Publications

3887

‘Candidatus Liberibacter asiaticus’ (CLas) is an unculturable α-proteobacterium associated with citrus Huanglongbing (HLB; yellow shoot disease). PCR procedures that accurately confirm or exclude CLas infection in citrus tissue/Asian citrus psyllid (ACP) samples are critical for HLB management. When CLas was described in 1994, a 23-bp signature oligonucleotide sequence (OI1) in the 16S rRNA gene (rrs, three genomic copies) was identified based on Sanger sequencing. OI1 contains single nucleotide polymorphisms (SNPs) distinguishing CLas from non-CLas species. The SNPs were used to design the primer HLBas, a key primer for a commonly used TaqMan PCR system (HLBas-PCR) for CLas detection. Recent developments in next-generation sequencing technology have led to the identification of 15 CLas whole genome sequence strains (WGSs). Analyses of CLas WGSs have generated a significant amount of biological information that could help to improve CLas detection. Utilizing the WGS information, this study re-evaluated the sequence integrity of OI1/HLBas and identified and/or confirmed a missing nucleotide G in the two primers. Replacement primers for OI1 and HLBas are proposed. At low cycle threshold (Ct) values (e.g., <30), HLBas-PCR remained reliable in CLas determination. At high Ct values (e.g., >30), HLBas-PCR alone was unreliable in differentiating whether samples contain low CLas titers or whether CLas is not present. The availability of ribonucleotide reductase (RNR)-PCR derived from the five-copy nrdB gene helped to resolve this problem. To further enhance low CLas titer detection, a 4CP-PCR system, based on a four-copy genomic locus, was developed. Evaluation of 107 HLB samples (94 citrus and 13 ACP) showed that 4CP-PCR was more sensitive than HLBas-PCR and shared similar sensitivity with RNR-PCR.

Genome analysis is one of the main criteria for description of new taxa. Availability of genome sequences for all the actinobacteria with a valid nomenclature will, however, require another decade’s works of sequencing. This paper describes the rearrangement of the higher taxonomic ranks of the members of the phylum ‘ Actinobacteria ’, using the phylogeny of 16S rRNA gene sequences and supported by the phylogeny of the available genome sequences. Based on the refined phylogeny of the 16S rRNA gene sequences, we could arrange all the members of the 425 genera of the phylum ‘ Actinobacteria ’ with validly published names currently in use into six classes, 46 orders and 79 families, including 16 new orders and 10 new families. The order Micrococcales Prévot 1940 (Approved Lists 1980) emend. Nouioui et al. 2018 is now split into 11 monophyletic orders: the emended order Micrococcales and ten proposed new orders Aquipuribacterales , Beutenbergiales , Bogoriellales , Brevibacteriales , Cellulomonadales , Demequinales , Dermabacterales , Dermatophilales , Microbacteriales and Ruaniales . Further, the class ‘ Actinobacteria ’ Stackebrandt et al. 1997 emend. Nouioui et al. 2018 was described without any nomenclature type, and therefore the name ‘ Actinobacteria ’ is deemed illegitimate. In accordance to Rule 8 of the International Code of Nomenclature of Prokaryotes, Parker et al. 2019, we proposed the name Actinomycetia which is formed by using the stem of the name Actinomycetales Buchanan 1917 (Approved Lists 1980) emend. Zhi et al. 2009, to replace the name ‘ Actinobacteria ’. The nomenclature type of the proposed new class Actinomycetia is the order Actinomycetales Buchanan 1917 (Approved Lists 1980) emend. Zhi et al. 2009.

Abstract Background Hemotropic mycoplasmas (hemoplasmas) infect animals and humans and can lead to clinical syndromes mainly characterized by hemolytic anemia. A novel pathogen, Candidatus Mycoplasma haemohominis, was recently associated with a case of human hemoplasmosis in Europe. Here we report the first detection of this pathogen in an Australian patient exhibiting persistent fever, hemolytic anemia, and pancytopenia over a 10-month period. Methods After exhaustive negative testing for human infectious diseases, whole genome sequencing (WGS) was performed on the patient’s bone marrow aspirate, using an Illumina NextSeq500 platform. Conventional polymerase chain reaction (PCR), followed by Sanger sequencing, was then performed on blood samples using novel Mycoplasma-specific primers targeting the 16S ribosomal RNA gene. In addition, a Mycoplasma-specific fluorescence in situ hybridization (FISH) assay was developed to differentiate Mycoplasma cells from other erythrocyte inclusions (eg, Pappenheimer and Howell-Jolly bodies) which are morphologically similar to bacterial cocci by light microscopy. Results WGS analysis revealed that approximately 0.04% of the total number of unmapped reads to human genome corresponded to Mycoplasma species. A 1-kb Mycoplasma 16S fragment was successfully amplified by conventional PCR, and sequence analyses revealed 100% identity with Candidatus Mycoplasma haemohominis. FISH confirmed that several (approximately 2%) epierythrocytic inclusions initially observed by light microscopy corresponded to Mycoplasma cells. Conclusions This represents the second report of hemolytic anemia associated with hemoplasma infection in a human, and the first report of human hemoplasmosis in Australia. This study highlights the importance of new and emerging diagnostic approaches and need for further investigations on the epidemiology of Candidatus Mycoplasma haemohominis in Australia.

An obligately anaerobic, Gram-stain-positive and coccus-shaped bacterium, designated strain YH-panp20T, was isolated from pig faeces. Phylogenetic analysis based on 16S rRNA gene sequencing indicated that the isolate belongs to the family Erysipelotrichaceae , and is most closely related to Catenisphaera adipataccumulans KCTC 15517T (93.5 % sequence similarity), followed by Faecalitalea cylindroides KCTC 5815T (92.2 %), Faecalicoccus acidiformans KCTC 15521T (90.2 %) and Holdemanella biformis KCTC 5969T (89.6 %). Average nucleotide identity values between YH-panp20T and its closest relatives were lower than 71 %. The G+C content of the isolate was 38.4 mol%, and its cell-wall peptidoglycan was found to be of A1γ type and contained meso-diaminopimelic acid. The predominant fatty acids were C18 : 1 cis 9, C18 : 0 DMA and C16 : 0. The major end-products of glucose fermentation were lactate, acetate and formate. Therefore, based on the phenotypic, phylogenetic and chemotaxonomic properties, a novel genus and species, Absicoccus porci gen. nov., sp. nov., is proposed for isolate YH-panp20T (=KCTC 15747T=JCM 32769T).