666. Molecular Epidemiology of Methicillin-Resistant Staphylococcus aureus in Chile between 1999-2018

Background The global spread of methicillin-resistant Staphylococcus aureus (MRSA) is associated with distinct genetic lineages that predominate in specific geographical regions. Available evidence suggests the Chilean-Cordobes clone (ChC), an ST5-SCCmecI lineage, has largely predominated in Chilean hospitals since its first description in the late 1990’s. Although the circulation of other MRSA lineages, including community-associated clones, has been well documented, the dynamics of clonal replacement over time has not been explored. Therefore, we aimed to study the molecular epidemiology and dynamics of clonal replacement using a large collection of clinical MRSA strains recovered from Chile during the last two decades. Methods We used whole-genome sequencing (WGS) and core-based phylogenomic analysis to identify genetic lineages and explore their relationship in 798 MRSA isolates obtained between 1999-2018 from two tertiary-care Chilean hospitals. Results Overall, the most frequently identified clones were the ST5-SCCmecI ChC (n=476, 60%), followed by ST105-SCCmecII (n=119, 15%), ST72-SCCmecIV (n=74, 9%), and ST8-SCCmecII (n=26, 3%). Phylogenomic reconstruction demonstrated 7 major clades: Clade I (CC30); Clade II (CC22); Clade III (CC97); Clade IV (CC8); Clade V (ST72); Clade VI (CC5/ST225 and ST105) and Clade VII (CC5/ST5-SCCmecI) (Fig. 1). The ChC clone remained the most frequent MRSA lineage throughout the study period (Fig. 2). However, its relative abundance decreased from >90% of isolates in 1999 to ca. 40% in 2018. This decrease began around 2005 and was associated with a progressive expansion of the ST105-SCCmecII and ST72-SCCmecIV lineages (Fig. 2). A Bayesian molecular clock analysis established the most recent common ancestor in 1964 (95% HPD interval=1961.975-1966.218) and corroborated a CC5 expansion event starting in Chile in 1999 (Fig. 3). Interestingly, our analyses revealed two branches within the ST5-SCCmecI lineage: one predominating in 1999-2006, and a more recent branch (related to the ST105-SCCmecII clone) that emerged around 2008. Figure 1. Core genome phylogenomic reconstruction of the 798 MRSA isolates. The seven major clades are represented by colored sections. The Clade I (purple section) was composed of isolates belonging to the CC30. Clade II (cyan section) includes four isolates of CC22. Clade III (red section) is composed of isolates of CC97. Clade IV (blue section) grouped isolates of different ST239 and ST8, belonging to the CC8. Clade V (orange section) includes isolates of ST72. Clade VI (yellow section) includes isolates of ST225 and ST105, both belonging to CC5. Clade VII (green section) is mostly composed of isolates of ST5-SCCmecI. The inner ring shows the ST of the isolates; the external ring shows the staphylococcal chromosomal cassette mec (SCCmec) type. Figure 2. Relative frequency of MRSA clones from 1999 to 2018. The genomes were grouped according to their isolation dates. Most frequent MRSA clones are represented by colored sections. Figure 3. Maximum clade credibility tree from the molecular clock analysis of the 798 MRSA genomes. A Bayesian molecular clock analysis was performed with BEAST using the isolation date of each genome as a calibrator. The colored strip showed the most frequent clones. The red dot shows a major event of divergence in 2008. Conclusion The ChC clone remains the most prevalent MRSA in Chile. However, our data is consistent with the evolution of this clone and a progressive replacement of with ST105 and ST72 genetic lineages. Disclosures Lorena Diaz, PhD , Nothing to disclose Cesar A. Arias, M.D., MSc, Ph.D., FIDSA, Entasis Therapeutics (Grant/Research Support)MeMed Diagnostics (Grant/Research Support)Merk (Grant/Research Support)

Methicillin-Resistant Staphylococcus aureus (MRSA) Clonal Replacement in a Malaysian Teaching Hospital: Findings from an Eight-Year Interval Molecular Surveillance

Periodical surveillance on nosocomial pathogens is important for antimicrobial stewardship and infection control. The first methicillin-resistant Staphylococcus aureus (MRSA) molecular surveillance in Hospital Canselor Tuanku Muhriz (HCTM), a Malaysian teaching hospital, was performed in 2009. The dominant clone was identified as an MRSA carrying SCCmec type III-SCCmercury with ccrC and sea+cna toxin genes. In this study, we report the findings of the second HCTM MRSA surveillance carried out in 2017, after an interval of 8 years. Antibiotic susceptibility testing, SCCmec, toxin gene, and spa typing were performed for 222 MRSA strains isolated in 2017. Most strains were resistant to ciprofloxacin, erythromycin, clindamycin, cefoxitin, and penicillin (n = 126, 56.8%), belong to SCCmec type IV (n = 205, 92.3%), spa type t032 (n = 160, 72.1%) and harboured seg+sei toxin genes (n = 172, 77.5%). There was significant association between resistance of the aforementioned antibiotics with SCCmec type IV (p < 0.05), t032 (p < 0.001), and seg+sei carriage (p < 0.05). Results from this second MRSA surveillance revealed the occurrence of clonal replacement in HCTM during an interval of not more than 8 years. Investigation of the corresponding phenotype changes in this new dominant MRSA clone is currently on-going.

Evolutionary histories and antimicrobial resistance in Shigella flexneri and Shigella sonnei in Southeast Asia

Conventional disease surveillance for shigellosis in developing country settings relies on serotyping and low-resolution molecular typing, which fails to contextualise the evolutionary history of the genus. Here, we interrogated a collection of 1,804 Shigella whole genome sequences from organisms isolated in four continental Southeast Asian countries (Thailand, Vietnam, Laos, and Cambodia) over three decades to characterise the evolution of both S. flexneri and S. sonnei. We show that S. sonnei and each major S. flexneri serotype are comprised of genetically diverse populations, the majority of which were likely introduced into Southeast Asia in the 1970s–1990s. Intranational and regional dissemination allowed widespread propagation of both species across the region. Our data indicate that the epidemiology of S. sonnei and the major S. flexneri serotypes were characterised by frequent clonal replacement events, coinciding with changing susceptibility patterns against contemporaneous antimicrobials. We conclude that adaptation to antimicrobial pressure was pivotal to the recent evolutionary trajectory of Shigella in Southeast Asia.

Conventional Chemotherapy Treatment Induces Unique Patterns of Clonal Evolution in a North American Adult T Cell Leukemia/Lymphoma Cohort

Adult T cell leukemia / lymphoma is a rare neoplasm with dismal cure rates and poor response to chemotherapy. The genetic basis of refractoriness against conventional agents is not known. We follow one of the largest cohorts of ATLL in the US and recently showed that North American ATLL (NA-ATLL) has a distinct mutational profile from Japanese ATLL (J-ATLL) with a higher incidence of epigenetic mutations (Shah et al, Blood, 2018; 132(14):1507-1518). Here, we analyze the mutational landscape of ATLL clones at diagnosis and at relapse in a pilot study of 7 NA-ATLL patients using deep targeted sequencing of 236 recurrently mutated genes. Our goal was to examine clonal evolution patterns that may result as a consequence of the selective pressure of chemotherapy by determining changes in mutational profiles as well as clonal abundance on patients with aggressive ATLL [acute and lymphomatous] (Table 1). All patients were treated with EPOCH (Etoposide, Prednisone, Vincristine, Cyclophosphamide and Doxorubicin) chemotherapy after initial diagnosis except one patient who received interferon therapy. Distinct patterns of clonal evolution were observed in patients with primary refractory disease when compared to those who relapsed after an initial remission. Group 1 has 3 cases (Pts 1 to 3), all of which experienced early relapse and showed mutation patterns suggestive of clonal replacement by a new emerging clone at relapse. Group 2 has 3 patients (Pts 4-6) that are characterized by worsening of primary refractory disease. Mutation patterns in the before-and-after sample pairs suggest linear progression from a previously existing clone during therapy. The clonal evolution status was not resolved in our third group (Pt#7) indicating that mutations outside the 236 genes evaluated might have been present. Frequent epigenetic mutations were found in both diagnostic and refractory/relapsed samples. Surprisingly, 4 epigenetic mutations (KMT2D, EP300, SPEN, SETBP1) identified in 4 cases were confirmed or suspected of germline origin. This suggests that ancestral genomic differences between the Caribbean and Japanese populations could contribute to the mutational and clinical disparities between NA- and J-ATLL. Multiple mutations with similar variant allele frequencies (VAF) at the time of relapse were identified, suggesting simultaneous acquisition of several mutations during clonal evolution. Clonal replacement by an emerging clone (Group 1) such as GATA3 (Pt#3) or KMT2D (Pt#1) (either germline or founding mutation) was observed in patients who achieved an initial response. On the other hand, in primary refractory disease (Group 2), relapse was driven by dominant TBL1XR1 mutations in 2 out of the 3 cases either alone (Pt#6) or in combination with mutated SMARCB1 (Pt#5). The protein encoded by TBL1XR1 is a negative regulator of the nuclear receptor corepressor (NCoR) /histone deacetylase 3 (HDAC3) complex. Therefore, TBL1XR1 inactivation is expected to result in hyper-activity of NCoR/HDAC3, an epigenetic abnormality targetable by HDAC inhibitors. SMARCB1 is also an epigenetic regulator associated with several malignancies and has been targeted with alisertib. A subclonal expansion of EP300 (a histone acetyltransferase) appears to drive relapse in the other case of this group (Pt#4). Therefore, our primary refractory cases show that epigenetic mutations seem to be of utmost importance not only at diagnosis as we have previously shown, but as the driving force behind chemotherapy refractoriness and subsequent relapse. Conclusion: To our knowledge, this is the first mutation-based clonal evolution study aimed to examine dynamic changes induced by chemotherapy among NA-ATLL patients. Early relapse appears to be driven by the emergence of new mutant clones. On the other hand, primary refractory disease appears to have epigenetic drivers that convey chemotherapy refractoriness and evolve from linear progression of a previously existing clone. Serial mutational testing can provide critical information on clonal architecture and identify actionable molecular vulnerabilities in a cohort without effective therapeutic options and a dismal prognosis. Disclosures Sica: Physician's Education Resource (PER): Honoraria. Shah:Physicians' Education Resource: Honoraria. Steidl:Aileron Therapeutics: Consultancy, Research Funding; Stelexis Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Other: Scientific Co-Founder; Pieries Pharmaceuticals: Consultancy; BayerHealthcare: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; GlaxoSmithKline: Research Funding; Celgene: Consultancy. Verma:Stelexis: Equity Ownership, Honoraria; Acceleron: Honoraria; Celgene: Honoraria; BMS: Research Funding; Janssen: Research Funding.