Molecular detection of Campylobacter species from human and cattle faecal samples in Kilosa District, Tanzania

A growing number of Campylobacter species other than C. jejuni and C. coli have been considered as emerging human and animal pathogens but their contribution to human gastroenteritis is poorly documented. This study aimed at detecting Campylobacter species from human and cattle faecal samples in Kilosa District, Tanzania using molecular techniques without culture. Seventy (70) faecal samples were collected from five diarrheic and 65 non-diarrheic human patients attending Kilosa District Hospital in Tanzania from July to October 2019. During the same period, 30 faecal samples were also collected from healthy cattle in the same district. Genus and species identification of Campylobacter was conducted on the samples using molecular techniques [the polymerase chain reaction (PCR) and 16S rRNA sequencing]. Phylogenetic analysis was carried out by comparison of the 16S rRNA gene sequences to reference strains by the Neighbor-Joining method in MEGA X. Campylobacter species detection rate by PCR was 65.7% (46/70) and 20% (6/30) in humans and cattle, respectively. There were five human diarrheic cases, four of which were positive for Campylobacter and of these, two were children ≤15 years of age. In humans, 16S rRNA sequencing revealed that C. concisus was the most predominant species occurring at a frequency of 37.8% (14/37), followed by uncultured Campylobacter spp. 24.3% (9/37) and C. hominis 21.6% (8/37). The least represented species were C. jejuni and C. lanienae, all occurring at 2.7% (1/37). In cattle, five (100%) sequenced PCR products matched with C. lanienae. Phylogenetic analysis revealed that with the exception of C. lanienae, 16S rRNA sequences of Campylobacter species were closely related to the reference strains used (Percent identity: 90.51-96.56%). Based on our findings, we recommend that molecular techniques, mainly PCR be adopted for the direct detection of Campylobacter species during laboratory screening and surveillance studies.

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Molecular detection of Campylobacter species from human and cattle faecal samples in Kilosa district, Tanzania

10.21203/rs.3.rs-46746/v1 ◽

2020 ◽

Author(s):

Noel Gahamanyi ◽

Leonard E.G. Mboera ◽

Mecky I. Matee ◽

Dieudonné Mutangana ◽

Raghavendra G. Amachawadi ◽

...

Keyword(s):

Polymerase Chain Reaction ◽

Phylogenetic Analysis ◽

16S Rrna ◽

Rrna Gene ◽

Chain Reaction ◽

Faecal Samples ◽

Rrna Sequencing ◽

Polymerase Chain ◽

Campylobacter Species ◽

The 16S Rrna Gene

Abstract Background: A growing number of Campylobacter species other than C. jejuni and C. coli have been considered as emerging human and animal pathogens. However, the contribution of these species to human gastroenteritis is poorly documented. This study aimed at detecting Campylobacter species from human and cattle faecal samples in Kilosa district, Tanzania using Polymerase Chain Reaction (PCR) amplification of the 16S rRNA gene, and Sanger sequencing . Methods: A total number of 100 faecal samples (70 from human and 30 from cattle) were collected from diarrheic and non-diarrheic patients and healthy cattle in Kilosa district, Tanzania from July to October 2019. Species identification was conducted by PCR and 16S rRNA sequencing. The phylogenetic analysis was carried out by comparison of the 16S rRNA gene sequences to reference strains by the Neighbor-Joining method in MEGA X. Results: Campylobacter species detection rate by PCR was 65.7% (46/70) and 20% (6/30) in humans and cattle, respectively. There were five human diarrheic cases, four showed Campylobacter presence and two were from children ≤15 years of age. In humans, the 16S rRNA sequencing revealed that C. concisus was the most predominant species occurring at a frequency of 37.8% (14/37), followed by uncultured Campylobacter spp. 24.3% (9/37) and C. hominis 21.6% (8/37). The least represented species were C. jejuni and C. lanienae all occurring at 2.7% (1/37). In cattle, five (100%) sequenced PCR products matched with C. lanienae . Phylogenetic analysis revealed that Campylobacter 16S rRNA sequences were closely related to C. concisus , uncultured Campylobacter sp., C. hominis , and C. gracilis . Conclusion: The non- C. jejuni / C. coli species are present in human and cattle faecal samples and their true occurrence is probably under-reported due to shortcomings of conventional techniques used in most diagnostic microbiology laboratories. Based on our findings, we recommend that molecular techniques be adopted for direct detection of Campylobacter species during routine laboratory screening and surveillance studies. Keywords: Campylobacter , molecular diagnostics, polymerase chain reaction, sequencing, gastroenteritis, Tanzania

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Clinical optochin resistant Streptococcus pneumoniae and Streptococcus pseudopneumoniae strains in Tunisia

The Journal of Infection in Developing Countries ◽

10.3855/jidc.13106 ◽

2021 ◽

Vol 15 (05) ◽

pp. 672-677

Author(s):

Sonia Ktari ◽

Nour El Houda Ben Ayed ◽

Sonda Maalej ◽

Basma Mnif ◽

Faouzia Rhimi ◽

...

Keyword(s):

Streptococcus Pneumoniae ◽

16S Rrna ◽

Molecular Techniques ◽

16S Rrna Sequencing ◽

Easy Method ◽

Rrna Sequencing ◽

C Subunit ◽

Density Values ◽

A Subunit ◽

Human Infections

Introduction: Streptococcus pneumoniae can be responsible for severe human infections. Optochin resistance has been a potential cause of misidentification of pneumococcus and other members of the mitis group. Hence, rapid and easy optochin resistant (Optr) S. pneumoniae identification is essential. Methodology: Atypical pneumococci were characterized using optochin susceptibility, bile solubility based on spectrophotometric reading, serotyping, pulsed field gel electrophoresis (PFGE), 16S rRNA sequencing and PCR-based assays targeting pneumococcal genes lytA, ply, pspA, cpsA, Spn9802 and Spn9828. Results: Optical density values for the bile solubility test suggest the identification of four Optr S. pneumoniae and one Streptococcus pseudopneumoniae. All Optr pneumococci harbored cpsA, lytA, ply, Spn9802, Spn9828 and pspA genes. Only ply, spn9802 and Spn9828 genes were detected in S. pseudopneumoniae. The 16S rRNA sequencing differentiates between these two species. Optr S. pneumoniae strains belonged to different genotypes and serotypes (14, 19A, 3 and 9V). Three Optr S. pneumoniae isolates were typed as pspA family 2, while one belonged to pspA family 1. Sequencing of the atpA and atpC gene of the Optr variants revealed three mutations in the ATPase a-subunit (L99I, M23V and V52I) and one mutation in ATPase c-subunit (V48I). Conclusions: Our data indicate that bile OD-values provides an accurate, fast and easy method to discriminate between Optr S. pneumoniae and other Streptococcus mitis group. Moreover molecular techniques, confirming the bile test, can be used in order to prevent these atypical pneumococci and alert clinical microbiologists of the presence of these strains in the community.

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Molecular identification and phylogenetic analysis of chromium-resistant bacteria isolated from chromite mine area soil, Sukinda, India using 16S rRNA sequencing

Soil and Sediment Contamination An International Journal ◽

10.1080/15320383.2020.1771272 ◽

2020 ◽

Vol 29 (8) ◽

pp. 805-822

Author(s):

Sukanta Kumar Pradhan ◽

Nihar Ranjan Singh ◽

Sasmita Das ◽

Hrudayanath Thatoi

Keyword(s):

Phylogenetic Analysis ◽

16S Rrna ◽

Molecular Identification ◽

16S Rrna Sequencing ◽

Resistant Bacteria ◽

Mine Area ◽

Rrna Sequencing

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Reclassification of [Pasteurella] trehalosi as Bibersteinia trehalosi gen. nov., comb. nov.

INTERNATIONAL JOURNAL OF SYSTEMATIC AND EVOLUTIONARY MICROBIOLOGY ◽

10.1099/ijs.0.64521-0 ◽

2007 ◽

Vol 57 (4) ◽

pp. 666-674 ◽

Cited By ~ 62

Author(s):

P. J. Blackall ◽

Anders Miki Bojesen ◽

Henrik Christensen ◽

Magne Bisgaard

Keyword(s):

Phylogenetic Analysis ◽

16S Rrna ◽

16S Rrna Gene ◽

Type Strain ◽

Phenotypic Characterization ◽

Rrna Gene ◽

Field Isolates ◽

The Family ◽

Bibersteinia Trehalosi ◽

Reference Strains

[Pasteurella] trehalosi is an important pathogen of sheep, being primarily associated with serious systemic infections in lambs but also having an association with pneumonia. The aim of the present investigation was to characterize a broad collection of strains tentatively identified as [P.] trehalosi in order to reclassify and rename this taxon to support improvements in our understanding of the pathogenesis and epidemiology of this important organism. The type strain for [P.] trehalosi, strain NCTC 10370T, was included along with 42 field isolates from sheep (21), cattle (14), goats (1), roe deer (3) and unknown sources (3). An extended phenotypic characterization was performed on all 43 strains. Amplified fragment length polymorphism (AFLP) was also performed on the isolates. Two of the field isolates were subjected to 16S rRNA gene sequencing. These sequences, along with five existing sequences for [P.] trehalosi strains and 12 sequences for other taxa in the family Pasteurellaceae, were subjected to a phylogenetic analysis. All the isolates and the reference strains were identified as [P.] trehalosi. A total of 17 out of 22 ovine isolates produced acid from all glycosides, while only four out of 14 bovine isolates produced acid from all glycosides. All 22 ovine isolates were haemolytic and CAMP-positive, while no other isolate was haemolytic and only two bovine isolates were CAMP-positive. Nineteen AFLP types were found within the [P.] trehalosi isolates. All [P.] trehalosi isolates shared at least 70 % similarity in AFLP patterns. The largest AFLP type included the type strain and 7 ovine field isolates. Phylogenetic analysis indicated that the seven strains studied (two field isolates and the five serovar reference strains) are closely related, with 98.6 % or higher 16S rRNA gene sequence similarity. As both genotypic and phenotypic testing support the separate and distinct nature of these organisms, we propose the transfer of [P.] trehalosi to a new genus, Bibersteinia, as Bibersteinia trehalosi comb. nov. The type strain is NCTC 10370T (=ATCC 29703T). Bibersteinia trehalosi can be distinguished from the existing genera of the family by the observation of only nine characteristics; catalase, porphyrin, urease, indole, phosphatase, acid from dulcitol, (+)-d-galactose, (+)-d-mannose and (+)-d-trehalose.

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High-resolution microbiome analysis enabled by linking of 16S rRNA gene sequences with adjacent genomic contexts

Microbial Genomics ◽

10.1099/mgen.0.000624 ◽

2021 ◽

Vol 7 (9) ◽

Author(s):

Žana Kapustina ◽

Justina Medžiūnė ◽

Gediminas Alzbutas ◽

Irmantas Rokaitis ◽

Karolis Matjošaitis ◽

...

Keyword(s):

16S Rrna ◽

16S Rrna Gene ◽

16S Rrna Sequencing ◽

Gene Copy ◽

Rrna Gene ◽

Gene Sequences ◽

Copy Numbers ◽

Rrna Sequencing ◽

Metagenome Sequencing ◽

Gene Copy Numbers

Sequence-based characterization of bacterial communities has long been a hostage of limitations of both 16S rRNA gene and whole metagenome sequencing. Neither approach is universally applicable, and the main efforts to resolve constraints have been devoted to improvement of computational prediction tools. Here, we present semi-targeted 16S rRNA sequencing (st16S-seq), a method designed for sequencing V1–V2 regions of the 16S rRNA gene along with the genomic locus upstream of the gene. By in silico analysis of 13 570 bacterial genome assemblies, we show that genome-linked 16S rRNA sequencing is superior to individual hypervariable regions or full-length gene sequences in terms of classification accuracy and identification of gene copy numbers. Using mock communities and soil samples we experimentally validate st16S-seq and benchmark it against the established microbial classification techniques. We show that st16S-seq delivers accurate estimation of 16S rRNA gene copy numbers, enables taxonomic resolution at the species level and closely approximates community structures obtainable by whole metagenome sequencing.

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Phylogenetic analysis of Streptococcus saccharolyticus based on 16S rRNA sequencing

FEMS Microbiology Letters ◽

10.1111/j.1574-6968.1990.tb03828.x ◽

1990 ◽

Vol 71 (1-2) ◽

pp. 231-234 ◽

Cited By ~ 23

Author(s):

U. Rodrigues ◽

M.D. Collins

Keyword(s):

Phylogenetic Analysis ◽

16S Rrna ◽

16S Rrna Sequencing ◽

Rrna Sequencing

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Molecular Characterization of Sub-cuticular Bacteria in New Zealand Echinoderms

10.26686/wgtn.16958995 ◽

2021 ◽

Author(s):

◽

Scott Anthony Lawrence

Keyword(s):

Phylogenetic Analysis ◽

New Zealand ◽

16S Rrna ◽

Culture Media ◽

Molecular Techniques ◽

The Body ◽

Rrna Gene ◽

Associated Bacteria ◽

The Relationship

<p>Many echinoderms contain sub-cuticular bacteria (SCB), symbionts which reside in the lumen between the epidermal cells and the outer cuticle of the host. The relationship is very common, with ~60% of all echinoderms studied so far containing SCB. Currently, little is known about the function of the symbiosis, although it has been hypothesized that SCB may aid in host nutrition or antimicrobial defense. Whatever their function, the large numbers of SCB observed in many echinoderms (10 (to the power of 8) - 10 (to the power of 9) SCB g-1 AFDW host tissue) suggest that they may be important to the host. Factors contributing to the lack of knowledge about the echinoderm-SCB symbiosis include the difficulty associated with cultivating symbiotic bacteria, and the lack of studies identifying the SCB by molecular means. In this study, molecular techniques were employed to characterize the SCB of several common New Zealand echinoderms. The specific objectives of the study were to identify the SCB through sequencing of a region of the bacterial 16S rRNA gene, identify and locate SCB in situ through the use of fluorescence in situ hybridization (FISH), and cultivate SCB obtained from those echinoderms which were found to contain them. Phylogenetic analysis of 16S rRNA sequences obtained from echinoderm-associated bacteria resulted in the identification of four putative species of SCB. All four bacteria were isolated from samples of Stichopus mollis (class Holothuroidea), and two of the four were also found in samples of Patiriella sp (class Asteroidea). The first putative SCB belongs to the order Rhizobiales (a-proteobacteria), and is closely related to the SCB previously isolated from the brittle star Ophiactis balli. The second species belongs to the order Chromatiales (y-proteobacteria). Putative SCB species 3 falls within the Roseobacter clade (a-proteobacteria). The phylogenetic placement of the final putative SCB is more ambiguous, as this bacterium falls among members of the a- and y- subdivisions of the phylum Proteobacteria. The nearest relatives of this final bacterium are in the orders Rickettsiales and Thiotrichales. Results of FISH assays show that Patiriella sp. and S. mollis contain SCB, while a third species, Astrostole scabra (class Asteroidea) does not. The SCB community composition was found to vary between Patiriella sp. and S. mollis. In both species, the majority of the SCB present were found to belong to the a-subdivision of the phylum Proteobacteria (>80% in both species). However, in S. mollis, ~20% of the SCB community consists of bacteria belonging to the y-subdivision of the phylum Proteobacteria, whereas bacteria belonging to this subdivision were never observed in Patiriella sp. Cultivation experiments were carried out using a range of culture media, however results were inconclusive. Ten species of proteobacteria were successfully cultivated, three of which were obtained only from Patiriella sp. and S. mollis samples and were considered possible candidates for SCB. However, phylogenetic analysis of these three bacteria revealed that closely-related bacteria are predominantly free-living species. While the possibility remains that these three bacteria are in fact SCB, it seems more likely that they represent seawater or echinoderm surface-associated bacteria. This study contributes to the body of knowledge of the echinoderm-SCB symbiosis by identifying several potential SCB in Patiriella sp. and S. mollis, and is the first to identify SCB in situ through the use of FISH. An obvious goal in studies of the echinoderm-SCB symbiosis is to determine the function of the relationship. Potential functions of the symbiosis, based on the results obtained here, are discussed herein.</p>

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Identification of Mycobacterium neoaurumIsolated from a Neutropenic Patient with Catheter-Related Bacteremia by 16S rRNA Sequencing

Journal of Clinical Microbiology ◽

10.1128/jcm.38.9.3515-3517.2000 ◽

2000 ◽

Vol 38 (9) ◽

pp. 3515-3517 ◽

Cited By ~ 45

Author(s):

Patrick C. Y. Woo ◽

Hoi-Wah Tsoi ◽

Kit-Wah Leung ◽

Peggy N. L. Lum ◽

Andy S. P. Leung ◽

...

Keyword(s):

16S Rrna ◽

Lymphoblastic Leukemia ◽

Similarity Index ◽

16S Rrna Sequencing ◽

Identification System ◽

Rrna Gene ◽

Microbial Identification ◽

First Case ◽

Rrna Sequencing ◽

Mycobacterial Strain

A rapidly growing pigmented mycobacterial strain with an ambiguous biochemical profile was isolated from the blood culture taken through the Hickman catheter of a 9-year-old girl with acute lymphoblastic leukemia. Whole-cell fatty acid analysis showed that the best match profile was that of Mycobacterium aurum, but the similarity index was only 0.217, meaning that there were no good matches between the isolate and the organisms in the database of the Microbial Identification System. The 16S rRNA gene of the mycobacterial strain was amplified, agarose gel purified, and sequenced. There were 44 base differences between the gene sequence of the isolate and that ofM. aurum but only one base difference between the sequence of the isolate and that of Mycobacterium neoaurum, showing that the isolate was indeed a strain of M. neoaurum by using this “gold standard.” This represents the first case ofM. neoaurum infection documented by 16S rRNA sequencing.

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