Determination ofElizabethkingiaDiversity by MALDI-TOF Mass Spectrometry and Whole-Genome Sequencing

Abstract Background A number of episodes of nosocomial Agrobacterium spp. bacteremia (two cases per year) were observed at Bern University Hospital, Switzerland, from 2015 to 2017. This triggered an outbreak investigation. Methods Cases of Agrobacterium spp. bacteremias that occurred between August 2011 and February 2017 were investigated employing line lists, environmental sampling, rapid protein- (MALDI-TOF MS), and genome-based typing (pulsed field gel electrophoresis and whole genome sequencing) of the clinical isolates. Results We describe a total of eight bacteremia episodes due to A. radiobacter (n = 2), Agrobacterium genomovar G3 (n = 5) and A. pusense (n = 1). Two tight clusters were observed by WGS typing, representing the two A. radiobacter isolates (cluster I, isolated in 2015) and four of the Agrobacterium genomovar G3 isolates (cluster II, isolated in 2016 and 2017), suggesting two different point sources. The epidemiological investigations revealed two computer tomography (CT) rooms as common patient locations, which correlated with the two outbreak clusters. MALDI-TOF MS permitted faster evaluation of strain relatedness than DNA-based methods. High resolution WGS-based typing confirmed the MALDI-TOF MS clustering. Conclusions We report clinical and epidemiological characteristics of two outbreak clusters with Agrobacterium. spp. bacteremia likely acquired during CT contrast medium injection and highlight the use of MALDI-TOF MS as a rapid tool to assess relatedness of rare gram-negative pathogens in an outbreak investigation.

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Ornithobacterium rhinotracheale: MALDI-TOF MS and Whole Genome Sequencing Confirm That Serotypes K, L and M Deviate from Well-Known Reference Strains and Numerous Field Isolates

Microorganisms ◽

10.3390/microorganisms9051006 ◽

2021 ◽

Vol 9 (5) ◽

pp. 1006

Author(s):

Merima Alispahic ◽

Lukas Endler ◽

Michael Hess ◽

Claudia Hess

Keyword(s):

Whole Genome Sequencing ◽

Genome Sequencing ◽

Whole Genome ◽

Maldi Tof Ms ◽

Maldi Tof ◽

Identification And Characterization ◽

Reference Strains ◽

Ornithobacterium Rhinotracheale ◽

Tof Ms

Ornithobacterium rhinotracheale is one of the most important bacterial agents of respiratory diseases in poultry. For correct identification and characterization of this fastidious bacterium, reliable diagnostic tools are essential. Still, phenotypic tests are used to identify O. rhinotracheale and serotyping is the most common method for characterization, despite known drawbacks and disadvantages such as divergent results, cross-reactivity between strains, or the non-typeability of strains. The intention of the present study was to evaluate MALDI-TOF MS and whole genome sequencing for the identification and characterization of O. rhinotracheale. For this purpose, a selection of 59 well-defined reference strains and 47 field strains derived from outbreaks on Austrian turkey farms were investigated by MALDI-TOF MS. The field strains originated from different geographical areas in Austria with some of the isolates derived from multiple outbreaks on farms within a year, or recurrent outbreaks over several years. MALDI-TOF MS proved a suitable method for identification of O. rhinotracheale to genus or species level except for 3 strains representing serotypes M, K and F. Phylogenetic analysis showed that most strains grouped within one cluster even though they were comprised of different serotypes, while serotypes F, K, and M clearly formed a different cluster. All field isolates from turkey farms clustered together, independent of the origin of the isolates, e.g., geographical area, multiple outbreaks within a year or recurrent outbreaks over several years. Whole genome sequencing of serotype M, K and F strains confirmed the extraordinary status and deviation from known fully-sequenced strains due to a lack of sequence similarity. This was further confirmed by alignments of single genes (16S-RNA and rpoB) and multilocus sequence typing although the demarcation was less obvious. Altogether, the results indicate that these three serotypes belong to a different species than O. rhinotracheale, and might even be members of multiple new species.

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Phenotypic H-Antigen Typing by Mass Spectrometry Combined with Genetic Typing of H Antigens, O Antigens, and Toxins by Whole-Genome Sequencing Enhances Identification of Escherichia coli Isolates

Journal of Clinical Microbiology ◽

10.1128/jcm.00422-16 ◽

2016 ◽

Vol 54 (8) ◽

pp. 2162-2168 ◽

Cited By ~ 4

Author(s):

Keding Cheng ◽

Huixia Chui ◽

Larissa Domish ◽

Angela Sloan ◽

Drexler Hernandez ◽

...

Keyword(s):

Mass Spectrometry ◽

Escherichia Coli ◽

Whole Genome Sequencing ◽

Genome Sequencing ◽

Shiga Toxin ◽

Gene Pair ◽

Whole Genome ◽

Genetic Typing ◽

Content Type ◽

O Antigens

Mass spectrometry-based phenotypic H-antigen typing (MS-H) combined with whole-genome-sequencing-based genetic identification of H antigens, O antigens, and toxins (WGS-HOT) was used to type 60 clinicalEscherichia coliisolates, 43 of which were previously identified as nonmotile, H type undetermined, or O rough by serotyping or having shown discordant MS-H and serotyping results. Whole-genome sequencing confirmed that MS-H was able to provide more accurate data regarding H antigen expression than serotyping. Further, enhanced and more confident O antigen identification resulted from gene cluster based typing in combination with conventional typing based on the gene pair comprisingwzxandwzyand that comprisingwzmandwzt. The O antigen was identified in 94.6% of the isolates when the two genetic O typing approaches (gene pair and gene cluster) were used in conjunction, in comparison to 78.6% when the gene pair database was used alone. In addition, 98.2% of the isolates showed the existence of genes for various toxins and/or virulence factors, among which verotoxins (Shiga toxin 1 and/or Shiga toxin 2) were 100% concordant with conventional PCR based testing results. With more applications of mass spectrometry and whole-genome sequencing in clinical microbiology laboratories, this combined phenotypic and genetic typing platform (MS-H plus WGS-HOT) should be ideal for pathogenicE. colityping.

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Lactobacillus fermentum 90 TC-4 taxonomic status confirmation using whole genome sequencing and MALDI TOF mass spectrum

Russian Journal of Genetics ◽

10.1134/s1022795416090039 ◽

2016 ◽

Vol 52 (9) ◽

pp. 907-913 ◽

Cited By ~ 2

Author(s):

I. V. Belova ◽

A. G. Tochilina ◽

I. V. Solovyeva ◽

E. I. Efimov ◽

I. S. Gorlova ◽

...

Keyword(s):

Mass Spectrum ◽

Whole Genome Sequencing ◽

Genome Sequencing ◽

Taxonomic Status ◽

Lactobacillus Fermentum ◽

Whole Genome ◽

Maldi Tof

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Mass Spectrometry–Based Escherichia coli H Antigen/Flagella Typing: Validation and Comparison with Traditional Serotyping

Clinical Chemistry ◽

10.1373/clinchem.2015.244236 ◽

2016 ◽

Vol 62 (6) ◽

pp. 839-847 ◽

Cited By ~ 11

Author(s):

Keding Cheng ◽

Yi-Min She ◽

Huixia Chui ◽

Larissa Domish ◽

Angela Sloan ◽

...

Keyword(s):

Mass Spectrometry ◽

Escherichia Coli ◽

Whole Genome Sequencing ◽

Genome Sequencing ◽

Close Correlation ◽

Clinical Isolates ◽

Whole Genome ◽

Sequence Coverage ◽

E Coli ◽

Reference Strains

Abstract BACKGROUND Escherichia coli H antigen typing with antisera, a useful method for flagella clinical identification and classification, is a time-consuming process because of the need to induce flagella growth and the occurrence of undetermined strains. We developed an alternative rapid and analytically sensitive mass spectrometry (MS) method, termed MS-based H antigen typing (MS-H), and applied it at the protein sequence level for H antigen typing. We also performed a comparison with traditional serotyping on reference strains and clinical isolates. METHODS On the basis of international guidelines, the analytical selectivity and sensitivity, imprecision, correlation, repeatability, and reproducibility of the MS-H platform was evaluated using reference strains. Comparison of MS-H typing and serotyping was performed using 302 clinical isolates from 5 Canadian provinces, and discrepant results between the 2 platforms were resolved through whole genome sequencing. RESULTS Repeated tests on reference strain EDL933 demonstrated a lower limit of the measuring interval at the subsingle colony (16.97 μg or 1.465 × 107 cells) level and close correlation (r2 > 0.99) between cell culture biomass and sequence coverage. The CV was <10.0% among multiple repeats with 4 reference strains. Intra- and interlaboratory tests demonstrated that the MS-H method was robust and reproducible under various sample preparation and instrumentation conditions. Using discrepancy analysis via whole genome sequencing, performed on isolates with discrepant results, MS-H accurately identified 12.3% more isolates than conventional serotyping. CONCLUSIONS MS-H typing of E. coli is useful for fast and accurate flagella typing and could be very useful during E. coli outbreaks.

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Methicillin-Resistant and Methicillin-Susceptible Staphylococcus from Vervet Monkeys (Chlorocebus sabaeus) in Saint Kitts

Antibiotics ◽

10.3390/antibiotics10030290 ◽

2021 ◽

Vol 10 (3) ◽

pp. 290

Author(s):

Andreas Hoefer ◽

Filip Boyen ◽

Amy Beierschmitt ◽

Arshnee Moodley ◽

Marilyn C. Roberts ◽

...

Keyword(s):

Whole Genome Sequencing ◽

Genome Sequencing ◽

Mobile Genetic Elements ◽

Pcr Analysis ◽

Whole Genome ◽

Vervet Monkeys ◽

Methicillin Resistant ◽

Genetic Elements ◽

Maldi Tof ◽

Staphylococcus Spp

Antimicrobial resistance has been described in all ecosystems, including wildlife. Here we investigated the presence of methicillin-resistant and susceptible staphylococci in both colony-born and wild vervet monkeys (Chlorocebus sabaeus). Through selective isolation, PCR, MALDI-TOF, and whole-genome sequencing, methicillin-resistant and susceptible Staphylococcus spp. isolated from vervet monkeys were characterized. We obtained putatively methicillin-resistant staphylococci from 29 of the 34 nasal samples collected. Strains were identified by MALDI-TOF analysis. Staphylococcus cohnii (n = 15) was the most commonly isolated species, while nine other species were isolated one or two times. PCR analysis indicated that eight [28%] strains were mecA positive. The whole-genome sequencing [WGS] included eight methicillin-resistant strains (S. epidermidis (n = 2), S. cohnii (n = 3), S. arlettae (n = 2) and S. hominis (n = 1)), nine additional S. cohnii strains and two strains that could not be identified by MALDI-TOF, but genetically characterized as one S. cohnii and one S. warneri. Different resistance genes carried by different mobile genetic elements, mainly blaZ (n = 10) and tet(K) (n = 5) were found, while msr(A), cat, fosB, dfrG, erm(C), mph(C) and str were identified in one to three strains. Phylogenetic analysis of the S. cohnii strains based on SNPs indicated four clusters associated with colony born or wild. In addition, one singleton S. cohnii isolated did not form a separate group and clustered within other S. cohnii strains submitted to the NCBI. In this study, we demonstrated the presence of AMR and mobile genetic elements to both colony-born and wild vervet monkeys. We also identified a previously undescribed prevalence of S. cohnii in the nasal flora of these monkeys, which merits further investigation.

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Evaluating transmission dynamics of Shiga toxin-producing E. coli (STEC) in New Zealand cattle from farm to slaughter using PCR/MALDI-TOF and genome sequencing

Applied and Environmental Microbiology ◽

10.1128/aem.02907-20 ◽

2021 ◽

Author(s):

A. Springer Browne ◽

Anne C. Midwinter ◽

Helen Withers ◽

Adrian L. Cookson ◽

Patrick J. Biggs ◽

...

Keyword(s):

New Zealand ◽

Whole Genome Sequencing ◽

Genome Sequencing ◽

Shiga Toxin ◽

Whole Genome ◽

Meat Processing ◽

Serious Illness ◽

E Coli ◽

Maldi Tof ◽

Processing Plants

Cattle are asymptomatic carriers of Shiga toxin-producing E. coli (STEC) that can cause serious illness or death in humans. In New Zealand, contact with cattle feces and living near cattle populations are known risk factors for human STEC infection. Contamination of fresh meat with STECs also has the potential for rejection of consignments by importing countries. We used a combination of PCR/MALDI-TOF and whole genome sequencing to evaluate the presence and transmission of STEC on farms and in processing plants to better understand the potential pathways for human exposure and thus mitigate risk. Animal and environmental samples (n=2,580) were collected from six farms and three meat processing plants in New Zealand during multiple sampling sessions in spring of 2015 and 2016. PCR/MALDI-TOF analysis revealed 6.2% were positive for ‘Top 7’ STEC. ‘Top 7’ STEC were identified in all sample sources (n=17) tested. A marked increase in ‘Top 7’ STEC prevalence was observed between calf hides on-farm (6.3% prevalence), and calf hides at processing plants (25.1% prevalence). Whole genome sequencing was performed on ‘Top 7’ STEC bacterial isolates (n=40). Analysis of STEC O26 (n=25 isolates) revealed relatively low genetic diversity on individual farms, consistent with the presence of a resident strain disseminated within the farm environment. Public health efforts should focus on minimizing human contact with fecal material on farms and during handling, transport and slaughter of calves. Meat processing plants should focus on minimizing cross-contamination between the hides of calves in a cohort during transport, lairage and slaughter. Importance Cattle are asymptomatic carriers of Shiga toxin-producing E. coli (STEC), which can cause serious illness or death in humans. Contact with cattle feces and living near cattle are known risk factors for human STEC infection. This study evaluated STEC carriage in young calves and the farm environment with an in-depth evaluation of six farms and three meat processing plants over two years. An advanced molecular detection method and whole genome sequencing were used to provide a detailed evaluation of the transmission of STEC both within and between farms. The study revealed widespread STEC contamination within the farm environment, but no evidence of recent spread between farms. Contamination of young dairy calf hides increased following transport and holding at meat processing plants. The elimination of STEC in farm environments may be very difficult given the multiple transmission routes; interventions should be targeted at decreasing fecal contamination of calf hides during transport, lairage, and processing.

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Comparison of the Multiple Platforms to Identify Various Aeromonas Species

Frontiers in Microbiology ◽

10.3389/fmicb.2020.625961 ◽

2021 ◽

Vol 11 ◽

Author(s):

Xiaoli Du ◽

Mengyu Wang ◽

Haijian Zhou ◽

Zhenpeng Li ◽

Jialiang Xu ◽

...

Keyword(s):

Mass Spectrometry ◽

Whole Genome Sequencing ◽

Multiplex Pcr ◽

Genome Sequencing ◽

Pcr Amplification ◽

Identification Accuracy ◽

Aeromonas Species ◽

Whole Genome ◽

Mass Spectrometry Identification ◽

Mass Spectrometric Identification

We compared several identification methods for Aeromonas genus members, including traditional biochemical testing, multiplex-PCR amplification, mass spectrometry identification, whole-genome sequencing, multilocus phylogenetic analysis (MLPA), and rpoD, gyrA, and rpoD-gyrA gene sequencing. Isolates (n = 62) belonging to the Aeromonas genus, which were came from the bacterial bank in the laboratory, were used to assess the identification accuracy of the different methods. Whole-genome sequencing showed that the Aeromonas spp. isolates comprised A. caviae (n = 21), A. veronii (n = 18), A. dhakensis (n = 8), A. hydrophila (n = 7), A. jandaei (n = 5), A. enteropelogenes (n = 2), and A. media (n = 1). Using the whole-genome sequencing results as the standard, the consistency of the other methods was compared with them. The results were 46.77% (29/62) for biochemical identification, 83.87% (52/62) for mass spectrometric identification, 67.74% (42/62) for multiplex-PCR, 100% (62/62) for MLPA typing, 72.58% for gyrA, and 59.68% for rpoD and gyrA-rpoD. MLPA was the most consistent, followed by mass spectrometry. Therefore, in the public health laboratory, both MLPA and whole-genome sequencing methods can be used to identify various Aeromonas species. However, rapid and relatively accurate mass spectrometry is recommended for clinical lab.

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Screening of triploid with low-coverage whole-genome sequencing by a single-nucleotide polymorphism-based test in miscarriage tissue

Journal of Assisted Reproduction and Genetics ◽

10.1007/s10815-019-01588-6 ◽

2019 ◽

Vol 36 (12) ◽

pp. 2525-2531

Author(s):

Qian Geng ◽

Xiaoli Cui ◽

Yaqi Zhang ◽

Lijuan Zhang ◽

Cai Zhang ◽

...

Keyword(s):

Mass Spectrometry ◽

Single Nucleotide Polymorphism ◽

Whole Genome Sequencing ◽

Genome Sequencing ◽

Uniparental Disomy ◽

Whole Genome ◽

Nucleotide Polymorphism ◽

Single Nucleotide ◽

Quadratic Curve ◽

Low Coverage

Abstract Purpose To establish a single-nucleotide polymorphism-based analysis (SBA) method to identify triploidy in the miscarriage tissue by using low-coverage whole-genome sequencing (LC-WGS). Methods The method was established by fitting a quadratic curve model by counting the distribution of three heterozygous mutation content intervals. The triploid test result was mainly determined by the opening direction and the axis of symmetry of the quadratic curve, and Z test between the same batch samples was also used for auxiliary judgment. Results Two hundred thirteen diploid samples and 8 triploid samples were used for establishment of the analytical method and 203 unknown samples were used for blind testing. In the blind testing, we found 2 cases positive for triploidy. After chromosome microarray analysis (CMA) and mass spectrometry verification, we found that both samples were true positives. We randomly selected 5 samples from the negative samples for mass spectrometry verification, and the results showed that these samples were all true negatives. Conclusions Our method achieved accurate detection of triploidy in the miscarriage tissue and has the potential to detect more chromosomal abnormality types such as uniparental disomy (UPD) using a single LC-WGS approach.

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