Isolation and characterization of microsatellite markers in Garcinia gummi-gutta by next-generation sequencing and cross-species amplification.

Garcinia gummi-gutta (L.) Roxb. (Clusiaceae) is an endemic, semidomesticated, fruit-yielding tree species distributed in the Western Ghats of India and Sri Lanka. Various bioactive phytochemicals, such as garcinol, benzophenones and xanthones are isolated from G. gummi-gutta and have shown antibacterial, antiviral and antioxidant activities. We sequenced the total genomic DNA using Illumina Hiseq 2000 platform and examined 241,141,804 bp high quality data, assembled into 773,889 contigs. In these contigs, 27,313 simple-sequence repeats (SSRs) were identified, among which mononucleotide repeats were predominant (44.98%) followed by dinucleotide and trinucleotide repeats. Primers were designed for 9964 microsatellites among which 32 randomly selected SSR primer pairs were standardized for amplification. Polymerase chain reaction (PCR) amplification of genomic DNA in 30 G. gummi-gutta genotypes revealed polymorphic information content (PIC) across all 32 loci ranging from 0.867 to 0.951, with a mean value of 0.917. The observed and expected heterozygosity ranged from 0.00 to 0.63 and 0.896 to 0.974, respectively. Alleles per locus ranged from 12 to 27. This is the first report on the development of genomic SSR markers in G. gummi-gutta using next-generation sequencing technology. The genomic SSR markers developed in this study will be useful in identification, mapping, diversity and breeding studies.

Dynamics of MDR Enterobacter cloacae outbreaks in a neonatal unit in Nepal: insights using wider sampling frames and next-generation sequencing.

OBJECTIVES: There are limited data on Enterobacter cloacae outbreaks and fewer describing these in association with NDM-1. With whole-genome sequencing, we tested the hypothesis that a cluster of 16 E. cloacae bacteraemia cases in a Nepali neonatal unit represented a single clonal outbreak, using a wider set of epidemiologically unrelated clinical E. cloacae isolates for comparison. METHODS: Forty-three isolates were analysed, including 23 E. cloacae and 3 Citrobacter sp. isolates obtained from blood cultures from 16 neonates over a 3 month period. These were compared with two contemporaneous community-associated drug-resistant isolates from adults, a unit soap dispenser isolate and a set of historical invasive isolates (n=14) from the same geographical locality. RESULTS: There were two clear neonatal outbreaks and one isolated case in the unit. One outbreak was associated with an NDM-1 plasmid also identified in a historical community-associated strain. The smaller, second outbreak was likely associated with a contaminated soap dispenser. The two community-acquired adult cases and three sets of historical hospital-associated neonatal isolates represented four additional genetic clusters. CONCLUSIONS: E. cloacae infections in this context represent several different transmission networks, operating at the community/hospital and host strain/plasmid levels. Wide sampling frames and high-resolution typing methods are needed to describe the complex molecular epidemiology of E. cloacae outbreaks, which is not appropriately reflected by routine susceptibility phenotypes. Soap dispensers may represent a reservoir for E. cloacae and bacterial strains and plasmids may persist in hospitals and in the community for long periods, sporadically being involved in outbreaks of disease.

Genome sequencing of an extended series of NDM-producing Klebsiella pneumoniae isolates from neonatal infections in a Nepali hospital characterizes the extent of community- versus hospital-associated transmission in an endemic setting.

NDM-producing Klebsiella pneumoniae strains represent major clinical and infection control challenges, particularly in resource-limited settings with high rates of antimicrobial resistance. Determining whether transmission occurs at a gene, plasmid, or bacterial strain level and within hospital and/or the community has implications for monitoring and controlling spread. Whole-genome sequencing (WGS) is the highest-resolution typing method available for transmission epidemiology. We sequenced carbapenem-resistant K. pneumoniae isolates from 26 individuals involved in several infection case clusters in a Nepali neonatal unit and 68 other clinical Gram-negative isolates from a similar time frame, using Illumina and PacBio technologies. Within-outbreak chromosomal and closed-plasmid structures were generated and used as data set-specific references. Three temporally separated case clusters were caused by a single NDM K. pneumoniae strain with a conserved set of four plasmids, one being a 304,526-bp plasmid carrying bla(NDM-1). The plasmids contained a large number of antimicrobial/heavy metal resistance and plasmid maintenance genes, which may have explained their persistence. No obvious environmental/human reservoir was found. There was no evidence of transmission of outbreak plasmids to other Gram-negative clinical isolates, although bla(NDM) variants were present in other isolates in different genetic contexts. WGS can effectively define complex antimicrobial resistance epidemiology. Wider sampling frames are required to contextualize outbreaks. Infection control may be effective in terminating outbreaks caused by particular strains, even in areas with widespread resistance, although this study could not demonstrate evidence supporting specific interventions. Larger, detailed studies are needed to characterize resistance genes, vectors, and host strains involved in disease, to enable effective intervention.