Adaptation of gltA and ssrA assays for diversity profiling by Illumina sequencing to identify Bartonella henselae, B. clarridgeiae and B. koehlerae

2021 ◽  
Vol 70 (7) ◽  
Author(s):  
Rosemonde Isabella Power ◽  
Nichola Elisa Davies Calvani ◽  
Yaarit Nachum-Biala ◽  
Harold Salant ◽  
Shimon Harrus ◽  
...  
Keyword(s):  
Illumina Sequencing ◽  
Type Species ◽  
Bacterial Infections ◽  
Sanger Sequencing ◽  
Bartonella Henselae ◽  
Diagnostic Methods ◽  
Mixed Infections ◽  
Accurate Identification ◽  
Content Type ◽  
Link Type

Introduction. Bartonellosis is an emerging zoonotic disease caused by bacteria of the genus Bartonella . Mixed Bartonella infections are a well-documented phenomenon in mammals and their ectoparasites. The accurate identification of Bartonella species in single and mixed infections is valuable, as different Bartonella species have varying impacts on infected hosts. Gap Statement. Current diagnostic methods are inadequate at identifying the Bartonella species present in mixed infections. Aim. The aim of this study was to adopt a Next Generation Sequencing (NGS) approach using Illumina sequencing technology to identify Bartonella species and demonstrate that this approach can resolve mixed Bartonella infections. Methodology. We used Illumina PCR amplicon NGS to target the ssrA and gltA genes of Bartonella in fleas collected from cats, dogs and a hedgehog in Israel. We included artificially mixed Bartonella samples to demonstrate the ability for NGS to resolve mixed infections and we compared NGS to traditional Sanger sequencing. Results. In total, we identified 74 Ctenocephalides felis, two Ctenocephalides canis, two Pulex irritans and three Archaeopsylla e. erinacei fleas. Real-time PCR of a subset of 48 fleas revealed that twelve were positive for Bartonella , all of which were cat fleas. Sanger sequencing of the ssrA and gltA genes confirmed the presence of Bartonella henselae , Bartonella clarridgeiae and Bartonella koehlerae . Illumina NGS of ssrA and gltA amplicons further confirmed the Bartonella species identity in all 12 flea samples and unambiguously resolved the artificially mixed Bartonella samples. Conclusion. The adaptation and multiplexing of existing PCR assays for diversity profiling via NGS is a feasible approach that is superior to traditional Sanger sequencing for Bartonella speciation and resolving mixed Bartonella infections. The adaptation of other PCR primers for Illumina NGS will be useful in future studies where mixed bacterial infections may be present.

Emergence of a plasmid-borne tigecycline resistance in Klebsiella pneumoniae in Vietnam

2021 ◽  
Author(s):  
Aki Hirabayashi ◽  
Van Thi Thu Ha ◽  
An Van Nguyen ◽  
Son Thai Nguyen ◽  
Keigo Shibayama ◽  
...  
Keyword(s):  
Klebsiella Pneumoniae ◽  
Gene Cluster ◽  
Type Species ◽  
Bacterial Infections ◽  
Efflux Pump ◽  
Whole Genome Sequence ◽  
High Sequence Identity ◽  
Pump System ◽  
Content Type ◽  
Link Type

Tigecycline is a last-resort antimicrobial used to treat multidrug-resistant Gram-negative bacterial infections. One of the common antimicrobial resistance mechanisms is the efflux pump system composed of membrane protein complexes to excrete xenobiotic substrates. Recently, a novel gene cluster, tmexCD1-toprJ1, encoding the resistance–nodulation–cell division (RND) efflux pump was identified on plasmids in Klebsiella pneumoniae isolates in China. TMexCD1-TOprJ1 was found to be capable of excreting multiple antimicrobials, including tigecycline, which contributed to the strain's resistance. In this study, we identified K. pneumoniae isolates harbouring the tmexCD1-toprJ1 genes outside of China for the first time. Two tigecycline-resistant K. pneumoniae isolates belonging to ST273 by multilocus sequence typing were collected from different patients in a medical institution in Hanoi, Vietnam, in 2015. Whole-genome sequence analysis revealed that these isolates harboured a 288.0 kb tmexCD1-toprJ1–carrying plasmid with IncFIB and IncHI1B replicons. The tmexCD1-toprJ1 gene cluster was surrounded by several mobile gene elements, including IS26, and the plasmids had high sequence identity with that of K. pneumoniae isolated in China. Our finding suggests that the horizontal spread of tigecycline resistance mediated by tmexCD1-toprJ1–carrying plasmids has occurred in Vietnam and other countries, and raises concern about the further global dissemination.

scholarly journals Intraspecies plasmid and genomic variation of Mycobacterium kubicae revealed by the complete genome sequences of two clinical isolates

2020 ◽  
Author(s):  
Jo Hendrix ◽  
L. Elaine Epperson ◽  
David Durbin ◽  
Jennifer R. Honda ◽  
Michael Strong
Keyword(s):  
Type Species ◽  
Mycobacterium Abscessus ◽  
Genomic Variation ◽  
Diagnostic Methods ◽  
Pathogenic Potential ◽  
Circular Chromosome ◽  
Content Type ◽  
Link Type ◽  
Complete Genomes ◽  
Long Read

Mycobacterium kubicae is 1 of nearly 200 species of nontuberculous mycobacteria (NTM), environmental micro-organisms that in some situations can infect humans and cause severe lung, skin and soft tissue infections. Although numerous studies have investigated the genetic variation among prevalent clinical NTM species, including Mycobacterium abscessus and Mycobacterium avium , many of the less common but clinically relevant NTM species, including M. kubicae , still lack complete genomes to serve as a comparative reference. Well-characterized representative genomes for each NTM species are important both for investigating the pathogenic potential of NTM, as well as for use in diagnostic methods, even for species that less frequently cause human disease. Here, we report the complete genomes of two M. kubicae strains, isolated from two unrelated patients. Hybrid short-read and long-read sequencing and assembly, using sequence reads from Illumina and Oxford Nanopore Technologies platforms, were utilized to resolve the chromosome and plasmid sequences of each isolate. The genome of NJH_MKUB1 had 5135 coding sequences (CDSs), a circular chromosome of length 5.3 Mb and two plasmids. The genome of NJH_MKUB2 had 5957 CDSs, a circular chromosome of 6.0 Mb and five plasmids. We compared our completed genomic assemblies to four recently released draft genomes of M. kubicae in order to better understand intraspecies genomic conservation and variability. We also identified genes implicated in drug resistance, virulence and persistence in the M. kubicae chromosome and plasmids. Virulence factors encoded in the genome and in the plasmids of M. kubicae provide a foundation for investigating how opportunistic environmental NTM may cause disease.

scholarly journals Rapid molecular testing for Staphylococcus aureus bacteraemia improves clinical management

2020 ◽  
Vol 69 (4) ◽  
pp. 552-557
Author(s):  
Martin P. McHugh ◽  
Benjamin J. Parcell ◽  
Fiona M. MacKenzie ◽  
Kate E. Templeton ◽  
Keyword(s):  
Staphylococcus Aureus ◽  
Blood Culture ◽  
Positive Blood Culture ◽  
Type Species ◽  
Patient Management ◽  
Blood Cultures ◽  
Diagnostic Methods ◽  
Content Type ◽  
Link Type ◽  
The Impact

Introduction. Staphylococcus aureus bacteraemia (SAB) causes significant morbidity and mortality. Standard diagnostic methods require 24–48 h to provide results, during which time management is guideline-based and may be suboptimal. Aim. Evaluate the impact of rapid molecular detection of S. aureus in positive blood culture bottle fluid on patient management. Methodology. Samples were tested prospectively at two clinical centres. Positive blood cultures with Gram-positive cocci in clusters on microscopy were tested with the Xpert MRSA/SA blood culture assay (Cepheid), as well as standard culture-based identification and antimicrobial sensitivity tests. Results were passed to clinical microbiologists in real time and used for patient management. Results. Of 264 blood cultures tested (184 and 80 from each centre), S. aureus was grown from 39 (14.8 %) with one identified as methicillin-resistant S. aureus ; all Xpert results agreed with culture results. Median turnaround time from culture flagging positive to result reporting for Xpert was 1.7 h, compared to 25.7 h for species identification by culture. Xpert results allowed early changes to management in 40 (16.8 %) patients, with Xpert positive patients starting specific therapy for SAB and Xpert negative patients stopping or avoiding empiric antimicrobials for SAB. Conclusion. Rapid and accurate detection of S. aureus with the Xpert MRSA/SA BC assay in positive blood culture bottles allowed earlier targeted patient management. Negative Xpert results are suggestive of coagulase negative staphylococci, allowing de-escalation of antimicrobial therapy if clinically appropriate.

scholarly journals Duplex real-time PCR assay for the simultaneous detection of Achromobacter xylosoxidans and Achromobacter spp.

2020 ◽  
Vol 6 (7) ◽  
Author(s):  
Erin P. Price ◽  
Valentina Soler Arango ◽  
Timothy J. Kidd ◽  
Tamieka A. Fraser ◽  
Thuy-Khanh Nguyen ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Type Species ◽  
Simultaneous Detection ◽  
Diagnostic Methods ◽  
Achromobacter Xylosoxidans ◽  
Resistant Bacteria ◽  
Pcr Assay ◽  
Content Type ◽  
Link Type

Several members of the Gram-negative environmental bacterial genus Achromobacter are associated with serious infections, with Achromobacter xylosoxidans being the most common. Despite their pathogenic potential, little is understood about these intrinsically drug-resistant bacteria and their role in disease, leading to suboptimal diagnosis and management. Here, we performed comparative genomics for 158 Achromobacter spp. genomes to robustly identify species boundaries, reassign several incorrectly speciated taxa and identify genetic sequences specific for the genus Achromobacter and for A. xylosoxidans . Next, we developed a Black Hole Quencher probe-based duplex real-time PCR assay, Ac-Ax, for the rapid and simultaneous detection of Achromobacter spp. and A. xylosoxidans from both purified colonies and polymicrobial clinical specimens. Ac-Ax was tested on 119 isolates identified as Achromobacter spp. using phenotypic or genotypic methods. In comparison to these routine diagnostic methods, the duplex assay showed superior identification of Achromobacter spp. and A. xylosoxidans , with five Achromobacter isolates failing to amplify with Ac-Ax confirmed to be different genera according to 16S rRNA gene sequencing. Ac-Ax quantified both Achromobacter spp. and A. xylosoxidans down to ~110 genome equivalents and detected down to ~12 and ~1 genome equivalent(s), respectively. Extensive in silico analysis, and laboratory testing of 34 non- Achromobacter isolates and 38 adult cystic fibrosis sputa, confirmed duplex assay specificity and sensitivity. We demonstrate that the Ac-Ax duplex assay provides a robust, sensitive and cost-effective method for the simultaneous detection of all Achromobacter spp. and A. xylosoxidans and will facilitate the rapid and accurate diagnosis of this important group of pathogens.

scholarly journals Genomics reveals the novel species placement of industrial contaminant isolates incorrectly identified as Burkholderia lata

2021 ◽  
Vol 7 (4) ◽  
Author(s):  
Edward Cunningham-Oakes ◽  
Tom Pointon ◽  
Barry Murphy ◽  
Stuart Campbell-Lee ◽  
Gordon Webster ◽  
...  
Keyword(s):  
Burkholderia Cepacia ◽  
Type Species ◽  
Manufacturing Industry ◽  
Core Gene ◽  
Resistant Bacteria ◽  
Accurate Identification ◽  
Content Type ◽  
Link Type ◽  
Industrial Contaminants ◽  
Industrial Strains

The Burkholderia cepacia complex (Bcc) is a closely related group of bacteria, composed of at least 20 different species, the accurate identification of which is essential in the context of infectious diseases. In industry, they can contaminate non-food products, including home and personal care products and cosmetics. The Bcc are problematic contaminants due to their ubiquitous presence and intrinsic antimicrobial resistance, which enables them to occasionally overcome preservation systems in non-sterile products. Burkholderia lata and Burkholderia contaminans are amongst the Bcc bacteria encountered most frequently as industrial contaminants, but their identification is not straightforward. Both species were historically established as a part of a group known collectively as taxon K, based upon analysis of the recA gene and multilocus sequence typing (MLST). Here, we deploy a straightforward genomics-based workflow for accurate Bcc classification using average nucleotide identity (ANI) and core-gene analysis. The workflow was used to examine a panel of 23 Burkholderia taxon K industrial strains, which, based on MLST, comprised 13 B. lata, 4 B. contaminans and 6 unclassified Bcc strains. Our genomic identification showed that the B. contaminans strains retained their classification, whilst the remaining strains were reclassified as Burkholderia aenigmatica sp. nov. Incorrect taxonomic identification of industrial contaminants is a problematic issue. Application and testing of our genomic workflow allowed the correct classification of 23 Bcc industrial strains, and also indicated that B. aenigmatica sp. nov. may have greater importance than B. lata as a contaminant species. Our study illustrates how the non-food manufacturing industry can harness whole-genome sequencing to better understand antimicrobial-resistant bacteria affecting their products.

scholarly journals Ciprofloxacin-induced persister-cells in Campylobacter jejuni

Microbiology ◽  
2020 ◽  
Vol 166 (9) ◽  
pp. 849-853 ◽  
Author(s):  
Armen Ovsepian ◽  
Marianne Halberg Larsen ◽  
Christina Skovgaard Vegge ◽  
Hanne Ingmer
Keyword(s):  
Campylobacter Jejuni ◽  
Type Species ◽  
Bacterial Infections ◽  
Bacterial Species ◽  
Cell Formation ◽  
Fluoroquinolone Resistance ◽  
Persister Cells ◽  
Content Type ◽  
Link Type ◽  
Persister Cell

Campylobacter jejuni is a major bacterial foodborne-pathogen. Ciprofloxacin is an important antibiotic for the treatment of C. jejuni , albeit high rates of fluoroquinolone resistance have limited its usefulness. Persister-cells are transiently antibiotic-tolerant fractions of bacterial populations and their occurrence has been associated with recalcitrant and persistent bacterial infections. Here, time-kill assays with ciprofloxacin (200×MIC, 25 µg ml−1) were performed in C. jejuni strains 81–176 and RM1221 and persister-cells were found. The frequency of survivors after 8 h of ciprofloxacin exposure was approx. 10−3 for both strains, while after 22 h the frequency was between 10−5–10−7, depending on the strain and growth-phase. Interestingly, the stationary-phase cultures did not display more persister-cells compared to exponential-phase cultures, in contrast to what has been observed in other bacterial species. Persister-cells after ampicillin exposure (100×MIC, 200 µg ml−1) were not detected, implying that persister-cell formation in C. jejuni is antibiotic-specific. In attempts to identify the mechanism of ciprofloxacin persister-cell formation, stringent or SOS responses were not found to play major roles. Overall, this study reports ciprofloxacin persister-cells in C. jejuni and challenges the notion of persister-cells as plainly dormant non-growing cells.

scholarly journals Faecalicoccus acidiformans gen. nov., sp. nov., isolated from the chicken caecum, and reclassification of Streptococcus pleomorphus (Barnes et al. 1977), Eubacterium biforme (Eggerth 1935) and Eubacterium cylindroides (Cato et al. 1974) as Faecalicoccus pleomorphus comb. nov., Holdemanella biformis gen. nov., comb. nov. and Faecalitalea cylindroides gen. nov., comb. nov., respectively, within the family Erysipelotrichaceae

2014 ◽  
Vol 64 (Pt_11) ◽  
pp. 3877-3884 ◽  
Author(s):  
Celine De Maesschalck ◽  
Filip Van Immerseel ◽  
Venessa Eeckhaut ◽  
Siegrid De Baere ◽  
Margo Cnockaert ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Type Species ◽  
Gene Sequence ◽  
Sequence Similarity ◽  
16S Rrna Gene Sequence ◽  
Rrna Gene ◽  
Rrna Gene Sequence ◽  
Content Type ◽  
Link Type

Strains LMG 27428T and LMG 27427 were isolated from the caecal content of a chicken and produced butyric, lactic and formic acids as major metabolic end products. The genomic DNA G+C contents of strains LMG 27428T and LMG 27427 were 40.4 and 38.8 mol%. On the basis of 16S rRNA gene sequence similarity, both strains were most closely related to the generically misclassified Streptococcus pleomorphus ATCC 29734T. Strain LMG 27428T could be distinguished from S. pleomorphus ATCC 29734T based on production of more lactic acid and less formic acid in M2GSC medium, a higher DNA G+C content and the absence of activities of acid phosphatase and leucine, arginine, leucyl glycine, pyroglutamic acid, glycine and histidine arylamidases, while strain LMG 27428 was biochemically indistinguishable from S. pleomorphus ATCC 29734T. The novel genus Faecalicoccus gen. nov. within the family Erysipelotrichaceae is proposed to accommodate strains LMG 27428T and LMG 27427. Strain LMG 27428T ( = DSM 26963T) is the type strain of Faecalicoccus acidiformans sp. nov., and strain LMG 27427 ( = DSM 26962) is a strain of Faecalicoccus pleomorphus comb. nov. (type strain LMG 17756T = ATCC 29734T = DSM 20574T). Furthermore, the nearest phylogenetic neighbours of the genus Faecalicoccus are the generically misclassified Eubacterium cylindroides DSM 3983T (94.4 % 16S rRNA gene sequence similarity to strain LMG 27428T) and Eubacterium biforme DSM 3989T (92.7 % 16S rRNA gene sequence similarity to strain LMG 27428T). We present genotypic and phenotypic data that allow the differentiation of each of these taxa and propose to reclassify these generically misnamed species of the genus Eubacterium formally as Faecalitalea cylindroides gen. nov., comb. nov. and Holdemanella biformis gen. nov., comb. nov., respectively. The type strain of Faecalitalea cylindroides is DSM 3983T = ATCC 27803T = JCM 10261T and that of Holdemanella biformis is DSM 3989T = ATCC 27806T = CCUG 28091T.

scholarly journals Pseudoseohaeicola caenipelagi gen. nov., sp. nov., isolated from a tidal flat

2015 ◽  
Vol 65 (Pt_6) ◽  
pp. 1819-1824 ◽  
Author(s):  
Sooyeon Park ◽  
Ji-Min Park ◽  
Chul-Hyung Kang ◽  
Song-Gun Kim ◽  
Jung-Hoon Yoon
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Tidal Flat ◽  
Type Strain ◽  
Type Species ◽  
Phylogenetic Trees ◽  
Sequence Similarity ◽  
Rrna Gene ◽  
Content Type ◽  
Link Type

A Gram-stain-negative, non-motile, aerobic and pleomorphic bacterium, designated BS-W13T, was isolated from a tidal flat on the South Sea, South Korea, and its taxonomic position was investigated using a polyphasic approach. Strain BS-W13T grew optimally at 25 °C, at pH 7.0–8.0 and in the presence of 1.0–2.0 % (w/v) NaCl. Neighbour-joining and maximum-parsimony phylogenetic trees based on 16S rRNA gene sequences showed that strain BS-W13T clustered with the type strain of Seohaeicola saemankumensis , showing the highest sequence similarity (95.96 %) to this strain. Strain BS-W13T exhibited 16S rRNA gene sequence similarity values of 95.95, 95.91, 95.72 and 95.68 % to the type strains of Sulfitobacter donghicola , Sulfitobacter porphyrae , Sulfitobacter mediterraneus and Roseobacter litoralis , respectively. Strain BS-W13T contained Q-10 as the predominant ubiquinone and C18 : 1ω7c as the major fatty acid. The polar lipid profile of strain BS-W13T, containing phosphatidylcholine, phosphatidylglycerol, phosphatidylethanolamine, one unidentified aminolipid and one unidentified lipid as major components, was distinguishable from those of some phylogenetically related taxa. The DNA G+C content of strain BS-W13T was 58.1 mol%. The phylogenetic data and differential chemotaxonomic and other phenotypic properties revealed that strain BS-W13T constitutes a novel genus and species within family Rhodobacteraceae of the class Alphaproteobacteria , for which the name Pseudoseohaeicola caenipelagi gen. nov., sp. nov. is proposed. The type strain is BS-W13T ( = KCTC 42349T = CECT 8724T).

Litorilinea aerophila gen. nov., sp. nov., an aerobic member of the class Caldilineae , phylum Chloroflexi , isolated from an intertidal hot spring

2013 ◽  
Vol 63 (Pt_3) ◽  
pp. 1149-1154 ◽  
Author(s):  
Varsha Kale ◽  
Snædís H. Björnsdóttir ◽  
Ólafur H. Friðjónsson ◽  
Sólveig K. Pétursdóttir ◽  
Sesselja Ómarsdóttir ◽  
...  
Keyword(s):  
Type Species ◽  
Hot Spring ◽  
Sequence Similarity ◽  
Rrna Gene ◽  
Content Type ◽  
Phenotypic Differences ◽  
Link Type ◽  
Fermentative Growth ◽  
Distinct Lineage ◽  
Ph Range

A thermophilic, aerobic, Gram-stain-negative, filamentous bacterium, strain PRI-4131T, was isolated from an intertidal hot spring in Isafjardardjup, NW Iceland. The strain grew chemo-organotrophically on various carbohydrates. The temperature range for growth was 40–65 °C (optimum 55 °C), the pH range was pH 6.5–9.0 (optimum pH 7.0) and the NaCl range was 0–3 % (w/v) (optimum 0.5 %). Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain PRI-4131T represented a distinct lineage within the class Caldilineae of the phylum Chloroflexi. The highest levels of sequence similarity, about 91 %, were with Caldilinea aerophila STL-6-O1T and Caldilinea tarbellica D1-25-10-4T. Fermentative growth was not observed for strain PRI-4131T, which, in addition to other characteristics, distinguished it from the two Caldilinea species. Owing to both phylogenetic and phenotypic differences from the described members of the class Caldilineae , we propose to accommodate strain PRI-4131T in a novel species in a new genus, Litorilinea aerophila gen. nov., sp. nov. The type strain of Litorilinea aerophila is PRI-4131T ( = DSM 25763T  = ATCC BAA-2444T).

Catenulispora graminis sp. nov., a rhizobacterium from bamboo (Phyllostachys nigro var. henonis) rhizosphere soil

2012 ◽  
Vol 62 (Pt_11) ◽  
pp. 2589-2592 ◽  
Author(s):  
Hyo-Jin Lee ◽  
Song-Ih Han ◽  
Kyung-Sook Whang
Keyword(s):  
Type Species ◽  
Rhizosphere Soil ◽  
Major Fatty Acid ◽  
Rrna Gene ◽  
Phenotypic Data ◽  
Content Type ◽  
Dna Relatedness ◽  
Link Type ◽  
Isoprenoid Quinones ◽  
Type Strains

A novel actinobacterium, designated strain BR-34T, was isolated from rhizosphere soil of bamboo (Phyllostachys nigro var. henonis) sampled in Damyang, Korea. The strain was found to have morphological and chemotaxonomic characteristics typical of the genus Catenulispora . The strain contained iso-C16 : 0 as the major fatty acid and MK-9(H4), MK-9(H6) and MK-9(H8) as major isoprenoid quinones. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain BR-34T formed a cluster separate from members of the genus Catenulispora and was related most closely to Catenulispora acidiphila ID139908T (97.4 % similarity), Catenulispora rubra Aac-30T (97.3 %), Catenulispora yoronensis TT N02-20T (97.3 %) and Catenulispora subtropica TT 99-48T (97 %). However, the level of DNA–DNA relatedness between strain BR-34T and C. acidiphila ID139908T was only 45.32 %. Based on DNA–DNA relatedness, morphological and phenotypic data, strain BR-34T could be distinguished from the type strains of phylogenetically related species. It is therefore considered to represent a novel species of the genus Catenulispora , for which the name Catenulispora graminis sp. nov. is proposed. The type strain is BR-34T ( = KACC 15070T = NBRC 107755T).

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