Socioeconomic indices guided linear mixed-effects and meta-regression modelling of the temporal, global and regional prevalence of Helicobacter pylori in environmental waters: A class I carcinogen.

Environmental waters (EW) substantially lend to the transmission of Helicobacter pylori (Hp). But the increase in Hp infections and antimicrobial resistance is often attributed to socioeconomic status. The connection between socioeconomic status and Hp prevalence in EW is however yet to be investigated. This study aimed to assess the impacts of socioeconomic indices (SI: continent, world bank region (WBR), world bank income (WBI), WHO region, Socio-demographic Index (SDI quintile), Sustainable Development Index (SuDI), and Human Development Index (HDI)) on the prevalence of Hp in EW. Hp-EW data were fitted to a generalized linear mixed-effects model and SI-guided meta-regression models with a 1000-resampling test. The worldwide prevalence of Hp in EW was 21.76% [95% confidence interval [CI]: 10.29-40.29], which declined significantly from 59.52% [43.28-74.37] in 1990-99 to 19.36% [3.99-58.09] in 2010-19 and with increasing trend in 2020-22 (33.33%, 22.66-45.43). Hp prevalence in EW was highest in North America (45.12%, 17.07-76.66), then Europe (22.38%, 5.96-56.74), South America (22.09%, 13.76-33.49), Asia (2.98%, 0.02-85.17), and Africa (2.56%, 0.00-99.99). It was negligibly different among sampling settings, WBI, and WHO regions demonstrating highest prevalence in rural location [42.62%, 3.07-94.56], HIEs [32.82%, 13.19-61.10], and AMR [39.43%, 19.92-63.01], respectively. However, HDI, sample size, and microbiological method robustly predict Hp prevalence in EW justifying 26.08%, 21.15%, and 16.44% of the true difference, respectively. In conclusion, Hp is highly prevalence in EW across regional/socioeconomic strata and thus challenged the uses of socioeconomic status as surrogate for hygienic/sanitary practices in estimating Hp infection prevalence.

The Viable but Non-Culturable (VBNC) State, a Poorly Explored Aspect of Beneficial Bacteria.

Many bacteria have the ability to survive in challenging environments; however, they cannot all grow on standard culture media, a phenomenon known as the viable but non-culturable (VBNC) state. Bacteria commonly enter the VBNC state under nutrient-poor environments or under stressful conditions. This review explores the concept of the VBNC state, providing insights into the beneficial bacteria known to employ this strategy. The investigation covers different chemical and physical factors that can induce the latency state, cell features, and gene expression observed in cells in the VBNC state. The review also covers the significance and applications of beneficial bacteria, methods of evaluating bacterial viability, the ability of bacteria to persist in environments associated with higher organisms, and the factors that facilitate the return to the culturable state. Knowledge about beneficial bacteria capable of entering the VBNC state remains limited; however, beneficial bacteria in this state could face adverse environmental conditions and return to a culturable state when the conditions become suitable and continue to exert their beneficial effects. Likewise, this unique feature positions them as potential candidates for healthcare applications, such as the use of probiotic bacteria to enhance human health, applications in industrial microbiology for the production of prebiotics and functional foods, and in the beer and wine industry. Moreover, their use in formulations to increase crop yields and for bacterial bioremediation offers an alternative pathway to harness their beneficial attributes.

Engineered Fluorescent Strains of Cryptococcus neoformans: a Versatile Toolbox for Studies of Host-Pathogen Interactions and Fungal Biology, Including the Viable but Nonculturable State.

Cryptococcus neoformans is an opportunistic fungal pathogen known for its remarkable ability to infect and subvert phagocytes. This ability provides survival and persistence within the host and relies on phenotypic plasticity. The viable but nonculturable (VBNC) phenotype was recently described in C. neoformans, whose study is promising in understanding the pathophysiology of cryptococcosis. The use of fluorescent strains is improving host interaction research, but it is still underexploited. Here, we fused histone H3 or the poly(A) binding protein (Pab) to enhanced green fluorescent protein (eGFP) or mCherry, obtaining a set of C. neoformans transformants with different colors, patterns of fluorescence, and selective markers (hygromycin B resistance [Hygr] or neomycin resistance [Neor]). We validated their similarity to the parental strain in the stress response, the expression of virulence-related phenotypes, mating, virulence in Galleria mellonella, and survival within murine macrophages. PAB-GFP, the brightest transformant, was successfully applied for the analysis of phagocytosis by flow cytometry and fluorescence microscopy. Moreover, we demonstrated that an engineered fluorescent strain of C. neoformans was able to generate VBNC cells. GFP-tagged Pab1, a key regulator of the stress response, evidenced nuclear retention of Pab1 and the assembly of cytoplasmic stress granules, unveiling posttranscriptional mechanisms associated with dormant C. neoformans cells. Our results support that the PAB-GFP strain is a useful tool for research on C. neoformans. IMPORTANCE Cryptococcus neoformans is a human-pathogenic yeast that can undergo a dormant state and is responsible for over 180,000 deaths annually worldwide. We engineered a set of fluorescent transformants to aid in research on C. neoformans. A mutant with GFP-tagged Pab1 improved fluorescence-based techniques used in host interaction studies. Moreover, this mutant induced a viable but nonculturable phenotype and uncovered posttranscriptional mechanisms associated with dormant C. neoformans. The experimental use of fluorescent mutants may shed light on C. neoformans-host interactions and fungal biology, including dormant phenotypes.

Occurrence of Helicobacter spp. and Fecal Bacterial Contamination in High-altitude Aquatic Environments from the Andes.

The occurrence of Helicobacter spp. and fecal bacterial contamination was investigated in high-altitude environments from the Northeastern Andes of Venezuela. Helicobacter DNA was detected by PCR in streams, drinking and irrigation waters, and one culture from drinking water by the HP enrichment medium for selection of Helicobacter pylori, which displayed 98.98% homology to this pathogen based on 16S rRNA gene sequencing. FISH demonstrated predominant coccoid cells of the target bacteria indicative of the viable but nonculturable (VBNC) state in all water samples and HP cultures. Our work reveals for the first time Helicobacter spp. in waters from one of the highest places in the world. These results, together with the presence of fecal coliforms (2-160 MPN/100 mL) from the headwaters of rivers to drinking and irrigation waters, alert fecal contamination and epidemiological implications in this area of ecological and economic importance for the region.

Behavior of Salmonella Enteritidis and Shigella flexneri during induction and recovery of the viable but nonculturable state.

Bacteria may enter into a viable but nonculturable (VBNC) state as a response to stresses, such as those found in food processing. Cells in the VBNC state lose the ability to grow in a conventional culture medium but man recover culturability. The viability, culturability and intracellular reactive oxygen species (ROS) of Salmonella Enteritidis and Shigella flexneri were evaluated under stress conditions to induce a VBNC state. Cells were maintained under nutritional, osmotic and cold stresses (long-term induction) in Butterfield's phosphate solution plus 1.2 M of NaCl at 4°C and under nutritional and oxidative stresses (short-term induction) in 10 mM of H2O2. Culture media, recovery agents, sterilization methods of media and incubation temperature, were combined and applied to recover the culturability of the VBNC cells. Salmonella entered in the VBNC state after 135 days under long-term induction, while Shigella maintained culturability after 240 days. Under short-term induction, Salmonella and Shigella lose culturability after 135 and 240 min, respectively. Flow cytometric analysis revealed viable cells and intracellular ROS in both species in VBNC. It was not possible to recover the culturability of VBNC cells using the 42 combinations of different factors.

Resistance and induction of viable but non culturable states (VBNC) during inactivation of E. coli and Klebsiella pneumoniae by addition of H2O2 to natural well water under simulated solar irradiation.

Inactivation of E. coli and Klebsiella pneumoniae by addition of H2O2 10 mg L-1 into natural well water samples containing natural total iron concentrations (around 0.3 mg L-1) under simulated solar light was followed by bacterial culturability (plate count) and viability (DVC-FISH). Results showed that culturability of both bacteria was totally reduced while viability was only completely depleted for E. coli in well water samples depending of total iron concentration. Post-irradiation effects in presence of residual H2O2 showed that viability of both bacteria kept dropping being totally reduced for E. coli cells while K. pneumoniae decreased only 1-log. SEM micrographs showed that E. coli and K. pneumoniae cells underwent morphological changes and size reduction according to VBNC states. Different dark and photo-induced processes where physical-chemical features of groundwater samples play an important role could be responsible of bacteria abatement.

Persistence in Phytopathogenic Bacteria: Do We Know Enough?

Phytopathogenic bacteria affect a wide range of crops worldwide and have a negative impact in agriculture due to their associated economic losses and environmental impacts. Together with other biotic and abiotic stress factors, they pose a threat to global food production. Therefore, understanding bacterial survival strategies is an essential step toward the development of new strategies to control plant diseases. One mechanism used by bacteria to survive under stress conditions is the formation of persister cells. Persisters are a small fraction of phenotypic variants within an isogenic population that exhibits multidrug tolerance without undergoing genetic changes. They are dormant cells that survive treatment with antimicrobials by inactivating the metabolic functions that are disrupted by these compounds. They are thus responsible for the recalcitrance of many human diseases, and in the same way, they are thought to contribute to the survival of bacterial phytopathogens under a range of stresses they face in the environment. It is believed that persister cells of bacterial phytopathogens may lead to the reoccurrence of disease by recovering growth and recolonizing the host plant after the end of stress. However, compared to human pathogens, little is known about persister cells in phytopathogens, especially about their genetic regulation. In this review, we describe the overall knowledge on persister cells and their regulation in bacterial phytopathogens, focusing on their ability to survive stress conditions, to recover from dormancy and to maintain virulence.

Effects of ozone and chlorine disinfection on VBNC Helicobacter pylori by molecular techniques and FESEM images.

Helicobacter pylori is a pathogen bacteria associated with chronic gastritis, peptic ulceration, and gastric carcinoma. H. pylori has a spiral morphology, which under certain conditions of stress becomes a coccoid form. This type of morphology has been linked to a viable but non-culturable (VBNC) state, which is thought to allow its persistence in the environment. Membrane damage in VBNC H. pylori in water as a mechanism for inactivation using ozone (O3) and chlorine disinfection has not been reported in the literature. In this paper, disinfection assays with ozone and chlorine were conducted to evaluate their effects on VBNC H. pylori cells. The use of fluorescent dyes such as propidium monoazide (PMA) coupled with quantitative real-time polymerase chain reactions produced results necessary to assess the viability of the microorganism and demonstrate the effect of each disinfectant on the bacterial count. Applying ozone showed a 5-log bacterial reduction using a disinfectant concentration and exposure time (CT) of 4 mg min/L. Chlorine disinfection for the same 5-log reduction required a higher CT value. Field emission scanning electron microscope images of ozone-treated VBNC H. pylori also showed severe cell damage. The use of PMA revealed that chlorine produced physical damage in the membrane in addition to the known inhibiting effect on cell enzymatic processes. These findings are important for the detection and control of VBNC H. pylori cells in drinking water systems.

Survival, induction and resuscitation of vibrio cholerae from the viable but non-culturable state in the Southern Caribbean sea / Supervivencia, inducción y resucitación de Vibrio cholerae del estado viable no cultivable en el sur del Mar Caribe

The causative agent of cholera, Vibrio cholerae, can enter into a viable but non-culturable (VBNC) state in response to unfavorable conditions. The aim of this study was to evaluate the in situ survival of V. cholerae in an aquatic environment of the Southern Caribbean Sea, and its induction and resuscitation from the VBNC state. V. cholerae non-O1, non-O139 was inoculated into diffusion chambers placed at the Cuare Wildlife Refuge, Venezuela, and monitored for plate, total and viable cells counts. At 119 days of exposure to the environment, the colony count was < 10 CFU/mL and a portion of the bacterial population entered the VBNC state. Additionally, the viability decreased two orders of magnitude and morphological changes occurred from rod to coccoid cells. Among the aquatic environmental variables, the salinity had negative correlation with the colony counts in the dry season. Resuscitation studies showed significant recovery of cell cultivability with spent media addition (p < 0.05). These results suggest that V. cholerae can persist in the VBNC state in this Caribbean environment and revert to a cultivable form under favorable conditions. The VBNC state might represent a critical step in cholera transmission in susceptible areas.

El agente causal del cólera, Vibrio cholerae, puede entrar a un estado viable no cultivable (VNC) en respuesta a condiciones desfavorables. El objetivo de este estudio fue evaluar la supervivencia in situ de V. cholerae en un ambiente acuático al sur del Mar Caribe y su inducción y resucitación del estado VBNC. V. cholerae no-O1, no-O139 fue inoculado en cámaras de difusión ubicadas en el Refugio de Fauna Cuare, Venezuela, y monitoreado para contaje de colonias, células totales y viables. En 119 días de exposición al ambiente, el contaje de colonias fue < 10 UFC/mL y una fracción de la población bacteriana entró al estado VBNC. Adicionalmente, la viabilidad disminuyó dos órdenes de magnitud y ocurrieron cambios morfológicos de células bacilares a cocoides. Entre las variables del ambiente acuático, la salinidad presentó correlación negativa con el contaje de colonias. Los estudios de resucitación mostraron recuperación significativa de la cultivabilidad celular con adición de sobrenadantes de cultivos en crecimiento activo (p < 0.05). Estos resultados sugieren que V. cholerae puede persistir en estado VBNC en este ambiente de Caribe y revertir a una forma cultivable bajo condiciones favorables. El estado VBNC podría representar un paso crítico en la transmisión del cólera en áreas susceptibles.

Efecto de medios de cultivo preparados con agua de mar sobre indicadores sanitarios en aguas marinas de balnearios de Chichiriviche, estado Falcón, Venezuela / Effect of culture media prepared with sea water over health indicators in sea water of the Chichiriviche resort, Falcon State, Venezuela

Debido a su uso por humanos y para preservar la salud pública, los ambientes marinos recreacionales, deberían cumplir requisitos de calidad fisico-química y microbiológica. Sin embargo, actividades antropogénicas, tales como agricultura, ganadería, industriales, turísticas y domésticas, pueden descargar aguas servidas en playas marinas. La metodología vigente para evaluar la carga bacteriana de aguas recreacionales, no pareciera satisfactoria, particularmente en la detección de microorganismos patógenos debido a la presencia, en aguas marinas, de bacterias viables pero no cultivables (VBNC). Este estudio evaluó la carga bacteriana de aerobios mesófilos, coliformes totales/fecales y enterococos, en playas de Chichiriviche, Falcón, Venezuela y el efecto de concentraciones de agua marina sobre las densidades bacterianas. Las determinaciones se efectuaron en agar nutritivo, Mac Conkey y KF, preparados con agua destilada (MAD) o con agua de mar 10% v/v, (MAM). La carga bacteriana se incrementó entre 20 y 47 veces cuando los medios de cultivo se suplementaron con agua de mar. Las diferencias en las cargas bacterianas entre MAD y MAM, para aerobios mesófilos, coliformes totales y fecales fueron estadísticamente significativas (p≤0,05). El oxígeno disuelto y pH, en ambos medios presentaron valores permisibles.

Due to their use by humans and to preserve public health, recreational marine environments should fulfill physico-chemical and microbiological quality prerequisites. Nevertheless, anthropogenic activities such as agriculture, cattle raising, industrial, touristic and domestic, can discharge contaminated water at marine beaches. The present methods for evaluating the bacterial load of recreational waters does not appear satisfactory, especially concerning the detection of pathogenic microorganisms due to the presence, in marine waters, of viable but not cultivable bacteria (VNCB). This study evaluated the bacterial load of mesophilic aerobials, total and fecal coliforms, and enterococci at the Chichiriviche beaches, Falcon State, Venezuela, and the effect of marine water concentrations over the bacterial densities. The determinations were done in nutrient, Mac Conkey and KF agar prepared with distilled water (MAD) or with sea water (MSW). The bacterial load increased between 27 and 47 times when the culture media were supplement with sea water. The differences of the bacterial loads between MAD and MSW for mesophilic aerobials, total and fecal coliforms were statistically significant (p≤0.05). Dissolved oxygen and pH presented permissible values in both media.