Objectives: This study investigated the occurrence of extended spectrum cephalosporinase (ESC)-producing Escherichia coli in a broiler production with no cephalosporin use and a low use of antimicrobials in general. Furthermore, it investigated whether the current consumption of aminopenicillins selects for ESC-producing E. coli and whether certain clones or plasmids spread from imported parent flocks to the meat. Materials and Methods: ESC-producing E. coli was isolated using MacConkey broth with 1 mg/L of ceftriaxone. ESC genes were identified using polymerase chain reaction and sequencing. Isolates with bla CMY-2 were subtyped by pulsed-field gel electrophoresis (PFGE), phylotyping, and antimicrobial susceptibility testing. Selected isolates were used as donors in filter-mating experiments, multilocus sequence typing (MLST), and plasmid replicons were typed. Aminopenicillin use at the farm (not flock) level was obtained from VetStat, a database for mandatory registration of veterinary prescriptions in Denmark. Results: ESC-producing E. coli occurred in 93% (27/29) of broiler parent farms in 2011, 27% (53/197) of broiler flocks in 2010, and 3.3% (4/121) of Danish retail broiler meat in 2009 and 8.6% (16/187) in 2010. The ESC producing E. coli contained bla CMY-2 , bla SHV-2 or bla CTX-M-1 . Isolates with bla CMY-2 represented 35 PFGE groups. One group dominated (39 isolates) and included isolates with indistinguishable PFGE patterns from parents, broilers, and meat. Most bla CMY-2 isolates were susceptible to non-b-lactams, and bla CMY-2 was mostly present on horizontally transferable incI1 or incK plasmids. Phylogroup D was most common and E. coli MLST types previously found in humans were observed. Broiler farms with registered aminopenicillin use had significantly higher occurrence of ESC E. coli. Conclusions: ESC-producing E. coli from flocks of imported broiler parents spread clonally and horizontally to broiler meat (including potentially human pathogenic types) even in a country with no cephalosporin use. Use of aminopenicillins may influence the persistence of ESC-producing E. coli in the broiler production, but other factors should be investigated.
dRetrospectively, we investigated the epidemiology of a massive Salmonella enterica serovar Typhi outbreak in Zambia during 2010 to 2012. Ninety-four isolates were susceptibility tested by MIC determinations. Whole-genome sequence typing (WGST) of 33 isolates and bioinformatic analysis identified the multilocus sequence type (MLST), haplotype, plasmid replicon, antimicrobial resistance genes, and genetic relatedness by single nucleotide polymorphism (SNP) analysis and genomic deletions. The outbreak affected 2,040 patients, with a fatality rate of 0.5%. Most (83.0%) isolates were multidrug resistant (MDR). The isolates belonged to MLST ST1 and a new variant of the haplotype, H58B. Most isolates contained a chromosomally translocated region containing seven antimicrobial resistance genes, catA1, bla TEM-1 , dfrA7, sul1, sul2, strA, and strB, and fragments of the incompatibility group Q1 (IncQ1) plasmid replicon, the class 1 integron, and the mer operon. The genomic analysis revealed 415 SNP differences overall and 35 deletions among 33 of the isolates subjected to whole-genome sequencing. In comparison with other genomes of H58, the Zambian isolates separated from genomes from Central Africa and India by 34 and 52 SNPs, respectively. The phylogenetic analysis indicates that 32 of the 33 isolates sequenced belonged to a tight clonal group distinct from other H58 genomes included in the study. The small numbers of SNPs identified within this group are consistent with the short-term transmission that can be expected over a period of 2 years. The phylogenetic analysis and deletions suggest that a single MDR clone was responsible for the outbreak, during which occasional other S. Typhi lineages, including sensitive ones, continued to cocirculate. The common view is that the emerging global S. Typhi haplotype, H58B, containing the MDR IncHI1 plasmid is responsible for the majority of typhoid infections in Asia and sub-Saharan Africa; we found that a new variant of the haplotype harboring a chromosomally translocated region containing the MDR islands of IncHI1 plasmid has emerged in Zambia. This could change the perception of the term "classical MDR typhoid" currently being solely associated with the IncHI1 plasmid. It might be more common than presently thought that S. Typhi haplotype H58B harbors the IncHI1 plasmid or a chromosomally translocated MDR region or both.
Long binary sequences related to cyclic difference sets are investigated. Among all known constructions of cyclic difference sets we show that only sequences constructed from Hadamard difference sets can have an asymptotic nonzero merit factor. Maximal length shift register sequences, Legendre, and twin-prime sequences are all constructed from Hadamard difference sets. We prove that the asymptotic merit factor of any maximal length shift register sequence is three. For twin-prime sequences it is shown that the best asymptotic merit factor is six. This value is obtained by shifting the twin-prime sequence one quarter of its length. It turns out that Legendre sequences and twin-prime sequences have similar behavior. Based on the Jacobi symbol we investigate Jacobi sequences. The best asymptotic merit factor is shown to be six. Through the introduction of product sequences, it is argued that the maximal merit factor among all sequences of length N is at least six when N is large. We also demonstrate that it is fairly easy to construct sequences of moderate composite length with merit factor close to six.
Background Typhoid fever, caused by S. enterica ser. Typhi, continues to be a substantial health burden in developing countries. Little is known of the genotypic diversity of S. enterica ser. Typhi in Zimbabwe, but this is key for understanding the emergence and spread of this pathogen and devising interventions for its control. Objectives To report the molecular epidemiology of S. enterica ser. Typhi outbreak strains circulating from 2012 to 2019 in Zimbabwe, using comparative genomics. Methods : A review of typhoid cases records from 2012 to 2019 in Zimbabwe was performed. The phylogenetic relationship of outbreak isolates from 2012 to 2019 and emergence of antibiotic resistance was investigated by whole-genome sequence analysis. Results A total 22 479 suspected typhoid cases, 760 confirmed cases were reported from 2012 to 2019 and 29 isolates were sequenced. The majority of the sequenced isolates were predicted to confer resistance to aminoglycosides, β-lactams, phenicols, sulphonamides, tetracycline and fluoroquinolones (including qnrS detection). The qnrS1 gene was associated with an IncN (subtype PST3) plasmid in 79% of the isolates. Whole-genome SNP analysis, SNP-based haplotyping and resistance determinant analysis showed that 93% of the isolates belonged to a single clade represented by multidrug-resistant H58 lineage I (4.3.1.1), with a maximum pair-wise distance of 22 SNPs. Conclusions This study has provided detailed genotypic characterization of the outbreak strain, identified as S. Typhi 4.3.1.1 (H58). The strain has reduced susceptibility to ciprofloxacin due to qnrS carried by an IncN (subtype PST3) plasmid resulting from ongoing evolution to full resistance.
f One unreported case of extended-spectrum-beta-lactamase (ESBL)-producing Salmonella enterica serovar Typhi was identified, whole-genome sequence typed, among other analyses, and compared to other available genomes of S. Typhi. The reported strain was similar to a previously published strain harboring bla SHV-12 from the Philippines and likely part of an undetected outbreak, the first of ESBL-producing S. Typhi. The occurrence of extended-spectrum-beta-lactamase (ESBL)-producing Salmonella enterica serovar Typhi is an alarming development which may significantly complicate the treatment of typhoid fever. To date, ESBL-producing S. enterica serovar Typhi has only been reported from Bangladesh, Egypt, India, Iran, Iraq, Pakistan, and the Philippines (1). A subset of strains (from India, Iraq, and the Philippines) has been independently confirmed, and an assortment of ESBL genes (bla , bla , and bla CMY-2 ) have been identified and sequenced.The purpose of the present study was to identify potentially unreported cases of typhoid fever caused by ESBL-producing S. Typhi at a global level, to confirm ESBL production phenotypically, and to identify the responsible ESBL genes. Furthermore, we wanted to investigate the genetic relatedness to other available ESBL-producing S. Typhi isolates using whole-genome sequence typing (WGST) and a variety of molecular and genomic studies and to test the hypothesis that impaired restriction modification (RM) systems could be a factor for the development of ESBL resistance.On 4 May 2012, an electronic message requesting information about any confirmed or suspected ESBL-producing S. Typhi isolates was sent to the members of the World Health Organization (WHO) Global Foodborne Infections Network (GFN). Of 1,062 recipients, 3 members (0.28%) responded. We believe that the low response rate reflects a true low prevalence as the members frequently receive and respond to messages.One strain (strain 1107-3567) confirmed as an ESBL-producing S. Typhi strain was submitted by The Norwegian Institute of Public Health. The Norwegian patient in question and a previously published case from the Netherlands (strain TY5359) (2) had travel histories to the Philippines in late 2007 that were almost identical. The Norwegian patient had gastroenteritis, whereas the Dutch patient was admitted to a hospital with typhoid fever caused by an ESBL-producing S. Typhi containing the bla gene. The Dutch patient was treated successfully with ciprofloxacin; the treatment of the Norwegian patient was unknown.The isolates were sequenced using the MiSeq platform (Illumina, Inc., San Diego, CA) (see Methods in the supplemental material). The raw reads were assembled using the Assembler pipeline (version 1.0) available from the Center for Genomic Epidemiology (CGE) (http://cge.cbs.dtu.dk/services/all.php) and submitted to the European Nucleotide Archive (http://www.ebi .ac.uk/ena/data/view/PRJEB6961) (accession no. ERS525820 and ERS525821). A complete list of genomic sequence data is available in Table S1A in the s...
BackgroundPlasmodium falciparum malaria remains a major health burden and genomic research represents one of the necessary approaches for continued progress towards malaria control and elimination. Sample acquisition for this purpose is troublesome, with the majority of malaria-infected individuals living in rural areas, away from main infrastructure and the electrical grid. The aim of this study was to describe a low-tech procedure to sample P. falciparum specimens for direct whole genome sequencing (WGS), without use of electricity and cold-chain.MethodsVenous blood samples were collected from malaria patients in Bandim, Guinea-Bissau and leukocyte-depleted using Plasmodipur filters, the enriched parasite sample was spotted on Whatman paper and dried. The samples were stored at ambient temperatures and subsequently used for DNA-extraction. Ratios of parasite:human content of the extracted DNA was assessed by qPCR, and five samples with varying parasitaemia, were sequenced. Sequencing data were used to analyse the sample content, as well as sample coverage and depth as compared to the 3d7 reference genome.ResultsqPCR revealed that 73% of the 199 samples were applicable for WGS, as defined by a minimum ratio of parasite:human DNA of 2:1. WGS revealed an even distribution of sequence data across the 3d7 reference genome, regardless of parasitaemia. The acquired read depths varied from 16 to 99×, and coverage varied from 87.5 to 98.9% of the 3d7 reference genome. SNP-analysis of six genes, for which amplicon sequencing has been performed previously, confirmed the reliability of the WGS-data.ConclusionThis study describes a simple filter paper based protocol for sampling P. falciparum from malaria patients for subsequent direct WGS, enabling acquisition of samples in remote settings with no access to electricity.Electronic supplementary materialThe online version of this article (10.1186/s12936-018-2232-6) contains supplementary material, which is available to authorized users.
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