeneous vancomycin-intermediate Staphylococcus aureus (hVISA) compromise the clinical efficacy of vancomycin. The hVISA isolates spontaneously produce vancomycin-intermediate Staphylococcus aureus (VISA) cells generated by diverse and intriguing mechanisms. OBJECTIVE: To characterize the biomolecular profile of clinical hVISA applying genomic, transcriptomic and metabolomic approaches. METHODS: 39 hVISA and 305 VSSA and their genomes were included. Core genome-based Bayesian phylogenetic reconstructions were built and alterations in predicted proteins in VISA/hVISA were interrogated. Linear discriminant analysis and a Genome-Wide Association Study were performed. Differentially expressed genes were identified in hVISA-VSSA by RNA-sequencing. The undirected profiles of metabolites were determined by liquid chromatography and hydrophilic interaction in six CC5-MRSA. RESULTS: Genomic relatedness of MRSA associated to hVISA phenotype was not detected. The change Try38 → His in Atl (autolysin) was identified in 92% of the hVISA. We identified SNPs and k-mers associated to hVISA in 11 coding regions with predicted functions in virulence, transport systems, carbohydrate metabolism and tRNA synthesis. Further, capABCDE, sdrD, esaA, esaD, essA and ssaA genes were overexpressed in hVISA, while lacABCDEFG genes were downregulated. Additionally, valine, threonine, leucine tyrosine, FAD and NADH were more abundant in VSSA, while arginine, glycine and betaine were more abundant in hVISA. Finally, we observed altered metabolic pathways in hVISA, including purine and pyrimidine pathway, CoA biosynthesis, amino acid metabolism and aminoacyl tRNA biosynthesis. CONCLUSIONS: Our results show that the mechanism of hVISA involves major changes in regulatory systems, expression of virulence factors and reduction in glycolysis via TCA cycle. This work contributes to the understanding of the development of this complex resistance mechanism in regional strains.

Selective digestive decontamination with oral colistin plus gentamicin for persistent bacteraemia caused by non-carbapenemase-producing carbapenem-resistant Klebsiella pneumoniae in a neutropenic patient.

BACKGROUND: Carbapenem-resistant Klebsiella pneumoniae (CRKp) have become an increasing public health problem worldwide. While most CRKp around the world harbour a carbapenemase enzyme, the clinical relevance of non-carbapenemase-producing CRKp (non-CP-CRKp) is increasingly recognized. Selective digestive decontamination (SDD) has been proven successful as a decolonization strategy for patients colonized with Gram-negatives in the ICU. However, it is not regularly used to treat invasive infections. OBJECTIVES: To report the use of SDD as a useful strategy for managing recalcitrant CRKp bloodstream infections. PATIENTS AND METHODS: We present a neutropenic patient with a recalcitrant bloodstream infection with non-CP-CRKp treated with SDD. Besides, genomic analyses of five isolates of non-CP-CRKp was performed. RESULTS: After 11 days of SDD treatment with oral colistin and gentamicin, bacteraemia was successfully eradicated. Genomic analysis indicates a fully carbapenem-resistant phenotype evolved in vivo and suggests that the mechanism of carbapenem resistance in our strains relates to gene amplification of narrow-spectrum ß-lactamases. CONCLUSIONS: Our report highlights that SDD might be a useful strategy to manage CRKp bloodstream infections, when intestinal translocation is the likely source of the bacteraemia. In addition, the development of a resistant phenotype during therapy is worrisome as therapies directed against these organisms are likely to favour the amplification process.

Dynamics of blaKPC-2 Dissemination from Non-CG258 Klebsiella pneumoniae to Other Enterobacterales via IncN Plasmids in an Area of High Endemicity.

Carbapenem-resistant Enterobacterales (CRE) pose a significant threat to global public health. The most important mechanism for carbapenem resistance is the production of carbapenemases. Klebsiella pneumoniae carbapenemase (KPC) represents one of the main carbapenemases worldwide. Complex mechanisms of blaKPC dissemination have been reported in Colombia, a country with a high endemicity of carbapenem resistance. Here, we characterized the dynamics of dissemination of blaKPC gene among CRE infecting and colonizing patients in three hospitals localized in a highly endemic area of Colombia (2013 and 2015). We identified the genomic characteristics of KPC-producing Enterobacterales recovered from patients infected/colonized and reconstructed the dynamics of dissemination of blaKPC-2 using both short and long read sequencing. We found that spread of blaKPC-2 among Enterobacterales in the participating hospitals was due to intra- and interspecies horizontal gene transfer (HGT) mediated by promiscuous plasmids associated with transposable elements that was originated from a multispecies outbreak of KPC-producing Enterobacterales in a neonatal intensive care unit. The plasmids were detected in isolates recovered in other units within the same hospital and nearby hospitals. The gene "epidemic" was driven by IncN-pST15-type plasmids carrying a novel Tn4401b structure and non-Tn4401 elements (NTEKPC) in Klebsiella spp., Escherichia coli, Enterobacter spp., and Citrobacter spp. Of note, mcr-9 was found to coexist with blaKPC-2 in species of the Enterobacter cloacae complex. Our findings suggest that the main mechanism for dissemination of blaKPC-2 is HGT mediated by highly transferable plasmids among species of Enterobacterales in infected/colonized patients, presenting a major challenge for public health interventions in developing countries such as Colombia.

Extensively Drug-Resistant Pseudomonas aeruginosa ST309 Harboring Tandem Guiana Extended Spectrum ß-Lactamase Enzymes: A Newly Emerging Threat in the United States.

BACKGROUND: Treatment of serious infections due to multidrug-resistant (MDR) Pseudomonas aeruginosa remains a challenge, despite the introduction of novel therapeutics. In this study, we report 2 extensively drug-resistant clinical isolates of sequence type (ST) 309 P aeruginosa resistant to all ß-lactams, including the novel combinations ceftolozane/tazobactam, ceftazidime/avibactam, and meropenem/vaborbactam. METHODS: Isolates were sequenced using both short-read (Illumina) and long-read technology to identify resistance determinants, polymorphisms (compared with P aeruginosa PAO1), and reconstruct a phylogenetic tree. A pair of ß-lactamases, Guiana extended spectrum ß-lactamase (GES)-19 and GES-26, were cloned and expressed in a laboratory strain of Escherichia coli to examine their relative impact on resistance. Using cell lysates from E coli expressing the GES genes individually and in tandem, we determined relative rates of hydrolysis for nitrocefin and ceftazidime. RESULTS: Two ST309 P aeruginosa clinical isolates were found to harbor the extended spectrum ß-lactamases GES-19 and GES-26 clustered in tandem on a chromosomal class 1 integron. The presence of both enzymes in E coli was associated with significantly elevated minimum inhibitory concentrations to aztreonam, cefepime, meropenem, ceftazidime/avibactam, and ceftolozane/tazobactam, compared with those expressed individually. The combination of ceftazidime/avibactam plus aztreonam was active in vitro and used to achieve cure in one patient. Phylogenetic analysis revealed ST309 P aeruginosa are closely related to MDR strains from Mexico also carrying tandem GES. CONCLUSIONS: The presence of tandem GES-19 and GES-26 is associated with resistance to all ß-lactams, including ceftolozane/tazobactam. Phylogenetic analysis suggests that ST309 P aeruginosa may be an emerging threat in the United States.

An Analysis of the Epidemic of Klebsiella pneumoniae Carbapenemase-Producing K. pneumoniae: Convergence of Two Evolutionary Mechanisms Creates the "Perfect Storm".

Background: Carbapenem resistance is a critical healthcare challenge worldwide. Particularly concerning is the widespread dissemination of Klebsiella pneumoniae carbapenemase (KPC). Klebsiella pneumoniae harboring blaKPC (KPC-Kpn) is endemic in many areas including the United States, where the epidemic was primarily mediated by the clonal dissemination of Kpn ST258. We postulated that the spread of blaKPC in other regions occurs by different and more complex mechanisms. To test this, we investigated the evolution and dynamics of spread of KPC-Kpn in Colombia, where KPC became rapidly endemic after emerging in 2005. Methods: We sequenced the genomes of 133 clinical isolates recovered from 24 tertiary care hospitals located in 10 cities throughout Colombia, between 2002 (before the emergence of KPC-Kpn) and 2014. Phylogenetic reconstructions and evolutionary mapping were performed to determine temporal and genetic associations between the isolates. Results: Our results indicate that the start of the epidemic was driven by horizontal dissemination of mobile genetic elements carrying blaKPC-2, followed by the introduction and subsequent spread of clonal group 258 (CG258) isolates containing blaKPC-3. Conclusions: The combination of 2 evolutionary mechanisms of KPC-Kpn within a challenged health system of a developing country created the "perfect storm" for sustained endemicity of these multidrug-resistant organisms in Colombia.