Characterization of the Pilosebaceous Microbiota and Biofilm-Forming Capacity of Bacteria Isolated from Healthy Individuals

Background: Understanding of the biological factors responsible for prevalence and persistence of Acinetobacter baumannii in hospital settings is critical to prevent and control the corresponding nosocomial infections. Objectives: This study was conducted to investigate whether the biofilm-forming ability is associated with the emergence of different antibiotic resistance phenotypes [multidrug resistance (MDR)/extensively drug resistance (XDR) and non-MDR] of A. baumannii. Methods: The capacities of biofilm formation in 80 clinical A. baumannii strains isolated from hospitalized burn patients in Bushehr, Iran, were assessed using the crystal violet staining. Results: The statistical analysis of the relationship between biofilm-forming ability and antibiotic resistance phenotypes among all clinical A. baumannii strains using one-way ANOVA test indicated that biofilm formation capacity of non-MDR A. baumannii isolates was significantly higher than that of MDR and XDR ones (P < 0.001), suggesting an inverse relationship between biofilm formation capacity and the acquisition of MDR/XDR phenotypes. Major international clonal types (ICI and ICII) also exhibited such a significant relationship (P < 0.0001). However, the investigation of A. baumannii IC variants showed no significant relationship between these phenotypes. Conclusions: Given that non-MDR A. baumannii isolates in major IC types were observed to form a strong biofilm compared to MDR/XDR ones, it seems that biofilm may play a key role in the persistence and survival of A. baumannii isolates with an inadequate level of antibiotic resistance. Furthermore, the results showed that the relationship between biofilm and antibiotic resistance phenotypes might be affected by the IC types (major IC types or IC variants).

Molecular characteristics and biofilm formation capacity of nontypeable Haemophilus influenza strains isolated from lower respiratory tract in children

Innate lymphoid cells type 1 suffice to induce hallmarks of alopecia areata phenotype in organ-cultured human scalp hair follicles ex vivo and in human scalp skin xenotransplants in vivo, suggesting that these cells play a role in early AA pathogenesis.

Innate lymphoid cells type 1 suffice to induce hallmarks of alopecia areata phenotype in organ-cultured human scalp hair follicles ex vivo and in human scalp skin xenotransplants in vivo, suggesting that these cells play a role in early AA pathogenesis.

Microbiome studies of the lower airways based on bacterial 16S rRNA gene sequencing assess microbial community structure but can only infer functional characteristics. Microbial products, such as short-chain fatty acids (SCFAs), in the lower airways have significant impact on the host's immune tone. Thus, functional approaches to the analyses of the microbiome are necessary. Here we used upper and lower airway samples from a research bronchoscopy smoker cohort. In addition, we validated our results in an experimental mouse model. We extended our microbiota characterisation beyond 16S rRNA gene sequencing with the use of whole-genome shotgun (WGS) and RNA metatranscriptome sequencing. SCFAs were also measured in lower airway samples and correlated with each of the sequencing datasets. In the mouse model, 16S rRNA gene and RNA metatranscriptome sequencing were performed. Functional evaluations of the lower airway microbiota using inferred metagenome, WGS and metatranscriptome data were dissimilar. Comparison with measured levels of SCFAs shows that the inferred metagenome from the 16S rRNA gene sequencing data was poorly correlated, while better correlations were noted when SCFA levels were compared with WGS and metatranscriptome data. Modelling lower airway aspiration with oral commensals in a mouse model showed that the metatranscriptome most efficiently captures transient active microbial metabolism, which was overestimated by 16S rRNA gene sequencing. Functional characterisation of the lower airway microbiota through metatranscriptome data identifies metabolically active organisms capable of producing metabolites with immunomodulatory capacity, such as SCFAs.

Arthrospira platensis and Arthrospira maxima are species of cyanobacteria used in health foods, animal feed, food additives, and fine chemicals. This study conducted a comparison of the 16S rRNA gene and cpcBA-intergenic spacer (cpcBA-IGS) sequences in Arthrospira strains from culture collections around the world. A cluster analysis divided the 10 Arthrospira strains into two main genotypic clusters, designated I and II, where Group I contained A. platensis SAG 86.79, UTEX 2340, A. maxima KCTC AG30054, and SAG 49.88, while Group II contained A. platensis PCC 9108, NIES 39, NIES 46, and SAG 257.80. However, although A. platensis PCC 9223 belonged to Group II-2 based on its cpcBA-IGS sequence, this strain also belonged to Group I based on its 16S rRNA gene sequence. Phylogenetic analyses based on the 16S rRNA gene and cpcBA-IGS sequences showed no division between A. platensis and A. maxima, plus the 16S rRNA gene and cpcBAIGS sequence clusters did not indicate any well-defined geographical distribution, instead overlapping in a rather inter esting way. Therefore, the current study supports some previous conclusions based on 16S rRNA gene and cpcBA-IGS sequences, which found that Arthrospira taxa are monophyletic. However, when compared with 16S rRNA sequences, cpcBA-IGS sequences may be better suited to resolve close relationships and intraspecies variability.

Bacterial vaginosis (BV) is the most common infection of the lower reproductive tract among women of reproductive age. Recurrent infections and antibiotic resistance associated with biofilms remain significant challenges for BV treatment. Gardnerella species are commonly found in women with and without BV, indicating that genetic differences among Gardnerella isolates may distinguish pathogenic from commensal subgroups. This study isolated 11 Gardnerella strains from vaginal samples obtained from women with BV before or after treatment. The biofilm formation ability of each strain was examined by crystal violet staining. Eight strains were selected using phylogenetic analysis of the cpn60 sequences and classified as subgroups A (6/8), B (1/8), and D (1/8). The biofilm formation ability and antibiotic resistance profile of these strains was compared among the subgroups. Subgroup D had the strongest biofilm formation ability. Six of the planktonic strains exhibited resistance to the first-line BV drug, metronidazole, and one to clindamycin. Moreover, biofilm formation in vitro increased strain resistance to clindamycin. Two strains with strong biofilm ability, S20 and S23, and two with weak biofilm ability, S24 and S25, were selected for comparative genomic analysis. S20 and S23 were found to contain four key genes associated with biofilm formation and more genes involved in carbohydrate synthesis and metabolism than S24 and S25. Identifying differences in the expression of virulence factors between Gardnerella subgroups could inform the development of novel treatments for BV.

The current classification of non-pigmented and late-pigmenting rapidly growing mycobacteria (RGM) capable of producing disease in humans and animals consists primarily of three groups, the Mycobacterium fortuitum group, the Mycobacterium chelonae-abscessus group and the Mycobacterium smegmatis group. Since 1995, eight emerging species have been tentatively assigned to these groups on the basis of their phenotypic characters and 16S rRNA gene sequence, resulting in confusing taxonomy. In order to assess further taxonomic relationships among RGM, complete sequences of the 16S rRNA gene (1483-1489 bp), rpoB (3486-3495 bp) and recA (1041-1056 bp) and partial sequences of hsp65 (420 bp) and sodA (441 bp) were determined in 19 species of RGM. Phylogenetic trees based upon each gene sequence, those based on the combined dataset of the five gene sequences and one based on the combined dataset of the rpoB and recA gene sequences were then compared using the neighbour-joining, maximum-parsimony and maximum-likelihood methods after using the incongruence length difference test. Combined datasets of the five gene sequences comprising nearly 7000 bp and of the rpoB+recA gene sequences comprising nearly 4600 bp distinguished six phylogenetic groups, the M. chelonae-abscessus group, the Mycobacterium mucogenicum group, the M. fortuitum group, the Mycobacterium mageritense group, the Mycobacterium wolinskyi group and the M. smegmatis group, respectively comprising four, three, eight, one, one and two species. The two protein-encoding genes rpoB and recA improved meaningfully the bootstrap values at the nodes of the different groups. The species M. mucogenicum, M. mageritense and M. wolinskyi formed new groups separated from the M. chelonae-abscessus, M. fortuitum and M. smegmatis groups, respectively. The M. mucogenicum group was well delineated, in contrast to the M. mageritense and M. wolinskyi groups. For phylogenetic organizations derived from the hsp65 and sodA gene sequences, the bootstrap values at the nodes of a few clusters were <70 %. In contrast, phylogenetic organizations obtained from the 16S rRNA, rpoB and recA genes were globally similar to that inferred from combined datasets, indicating that the rpoB and recA genes appeared to be useful tools in addition to the 16S rRNA gene for the investigation of evolutionary relationships among RGM species. Moreover, rpoB gene sequence analysis yielded bootstrap values higher than those observed with recA and 16S rRNA genes. Also, molecular signatures in the rpoB and 16S rRNA genes of the M. mucogenicum group showed that it was a sister group of the M. chelonae-abscessus group. In this group, M. mucogenicum ATCC 49650(T) was clearly distinguished from M. mucogenicum ATCC 49649 with regard to analysis of the five gene sequences. This was in agreement with phenotypic and biochemical characteristics and suggested that these strains are representatives of two closely related, albeit distinct species.

In this study, we attempted species-level identification of frog specimen collected from Faridpur district of Bangladesh beyond it’s location outside Orissa, India. Specimen was identified morphologically at genus level as Fejervarya sp. belonging to the family Dicroglossidae using finger formula F3&gt;F4&gt;F1=F1 where F denote as toe finger. From the study two nucleotide sequences of 16S and 12S rRNA genes were obtained which contained 508bp and 408bp respectively. The Sequences were submitted to Gene Bank database with the accession number OQ231604 and OQ240197 for 16S and 12S rRNA gene sequences. Furthermore, the 16S rRNA gene sequence was used as molecular bar-code for the identified Orissa frog F. orissaensis species from Bangladesh. GC content of partial 12S and 16S rRNA genes have been calculated as 44% and 45% respectively. For 16S rRNA gene sequence there was no intra specific divergence. Whereas the inter specific polymorphic divergence were calculated 4.13% and 6.3% when the collected Orissa frog F. orissaensis was compared with that of F. iskandari and F. kupitzi, respectively. In case of 12S rRNA gene intra specific divergence was found 2.45% where the inter specific divergence were 4.41% and 6.86% when the collected Orissa frog F. orissaensis was compared with that of F. iskandari and F. limnocaris, respectively. Maximum likelihood tree also indicates that our sample Orissa frog formed a monophyletic group with F. orissaensis in both the cases of 16S and 12S rRNA genes and thus can be concluded as closely related. Therefore, the collected specimen was identified to be belonging to Fejervarya orissaensis which would be first report from Bangladesh outside Orissa, India. Bangladesh J. Zool. 51(3): 301-313, 2023

Traditional morphology-based ciliate classification is often time-consuming and inaccurate, necessitating molecular approaches. Although 18S rRNA gene sequencing is widely used for taxonomic analyses of ciliates, its high degree of conservation makes it challenging to achieve species-level resolution. This study explores the potential of internal transcribed spacers (ITS1 and ITS2) and the 28S rRNA gene to improve taxonomic resolution beyond that offered by 18S rRNA gene in free-living and host-associated ciliates. A comparative analysis of ITS, the 18S, and 28S rRNA gene sequences retrieved from public databases indicated that ITS regions exhibit greater inter- and intra-specific sequence dissimilarity compared to 18S rRNA gene, supporting existing literature. We then designed universal primers targeting the ITS and 28S rRNA gene for freshwater and rumen ciliates. These primers were rigorously evaluated for their inclusiveness, specificity, and amplification efficiency using in-silico PCR, experimental PCR, followed by sequencing and metataxonomic analyses of the ciliate communities. In-silico analyses revealed inclusiveness exceeding 80%, while experimental analyses validated their specificity. Metataxonomic analyses of ciliates demonstrated that the ITS and 28S rRNA gene captured significantly greater taxonomic diversity than 18S rRNA gene. Also, ITS1 offered superior taxonomic resolution by detecting the most ciliate species that went unnoticed by the 18S rRNA gene. These findings underscore the superiority of ITS1, and to a lesser extent ITS2, as taxonomic markers for enhancing the resolution of freshwater and rumen ciliate communities. We recommend ITS1 as an alternative marker to overcome the limitations of 18S rRNA gene-based approaches in free-living and host-associated ciliate taxonomy.

ABSTRACTPropionibacterium acnes constitutes a major part of the skin microbiome and contributes to human health. However, it has also been implicated as a pathogenic factor in several diseases, including acne, one of the most common skin diseases. Its pathogenic role, however, remains elusive. To better understand the genetic landscape and diversity of the organism and its role in human health and disease, we performed a comparative genome analysis of 82 P. acnes strains, 69 of which were sequenced by our group. This collection covers all known P. acnes lineages, including types IA, IB, II, and III. Our analysis demonstrated that although the P. acnes pan-genome is open, it is relatively small and expands slowly. The core regions, shared by all the sequenced genomes, accounted for 88% of the average genome. Comparative genome analysis showed that within each lineage, the strains isolated from the same individuals were more closely related than the ones isolated from different individuals, suggesting that clonal expansions occurred within each individual microbiome. We also identified the genetic elements specific to each lineage. Differences in harboring these elements may explain the phenotypic and functional differences of P. acnes in functioning as a commensal in healthy skin and as a pathogen in diseases. Our findings of the differences among P. acnes strains at the genome level underscore the importance of identifying the human microbiome variations at the strain level in understanding its association with diseases and provide insight into novel and personalized therapeutic approaches for P. acnes-related diseases.

Genomic analysis has revealed heterogeneity among bacterial 16S rRNA gene sequences within a single species; yet the cause(s) remains uncertain. Generalized transducing bacteriophages have recently gained recognition for their abundance as well as their ability to affect lateral gene transfer and to harbor bacterial 16S rRNA gene sequences. Here, we demonstrate the ability of broad-host-range, generalized transducing phages to acquire 16S rRNA genes and gene sequences. Using PCR and primers specific to conserved regions of the 16S rRNA gene, we have found that generalized transducing phages (D3112, UT1, and SN-T), but not specialized transducing phages (D3), acquired entire bacterial 16S rRNA genes. Furthermore, we show that the broad-host-range, generalized transducing phage SN-T is capable of acquiring the 16S rRNA gene from two different genera: Sphaerotilus natans, the host from which SN-T was originally isolated, and Pseudomonas aeruginosa. In sequential infections, SN-T harbored only 16S rRNA gene sequences of the final host as determined by restriction fragment length polymorphism analysis. The frequency of 16S rRNA gene sequences in SN-T populations was determined to be 1 x 10(-9) transductants/PFU. Our findings further implicate transduction in the horizontal transfer of 16S rRNA genes between different species or genera of bacteria.

Phylogenetic analyses of the genus Glaciecola were performed using the sequences of the 16S rRNA gene and the GyrB protein to establish its taxonomic status. The results indicated a consistent clustering of the genus Glaciecola into two clades, with significant bootstrap values, with all the phylogenetic methods employed. Clade 1 was represented by seven species, Glaciecola agarilytica, G. aquimarina, G. arctica, G. chathamensis, G. mesophila, G. polaris and G. psychrophila, while clade 2 consisted of only three species, Glaciecola nitratireducens, G. pallidula and G. punicea. Evolutionary distances between species of clades 1 and 2, based on 16S rRNA gene and GyrB protein sequences, ranged from 93.0 to 95.0 % and 69.0 to 73.0 %, respectively. In addition, clades 1 and 2 possessed 18 unique signature nucleotides, at positions 132, 184 : 193, 185 : 192, 230, 616 : 624, 631, 632, 633, 738, 829, 1257, 1265, 1281, 1356 and 1366, in the 16S rRNA gene sequence and can be differentiated by the occurrence of a 15 nt signature motif 5'-CAAATCAGAATGTTG at positions 1354-1368 in members of clade 2. Robust clustering of the genus Glaciecola into two clades based on analysis of 16S rRNA gene and GyrB protein sequences, 16S rRNA gene sequence similarity of ≤95.0 % and the occurrence of signature nucleotides and signature motifs in the 16S rRNA gene suggested that the genus should be split into two genera. The genus Paraglaciecola gen. nov. is therefore created to accommodate the seven species of clade 1, while the name Glaciecola sensu stricto is retained to represent species of clade 2. The species of clade 1 are transferred to the genus Paraglaciecola as Paraglaciecola mesophila comb. nov. (type strain DSM 15026(T) = KMM 241(T)), P. agarilytica comb. nov. (type strain NO2(T) = KCTC 12755(T) = LMG 23762(T)), P. aquimarina comb. nov. (type strain GGW-M5(T) = KCTC 32108(T) = CCUG 62918(T)), P. arctica comb. nov. (type strain BSs20135(T) = CCTCC AB 209161(T) = KACC 14537(T)), P. chathamensis comb. nov. (type strain E3(T) = CGMCC 1.7001(T) = JCM 15139(T)), P. polaris comb. nov. (type strain ARK 150(T) = CIP 108324(T) = LMG 21857(T)) and P. psychrophila comb. nov. (type strain 170(T) = CGMCC1.6130(T) = JCM 13954(T)). The type species of the genus Paraglaciecola is Paraglaciecola mesophila. An emended description of the genus Glaciecola is provided. In addition, a novel strain, 162Z-12(T), was isolated from seawater collected as part of an iron fertilization experiment (LOHAFEX) conducted in the Southern Ocean in 2009 and was subjected to polyphasic taxonomic characterization. Cells of 162Z-12(T) were Gram-negative, aerobic, motile, ovoid to short rod-shaped, obligatorily halophilic and possessed all the characteristics of the genus Paraglaciecola. Strain 162Z-12(T) shared the highest 16S rRNA gene sequence similarity with the type strains of P. agarilytica (99.7 %), P. chathamensis (99.7 %), P. mesophila (98.5 %) and P. polaris (98.3 %). However, it exhibited DNA-DNA relatedness of less than 70.0 % with its nearest phylogenetic relatives, well below the threshold value for species delineation. Further, strain 162Z-12(T) differed from the nearest species in several phenotypic characteristics, in addition to the occurrence of unique nucleotides G, T, T and T at positions 1194, 1269, 1270 and 1271 of the 16S rRNA gene. Based on the cumulative differences it exhibited from its nearest phylogenetic neighbours, strain 162Z-12(T) was identified as a novel member of the genus Paraglaciecola and assigned to the novel species Paraglaciecola oceanifecundans sp. nov. The type strain of Paraglaciecola oceanifecundans is 162Z-12(T) ( = KCTC 32337(T) = LMG 27453(T)).

create a niche for M. tuberculosis

Evidence suggests that there is a rich and diverse microbial community in the semen of mammals, which may be important in reproductive health and fertility. However, the composition of ram seminal microbiota remains under-characterized, with factors shaping it still largely unknown. The objectives of this study were to; 1) characterize the seminal microbiota of mature rams and their ram lambs using 16S rRNA gene sequencing; 2) evaluate whether managing the mature rams on divergent planes of nutrition can influence their seminal microbiota and that of their male offspring; and 3) compare the seminal microbiota between mature rams and ram lamb cohorts to identify age-related microbiota characteristics. For this, mature rams (n = 24) were assigned to one of the 3 nutritional planes: 1) Positive (POS), to gain 12% of initial body weight (BW) (n = 8), 2) maintenance (MAINT), to maintain BW (n = 8), and 3) negative (NEG), to lose 12% BW (n = 8) over an 84-d period. Semen samples were collected from the mature rams (F0) after 28-d, 56-d, and 84-d from the start of the trial. Following the 84-d period, the 24 rams were used to breed 240 mature ewes over 28-d. After lambing, the ram lambs (F1) sired by POS, MAINT, and NEG rams were maintained on the same diet until 11 months of age, at which semen samples were collected. Genomic DNA was extracted from the semen, and the microbiota was analyzed using 16S rRNA gene (V3-V4) sequencing. Overall, there was a relatively diverse and dynamic bacterial microbial community in the ram semen, mainly dominated by Actinobacteriota, Bacillota, Bacteroidota, and Proteobacteria phyla. The predominant genera identified included Fastidiosipila, Corynebacterium, Trueperella, Arthrobacter, Dietzia, and Bifidobacterium. The seminal microbial community structure, composition, and alpha diversity of F0 rams was influenced by diet during the first 28-d, but these diet influences later diminished. The paternal plane of nutrition did not influence the seminal microbiota of offspring ram lambs. The mature rams and ram lambs had distinct seminal microbiota, with young rams showing greater microbial richness and diversity (P < 0.005). Our results suggest that there is a relatively diverse and dynamic microbial community present in the semen of both mature rams and ram lambs, and that this microbiota is transiently influenced by diet and age. Managing rams on divergent planes of nutrition may not affect their offspring's seminal microbiota.