[Show abstract][Hide abstract] ABSTRACT: Identification of bacteria may be based on sequencing and molecular analysis of a specific locus such as 16S rRNA, or a set of loci such as in multilocus sequence typing. In the near future, healthcare institutions and routine diagnostic microbiology laboratories may need to sequence the entire genome of microbial isolates. Therefore we have developed Reads2Type, a web-based tool for taxonomy identification based on whole bacterial genome sequence data.
Raw sequencing data provided by the user are mapped against a set of marker probes that are derived from currently available bacteria complete genomes. Using a dataset of 1003 whole genome sequenced bacteria from various sequencing platforms, Reads2Type was able to identify the species with 99.5 % accuracy and on the minutes time scale.
In comparison with other tools, Reads2Type offers the advantage of not needing to transfer sequencing files, as the entire computational analysis is done on the computer of whom utilizes the web application. This also prevents data privacy issues to arise. The Reads2Type tool is available at http://www.cbs.dtu.dk/~dhany/reads2type.html.
[Show abstract][Hide abstract] ABSTRACT: Pandemic methicillin-resistant
(MRSA) Clonal Complex (CC)97 MRSA lineages originated from livestock-to-human host jumps. In recent years CC97 has become one of the major MRSA lineages detected in Italian farmed animals. The aim of the study was to characterize and analyze differences of MRSA and methicillin-susceptible
(MSSA) mainly of swine and bovine origin. Forty-seven CC97 isolates, 35 MRSA and 6 MSSA from different Italian pig and cattle holdings, 5 pig MRSA from Germany, one human MSSA from Spain, were characterized by macro-restriction-PFGE analysis, MLST,
typing, antimicrobial resistance pattern. Virulence and resistance genes were investigated by PCR and microarray. Most of isolates were
V, except two German MRSA (
III). Five main clusters were identified by PFGE, with the German isolates (cluster I and II) showing 60.5%, similarity with the Italian isolates, which mostly (68.1%) grouped in cluster V. All CC97 were PVL-negative and few (n=7) tested positive for
. All MRSA were multidrug resistant (MDR), and the main features were:
(C)-mediated (n=18) macrolide-lincosamide-streptogramin B and
(A)-mediated (n=37) pleuromutilin resistance; fluoroquinolone resistance (n=33);
(K) in 32/37
(M)-positive isolates, and
in almost all MRSA. Few host-associated differences were detected among CC97 MRSA: its extensive MDR nature both in pigs and dairy cattle may be a consequence of a spill-back from pigs of a MRSA lineage originated in cattle as MSSA, and needs further investigation. Measures should be implemented at farm level to prevent spill-over to humans in intensive farming areas.
Applied and Environmental Microbiology 11/2015; DOI:10.1128/AEM.02854-15 · 3.67 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Objectives: Methicillin-resistant Staphylococcus aureus (MRSA), in particular clonal complex (CC) 398, is increasingly found in livestock. Recently, MRSA CC30 was identified in Danish pigs. We determined the susceptibility of porcine S. aureus isolates of CC398 and CC30 to disinfectants used in pig farming (benzalkonium chloride, hydrogen peroxide, formaldehyde, sodium hypochlorite, and caustic soda). Furthermore, efflux pump activity, antimicrobial resistance profiles, hemolysis properties, and the presence of toxic shock syndrome toxin-1 (TSST-1) and Panton-Valentine Leukocidin (PVL)-encoding virulence factors were investigated. Methods: Susceptibilities to biocides and antimicrobial agents of 79 porcine S. aureus isolates were determined by the microdilution method. Isolates comprised 21 methicillin-sensitive S. aureus (MSSA) and 40 MRSA isolates belonging to CC398 and 13 MSSA and 5 MRSA isolates belonging to CC30. The presence of quaternary ammonium compound (QAC) resistance efflux pumps was analyzed using an ethidium bromide accumulation assay. The presence of qac resistance genes in active efflux pump positive isolates was determined by whole-genome sequencing data. All isolates were screened for lukPV and tst genes with PCR, and hemolytic activities were determined using an agar plate assay. Results: S. aureus isolates did not show reduced susceptibility to the biocides tested. However, the QAC resistance gene, qacG, was detected in three MRSA CC30 isolates and the qacC in one MRSA CC30 isolate. CC30 isolates were generally more susceptible to non-beta-lactam antibiotics than CC398. Isolates generally had low hemolytic activity and none encoded PVL or TSST-1. Conclusion: The presence of qac genes in European porcine S. aureus isolates and in livestock-associated MRSA CC30 is for the first time described in this study. This finding is concerning as it ultimately may compromise disinfection with QACs and thereby contribute to the selection and spread of MRSA CC30.
Microbial Drug Resistance 10/2015; 21(5):527-536. DOI:10.1089/mdr.2014.0215 · 2.49 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Methicillin-resistant Staphylococcus aureus (MRSA) Sequence Type (ST)1, Clonal Com-plex(CC)1, SCCmec V is one of the major Livestock-Associated (LA-) lineages in pig farming industry in Italy and is associated with pigs in other European countries. Recently, it has been increasingly detected in Italian dairy cattle herds. The aim of this study was to analyse the differences between ST1 MRSA and methicillin-susceptible S. aureus (MSSA) from cattle and pig herds in Italy and Europe and human isolates. Sixty-tree animal isolates from different holdings and 20 human isolates were characterized by pulsed-field gel electrophoresis (PFGE), spa-typing, SCCmec typing, and by micro-array analysis for several virulence, antimicrobial resistance, and strain/host-specific marker genes. Three major PFGE clusters were detected. The bovine isolates shared a high (90% to 100%) similarity with human isolates and carried the same SCCmec type IVa. They often showed genetic features typical of human adaptation or present in human-associated CC1: Immune evasion cluster (IEC) genes sak and scn, or sea; sat and aphA3-mediated aminoglycoside resistance. Contrary, typical markers of porcine origin in Italy and Spain, like erm(A) mediated macrolide-lincosamide-streptograminB, and of vga(A)-mediated pleuromutilin resistancewere always absent in human and bovine isolates. Most of ST(CC)1 MRSA from dairy cattle were multidrug-resistant and contained virulence and immunomodulatory genes associated with full capability of colonizing humans. As such, these strains may represent a greater human hazard than the porcine strains. The zoonotic capacity of CC1 LA-MRSA from livestock must be taken seriously and measures should be implemented at farm-level to prevent spill-over.
PLoS ONE 08/2015; 10(8). DOI:10.1371/journal.pone.0137143 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Human populations worldwide are increasingly confronted with infectious diseases and antimicrobial resistance spreading faster and appearing more frequently. Knowledge regarding their occurrence and worldwide transmission is important to control outbreaks and prevent epidemics. Here, we performed shotgun sequencing of toilet waste from 18 international airplanes arriving in Copenhagen, Denmark, from nine cities in three world regions. An average of 18.6 Gb (14.8 to 25.7 Gb) of raw Illumina paired end sequence data was generated, cleaned, trimmed and mapped against reference sequence databases for bacteria and antimicrobial resistance genes. An average of 106,839 (0.06%) reads were assigned to resistance genes with genes encoding resistance to tetracycline, macrolide and beta-lactam resistance genes as the most abundant in all samples. We found significantly higher abundance and diversity of genes encoding antimicrobial resistance, including critical important resistance (e.g. blaCTX-M) carried on airplanes from South Asia compared to North America. Presence of Salmonella enterica and norovirus were also detected in higher amounts from South Asia, whereas Clostridium difficile was most abundant in samples from North America. Our study provides a first step towards a potential novel strategy for global surveillance enabling simultaneous detection of multiple human health threatening genetic elements, infectious agents and resistance genes.
[Show abstract][Hide abstract] ABSTRACT: Six strains of Enterococcus faecalis (S1, S12, S17, S18, S19 and S32) were isolated from copper fed pigs in Denmark. These Gram-positive bacteria within the genus Enterococcus are able to survive a variety of physical and chemical challenges by the acquisition of diverse genetic elements. The genome of strains S1, S12, S17, S18, S19 and S32 contained 2,615, 2,769, 2,625, 2,804, 2,853 and 2,935 protein-coding genes, with 41, 42, 27, 42, 32 and 44 genes encoding antibiotic and metal resistance, respectively. Differences between Cu resistant and sensitive E. faecalis strains, and possible co-transfer of Cu and antibiotic resistance determinants were detected through comparative genome analysis.
[Show abstract][Hide abstract] ABSTRACT: Probiotics are increasingly used in aquaculture to control diseases and improve feed digestion and pond water quality; however, little is known about the antimicrobial resistance properties of such probiotic bacteria and to what extent they may contribute to the development of bacterial resistance in aquaculture ponds. Concerns have been raised that the declared information on probiotic product labels are incorrect and information on bacterial composition are often missing. We therefore evaluated seven probiotics commonly used in Vietnamese shrimp culture for their bacterial species content, phenotypic antimicrobial resistance and associated transferable resistance genes. The bacterial species was established by 16S rRNA sequence analysis of 125 representative bacterial isolates. MIC testing was done for a range of antimicrobials and whole genome sequencing of six multiple antimicrobial resistant Bacillus spp. used to identify resistance genes and genetic elements associated with horizontal gene transfer. Thirteen bacterial species declared on the probiotic products could not be identified and 11 non-declared Bacillus spp. were identified. Although our culture-based isolation and identification may have missed a few bacterial species present in the tested products this would represent minor bias, but future studies may apply culture independent identification methods like pyro sequencing. Only 6/60 isolates were resistant to more than four antimicrobials and whole genome sequencing showed that they contained macrolide (ermD), tetracycline (tetL), phenicol (fexA) and trimethoprim (dfrD, dfrG and dfrK) resistance genes, but not known structures associated with horizontal gene transfer. Probiotic bacterial strains used in Vietnamese shrimp culture seem to contribute with very limited types and numbers of resistance genes compared to the naturally occurring bacterial species in aquaculture environments. Approval procedures of probiotic products must be strengthened through scientific-based efficacy trials and product labels should allow identification of individual bacterial strains and inform the farmer on specific purpose, dosage and correct application measures.
PLoS ONE 07/2015; 10(7):e0132338. DOI:10.1371/journal.pone.0132338 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Pasteurella multocida inhabits the upper respiratory tract of many animals. It can cause skin and soft tissue infections in humans, usually in association with animal bites. We present a case of a 66-year-old chemotherapy-induced immunocompromised patient with lung cancer, who was treated for pneumonia and septicaemia due to P. multocida. There was no anamnestic contact with animals, which underlines the fact that immunocompromised patients can suffer from serious systemic infections due to P. multocida - even with no known animal contact.
[Show abstract][Hide abstract] ABSTRACT: Retrospectively, 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 MLST, haplotype, plasmid replicon, antimicrobial resistance genes, and the 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 isolates (83.0%) were multi-drug 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, blaTEM-1, dfrA7, sul1, sul 2, strA, and strB, fragments of incQ1plasmid replicon, class 1 integron, and the mer operon. The genomic analysis revealed an overall 415 SNPs difference and 35 deletions among 33 of the isolates whole genome sequenced. In comparison with other genomes of H58, the Zambian isolates separated from genomes from Central Africa and India with 34 and 52 SNPs, respectively. The phylogenetic analysis indicates that 32 isolates of the 33 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 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 co-circulate. 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 harbouring a chromosomally translocated region containing the MDR islands of incHI1 plasmid emerged in Zambia. This could chance the perception of the term "classical MDR typhoid" currently being solely associated with the incHI1 plasmid. It might be more common than anticipated that S. Typhi haplotype; H58B harbour either the incHI1 plasmid and /or a chromosomally translocated MDR region.
[Show abstract][Hide abstract] ABSTRACT: The rapid evolution of bacteria is crucial to their survival and is caused by exchange, transfer, and uptake of DNA, among
other things. Conjugation is one of the main mechanisms by which bacteria share their DNA, and it is thought to be controlled
by varied bacterial immune systems. Contradictory results about restriction-modification systems based on phenotypic studies
have been presented as reasons for a barrier to conjugation with and other means of uptake of exogenous DNA. In this study,
we show that inactivation of the R.EcoKI restriction enzyme in strain Escherichia coli K-12 strain MG1655 increases the conjugational transfer of plasmid pOLA52, which carriers two EcoKI recognition sites. Interestingly,
the results were not absolute, and uptake of unmethylated pOLA52 was still observed in the wild-type strain (with an intact
hsdR gene) but at a reduction of 85% compared to the uptake of the mutant recipient with a disrupted hsdR gene. This leads to the conclusion that EcoKI restriction-modification affects the uptake of DNA by conjugation but is not
a major barrier to plasmid transfer.
Journal of Bacteriology 11/2014; 197(2). DOI:10.1128/JB.02418-14 · 2.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The draft genome sequences of two copper-resistant Escherichia coli strains were determined. These had been isolated from copper-fed pigs and contained additional putative operons conferring
copper and other metal and metalloid resistances.
[Show abstract][Hide abstract] ABSTRACT: Salmonella typhimurium is the causative agent of typhoid fever, which causes nearly 21.7 million illnesses and 217,000 deaths around the world each
year. Here, we describe the draft genome sequences of the Salmonella typhimurium strains S7, S15, and S23, isolated from copper-fed pigs in Denmark and containing additional putative determinants conferring
resistances to copper and other metals and metalloids.