HIV-1 currently comprises four lineages: group M, for major; group N, for non-M non-O; group O, for outlier; and group P, named to continue in alphabetical order. Group M is by far the most prevalent lineage and the only pandemic strain, believed to have originated in Kinshasa, the capital of the DRC, with the emergence of HIV-1 estimated to have occurred around the 1920s in the DRC. Within group M, ten subtypes have been delineated, labeled A through D, F through H, and K through L, along with nine sub-subtypes such as A1 through A8 and F1 and F2. These subtypes are thought to have diverged from one or a few ancestral HIV-1 strains after a single chimpanzee-to-human transmission, some of them around the 1950s. The most recently recognized subtype, L, was formally established in 2020 on the basis of complete genome sequences identified in the DRC between 1983 and 2001.

The designation of a new HIV-1 subtype is governed by strict criteria. Researchers must identify at least three viral strains or complete genomes from distinct, epidemiologically unlinked individuals. Phylogenetically, each subtype should form a distinct monophyletic clade, with approximately equal genetic distances separating it from the other subtypes, producing a characteristic star-like phylogeny. Established group M subtypes differ by a mean nucleotide divergence of approximately 15 percent at the genome level, with considerable variation between genes. In addition, genomes classified within a given subtype must be non-mosaic, distinguishing them from recombinant genomes that arise when viruses of different subtypes exchange genetic material inside coinfected cells.

The new report describes an HIV-1 genome recovered from residues of peripheral blood mononuclear cells from a single blood sample collected during the summer of 2023. The team extracted viral DNA using a commercial kit and amplified the genome with conventional polymerase chain reaction primers as well as a whole-genome HIV-1 genotyping assay. Sequencing was performed using three complementary platforms: Sanger sequencing, Oxford Nanopore technology on a GridION instrument, and Illumina technology on a MiSeq instrument. The resulting near-full-length consensus genome, deposited in GenBank under accession number PZ307881, was 8,950 nucleotides long and covered 92 percent of the subtype B reference genome, with the shortfall mostly due to sequencing defects at the 5-prime and 3-prime ends.

Comparative genomic analysis revealed that the ten best matches in the GenBank database exhibited nucleotide similarities of only 83.29 to 83.60 percent to the new sequence. These closest genomes had been collected from patients in the DRC, the United States, the Netherlands, and Gabon between 1987 and 2018. When aligned against representative genomes of the ten established subtypes, the new genome showed a mean nucleotide divergence of 12.69 percent, ranging between 11.87 and 13.28 percent, while the ten established subtypes diverged from one another by a mean of 12.64 percent, ranging between 10.01 and 13.73 percent. In other words, the candidate subtype is as distant from every known subtype as those subtypes are from each other, and pairwise comparisons showed no statistically significant differences in divergence. Similar results held when the genomes were cut into eight fragments corresponding to different genomic regions, with regional divergences for the candidate genome ranging from 8.55 to 20.23 percent, closely mirroring the 8.49 to 20.25 percent range observed among established subtypes.

Phylogenetic analyses reinforced this picture. Using IQ-TREE and Nextclade software, with a simian immunodeficiency virus genome from Gabon as an outgroup, the researchers found that the candidate genome formed a distinct branch within an overall star-like tree, the hallmark topology of genuine subtypes. The closest established subtype was J, supported by a bootstrap value of 99 percent. Independent trees built from each of the eight genome fragments likewise showed star-like topologies with the candidate sequence on its own branch. Bootscan analysis, which uses a sliding window of 200 nucleotides to detect recombination, found no recombination events, satisfying another key criterion for subtype status. Nextclade classified the genome as an other clade, and a tree incorporating 1,020 genomes from the Los Alamos National Laboratory HIV Sequence Database placed it on a new node. Taken together, the researchers concluded that the genome belongs to a candidate new subtype, which they named M following subtype L in alphabetical order, while acknowledging the potential nomenclatural ambiguity of reusing the letter that already designates the pandemic group.

The clinical course of the patient offers an intriguing backdrop to the genomic findings. At the start of antiretroviral treatment, her CD4-positive T lymphocyte count was 351 cells per cubic millimeter with a CD4-to-CD8 ratio of 0.23, and plasma HIV-1 RNA stood at 5.03 log10 copies per milliliter. Subsequently, her CD4 count fluctuated between 406 cells per cubic millimeter in 1999 and 2,394 cells per cubic millimeter in 2008, and has remained above 1,000 since 2007. Plasma viral load has been undetectable during regular monitoring from 2008 onward, except for a reading of 2.2 log10 copies per milliliter in 2024. Notably, a Western blot performed in 2023 showed antibody reactivities only to envelope and capsid proteins, with no response to Pol proteins. The authors suggest this unusual profile may partly reflect reduced antibody affinity for antigenically divergent proteins, or an overall weak antibody response to HIV-1 antigens, which particularly affects Pol-encoded proteins such as reverse transcriptase, protease, and integrase, especially after more than three decades of infection and long-term antiretroviral therapy.

Genotypic resistance analysis using algorithms from the French National Agency for AIDS Research and the Stanford University HIV drug resistance database found no naturally present amino acid mutations conferring resistance to any currently available antiretroviral drug, apart from two substitutions acquired during treatment failures: Y181C in reverse transcriptase, detected transiently in 2003 during a virological failure episode on nevirapine, and I84V in protease, detected in 2007 during failure on atazanavir and ritonavir. Viral tropism was predicted to be R5, indicating use of the CCR5 co-receptor. The researchers cautioned that the absence of natural resistance mutations should be interpreted carefully given the markedly different genetic background of this genome, though the finding is congruent with observations for both rare and prevalent subtypes.

The team also turned to molecular modeling to explore how the roughly 40 amino acid differences in the candidate subtype’s reverse transcriptase, the central enzyme of HIV replication and a prime drug target, might alter its structure. Mapping the mutations onto the reference enzyme structure revealed changes predicted to reshape several surface loops, including a closure of the F130 to P150 buckle and a flattening of the K281 to P294 loop. Alterations in the N57 to D76 region, corresponding to the functionally important beta3-beta4 loop within the fingers subdomain, were associated with a greater space between the fingers and palm subdomains. The surface electrostatic potential was modified but remained globally electropositive, particularly in the ligand-interacting area. The modeled enzyme-ligand interaction energy decreased slightly, from minus 646 to minus 512 kilojoules per mole, because the number of amino acids involved in ligand interaction fell from 15 to 7. The authors speculate that such changes could modulate enzyme processivity and, by promoting dissociation and re-association of the enzyme-substrate complex, potentially increase template switching and genetic recombination, though they stress these hypotheses derive entirely from in silico modeling and require in vitro biochemical validation.

The discovery underscores that HIV-1 diversity remains far from fully charted and reinforces the status of the DRC as the epicenter of the group M pandemic and a region where rare subtypes continue to circulate. Rare established subtypes, including F, H, J, K, and L, each account for less than 1 percent of global HIV-1 infections and circulate primarily in the DRC, Angola, and Cameroon, with limited data on their transmissibility, virulence, and pathogenicity. The researchers also observed notable intra-sample diversity, with mean intra-host nucleotide divergence of 1.61 percent and a maximum of 5.52 percent, suggesting intra-patient viral evolution or, less likely given the candidate subtype’s apparent rarity, superinfection with multiple variants during the patient’s decades of stays in the DRC. Formal recognition of the new subtype will require detecting it in at least two more epidemiologically unlinked individuals, and the authors call for further studies, primarily in the DRC, to establish its prevalence and genotypic and phenotypic characteristics.

Subject of Research: Discovery of a candidate new HIV-1 group M subtype in a patient from the Democratic Republic of the Congo

Article Title: A case of infection with a candidate new subtype, tentatively termed M, of human immunodeficiency virus type 1 of group M

Article References: COLSON, P., BOSCHI, C., BEDOTTO, M., MOTTE, A., SOLAS, C., LEFEBVRE, M., MILLION, M., TOMEI, C., DELERCE, J., & FANTINI, J. (2026). A case of infection with a candidate new subtype, tentatively termed M, of human immunodeficiency virus type 1 of group M. New Microbes and New Infections, Article 101863. https://doi.org/10.1016/j.nmni.2026.101863

Image Credits: AI Generated

DOI: 10.1016/j.nmni.2026.101863

Keywords: HIV-1, subtype M, Democratic Republic of the Congo, viral phylogenetics, whole-genome sequencing, reverse transcriptase, group M, subtype L, molecular modeling, antiretroviral resistance, HIV diversity, New Microbes and New Infections

The genus Pseudomonas is one of the most versatile groups of bacteria known to science. First defined in 1894 by the German mycologist Walter Migula, it comprises Gram-negative, aerobic, rod-shaped bacteria equipped with flagella. Over more than a century of microbiological discovery, the genus has grown to encompass 381 validly published species, according to the List of Prokaryotic names with Standing in Nomenclature, with Pseudomonas aeruginosa serving as the type species. Members of the genus colonize soil, rivers, plants and animal hosts, and their roles range from promoting plant growth and cleaning contaminated environments to causing serious opportunistic infections in vulnerable patients. Among the many niches these bacteria occupy is human milk, a biological fluid whose microbial community helps seed the newborn gut and shapes early immune and metabolic development.

Despite the growing interest in the milk microbiome, African populations remain markedly under-studied, and most surveys of milk bacteria rely on culture-independent methods that detect genetic signatures without ever yielding a living isolate. Motivated by this gap, the research team turned to a culture-based, genome-resolved strategy. Breast milk samples were collected from mothers of both malnourished and healthy children in Niakhar, Senegal, with informed consent and approval from the Ethical and Scientific Committee of the Senegalese Ministry of Health and Public Hygiene. Ten to fifteen milliliters of milk from each participant were transported at four degrees Celsius to a laboratory in Dakar, aliquoted, frozen at minus eighty degrees Celsius, and later shipped on dry ice to Marseille for culturing, sequencing and analysis.

Two strains, designated Marseille-QA0332 and Marseille-QA0892, emerged from this effort. The first was obtained by direct plating of milk onto Columbia blood agar during routine colony counting, before any enrichment step. The second was recovered through a culturomics protocol, in which milk was inoculated into a series of liquid enrichment media and subcultured at scheduled intervals over thirty days; strain QA0892 appeared at the day-fifteen subculture from an aerobic blood-culture bottle containing MacConkey broth. Rigorous sterility controls, in which every batch of medium was tested for contamination before use, ensured that any bacterial growth traced back to the milk samples themselves rather than to laboratory reagents.

Initial identification attempts using MALDI-TOF mass spectrometry failed to match either isolate to a known species, prompting whole-genome sequencing. Comparison of 16S ribosomal RNA gene sequences showed that strain QA0332 shared its highest similarity, 97.98 percent, with Pseudomonas ceruminis, while strain QA0892 was 99 percent similar to Pseudomonas matsuisoli. Those figures alone might have suggested close relatives, but whole-genome comparisons told a different story. Average nucleotide identity values, which should exceed roughly 95 to 96 percent between strains of the same species, came in at just 81.1 to 81.4 percent for QA0332 against its nearest neighbors and 75.0 percent for QA0892 against P. matsuisoli. Digital DNA-DNA hybridization values, with a species threshold of 70 percent, were even lower, at 26.7 percent and 21.1 percent respectively. By every genomic yardstick, the two isolates represented lineages distinct from any previously described species.

The phenotypic portraits of the two bacteria are strikingly different. Pseudomonas senegalensis grows optimally at 37 degrees Celsius under aerobic conditions and forms circular, convex, yellowish, mucoid colonies. It is motile, non-spore-forming, oxidase- and catalase-positive, and capable of fermenting a broad range of sugars including glucose, xylose, arabinose and trehalose. It also produces urease and arginine dihydrolase, traits that help distinguish it from close relatives. Pseudomonas millioni, by contrast, prefers cooler temperatures, growing best at room temperature and 28 degrees Celsius in alkaline media, and its colonies mature from translucent punctiform dots into dry, filamentous, opaque mats. Uniquely among its comparisons, it reduces nitrate to nitrite and hydrolyzes esculin. Fatty acid profiling by gas chromatography further separated the two: the cell membranes of P. senegalensis are dominated by hexadecanoic acid and cis-9,10-methylene-hexadecanoic acid, while those of P. millioni feature a more balanced mixture of unsaturated and saturated fatty acids.

Genome architecture reinforced the case for two new species. Pseudomonas senegalensis carries a single circular chromosome of 4,577,173 base pairs with a GC content of 63.65 percent, encoding 4,173 genes, of which 4,079 are protein-coding. Pseudomonas millioni has a slightly larger chromosome of 4,658,126 base pairs, a GC content of 60.22 percent, and 4,327 genes. Neither genome contains plasmids, and functional annotation revealed repertoires dominated by amino acid transport, transcription, energy production and inorganic ion metabolism, consistent with metabolically versatile, free-living organisms. Mining of secondary metabolite biosynthetic gene clusters uncovered eleven candidate regions in P. senegalensis and eight in P. millioni, including a pseudomonine-like metallophore cluster in the former, suggesting a siderophore-based iron-scavenging system well suited to the iron-limited environment of milk. Both genomes also harbor carotenoid, hydrogen cyanide and N-acetylglutaminylglutamine amide clusters, although the researchers caution that these remain computational predictions rather than chemically validated products.

From a clinical standpoint, the news is reassuring. Screening against virulence factor databases found none of the hallmark weapons of Pseudomonas aeruginosa: no type III secretion system, no exotoxin A, no elastase or phospholipases, and no high-risk pathogenicity islands. No acquired resistance genes, such as extended-spectrum beta-lactamases or carbapenemases, were detected in either genome. The only resistance determinants identified were intrinsic chromosomal efflux pumps and regulators typical of the genus. One notable exception emerged in Pseudomonas millioni, which proved resistant to the antibiotic ceftazidime. A targeted search revealed a chromosomal class C beta-lactamase, bearing the three canonical catalytic motifs of Ambler class C enzymes, that accounts for this phenotype. Because the enzyme is intrinsic and not transmissible, the researchers conclude that both species pose a low biosafety risk, though they note that pathogenic potential cannot be formally excluded without infection models.

Perhaps the most intriguing evidence for the ecological identity of these bacteria comes from mining public metagenomic archives through the IMNGS platform. Both species were detected predominantly in human-associated ecosystems rather than environmental ones. Pseudomonas senegalensis appeared in 0.95 percent of human gut metagenomes and 0.69 percent of skin metagenomes, while Pseudomonas millioni was found in 1.53 percent of gut metagenomes and 0.50 percent of blood metagenomes. This pattern of low prevalence but consistent human association supports the idea that the two organisms are genuine members of the human microbiome rather than transient contaminants. The researchers argue that the combination of viable cultured isolates, verified sterile media and expected recovery of Pseudomonas from milk makes a laboratory origin for the strains implausible.

The discovery also carries a cautionary lesson about genome-based inference. Both genomes encode a carotenoid biosynthetic cluster, yet only Pseudomonas senegalensis forms visibly yellow colonies. This dissociation between genotype and phenotype demonstrates that the mere presence of a biosynthetic gene cluster does not guarantee its expression, tempering the functional conclusions that can be drawn from genome mining alone. With type strains deposited in the Collection de Souches de l’Unité des Rickettsies in Marseille and the Colección Española de Cultivos Tipo in Spain, and genome sequences available in GenBank, Pseudomonas senegalensis and Pseudomonas millioni now stand as formally recognized members of the bacterial tree of life, and as reminders of how much microbial diversity still hides in ordinary human fluids waiting to be cultured.

Subject of Research: Discovery and taxonomic description of two novel Pseudomonas species isolated from human breast milk in Senegal

Article Title: Pseudomonas senegalensis sp. nov. and Pseudomonas millioni sp. nov., two novel species isolated from the breast milk of Senegalese women

Article References: Sarr, S., Ndour, O., Seck, E. H., Abdoulaye, A. I., Beye, M., Diatta, G., Bassene, H., Diop, M., Bellali, S., Semmar, R., Alibar, S., Boukili, M., Kacel, I., Armstrong, N., Couderc, C., Thiakane, C., Alou, M. T., Sokhna, C., & Camara, M. (2026). Pseudomonas senegalensis sp. nov. and Pseudomonas millioni sp. nov., two novel species isolated from the breast milk of Senegalese women. New Microbes and New Infections, 73, Article 101846. https://doi.org/10.1016/j.nmni.2026.101846

Image Credits: AI Generated

DOI: 10.1016/j.nmni.2026.101846

Keywords: Pseudomonas senegalensis, Pseudomonas millioni, breast milk microbiota, new bacterial species, taxonogenomics, Senegal, culturomics, whole-genome sequencing, antimicrobial resistance, human microbiome, MALDI-TOF, New Microbes and New Infections