Acanthamoeba species are clinically relevant free-living amoebae (FLA) ubiquitously found in soil and water bodies. Metabolically active trophozoites graze on diverse microbes via phagocytosis. However, functional studies on Rab GTPases (Rabs), which are critical for controlling vesicle trafficking and maturation, are scarce for this FLA. This knowledge gap can be partly explained by the limited genetic tools available for Acanthamoeba cell biology. Here, we developed plasmids to generate fusions of A. castellanii strain Neff proteins to the N- or C-termini of mEGFP and mCherry2. Phylogenomic and structural analyses of the 11 Neff Rab7 paralogs found in the RefSeq assembly revealed that eight of them had non-canonical sequences. After correcting the gene annotation for the Rab7A ortholog, we generated a line stably expressing an mEGFP-Rab7A fusion, demonstrating its correct localization to acidified macropinocytic and phagocytic vacuoles using fluorescence microscopy live cell imaging (LCI). Direct labeling of live Stenotrophomonas maltophilia ESTM1D_MKCAZ16_6a (Sm18) cells with pHrodo Red, a pH-sensitive dye, demonstrated that they reside within acidified, Rab7A-positive vacuoles. We constructed new mini-Tn7 delivery plasmids and tagged Sm18 with constitutively expressed mScarlet-I. Co-culture experiments of Neff trophozoites with Sm18::mTn7TC1_Pc_mScarlet-I, coupled with LCI and microplate reader assays, demonstrated that Sm18 underwent multiple replication rounds before reaching the extracellular medium via non-lytic exocytosis. We conclude that S. maltophilia belongs to the class of bacteria that can use amoeba as an intracellular replication niche within a Stenotrophomonas -containing vacuole that interacts extensively with the endocytic pathway. IMPORTANCE: Diverse Acanthamoeba lineages (genotypes) are of increasing clinical concern, mainly causing amoebic keratitis and granulomatous amebic encephalitis among other infections. S. maltophilia ranks among the top 10 most prevalent multidrug-resistant opportunistic nosocomial pathogens and is a recurrent member of the microbiome hosted by Acanthamoeba and other free-living amoebae. However, little is known about the molecular strategies deployed by Stenotrophomonas for an intracellular lifestyle in amoebae and other professional phagocytes such as macrophages, which allow the bacterium to evade the immune system and the action of antibiotics. Our plasmids and easy-to-use microtiter plate co-culture assays should facilitate investigations into the cellular microbiology of Acanthamoeba interactions with Stenotrophomonas and other opportunistic pathogens, which may ultimately lead to the discovery of new molecular targets and antimicrobial therapies to combat difficult-to-treat infections caused by these ubiquitous microbes.

Industrial residues with high concentrations of hexavalent chromium [Cr(VI)], characterized by an alkaline pH (between 9 and 13) and high salinity (around 100 psu), were used as a source for extremophilic chromium-resistant and -reducing microorganisms. An investigation of biodiversity through MiSeq showed the presence of 20 bacterial classes, with Bacilli (47%), Negativicutes (15%), Bacteriodia (8%), Gammaproteobacteria (7%) and Clostridia (5%) being the most abundant. The bioprospection allowed the cultivation of 87 heterotrophic bacterial colonies and 17 bacterial isolates at the end of the isolation, and screening procedures were obtained. The isolates were related to Cellulosimicrobium aquatile, C. funkei, Acinetobacter radioresistens, Staphylococcus equorum, S. epidermis, Brachybacterium paraconglometratum, Glutamicibacter creatinolyticus, Pseudomonas songnenensis, Microbacterium algeriense and Pantoea eucalypti, most of them being resistant to Cr(VI). Resistances of up to 400 ${\mathrm{m}\mathrm{g}.\mathrm{L}}^{-1}$ of chromate were obtained for four related strains (QReMLB55A, QRePRA55, QReMLB33A and QReMLB44C). The C. aquatile strain QReMLB55A and the P. songnenensis strain QReMLB33A were exposed to ${\mathrm{K}}_2{\mathrm{C}\mathrm{r}}_2{\mathrm{O}}_7$ (200 ${\mathrm{m}\mathrm{g}.\mathrm{L}}^{-1}$) under optimal conditions, diminishing 94% and 24% of the Cr(VI) in 6 days, respectively. These strains exhibited a high potential for chromium remediation biotechnologies.

Stenotrophomonas maltophilia ESTM1D_MKCAZ16_6a (Sm18) is a multidrug-resistant strain isolated from river sediments in Morelos, Mexico. It is capable of replicating within acid Rab7A-positive Acanthamoeba castellanii phagosomes. The complete genome was assembled from Oxford Nanopore and paired-end Illumina reads, encoding 3,970 proteins and 91 RNAs on a single circular 4.48 Mbp chromosome.

Manganese homeostasis is essential for the environmental adaptability and pathogenic potential of Stenotrophomonas maltophilia , a bacterium that thrives across diverse and fluctuating niches. Here, we characterize the manganese homeostasis network of S. maltophilia strain Sm18, revealing a coordinated system that integrates conserved transporters with previously unrecognized components. Central to this system is an MntR-controlled miniregulon that includes the canonical Mn²⁺ importer MntH and exporter MntP, together with a TonB-dependent receptor (TBDR) and a periplasmic thioredoxin-fold protein (pTFP), both defining novel protein families with restricted phylogenetic distribution. Transcriptomic analyses under varying Mn²⁺ and Fe²⁺ conditions uncovered a tight interplay between these metals, highlighting the ferrophilic nature of S. maltophilia and the differential regulation of miniregulon components. Notably, the TBDR-pTFP module is strongly induced under combined Mn ²⁺ and Fe ²⁺ limitation, suggesting a specialized role in metal acquisition under nutrient-restricted conditions. Functional analyses demonstrated that MntP is required to prevent Mn toxicity even at sub-inhibitory concentrations, whereas MntH supports growth under oxidative stress and promotes intracellular replication within Acanthamoeba castellanii phagosomes. Together, these findings define a previously unrecognized Mn-responsive module that expands the MntR regulatory network and provides new insight into the mechanisms that enable S. maltophilia to adapt to metal-limited and host-associated environments.

• The S. maltophilia MntH importer contributes to H₂O₂ resistance and intracellular survival. • The Mn exporter MntP limits toxicity even at sub-inhibitory Mn²⁺ concentrations. • The S. maltophilia MntR regulated genes include the extracytoplasmic TBDR-pTFP module. • The TBDR-pTFP locus is specifically induced under combined Mn²⁺ and Fe²⁺ limitation. • S. maltophilia Mn homeostasis is linked to iron availability and oxidative stress responses.

Guanajuato town is an important touristic center of Mexico, and one of its main attractions is its tunnel network (subterranean streets). In this work the air quality inside this tunnel network was evaluated using samples of Tillandsia usneoides as a biomonitor. Twenty-one plastic passive samplers, containing 50 g of plants each, were installed at different points of this tunnel network. In addition, surface layer of the tunnel walls, close to the plant sampling sites, were collected. Estimates for the enrichment factor, using Fe as reference and standard metal crustal data, suggest that the origin of the suspended metals is probably geogenic for Cr, a mix for Cu, and anthropogenic for Zn and Hg. It is important to emphasize that the Guanajuato’s tunnel network is used both by pedestrians and vehicular traffic and, probably, the last one is acting as the main source for toxic elements for the tunnel’s air.