c Whole-genome sequencing (WGS) is becoming available as a routine tool for clinical microbiology. If applied directly on clinical samples, this could further reduce diagnostic times and thereby improve control and treatment. A major bottleneck is the availability of fast and reliable bioinformatic tools. This study was conducted to evaluate the applicability of WGS directly on clinical samples and to develop easy-to-use bioinformatic tools for the analysis of sequencing data. Thirty-five random urine samples from patients with suspected urinary tract infections were examined using conventional microbiology, WGS of isolated bacteria, and direct sequencing on pellets from the urine samples. A rapid method for analyzing the sequence data was developed. Bacteria were cultivated from 19 samples but in pure cultures from only 17 samples. WGS improved the identification of the cultivated bacteria, and almost complete agreement was observed between phenotypic and predicted antimicrobial susceptibilities. Complete agreement was observed between species identification, multilocus sequence typing, and phylogenetic relationships for Escherichia coli and Enterococcus faecalis isolates when the results of WGS of cultured isolates and urine samples were directly compared. Sequencing directly from the urine enabled bacterial identification in polymicrobial samples. Additional putative pathogenic strains were observed in some culture-negative samples. WGS directly on clinical samples can provide clinically relevant information and drastically reduce diagnostic times. This may prove very useful, but the need for data analysis is still a hurdle to clinical implementation. To overcome this problem, a publicly available bioinformatic tool was developed in this study.

A row for sample number 16 was erroneously included, and thus the agreement between the results for single isolates and direct sequencing is not correct for any of the subsequently listed samples. The body of the table should read as follows:

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.

We report the genetic characterization of 15 Klebsiella pneumoniae (KP) and 4 isolates of K. oxytoca (KO) from clinical cases in dogs and cats and showing extended-spectrum cephalosporin (ESC) resistance. Extended spectrum beta-lactamase (ESBL) and AmpC genes, plasmid-mediated quinolone resistance (PMQR) and co-resistances were investigated. Among KP isolates, ST101 clone was predominant (8/15, 53%), followed by ST15 (4/15, 27%). ST11 and ST340, belonging to Clonal Complex (CC)11, were detected in 2012 (3/15, 20%). MLST on KP isolates corresponded well with PFGE results, with 11 different PFGE patterns observed, including two clusters of two (ST340) and four (ST101) indistinguishable isolates, respectively. All isolates harbored at least one ESBL or AmpC gene, all carried on transferable plasmids (IncR, IncFII, IncI1, IncN), and 16/19 were positive for PMQR genes (qnr family or aac(6′)-Ib-cr). The most frequent ESBL was CTX-M-15 (11/19, 58%), detected in all KP ST101, in one KP ST15 and in both KP ST340. bla CTX-M-15 was carried on IncR plasmids in all but one KP isolate. All KP ST15 isolates harbored different ESC resistance genes and different plasmids, and presented the non-transferable bla SHV-28 gene, in association with bla CTX-M-15, bla CTX-M-1 (on IncR, or on IncN), bla SHV-2a (on IncR) or bla CMY-2 genes (on IncI1). KO isolates were positive for bla CTX-M-9 gene (on IncHI2), or for the bla SHV-12 and bla DHA-1 genes (on IncL/M). They were all positive for qnr genes, and one also for the aac(6′)-Ib-cr gene. All Klebsiella isolates showed multiresistance towards aminoglycosides, sulfonamides, tetracyclines, trimethoprim and amphenicols, mediated by strA/B, aadA2, aadB, ant (2")-Ia, aac(6′)-Ib, sul, tet, dfr and cat genes in various combinations. The emergence in pets of multidrug-resistant Klebsiella with ESBL, AmpC and PMQR determinants, poses further and serious challenges in companion animal therapy and raise concerns for possible bi-directional transmission between pets and humans, especially at household level.

Aim: To identify the sources of Salmonella contamination, distribution, prevalence and antimicrobial susceptibility patterns, which have significant impact on public and animal health, and international trade. Methods and Results: A total of 1888 samples were collected by stratified random sampling from 2009 to 2011 from cattle, camels, poultry, fish, vegetables and humans. All identified Salmonella isolates were serotyped and tested for antimicrobial susceptibility by MIC determinations. A total of 149 Salmonella isolates comprising 17 different serovars were obtained (7Á9% prevalence). Salmonella Hadar (37%), S. Eko (17%), S. Enteritidis (10%), S. Kentucky (7%) and S. Uganda (7%) were isolated from different sources. The occurrence of antimicrobial resistance was generally low, but S. Enteritidis and S. Eko showed variable antimicrobial resistance patterns, while all S. Kentucky isolates were resistant to seven of 17 tested antimicrobials, including ciprofloxacin and nalidixic acid. Three S. Hadar isolates revealed reduced susceptibility to ciprofloxacin and susceptibility to nalidixic acid and harboured the plasmid-mediated quinolone resistance gene qnrS1. Conclusions: Salmonella serovars Hadar, Enteritidis and the previously very rarely reported Eko were the major serovars associated with human infections, animal and environmental contamination in the north-eastern region of Nigeria. Significance and Impact of the Study: These serovars constitute a health risk to poultry, environment and human population in the region.