We benchmarked the hybrid assembly approaches of MaSuRCA, SPAdes, and Unicycler for bacterial pathogens using Illumina and Oxford Nanopore sequencing by determining genome completeness and accuracy, ...antimicrobial resistance (AMR), virulence potential, multilocus sequence typing (MLST), phylogeny, and pan genome. Ten bacterial species (10 strains) were tested for simulated reads of both mediocre- and low-quality, whereas 11 bacterial species (12 strains) were tested for real reads.
Unicycler performed the best for achieving contiguous genomes, closely followed by MaSuRCA, while all SPAdes assemblies were incomplete. MaSuRCA was less tolerant of low-quality long reads than SPAdes and Unicycler. The hybrid assemblies of five antimicrobial-resistant strains with simulated reads provided consistent AMR genotypes with the reference genomes. The MaSuRCA assembly of Staphylococcus aureus with real reads contained msr(A) and tet(K), while the reference genome and SPAdes and Unicycler assemblies harbored blaZ. The AMR genotypes of the reference genomes and hybrid assemblies were consistent for the other five antimicrobial-resistant strains with real reads. The numbers of virulence genes in all hybrid assemblies were similar to those of the reference genomes, irrespective of simulated or real reads. Only one exception existed that the reference genome and hybrid assemblies of Pseudomonas aeruginosa with mediocre-quality long reads carried 241 virulence genes, whereas 184 virulence genes were identified in the hybrid assemblies of low-quality long reads. The MaSuRCA assemblies of Escherichia coli O157:H7 and Salmonella Typhimurium with mediocre-quality long reads contained 126 and 118 virulence genes, respectively, while 110 and 107 virulence genes were detected in their MaSuRCA assemblies of low-quality long reads, respectively. All approaches performed well in our MLST and phylogenetic analyses. The pan genomes of the hybrid assemblies of S. Typhimurium with mediocre-quality long reads were similar to that of the reference genome, while SPAdes and Unicycler were more tolerant of low-quality long reads than MaSuRCA for the pan-genome analysis. All approaches functioned well in the pan-genome analysis of Campylobacter jejuni with real reads.
Our research demonstrates the hybrid assembly pipeline of Unicycler as a superior approach for genomic analyses of bacterial pathogens using Illumina and Oxford Nanopore sequencing.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Oxford Nanopore sequencing can be used to achieve complete bacterial genomes. However, the error rates of Oxford Nanopore long reads are greater compared to Illumina short reads. Long-read assemblers ...using a variety of assembly algorithms have been developed to overcome this deficiency, which have not been benchmarked for genomic analyses of bacterial pathogens using Oxford Nanopore long reads. In this study, long-read assemblers, namely Canu, Flye, Miniasm/Racon, Raven, Redbean, and Shasta, were thus benchmarked using Oxford Nanopore long reads of bacterial pathogens. Ten species were tested for mediocre- and low-quality simulated reads, and 10 species were tested for real reads. Raven was the most robust assembler, obtaining complete and accurate genomes. All Miniasm/Racon and Raven assemblies of mediocre-quality reads provided accurate antimicrobial resistance (AMR) profiles, while the Raven assembly of
with low-quality reads was the only assembly with an accurate AMR profile among all assemblers and species. All assemblers functioned well for predicting virulence genes using mediocre-quality and real reads, whereas only the Raven assemblies of low-quality reads had accurate numbers of virulence genes. Regarding multilocus sequence typing (MLST), Miniasm/Racon was the most effective assembler for mediocre-quality reads, while only the Raven assemblies of
O157:H7 and
with low-quality reads showed positive MLST results. Miniasm/Racon and Raven were the best performers for MLST using real reads. The Miniasm/Racon and Raven assemblies showed accurate phylogenetic inference. For the pan-genome analyses, Raven was the strongest assembler for simulated reads, whereas Miniasm/Racon and Raven performed the best for real reads. Overall, the most robust and accurate assembler was Raven, closely followed by Miniasm/Racon.
Metagenomics offers the highest level of strain discrimination of bacterial pathogens from complex food and water microbiota. With the rapid evolvement of assembly algorithms, defining an optimal ...assembler based on the performance in the metagenomic identification of foodborne and waterborne pathogens is warranted. We aimed to benchmark short-read assemblers for the metagenomic identification of foodborne and waterborne pathogens using simulated bacterial communities. Bacterial communities on fresh spinach and in surface water were simulated by generating paired-end short reads of Illumina HiSeq, MiSeq, and NovaSeq at different sequencing depths. Multidrug-resistant
Indiana SI43 and
PAO1 were included in the simulated communities on fresh spinach and in surface water, respectively. ABySS, IDBA-UD, MaSuRCA, MEGAHIT, metaSPAdes, and Ray Meta were benchmarked in terms of assembly quality, identifications of plasmids, virulence genes,
pathogenicity island, antimicrobial resistance genes, chromosomal point mutations, serotyping, multilocus sequence typing, and whole-genome phylogeny. Overall, MEGHIT, metaSPAdes, and Ray Meta were more effective for metagenomic identification. We did not obtain an optimal assembler when using the extracted reads classified as
or
for downstream genomic analyses, but the extracted reads showed consistent phylogenetic topology with the reference genome when they were aligned with
or
strains. In most cases, HiSeq, MiSeq, and NovaSeq were comparable at the same sequencing depth, while higher sequencing depths generally led to more accurate results. As assembly algorithms advance and mature, the evaluation of assemblers should be a continuous process.
A total of 764 retail meat including 515 chicken, 91 pork, 78 beef and 80 lamb samples were collected in Shaanxi Province of China in 2007–2008 to determine the prevalence of
Salmonella. The isolates ...were characterized using serotyping, antimicrobial susceptibility testing, and the presence of
bla
CMY-2 and
bla
TEM and class I integrons. Selective serovars were further subtyped using PFGE. Approximately 54% (276) of chicken, 31% (28) of pork, 17% (13) of beef and 20% (16) of lamb samples were positive of
Salmonella. Among 24 serovars identified, Enteritidis (31.5%) was most common, followed by Typhimurium (13.4%), Shubra (10.0%), Indiana (9.7%), Derby (9.5%) and Djugu (7.0%). Nearly 80% of the isolates (283) were resistant to at least one antimicrobial, and 53% (191) to more than three antimicrobials. Resistance was most frequently observed to sulfamethoxazole (67%), to trimethoprim/sulfamethoxazole (58%) and to tetracycline (56%). Furthermore, many isolates were resistant to nalidixic acid (35%), ciprofloxacin (21%) and ceftriaxone (16%). Most isolates of Shubra (89%) and Indiana (88%) were resistant to ≥
9 antimicrobials, compared to only 11% of Enteritidis and 9% of Infantis that showed similar resistance. Class I integrons were detected in 10% of the isolates, and contained
aadA,
tetR,
dhfr,
bla
PSE-1,
bla
DHA-1 and
bla
VEB-1 gene cassettes alone or various combinations. Ceftriaxone- and/or cefoperazone-resistant isolates (
n
=
62) carried
bla
TEM (51.6%) and/or
bla
CMY-2 (56.5%). A total of 116 PFGE patterns were generated among 210 selected isolates. Our findings indicated that
Salmonella contamination was common in retail meats, and that the
Salmonella isolates were phenotypically and genetically diverse. Additionally, many
Salmonella isolates were resistant to multiple antimicrobials.
To examine the distribution of all genes known to be responsible for resistance to quaternary ammonium compounds (QACs), and their association with resistance to QACs and other antimicrobials, in ...Escherichia coli recovered from retail meats.
A total of 570 strains of E. coli isolated from US retail meats in 2006 were screened for the presence of 10 QAC resistance genes qacE, qacEΔ1, qacF, qacG, emrE, sugE(c), sugE(p), mdfA and ydgE/ydgF. The MICs of six common disinfectants were determined using an agar dilution method. Possible associations between the presence of the gene and bacterial resistance to QACs and antimicrobials were investigated.
emrE, sugE(c), mdfA and ydgE/ydgF were commonly present (77.2%-100%) in the E. coli isolates, but qac and sugE(p) were less prevalent (0.4%-22.3%). emrE-mdfA-sugE(c)-ydgE/F was the most common QAC resistance gene profile. A significant association was found between antimicrobial resistance and the presence of sugE(p) and qacEΔ1 (P < 0.05). Antimicrobial-resistant E. coli isolates tended to contain more diverse combinations of disinfectant resistance genes than susceptible ones. All isolates showed reduced susceptibility to five of six disinfectants compared with the control strains. Higher MICs were generally associated with the presence of qac and sugE(p) genes.
The QAC resistance genes were commonly present among E. coli isolated from retail meats, and the qac and sugE(p) genes were highly associated with multidrug resistance phenotypes. Using QACs in the food industry may not be as effective as expected and could provide selection pressure for strains with acquired resistance to other antimicrobials.
Phylogeny is a powerful tool that can be incorporated into quantitative descriptions of community diversity, yet its use has been limited largely due to the difficulty in constructing phylogenies ...which incorporate the wide genomic diversity of microbial communities. Here, we describe the development of a web portal, PhyloPlus, which enables users to generate customized phylogenies that may be applied to any bacterial or archaeal communities. We demonstrate the power of phylogeny by comparing metrics that employ phylogeny with those that do not when applied to data sets from two metagenomic studies (fermented food,
= 58; human microbiome,
= 60). This example shows how inclusion of all bacterial species identified by taxonomic classifiers (Kraken2 and Kaiju) made the phylogeny perfectly congruent to the corresponding classification outputs. Our phylogeny-based approach also enabled the construction of more constrained null models which (i) shed light into community structure and (ii) minimize potential inflation of type I errors. Construction of such null models allowed for the observation of under-dispersion in 44 (75.86%) food samples, with the metacommunity defined as bacteria that were found in different food matrices. We also observed that closely related species with high abundance and uneven distribution across different sites could potentially exaggerate the dissimilarity between phylogenetically similar communities if they were measured using traditional species-based metrics (
= 0.003), whereas this effect was mitigated by incorporating phylogeny (
= 1). In summary, our tool can provide additional insights into microbial communities of interest and facilitate the use of phylogeny-based approaches in metagenomic analyses.
There has been an explosion of interest in how microbial diversity affects human health, food safety, and environmental functions among many other processes. Accurately measuring the diversity and structure of those communities is central to understanding their effects. Here, we describe the development of a freely available online tool, PhyloPlus, which allows users to generate custom phylogenies that may be applied to any data set, thereby removing a major obstacle to the application of phylogeny to metagenomic data analysis. We demonstrate that the genetic relatedness of the organisms within those communities is a critical feature of their overall diversity, and that using a phylogeny which captures and quantifies this diversity allows for much more accurate descriptions while preventing misleading conclusions based on estimates that ignore evolutionary relationships.
Using whole-genome sequence (WGS) data from the GenomeTrakr network, a globally distributed network of laboratories sequencing foodborne pathogens, we present a new phylogeny of
comprising 445 ...isolates from 266 distinct serovars and originating from 52 countries. This phylogeny includes two previously unidentified
subsp.
clades. Serovar Typhi is shown to be nested within clade A. Our findings are supported by both phylogenetic support, based on a core genome alignment, and Bayesian approaches, based on single-nucleotide polymorphisms. Serovar assignments were refined by
analysis using SeqSero. More than 10% of serovars were either polyphyletic or paraphyletic. We found variable genetic content in these isolates relating to gene mobilization and virulence factors which have different distributions within clades. Gifsy-1- and Gifsy-2-like phages appear more prevalent in clade A; other viruses are more evenly distributed. Our analyses reveal IncFII is the predominant plasmid replicon in
Few core or clade-defining virulence genes are observed, and their distributions appear probabilistic in nature. Together, these patterns demonstrate that genetic exchange within
is more extensive and frequent than previously realized, which significantly alters how we view the genetic structure of the bacterial species.
Rapid improvements in nucleotide sequencing access and affordability have led to a drastic increase in availability of genetic information. This information will improve the accuracy of molecular descriptions, including serovars, within
Although the concept of serovars continues to be useful, it may have more significant limitations than previously understood. Furthermore, the discrete absence or presence of specific genes can be an unstable indicator of phylogenetic identity. Whole-genome sequencing provides more rigorous tools for assessing the distributions of these genes. Our phylogenetic and genetic content analyses reveal how active genetic elements are dynamically distributed within a species, allowing us to better understand genetic reservoirs and underlying bacterial evolution.
We sequenced 25 isolates of phenotypically multidrug-resistant Salmonella Indiana (n = 11), Typhimurium (n = 8), and Enteritidis (n = 6) using both MinION long-read SQK-LSK109 and flow cell (R9.4.1) ...and MiSeq short-read (Nextera XT and MiSeq Reagent Kit v2) sequencing technologies to determine the advantages of each approach in terms of the characteristics of genome structure, antimicrobial resistance (AMR), virulence potential, whole-genome phylogeny, and pan-genome. The MinION reads were base-called in real-time using MinKnow 3.4.8 integrated with Guppy 3.0.7. The long-read-only assembly, Illumina-only assembly, and hybrid assembly pipelines of Unicycler 0.4.8 were used to generate the MinION, MiSeq, and hybrid assemblies, respectively. The MinION assemblies were highly contiguous compared to the MiSeq assemblies but lacked accuracy, a deficiency that was mitigated by adding the MiSeq short reads through the Unicycler hybrid assembly which corrected erroneous single nucleotide polymorphisms (SNPs). The MinION assemblies provided similar predictions of AMR and virulence potential compared to the MiSeq and hybrid assemblies, although they produced more total false negatives of AMR genotypes, primarily due to failure in identifying tetracycline resistance genes in 11 of the 19 MinION assemblies of tetracycline-resistant isolates. The MinION assemblies displayed a large genetic distance from their corresponding MiSeq and hybrid assemblies on the whole-genome phylogenetic tree, indicating that the lower read accuracy of MinION sequencing caused incorrect clustering. The pan-genome of the MinION assemblies contained significantly more accessory genes and less core genes compared to the MiSeq and hybrid assemblies, suggesting that although these assemblies were more contiguous, their sequencing errors reduced accurate genome annotations. Our research demonstrates that MinION sequencing by itself provides an efficient assessment of the genome structure, antimicrobial resistance, and virulence potential of Salmonella; however, it is not sufficient for whole-genome phylogenetic and pan-genome analyses. MinION in combination with MiSeq facilitated the most accurate genomic analyses.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
In this study, we explored the prevalence of Staphylococcus aureus (S. aureus) ST398 in retail foods and then investigated for their virulence and antimicrobial resistance genetic background. ...Fourteen out of 5103 (0.27%) samples were positive for methicillin susceptible S. aureus (MSSA) ST398. Resistance was most frequently observed to penicillin (PEN) (100%), followed by trimethoprim (TMP), erythromycin (ERY) and ampicillin (AMP) (each 86.7%), clindamycin (CLD, 80.0%), and tetracycline (TET, 26.7%). All ST398 isolates were susceptible to amikacin, chloramphenicol, cefoxitin, gentamicin, oxacillin, and vancomycin. Two predominant resistance patterns including TMP-ERY-CLD-PEN-AMP (60.0%) and TMP-ERY-TET-CLD-PEN-AMP (20.0%) were identified. Isolates harbored blaZ (86.7%) gene, followed by tet(L) and linA/linA′ (each 46.7%), ermB and msrA (each 33.3%), aph(3′)-IIIa and dfrK (each 26.7%), tet(K) (20.0%), ant(4′)-Ia, ermA and emrC (each 13.3%) and cat::pC221 (6.7%). No isolate carried mecA, tet(M), tet(O), fexA, aac(6′)/aph(2″), cfr, ermT, msrB, cat::pC194, cat::pC223, catpIp-501, dfrD, dfrG and dfrS1 genes. For virulence genes, hld (73.3%), seb and sed (each 66.7%), hla (60.0%), lukPV (33.3%), sej (26.7%), lukED and seg (each 3.3%) were detected. None of isolates contained sea, sec, see, seh, sei, tst, eta, etb, sek-ser, seu, lukM, hlg, and hlgv genes. Four spa types were found, including t571 (6/15), t034 (4/15), t2876 (3/15) and t1250 (2/15). All strains were non-typeable for agr locus. Our findings indicated that MSSA ST398 isolates had a low prevalence rate in retail foods, and these isolates harbored multiple virulence and antimicrobial resistance genes, and exhibited multiple antimicrobial resistance. Further studies are required to elucidate the possible role of MSSA ST398 as a source of human infection.
•The first comprehensive study on S. aureus ST398 isolated from foods in China.•Low prevalence of MSSA ST398 in food.•S. aureus ST398 harbored multiple resistance and virulence genes.•S. aureus ST398 exhibited a multi-resistance phenotype.•Features of ST398 overlapped with those causing human and animal infections.
Probiotics are recognized for outcompeting pathogenic bacteria by competitive receptor-mediated colonization and secretion of functional metabolites which are antimicrobial against certain microbes ...as well as improving host's gut health and immunity. Recently, we have constructed a bioactive Lactobacillus casei (LC) strain, LC
+mcra
, by inserting mcra (myosin cross-reactive antigen) gene, which stimulates the conversion of conjugated linoleic acids. In this study, we evaluated the modulation of gut microbiome and protective roles of LC
+mcra
against pathogenic Salmonella enterica serovar Typhimurium (ST) and enterohemorrhagic E. coli (EHEC) infections in BALB/cJ mice. We observed that LC
+mcra
colonized efficiently in mice gut intestine and competitively reduced the infection with ST and EHEC in various locations of small and large intestine, specifically cecum, jejunum, and ileum (p < 0.05). Positive modulation of the cecal microbiota, for example, higher relative abundances of Firmicutes, lower relative abundances of Proteobacteria, and increased bacterial species diversity/richness, was detected in ST-challenged mice pretreated with LC
+mcra
based on 16S metagenomic sequencing. Cytokine gene expression analysis indicated that mice pretreated with LC
+mcra
associated with attenuated bacterial pathogen-induced gut inflammation. Furthermore, mice fed daily with LC
+mcra
for one week could protect themselves from the impairments caused by enteric infections with ST or EHEC. These impairments include weight loss, negative hematological changes, intestinal histological alterations, and potential death. This in vivo study suggests that daily consumption of novel conjugated linoleic acids over-producing probiotic effectively improves intestinal microbiota composition and prevents/combats foodborne enteric bacterial infections with pathogenic Salmonella and diarrheagenic E. coli.
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