UNASSIGNED: as cholera, due to toxigenic bacteria Vibrio cholera (serogroups O1 and O139), is a major public health threat in Africa, the aim of this work was to investigate potentially pathogenic Vibrionaceae bacteria firstly from human stool samples, and secondly from various environmental water points of Saint-Louis city in Senegal.
UNASSIGNED: a hospital-based study was conducted between 2013 and 2015. Stool samples were taken and cultured from daily incoming patients or hospitalized for acute diarrhea at Saint-Louis´ regional hospital. For environment, a monthly longitudinal sampling from January to October 2016 was carried out at 10 sites in the city. We used total DNA extracted from APW (alkaline peptone water) broth solutions and on suspect bacterial colonies to run PCR Multiplex targeting specific DNA fragments to detect Vibrio genus and specific species. In case of positivity, a simplex PCR was performed to test for cholera toxins Ctx, and V. parahaemolyticus TRH and TDH.
UNASSIGNED: for 43 patients screened, bacterial culture was positive in 6% of cases but no strain of V. cholerae or other Vibrio sp. was isolated. PCR on 90 APW solutions were positive for Vibrio sp.(n = 43), V. cholera(n = 27), V. mimicus(n = 16), V. parahaemolyticus(8), V. alginolyticus(n = 4), and V. vulnificus(n = 2). Unlike for those on suspected colonies which were positive for a majority of V. parahaemolyticus (n = 40) and V. cholerae non-O1 / O139 (n = 35). Six strains of V. parahaemolyticus carried TRH gene, 3 of which expressed simultaneously virulence TRH and TDH genes. For physicochemical parameters, all temperatures varied similarly according to a unimodal seasonality, as well as salinity.
UNASSIGNED: despite the presence of natural populations of Vibrionaceae, even toxigenic ones, was noted in water environment, along with favorable habitat conditions that could play a role in transmission of Vibriosis in the Saint Louis population, we did not isolate any of them from patients screened at the hospital.

OBJECTIVE: The human oral microbiome, a complex ecosystem linked to oral and systemic health, harbors a diverse array of microbial populations, including antimicrobial resistance genes (ARGs). As a critical component of the One Health approach to tackle antibiotic resistance, comprehending the oral resistome\'s composition and diversity is imperative. The objective of this study was to investigate the impact of chemical cell lysis treatment using MetaPolyzyme on the detectability of the oral microbiome, resistome, and DNA quality and quantity.
METHODS: Saliva samples were collected from five healthy individuals, and each of the samples was subjected to DNA extraction with and without the treatment with MetaPolyzyme. Through metagenomic sequencing, we analyzed, assessed, and compared the microbial composition, resistome, and DNA characteristics between both groups of extracted DNA.
RESULTS: Our study revealed that MetaPolyzyme treatment led to significant shifts in the detectability of microbial composition, favoring Gram-positive bacteria, notably Streptococcus, over Gram-negative counterparts. Moreover, the MetaPolyzyme treatment also resulted in a distinct change in ARG distribution. This shift was characterized by an elevated proportion of ARGs linked to fluoroquinolones and efflux pumps, coupled with a reduction in the prevalence of tetracycline and β-lactam resistance genes when compared with the nontreated group. Alpha diversity analysis demonstrated altered species and ARG distribution without affecting overall diversity, while beta diversity analysis confirmed significant differences in the taxonomical composition and oral resistome between treated and nontreated groups.
CONCLUSIONS: These findings underscore the critical role of cell lysis treatment in optimizing oral metagenomic studies and enhance our understanding of the oral resistome\'s dynamics in the context of antimicrobial resistance.

Dental calculus is a microbial biofilm that contains biomolecules from oral commensals and pathogens, including those potentially related to cause of death (CoD). To assess the utility of calculus as a diagnostically informative substrate, in conjunction with paleopathological analysis, calculus samples from 39 individuals in the Smithsonian Institution\'s Robert J. Terry Collection with CoDs of either syphilis or tuberculosis were assessed via shotgun metagenomic sequencing for the presence of Treponema pallidum subsp. pallidum and Mycobacterium tuberculosis complex (MTBC) DNA. Paleopathological analysis revealed that frequencies of skeletal lesions associated with these diseases were partially inconsistent with diagnostic criteria. Although recovery of T. p. pallidum DNA from individuals with a syphilis CoD was elusive, MTBC DNA was identified in at least one individual with a tuberculosis CoD. The authenticity of MTBC DNA was confirmed using targeted quantitative PCR assays, MTBC genome enrichment, and in silico bioinformatic analyses; however, the lineage of the MTBC strain present could not be determined. Overall, our study highlights the utility of dental calculus for molecular detection of tuberculosis in the archaeological record and underscores the effect of museum preparation techniques and extensive handling on pathogen DNA preservation in skeletal collections.

Type I hypolithons are microbial communities dominated by Cyanobacteria. They adhere to the underside of semi-translucent rocks in desert pavements, providing them with a refuge from the harsh abiotic stresses found on the desert soil surface. Despite their crucial role in soil nutrient cycling, our understanding of their growth rates and community development pathways remains limited. This study aimed to quantify the dynamics of hypolithon formation in the pavements of the Namib Desert. We established replicate arrays of sterile rock tiles with varying light transmission in two areas of the Namib Desert, each with different annual precipitation regimes. These were sampled annually over 7 years, and the samples were analysed using eDNA extraction and 16S rRNA gene amplicon sequencing. Our findings revealed that in the zone with higher precipitation, hypolithon formation became evident in semi-translucent rocks 3 years after the arrays were set up. This coincided with a Cyanobacterial \'bloom\' in the adherent microbial community in the third year. In contrast, no visible hypolithon formation was observed at the array set up in the hyper-arid zone. This study provides the first quantitative evidence of the kinetics of hypolithon development in hot desert environments, suggesting that development rates are strongly influenced by precipitation regimes.

The human gut microbiota refers to a diverse community of microorganisms that symbiotically exist in the human intestinal system. Altered microbial communities have been linked to many human pathologies. However, there is a lack of rapid and efficient methods to assess gut microbiota signatures in practice. To address this, we established an appraisal system containing 45 quantitative real-time polymerase chain reaction (qPCR) assays targeting gut core microbes with high prevalence and/or abundance in the population. Through comparative genomic analysis, we selected novel species-specific genetic markers and primers for 31 of the 45 core microbes with no previously reported specific primers or whose primers needed improvement in specificity. We comprehensively evaluated the performance of the qPCR assays and demonstrated that they showed good sensitivity, selectivity, and quantitative linearity for each target. The limit of detection ranged from 0.1 to 1.0 pg/µL for the genomic DNA of these targets. We also demonstrated the high consistency (Pearson\'s r = 0.8688, P < 0.0001) between the qPCR method and metagenomics next-generation sequencing (mNGS) method in analyzing the abundance of selected bacteria in 22 human fecal samples. Moreover, we quantified the dynamic changes (over 8 weeks) of these core microbes in 14 individuals using qPCR, and considerable stability was demonstrated in most participants, albeit with significant individual differences. Overall, this study enables the simple and rapid quantification of 45 core microbes in the human gut, providing a promising tool to understand the role of gut core microbiota in human health and disease. KEY POINTS: • A panel of original qPCR assays was developed to quantify human gut core microbes. • The qPCR assays were evaluated and compared with mNGS using real fecal samples. • This method was used to dynamically profile the gut core microbiota in individuals.

Pleural empyema is a serious complication of pneumonia in children. Negative bacterial cultures commonly impede optimal antibiotic therapy. To improve bacterial identification, we developed a molecular assay and evaluated its performance compared with bacterial culture. Our multiplex-quantitative PCR to detect Streptococcus pneumoniae, Streptococcus pyogenes, Staphylococcus aureus and Haemophilus influenzae was assessed using bacterial genomic DNA and laboratory-prepared samples (n = 267). To evaluate clinical performance, we conducted the Molecular Assessment of Thoracic Empyema (MATE) observational study, enrolling children hospitalised with empyema. Pleural fluids were tested by bacterial culture and multiplex-qPCR, and performance determined using a study gold standard. We determined clinical sensitivity and time-to-organism-identification to assess the potential of the multiplex-qPCR to reduce the duration of empiric untargeted antibiotic therapy. Using spiked samples, the multiplex-qPCR demonstrated 213/215 (99.1%) sensitivity and 52/52 (100%) specificity for all organisms. During May 2019-March 2023, 100 children were enrolled in the MATE study; median age was 3.9 years (IQR 2-5.6). A bacterial pathogen was identified in 90/100 (90%) specimens by multiplex-qPCR, and 24/100 (24%) by bacterial culture (P <0.001). Multiplex-qPCR identified a bacterial cause in 68/76 (90%) culture-negative specimens. S. pneumoniae was the most common pathogen, identified in 67/100 (67%) specimens. We estimate our multiplex-qPCR would have reduced the duration of untargeted antibiotic therapy in 61% of cases by a median 20 days (IQR 17.5-23, range 1-55). Multiplex-qPCR significantly increased pathogen detection compared with culture and may allow for reducing the duration of untargeted antibiotic therapy.

Staphylococcus aureus is a major causative pathogen of osteomyelitis. Intracellular infections of resident bone cells including osteocytes can persist despite gold-standard clinical intervention. The mechanisms by which intracellular S. aureus evades antibiotic therapy are unknown. In this study, we utilised an in vitro S. aureus infection model of human osteocytes to investigate whether antibiotic-mediated dysregulation of autophagy contributes to this phenomenon. Infected or non-infected osteocyte-like cells were exposed to combinations of rifampicin, vancomycin, and modulators of autophagy. Intracellular bacterial growth characteristics were assessed using colony-forming unit (CFU) analysis, viable bacterial DNA abundance, and the rate of escape into antibiotic-free medium, together with measures of autophagic flux. Rifampicin, alone or in combination with vancomycin, caused a rapid decrease in the culturability of intracellular bacteria, concomitant with stable or increased absolute bacterial DNA levels. Both antibiotics significantly inhibited autophagic flux. However, modulation of autophagic flux did not affect viable bacterial DNA levels. In summary, autophagy was shown to be a factor in the host-pathogen relationship in this model, as its modulation affected the growth state of intracellular S. aureus with respect to both their culturability and propensity to escape the intracellular niche. While rifampicin and vancomycin treatments moderately suppressed autophagic flux acutely, this did not explain the paradoxical response of antibiotic treatment in decreasing S. aureus culturability whilst failing to clear bacterial DNA and hence intracellular bacterial load. Thus, off-target effects of rifampicin and vancomycin on autophagic flux in osteocyte-like cells could not explain the persistent S. aureus infection in these cells.

Nucleic acid amplification testing has great potential for point-of-need diagnostic testing with high detection sensitivity and specificity. Current sample preparation is limited by a tedious workflow requiring multiple steps, reagents and instrumentation, hampering nucleic acid testing at point of need. In this study, we present the use of mixed cellulose ester (MCE) paper for DNA binding by ionic interaction under molecular crowding conditions and fluid transport by wicking. The poly(ethylene) glycol-based (PEG) reagent simultaneously provides the high pH for alkaline lysis and crowding effects for ionic binding of the DNA under high salt conditions. In this study, we introduce Paper-based Abridged Solid-Phase Extraction with Alkaline Poly(ethylene) Glycol Lysis (PASAP). The anionic mixed cellulose ester (MCE) paper is used as solid phase and allows for fluid transport by wicking, eliminating the need for pipetting skills and the use of a magnet to retain beads. Following the release of DNA from the cells due to the lytic activity of the PASAP solution, the DNA binds to the anionic surface of the MCE paper, concentrating at the bottom while the sample matrix is transported towards the top by wicking. The paper was washed by dipping it in 40% isopropanol for 10 s. After air-drying for 30 s, the bottom section of the paper (3 mm × 4 mm) was snapped off using the cap of a PCR tube and immersed in the colourimetric loop-mediated isothermal amplification (cLAMP) solution for direct amplification and colourimetric detection. The total sample processing was completed in 15 min and ready for amplification. cLAMP enabled the detection of 102 CFU/mL of Escherichia coli (E. coli) from culture media and the detection of E. coli in milk < 103 CFU/mL (10 CFU) after incubation at 68 °C for 60 min, demonstrating applicability of the method to complex biological samples.

One Gram-negative, rod-shaped bacterial strain, isolated from an undescribed Heterorhabditis entomopathogenic nematode species was characterized to determine its taxonomic position. The 16S rRNA gene sequences indicate that it belongs to the class Gammaproteobacteria, to the family Morganellaceae, to the genus Photorhabdus, and likely represents a novel bacterial species. This strain, designated here as CRI-LCT, was therefore molecularly, biochemically, and morphologically characterized to describe the novel bacterial species. Phylogenetic reconstructions using 16S rRNA gene sequences show that CRI-LCT is closely related to P. laumondii subsp. laumondii TT01T and to P. laumondii subsp. clarkei BOJ-47T. The 16rRNA gene sequences between CRI-LCT and P. laumondii subsp. laumondii TT01T are 99.1% identical, and between CRI-LCT and P. laumondii subsp. clarkei BOJ-47T are 99.2% identical. Phylogenetic reconstructions using whole genome sequences show that CRI-LCT is closely related to P. laumondii subsp. laumondii TT01T and to P. laumondii subsp. clarkei BOJ-47T. Moreover, digital DNA-DNA hybridization (dDDH) values between CRI-LCT and its two relative species P. laumondii subsp. laumondii TT01T and P. laumondii subsp. clarkei BOJ-47T are 65% and 63%, respectively. In addition, we observed that average nucleotide identity (ANI) values between CRI-LCT and its two relative species P. laumondii subsp. laumondii TT01T and P. laumondii subsp. clarkei BOJ-47T are 95.8% and 95.5%, respectively. These values are below the 70% dDDH and the 95-96% ANI divergence thresholds that delimits prokaryotic species. Based on these genomic divergence values, and the phylogenomic separation, we conclude that CRI-LCT represents a novel bacterial species, for which we propose the name Photorhabdus africana sp. nov. with CRI-LCT (= CCM 9390T = CCOS 2112T) as the type strain. The following biochemical tests allow to differentiate P. africana sp. nov. CRI-LCT from other species of the genus, including its more closely related taxa: β-Galactosidase, citrate utilization, urease and tryptophan deaminase activities, indole and acetoin production, and glucose and inositol oxidation. Our study contributes to a better understanding of the taxonomy and biodiversity of this important bacterial group with great biotechnological and agricultural potential.

UNASSIGNED: Stenotrophomonas is a prominent genus owing to its dual nature. Species of this genus have many applications in industry and agriculture as plant growth-promoting rhizobacteria and microbial biological control agents, whereas species such as Stenotrophomonas maltophilia are considered one of the leading gram-negative multi-drug-resistant bacterial pathogens because of their high contribution to the increase in crude mortality and significant clinical challenge. Pathogenic Stenotrophomonas species and most clinical isolates belong to the Stenotrophomonas maltophilia complex (SMc). However, a strain highly homologous to S. terrae was isolated from a patient with pulmonary tuberculosis (TB), which aroused our interest, as S. terrae belongs to a relatively distant clade from SMc and there have been no human association reports.
UNASSIGNED: The pathogenicity, immunological and biochemical characteristics of 610A2T were systematically evaluated.
UNASSIGNED: 610A2T is a new species of genus Stenotrophomonas, which is named as Stenotrophomonas pigmentata sp. nov. for its obvious brown water-soluble pigment. 610A2T is pathogenic and caused significant weight loss, pulmonary congestion, and blood transmission in mice because it has multiple virulence factors, haemolysis, and strong biofilm formation abilities. In addition, the cytokine response induced by this strain was similar to that observed in patients with TB, and the strain was resistant to half of the anti-TB drugs.
UNASSIGNED: The pathogenicity of 610A2T may not be weaker than that of S. maltophilia. Its isolation extended the opportunistic pathogenic species to all 3 major clades of the genus Stenotrophomonas, indicating that the clinical importance of species of Stenotrophomonas other than S. maltophilia and potential risks to biological safety associated with the use of Stenotrophomonas require more attention.