Serotyping of sub-Saharan Africa Salmonella Strains Isolated from Different Sources Using Multiplex PCR and Capillary Electrophoresis Analysis and whole Genome Sequencing

Abstract BackgroundSalmonella enterica remains a leading cause of food-borne diseases worldwide. Serotype information is important in food safety and public health activities to reduce the burden of salmonellosis. In the current study, two methods were used to determine serotypes of 225 clinical, environmental, food and veterinary isolates of Salmonella from Burkina Faso. First, Salmonella Multiplex Assay for Rapid Typing (SMART) Polymerase Chain Reaction (PCR) was used to determine the serovars of the S. enterica isolates. Second, serovar prediction based on whole genome sequencing (WGS) data was performed using SeqSero 2.0.ResultsAmong the 225 Salmonella isolates, serotypes for 48 (21.33%) isolates were identified based on comparison to a panel of representative SMART codes previously determined for the 50 most common serovars in the United States. One hundred and seventy-six new SMART codes were developed for common and uncommon serotypes. Serotypes for a total of 205 (91.1%) isolates were identified using SeqSero 2.0 for serovar prediction based on WGS data.ConclusionWe determined that SeqSero 2.0 was more comprehensive for identifying Salmonella serotypes from Burkina Faso than SMART PCR.

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Genomic investigation of a suspected multi-drug resistant Klebsiella pneumoniae outbreak in a neonatal care unit in sub-Saharan Africa

10.1101/2020.08.06.236117 ◽

2020 ◽

Author(s):

Jennifer Cornick ◽

Patrick Musicha ◽

Chikondi Peno ◽

Ezgi Saeger ◽

Pui-ying Iroh Toh ◽

...

Keyword(s):

Klebsiella Pneumoniae ◽

Whole Genome Sequencing ◽

Genome Sequencing ◽

Resistance Genes ◽

Sub Saharan Africa ◽

Whole Genome ◽

Drug Resistant ◽

Sub Saharan ◽

Neonatal Care Unit ◽

Suspected Outbreak

ABSTRACTA suspected outbreak of multi-drug resistant (MDR) Klebsiella pneumoniae in a Malawian neonatal unit was investigated using whole-genome sequencing. Strain-types, virulence and resistance genes of K. pneumoniae isolated from patients from the hospital over a four-year period were identified. A MDR ST340 clone was implicated as the likely outbreak cause.

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New Variant of Multidrug-Resistant Salmonella enterica Serovar Typhimurium Associated with Invasive Disease in Immunocompromised Patients in Vietnam

mBio ◽

10.1128/mbio.01056-18 ◽

2018 ◽

Vol 9 (5) ◽

Cited By ~ 16

Author(s):

Alison E. Mather ◽

Tu Le Thi Phuong ◽

Yunfeng Gao ◽

Simon Clare ◽

Subhankar Mukhopadhyay ◽

...

Keyword(s):

Whole Genome Sequencing ◽

Genome Sequencing ◽

Invasive Disease ◽

Multidrug Resistant ◽

Sub Saharan Africa ◽

Whole Genome ◽

Phase 2 ◽

Serovar Typhimurium ◽

Sub Saharan

ABSTRACT Nontyphoidal Salmonella (NTS), particularly Salmonella enterica serovar Typhimurium, is among the leading etiologic agents of bacterial enterocolitis globally and a well-characterized cause of invasive disease (iNTS) in sub-Saharan Africa. In contrast, S. Typhimurium is poorly defined in Southeast Asia, a known hot spot for zoonotic disease with a recently described burden of iNTS disease. Here, we aimed to add insight into the epidemiology and potential impact of zoonotic transfer and antimicrobial resistance (AMR) in S. Typhimurium associated with iNTS and enterocolitis in Vietnam. We performed whole-genome sequencing and phylogenetic reconstruction on 85 human (enterocolitis, carriage, and iNTS) and 113 animal S. Typhimurium isolates isolated in Vietnam. We found limited evidence for the zoonotic transmission of S. Typhimurium. However, we describe a chain of events where a pandemic monophasic variant of S. Typhimurium (serovar I:4,[5],12:i:− sequence type 34 [ST34]) has been introduced into Vietnam, reacquired a phase 2 flagellum, and acquired an IncHI2 multidrug-resistant plasmid. Notably, these novel biphasic ST34 S. Typhimurium variants were significantly associated with iNTS in Vietnamese HIV-infected patients. Our study represents the first characterization of novel iNTS organisms isolated outside sub-Saharan Africa and outlines a new pathway for the emergence of alternative Salmonella variants into susceptible human populations. IMPORTANCE Salmonella Typhimurium is a major diarrheal pathogen and associated with invasive nontyphoid Salmonella (iNTS) disease in vulnerable populations. We present the first characterization of iNTS organisms in Southeast Asia and describe a different evolutionary trajectory from that of organisms causing iNTS in sub-Saharan Africa. In Vietnam, the globally distributed monophasic variant of Salmonella Typhimurium, the serovar I:4,[5],12:i:− ST34 clone, has reacquired a phase 2 flagellum and gained a multidrug-resistant plasmid to become associated with iNTS disease in HIV-infected patients. We document distinct communities of S. Typhimurium and I:4,[5],12:i:− in animals and humans in Vietnam, despite the greater mixing of these host populations here. These data highlight the importance of whole-genome sequencing surveillance in a One Health context in understanding the evolution and spread of resistant bacterial infections.

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Serotyping of sub-Saharan Africa Salmonella strains isolated from poultry feces using multiplex PCR and whole genome sequencing

BMC Microbiology ◽

10.1186/s12866-021-02085-6 ◽

2021 ◽

Vol 21 (1) ◽

Author(s):

Assèta Kagambèga ◽

Lari M. Hiott ◽

David S. Boyle ◽

Elizabeth A. McMillan ◽

Poonam Sharma ◽

...

Keyword(s):

Burkina Faso ◽

Whole Genome Sequencing ◽

Genome Sequencing ◽

The United States ◽

Sub Saharan Africa ◽

Whole Genome ◽

Chain Reaction ◽

Food Borne ◽

Polymerase Chain ◽

Sub Saharan

Abstract Background Salmonella enterica remains a leading cause of food-borne diseases worldwide. Serotype information is important in food safety and public health activities to reduce the burden of salmonellosis. In the current study, two methods were used to determine serotypes of 111 strains of Salmonella isolated from poultry feces in Burkina Faso. First, Salmonella Multiplex Assay for Rapid Typing (SMART) Polymerase Chain Reaction (PCR) was used to determine the serovars of the S. enterica isolates. Second, serovar prediction based on whole genome sequencing (WGS) data was performed using SeqSero 2.0. Results Among the 111 Salmonella isolates, serotypes for 17 (15.31%) isolates were identified based on comparison to a panel of representative SMART codes previously determined for the 50 most common serovars in the United States. Forty-four (44) new SMART codes were developed for common and uncommon serotypes. A total of 105 (94.59%) isolates were serotyped using SeqSero 2.0 for serovar prediction based on WGS data. Conclusion We determined that SeqSero 2.0 was more comprehensive for identifying Salmonella serotypes from Burkina Faso than SMART PCR.

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Whole-Genome Sequencing of Kaposi's Sarcoma-Associated Herpesvirus from Zambian Kaposi's Sarcoma Biopsy Specimens Reveals Unique Viral Diversity

Journal of Virology ◽

10.1128/jvi.01712-15 ◽

2015 ◽

Vol 89 (24) ◽

pp. 12299-12308 ◽

Cited By ~ 26

Author(s):

Landon N. Olp ◽

Adrien Jeanniard ◽

Clemence Marimo ◽

John T. West ◽

Charles Wood

Keyword(s):

Whole Genome Sequencing ◽

Genome Sequencing ◽

Kaposi's Sarcoma ◽

Sequence Data ◽

Kaposi’S Sarcoma ◽

Sub Saharan Africa ◽

Whole Genome ◽

Phylogenetic Clustering ◽

Western Countries ◽

Sub Saharan

ABSTRACTKaposi's sarcoma-associated herpesvirus (KSHV) is the etiological agent for Kaposi's sarcoma (KS). Both KSHV and KS are endemic in sub-Saharan Africa where approximately 84% of global KS cases occur. Nevertheless, whole-genome sequencing of KSHV has only been completed using isolates from Western countries—where KS is not endemic. The lack of whole-genome KSHV sequence data from the most clinically important geographical region, sub-Saharan Africa, represents an important gap since it remains unclear whether genomic diversity has a role on KSHV pathogenesis. We hypothesized that distinct KSHV genotypes might be present in sub-Saharan Africa compared to Western countries. Using a KSHV-targeted enrichment protocol followed by Illumina deep-sequencing, we generated and analyzed 16 unique Zambian, KS-derived, KSHV genomes. We enriched KSHV DNA over cellular DNA 1,851 to 18,235-fold. Enrichment provided coverage levels up to 24,740-fold; therefore, supporting highly confident polymorphism analysis. Multiple alignment of the 16 newly sequenced KSHV genomes showed low level variability across the entire central conserved region. This variability resulted in distinct phylogenetic clustering between Zambian KSHV genomic sequences and those derived from Western countries. Importantly, the phylogenetic segregation of Zambian from Western sequences occurred irrespective of inclusion of the highly variable genes K1 and K15. We also show that four genes within the more conserved region of the KSHV genome contained polymorphisms that partially, but not fully, contributed to the unique Zambian KSHV whole-genome phylogenetic structure. Taken together, our data suggest that the whole KSHV genome should be taken into consideration for accurate viral characterization.IMPORTANCEOur results represent the largest number of KSHV whole-genomic sequences published to date and the first time that multiple genomes have been sequenced from sub-Saharan Africa, a geographic area where KS is highly endemic. Based on our new sequence data, it is apparent that whole-genome KSHV diversity is greater than previously appreciated and differential phylogenetic clustering exists between viral genomes of Zambia and Western countries. Furthermore, individual genes may be insufficient for KSHV genetic characterization. Continued investigation of the KSHV genetic landscape is necessary in order to effectively understand the role of viral evolution and sequence diversity on KSHV gene functions and pathogenesis.

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A Case of IMP-4-, OXA-421-, OXA-96-, and CARB-2-Producing Acinetobacter pittii Sequence Type 119 in Australia

Journal of Clinical Microbiology ◽

10.1128/jcm.02726-14 ◽

2014 ◽

Vol 53 (2) ◽

pp. 727-730 ◽

Cited By ~ 15

Author(s):

Witchuda Kamolvit ◽

Petra Derrington ◽

David L. Paterson ◽

Hanna E. Sidjabat

Keyword(s):

Whole Genome Sequencing ◽

Multiplex Pcr ◽

Genome Sequencing ◽

Draft Genome ◽

Sequence Type ◽

Species Level ◽

Whole Genome ◽

Patient Identification ◽

Content Type ◽

Acinetobacter Pittii

An IMP-4-producingAcinetobacterpittiistrain coproducing oxacillinases was isolated from a leg wound of a 67-year-old female patient. Identification to the species level byrpoBandgyrBsequencing and multiplex-PCR-based analysis revealed that the isolate wasA. pittii. Whole-genome sequencing of thisA. pittiiisolate determined the presence ofblaOXA-96,blaCARB-2, and a novelblaOXA-421gene. The position of this novelblaOXA-421gene was similar to that ofblaOXA-51inA. baumannii, downstream of the phosphinothricinN-acetyltransferase gene and upstream offxsAin the chromosome. ThisA. pittiiisolate was found to belong to sequence type 119 (ST119). Here, we report the first isolation of IMP-4-producingA. pittiiST119 with a novelblaOXA-421gene from a patient in Australia and characterize its draft genome.

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Optimization of multiplex PCR composition to screen for SARS-CoV-2 variants of concern

Acta Biochimica Indonesiana ◽

10.32889/actabioina.58 ◽

2021 ◽

Vol 4 (2) ◽

pp. 58

Author(s):

Maya Savira ◽

Enikarmila Asni ◽

Rahmat Azhari Kemal

Keyword(s):

Whole Genome Sequencing ◽

Multiplex Pcr ◽

Genome Sequencing ◽

Screening Method ◽

Positive Control ◽

Whole Genome ◽

Rt Pcr ◽

Pcr Screening ◽

Single Target ◽

Alpha Variant

Background: The ongoing COVID-19 pandemic has led to the emergence of several variants of concern. To rapidly identify those variants, screening samples for whole-genome sequencing (WGS) prioritization could be performed. Objective: We optimized the polymerase chain reaction (PCR) screening method to identify the mutation in spike and ORF1a regions. Methods: We adopted primers targeting mutation in spike and ORF1a region from another study. We optimized the PCR screening method using kits readily available in Indonesia. Firstly, we compared N1 and N2 primers as internal positive control. We also compared GoTaq® 1-Step RT-qPCR System and Indonesia TFRIC-19 BioCOV-19 for the multiplex reaction. We used the optimized composition to screen SARS-CoV-2 positive samples from April – June 2021. Samples with spike and/or ORF1a target failure were subjected to whole genome sequencing (WGS). Results: The results demonstrated the N2 BioCOV-19 reaction as the optimized multiplex PCR composition for spike and ORF1a mutations screening. Whole-genome sequencing has shown that a sample with spike and ORF1a targets failure to be Alpha variant, while other samples with single target failure as non-variants of concern. Therefore, a multiplex RT-PCR composition has been optimized to detect mutation in spike and ORF1a regions. Conclusion: We have optimized a multiplex RT-PCR composition to detect mutation in spike and ORF1a regions.

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A population-specific reference panel for improved genotype imputation in African Americans

Communications Biology ◽

10.1038/s42003-021-02777-9 ◽

2021 ◽

Vol 4 (1) ◽

Author(s):

Jared O’Connell ◽

Taedong Yun ◽

Meghan Moreno ◽

Helen Li ◽

Nadia Litterman ◽

...

Keyword(s):

Whole Genome Sequencing ◽

Genome Sequencing ◽

African Ancestry ◽

Genotype Imputation ◽

Whole Genome Sequencing Data ◽

Specific Reference ◽

Whole Genome ◽

Sequencing Data ◽

High Quality ◽

Sub Saharan

AbstractThere is currently a dearth of accessible whole genome sequencing (WGS) data for individuals residing in the Americas with Sub-Saharan African ancestry. We generated whole genome sequencing data at intermediate (15×) coverage for 2,294 individuals with large amounts of Sub-Saharan African ancestry, predominantly Atlantic African admixed with varying amounts of European and American ancestry. We performed extensive comparisons of variant callers, phasing algorithms, and variant filtration on these data to construct a high quality imputation panel containing data from 2,269 unrelated individuals. With the exception of the TOPMed imputation server (which notably cannot be downloaded), our panel substantially outperformed other available panels when imputing African American individuals. The raw sequencing data, variant calls and imputation panel for this cohort are all freely available via dbGaP and should prove an invaluable resource for further study of admixed African genetics.

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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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