Bacterial Population Size Research Articles

Lettuce Genotype-Dependent Effects of Temperature on Escherichia coli O157:H7 Persistence and Plant Head Growth

Lettuce has been commonly associated with the contamination of human pathogens, such as Escherichia coli O157:H7 (hereafter O157:H7), which has resulted in serious foodborne illnesses. Contamination events may happen throughout the farm-to-fork chain, when O157:H7 colonizes edible tissues and closely interacts with the plant. Environmental conditions have a significant impact on many plant–microbe interactions; however, it is currently unknown whether temperature affects O157:H7 colonization of the lettuce phyllosphere. In this study, we investigated the relationship between elevated growth temperatures, O157:H7 persistence, and lettuce head growth using 25 lettuce genotypes. Plants were grown under optimal or elevated temperatures for 3.5 weeks before being inoculated with O157:H7. The bacterial population size in the phyllosphere and lettuce head area was estimated at 0- and 10-days postinoculation (DPI) to assess bacterial persistence and head growth during contamination. We found that growing temperature can have a positive, negative, or no effect on O157:H7 persistence depending on the lettuce genotype. Furthermore, temperature had a greater effect on head area size than the presence of O157:H7. The results suggested that the combination of plant genotype and temperature level is an important factor for O157:H7 colonization of lettuce and the possibility to combine desirable food safety traits with heat tolerance into the lettuce germplasm.

Bacterial lifestyle shapes pangenomes

Pangenomes vary across bacteria. Some species have fluid pangenomes, with a high proportion of genes varying between individual genomes. Other species have less fluid pangenomes, with different genomes tending to contain the same genes. Two main hypotheses have been suggested to explain this variation: differences in species' bacterial lifestyle and effective population size. However, previous studies have not been able to test between these hypotheses because the different features of lifestyle and effective population size are highly correlated with each other, and phylogenetically conserved, making it hard to disentangle their relative importance. We used phylogeny-based analyses, across 126 bacterial species, to tease apart the causal role of different factors. We found that pangenome fluidity was lower in i) host-associated compared with free-living species and ii) host-associated species that are obligately dependent on a host, live inside cells, and are more pathogenic and less motile. In contrast, we found no support for the competing hypothesis that larger effective population sizes lead to more fluid pangenomes. Effective population size appears to correlate with pangenome variation because it is also driven by bacterial lifestyle, rather than because of a causal relationship.

Alterations in cell arrangements of group B streptococcus due to virulence factor expression can bias estimates of bacterial populations based on colony count measures.

Group B streptococcus (GBS) is a chain-forming commensal bacterium and opportunistic pathogen that resides in the gastrointestinal and genitourinary tract of healthy adults. GBS can cause various infections and related complications in pregnant and nonpregnant women, adults, and newborns. Investigations of the mechanisms by which GBS causes disease pathogenesis often utilize colony count assays to estimate bacterial population size in experimental models. In other streptococci, such as group A streptococcus and pneumococcus, variation in the chain length of the bacteria that can occur naturally or due to mutation can affect facets of pathogenesis, such as adherence to or colonization of a host. No studies have reported a relationship between GBS chain length and pathogenicity. Here, we used GBS strain 874391 and several derivative strains displaying longer chain-forming phenotypes (874391pgapC, 874391ΔcovR, 874391Δstp1) to assess the impact of chain length on bacterial population estimates based on the colony-forming unit (c.f.u.) assay. Disruption of GBS chains via bead beating or sonication in conjunction with fluorescence microscopy was used to compare chaining phenotypes pre- and post-disruption to detect long- and short-chain forms, respectively. We used a murine model of GBS colonization of the female reproductive tract to assess whether chaining may affect bacterial colonization dynamics in the host during chronic infection in vivo. Overall, we found that GBS exhibiting long-chain form can significantly affect population size estimates based on the colony count assay. Additionally, we found that the length of chaining of GBS can affect virulence in the reproductive tract colonization model. Collectively, these findings have implications for studies of GBS that utilize colony count assays to measure GBS populations and establish that chain length can affect infection dynamics and disease pathogenesis for this important opportunistic pathogen.

Prevalence and Diversity of Antibiotic Resistance Heterotrophic Bacteria Found Along the Bintulu Rivers, Sarawak

The study aims (1) to isolate and characterise the heterotrophic bacteria from different rivers in Bintulu, Sarawak, (2) to investigate the hygienic condition of the rivers through faecal coliforms and (3) to determine the antibiotic resistance among the heterotrophic bacteria isolates. A total of 100 heterotrophic bacteria strains were identified from rivers of Bintulu, Sarawak. The characterisation of bacteria was performed using (GTG)5 fingerprinting to investigate their genetic distribution diversity and 16S rRNA gene sequencing. Microbiological variables tested including total viable count, coliform count, Escherichia coli confirmation test. Antibiotic susceptibility test was performed against 10 antibiotics. Sample collected from Sungai Teknik showed higher mean bacterial population size (log 4.48 ± 0.00 CFU/mL). Sungai Sibiu, Waterfront and Sungai Plan revealed the highest most probable number (MPN) index (>1,600 per mL). Each dendrogram showed 3 to 7 clusters of bacteria groups confirmed as Enterobacter spp. (40%), Acinetobacter spp. (13%), Bacillus spp. (13%), Klebsiella spp. (13%), Staphylococcus spp. (7%), Chromobacterium spp. (7%) and Citrobacter spp. (7%). All the heterotrophic bacteria isolated showed high resistance against ciprofloxacin (63.70 ± 33.40%), piperacillin (58.10 ± 31.37%), aztreonam (48.40 ± 30.95%) and more susceptible to tetracycline (3.30 ± 10.44%). Our findings highlight the multiple antibiotics resistance and microbiological analysis of heterotrophic bacteria found in polluted river water. The preservation of the river water is vital as hydrologic purposes, sustain the microbial composition, ecological integrity of the river.

Droplet-based methodology for investigating bacterial population dynamics in response to phage exposure

An alarming rise in antimicrobial resistance worldwide has spurred efforts into the search for alternatives to antibiotic treatments. The use of bacteriophages, bacterial viruses harmless to humans, represents a promising approach with potential to treat bacterial infections (phage therapy). Recent advances in microscopy-based single-cell techniques have allowed researchers to develop new quantitative methodologies for assessing the interactions between bacteria and phages, especially the ability of phages to eradicate bacterial pathogen populations and to modulate growth of both commensal and pathogen populations. Here we combine droplet microfluidics with fluorescence time-lapse microscopy to characterize the growth and lysis dynamics of the bacterium Escherichia coli confined in droplets when challenged with phage. We investigated phages that promote lysis of infected E. coli cells, specifically, a phage species with DNA genome, T7 (Escherichia virus T7) and two phage species with RNA genomes, MS2 (Emesvirus zinderi) and Qβ (Qubevirus durum). Our microfluidic trapping device generated and immobilized picoliter-sized droplets, enabling stable imaging of bacterial growth and lysis in a temperature-controlled setup. Temporal information on bacterial population size was recorded for up to 25 h, allowing us to determine growth rates of bacterial populations and helping us uncover the extent and speed of phage infection. In the long-term, the development of novel microfluidic single-cell and population-level approaches will expedite research towards fundamental understanding of the genetic and molecular basis of rapid phage-induced lysis and eco-evolutionary aspects of bacteria-phage dynamics, and ultimately help identify key factors influencing the success of phage therapy.

Growth phase estimation for abundant bacterial populations sampled longitudinally from human stool metagenomes

Longitudinal sampling of the stool has yielded important insights into the ecological dynamics of the human gut microbiome. However, human stool samples are available approximately once per day, while commensal population doubling times are likely on the order of minutes-to-hours. Despite this mismatch in timescales, much of the prior work on human gut microbiome time series modeling has assumed that day-to-day fluctuations in taxon abundances are related to population growth or death rates, which is likely not the case. Here, we propose an alternative model of the human gut as a stationary system, where population dynamics occur internally and the bacterial population sizes measured in a bolus of stool represent a steady-state endpoint of these dynamics. We formalize this idea as stochastic logistic growth. We show how this model provides a path toward estimating the growth phases of gut bacterial populations in situ. We validate our model predictions using an in vitro Escherichia coli growth experiment. Finally, we show how this method can be applied to densely-sampled human stool metagenomic time series data. We discuss how these growth phase estimates may be used to better inform metabolic modeling in flow-through ecosystems, like animal guts or industrial bioreactors.

Trends in Salmonella Dublin over time in Denmark from food and animal related isolates.

Salmonella enterica serovar Dublin is highly adapted to cattle and a relatively rare cause of human infections. In Denmark S. Dublin has been endemic in the cattle population for many years. A national surveillance program in the cattle population was established at herd-level to reduce the occurrence of S. Dublin. In this study, we analyzed 421S. Dublin genomes from cattle and food in order to determine the trend of S. Dublin's population size over time in Denmark and the impact of intervention in the cattle industry on the bacterial population size. A phylogenetic tree based on SNPs exhibited two major clades and one small cluster. All isolates were ST10. The temporal phylogenetic tree for the S. Dublin isolates showed that the most recent common ancestor was estimated to be in ∼1980 for the two major clades. An effective population size over time based on a Bayesian skyline plot showed that the population size of S. Dublin decreased significantly between 2014 and 2019 in both major clades. This result was concordant with the decrease of infected human cases by S. Dublin in Denmark. The strengthening of a surveillance program in Denmark could be the cause for the reduction of S. Dublin's effective population size. This study showed that whole genome sequencing combined with computer intensive phylogenetic analysis estimating the effective size of the S. Dublin's population over time is a strongly relevant measure with respect to assessing the impact of control measures aiming to reduce the bacterial population in the reservoir and the risk for human infection.

Optimal density of bacterial cells.

A substantial fraction of the bacterial cytosol is occupied by catalysts and their substrates. While a higher volume density of catalysts and substrates might boost biochemical fluxes, the resulting molecular crowding can slow down diffusion, perturb the reactions' Gibbs free energies, and reduce the catalytic efficiency of proteins. Due to these tradeoffs, dry mass density likely possesses an optimum that facilitates maximal cellular growth and that is interdependent on the cytosolic molecule size distribution. Here, we analyze the balanced growth of a model cell, accounting systematically for crowding effects on reaction kinetics. Its optimal cytosolic volume occupancy depends on the nutrient-dependent resource allocation into large ribosomal vs. small metabolic macromolecules, reflecting a tradeoff between the saturation of metabolic enzymes, favoring larger occupancies with higher encounter rates, and the inhibition of the ribosomes, favoring lower occupancies with unhindered diffusion of tRNAs. Our predictions across growth rates are quantitatively consistent with the experimentally observed reduction in volume occupancy on rich media compared to minimal media in E. coli. Strong deviations from optimal cytosolic occupancy only lead to minute reductions in growth rate, which are nevertheless evolutionarily relevant due to large bacterial population sizes. In sum, cytosolic density variation in bacterial cells appears to be consistent with an optimality principle of cellular efficiency.

O-Polysaccharides of LPS Modulate E. coli Uptake by Acanthamoeba castellanii.

Protozoan grazing is a major cause of bacterial mortality and controls bacterial population size and composition in the natural environment. To enhance their survival, bacteria evolved many defense strategies to avoid grazing by protists. Cell wall modification is one of the defense strategies that helps bacteria escape from recognition and/or internalization by its predators. Lipopolysaccharide (LPS) is the major component of Gram-negative bacterial cell wall. LPS is divided into three regions: lipid A, oligosaccharide core and O-specific polysaccharide. O-polysaccharide as the outermost region of E. coli LPS provides protection against predation by Acanthamoeba castellanii; however, the characteristics of O-polysaccharide contribute to this protection remain unknown. Here, we investigate how length, structure and composition of LPS affect E. coli recognition and internalization by A. castellanii. We found that length of O-antigen does not play a significant role in regulating bacterial recognition by A. castellanii. However, the composition and structure of O-polysaccharide play important roles in providing resistance to A. castellanii predation.

Erratum for Versluis et al., "A Multiscale Spatiotemporal Model Including a Switch from Aerobic To Anaerobic Metabolism Reproduces Succession in the Early Infant Gut Microbiota".

Volume 7, no. 5, e00446-22, 2022 , https://doi.org/10.1128/mSystems.00446-22. Page 19: Equation 3 should read as follows: F⃗ub(x⃗,t) = c⃗(x⃗,t)B(x⃗,t)where c⃗(x⃗,t) is the vector of metabolite concentrations at point x⃗, x⃗ indicates the location, and B(x⃗,t) is the size of the local bacterial population in population units.

Weather stressors correlate with Escherichia coli and Salmonella enterica persister formation rates in the phyllosphere: a mathematical modeling study

Enteric pathogens can enter a persister state in which they survive exposure to antibiotics and physicochemical stresses. Subpopulations of such phenotypic dormant variants have been detected in vivo and in planta in the laboratory, but their formation in the natural environment remains largely unexplored. We applied a mathematical model predicting the switch rate to persister cell in the phyllosphere to identify weather-related stressors associated with E. coli and S. enterica persister formation on plants based on their population dynamics in published field studies from the USA and Spain. Model outputs accurately depicted the bi-phasic decay of bacterial population sizes measured in the lettuce and spinach phyllosphere in these studies. Predicted E. coli persister switch rate on leaves was positively and negatively correlated with solar radiation intensity and wind velocity, respectively. Likewise, predicted S. enterica persister switch rate correlated positively with solar radiation intensity; however, a negative correlation was observed with air temperature, relative humidity, and dew point, factors involved in water deposition onto the phylloplane. These findings suggest that specific environmental factors may enrich for dormant bacterial cells on plants. Our model quantifiably links persister cell subpopulations in the plant habitat with broader physical conditions, spanning processes at different granular scales.

Bacterial diversity and community structure in the rhizosphere of the halophyte Halocnemum strobilaceum in an Algerian arid saline soil.

Hypersaline ecosystems host a particular microbiota, which can be specifically recruited by halophytes. In order to broaden our knowledge of hypersaline ecosystems, an in natura study was conducted on the microbiota associated with the halophyte Halocnemum strobilaceum from alkaline-saline arid soil in Algeria. We collected and identified a total of 414 strains isolated from root tissues (RT), root-adhering soil (RAS), non-adhering rhizospheric soil (NARS) and bulk soil (BS) using different NaCl concentrations. Our data showed that halophilic and halotolerant bacterial isolates in BS and the rhizosphere belonged to 32 genera distributed in Proteobacteria (49%), Firmicutes (36%), Actinobacteria (14%) and Bacteroidetes (1%). Bacterial population size and species diversity were greatly increased in the rhizosphere (factor 100). The reservoir of diversity in BS was dominated by the genera Bacillus and Halomonas. Bacillus/Halomonas ratio decreased with the proximity to the roots from 2.2 in BS to 0.3 at the root surface. Salt screening of the strains showed that species belonging to nine genera were able to grow up to 5.1M NaCl. Thus, we found that H. strobilaceum exerted a strong effect on the diversity of the recruited microbiota with an affinity strongly attributed to the genus Halomonas.

Phytochemical effects of Apium graveolens on the abundances of functional genes associated with PAH degradation in soil

The biodegradation of polycyclic aromatic hydrocarbons in soil is enhanced by chemical constituents of various plant species that support the growth and activity of PAH degrading bacteria in the rhizosphere. Here, we investigated the phytochemical effects of celeriac (Apium graveolens var. rapaceum) root tissues, a known stimulator of PAH degradation, on the copy numbers of nahAc, xylE, and 16S rRNA genes to determine the relative contributions of growth-linked degradation versus selective enrichment of PAH degraders. Real-time quantitative PCR analysis showed that the copy numbers of 16S rRNA genes, as a reporter of general bacterial population size, increased approximately 2- to 5-fold in soil amended with celeriac, but were not affected by exposure to naphthalene alone. In contrast, copy numbers of two functional genes (nahAc and xylE), commonly used as reporters for monitoring the population size of PAH degraders, were significantly increased following exposure to naphthalene but were differently affected by soil amendment with celeriac. As compared with the copy numbers of nahAc gene in unamended control soil, the nahAc copy numbers were increased by 460-fold in soil amended with naphthalene alone and by 610-fold in soil amended with both naphthalene and celeriac after 10 days of incubation. The copy numbers of xylE were increased by 20-fold in soil amended with naphthalene alone and increased by 200-fold in soil amended with both naphthalene and celeriac after 10 days of incubation. The result suggests that increased degradation rates of PAHs in the presence of celeriac involves both growth linked processes and selective enrichment of the degrader population.

Effects of 2,4-di-tert-butylphenol at different concentrations on soil functionality and microbial community structure in the Lanzhou lily rhizosphere

The autotoxicity of root exudates is an important factor that affects soil enzymes and microbes in rhizosphere soil. Previous studies have confirmed that 2,4-di-tert-butylphenol (2,4-DTBP) is a major autotoxin of the root exudates that severely hampers the yield and quality of Lanzhou lily. However, little is currently known regarding the mechanism of 2,4-DTBP. Here, we hypothesized that 2,4-DTBP has a direct effect on soil functionality and the microbial community structure in Lanzhou lily rhizosphere soil, thereby affecting the growth of Lanzhou lily. To test our hypothesis, various experiments were conducted in this study. We treated Lanzhou lily plants with 2,4-DTBP at different concentrations and analyzed the effects of 2,4-DTBP on rhizosphere soil enzyme activity and microbial community structure and function. The diversity of soil microbes was analyzed using high-throughput sequencing. The results showed that 2,4-DTBP altered rhizosphere soil enzyme activity and microbial community structure and function. Moreover, microbial biomass was stimulated with the addition of 0.5 mM 2,4-DTBP and inhibited with the addition of 2.0 mM 2,4-DTBP. Thus, the 2.0 mM 2,4-DTBP treatment was not suitable for the growth of soil microorganisms. We also found that the 2.0 mM 2,4-DTBP treatment resulted in an increase in the levels of fungal pathogens, such as Fusarium, and reduced the size of beneficial bacterial populations in the rhizosphere soil. Moreover, the effects of 2,4-DTBP on soil functionality and the microbial community structure differed based on the concentrations of 2,4-DTBP and the specific indexes of determination. Thus, we concluded that 2,4-DTBP could inhibit soil functionality and disturb the microbial community structure in the rhizosphere soil. This finding may be important for understanding the relationship between autotoxicity and barriers and clarifying the long-term continuous cropping problems of Lanzhou lily.

Epidermis as a Platform for Bacterial Transmission.

The epidermis constitutes a continuous external layer covering the body, offering protection against bacteria, the most abundant living organisms that come into contact with this barrier. The epidermis is heavily colonized by commensal bacterial organisms that help protect against pathogenic bacteria. The highly regulated and dynamic interaction between the epidermis and commensals involves the host’s production of nutritional factors promoting bacterial growth together to chemical and immunological bacterial inhibitors. Signal trafficking ensures the system’s homeostasis; conditions that favor colonization by pathogens frequently foster commensal growth, thereby increasing the bacterial population size and inducing the skin’s antibacterial response, eliminating the pathogens and re-establishing the normal density of commensals. The microecological conditions of the epidermis favors Gram-positive organisms and are unsuitable for long-term Gram-negative colonization. However, the epidermis acts as the most important host-to-host transmission platform for bacteria, including those that colonize human mucous membranes. Bacteria are frequently shared by relatives, partners, and coworkers. The epidermal bacterial transmission platform of healthcare workers and visitors can contaminate hospitalized patients, eventually contributing to cross-infections. Epidermal transmission occurs mostly via the hands and particularly through fingers. The three-dimensional physical structure of the epidermis, particularly the fingertips, which have frictional ridges, multiplies the possibilities for bacterial adhesion and release. Research into the biology of bacterial transmission via the hands is still in its infancy; however, tribology, the science of interacting surfaces in relative motion, including friction, wear and lubrication, will certainly be an important part of it. Experiments on finger-to-finger transmission of microorganisms have shown significant interindividual differences in the ability to transmit microorganisms, presumably due to genetics, age, sex, and the gland density, which determines the physical, chemical, adhesive, nutritional, and immunological status of the epidermal surface. These studies are needed to optimize interventions and strategies for preventing the hand transmission of microorganisms.

Effect of Supercritical Extract from Black Poplar and Basket Willow on the Quality of Natural and Probiotic Drinkable Yogurt.

Simple SummaryFermented milk products should be an important component of the human diet due to their high nutritional value and easy digestibility. In order to enhance the nutritional value of yogurts, additives in the form of plant-based raw materials are often used. Phytoextracts are a mixture of biologically active compounds that can have beneficial effects on the human body and influence both the manufacturing process and the quality of food products. The incorporation of extracts from the basket willow (Salix viminalis) into natural and probiotic yogurts reduced the fermentation time and increased their antioxidant activity. Sensory evaluation of the appearance and texture of yogurts enriched with extracts showed no significant differences compared to natural yogurts. The addition of plant extracts may enable the standardisation of yogurt quality and increase its biological activity.Yogurt is a fermented milk drink produced by Streptococcus thermophilus, Lactobacillus delbrüeckii ssp. bulgaricus, or Lactobacillus rhamnosus, which can be enriched with polyphenolic compounds to enhance its antioxidant properties. Supercritical (scCO2/H2O) extracts obtained from the mixture of bark and wood of black poplar (Populus nigra) and basket willow (Salix viminalis) are the source of bioactive compounds. The aim of the study was to assess the effect of supercritical extracts from the P. nigra and S. viminalis on the fermentation, quality, and bioactive properties of drinkable natural and probiotic yogurts. The incorporation of scCO2/H2O extracts at a dose of 0.01% (w/v) into milk for the production of natural and probiotic yogurts increases their functional properties by enhancing the antioxidant activity without causing negative effects on the physicochemical and organoleptic properties of products. The antioxidant activity of yogurt with scCO2/H2O extract from P. nigra and S. viminalis was higher than control yogurts by 1.3–13.2% and 4.4–37.5%, respectively. The addition of a supercritical S. viminalis extract reduced the time of natural and probiotic yogurt fermentation. Natural and probiotic yogurt with scCO2/H2O extracts added was characterised by a bacterial population size of over 7 log cfu/g, and the microflora was active throughout the cold storage period. FTIR analysis confirmed the presence of scCO2/H2O extracts from P. nigra or S. viminalis in both types of yogurt. A secondary structure analysis confirmed interactions between compounds of scCO2/H2O extract from P. nigra and S. viminalis extract with milk proteins. These interactions affect the compounds’ structural and functional properties by changing, e.g., their digestibility and antioxidant properties.

Mangrove microbiota along the urban-to-rural gradient of the Cayenne estuary (French Guiana, South America): Drivers and potential bioindicators

The microbial communities inhabiting the Atlantic-East Pacific (AEP) mangroves have been poorly studied, and mostly comprise chronically polluted mangroves. In this study, we characterized changes in the structure and diversity of microbial communities of mangroves along the urban-to-rural gradient of the Cayenne estuary (French Guiana, South America) that experience low human impact. The microbial communities were assigned into 50 phyla. Proteobacteria, Chloroflexi, Acidobacteria, Bacteroidetes, and Planctomycetes were the most abundant taxa. The environmental determinants found to significantly correlated to the microbial communities at these mangroves were granulometry, dieldrin concentration, pH, and total carbon (TC) content. Furthermore, a precise analysis of the sediment highlights the existence of three types of anthropogenic pressure among the stations: (i) organic matter (OM) enrichment due to the proximity to the city and its wastewater treatment plant, (ii) dieldrin contamination, and (iii) naphthalene contamination. These forms of weak anthropogenic pressure seemed to impact the bacterial population size and microbial assemblages. A decrease in Bathyarchaeota, “Candidatus Nitrosopumilus”, and Nitrospira genera was observed in mangroves subjected to OM enrichment. Mangroves polluted with organic contaminants were enriched in Desulfobacteraceae, Desulfarculaceae, and Acanthopleuribacteraceae (with dieldrin or polychlorobiphenyl contamination), and Chitinophagaceae and Geobacteraceae (with naphthalene contamination). These findings provide insights into the main environmental factors shaping microbial communities of mangroves in the AEP that experience low human impact and allow for the identification of several potential microbial bioindicators of weak anthropogenic pressure.

Evolution and emergence of multidrug-resistant Mycobacterium tuberculosis in Chisinau, Moldova.

The evolution and emergence of drug-resistant tuberculosis (TB) has been studied extensively in some contexts, but the ecological drivers of these two processes remain poorly understood. This study sought to describe the joint evolutionary and epidemiological histories of a novel multidrug-resistant Mycobacterium tuberculosis strain recently identified in the capital city of the Republic of Moldova (MDR Ural/4.2), where genomic surveillance of drug-resistant M. tuberculosis has been limited thus far. Using whole genome sequence data and Bayesian phylogenomic methods, we reconstruct the stepwise acquisition of drug resistance mutations in the MDR Ural/4.2 strain, estimate its historical bacterial population size over time, and infer the migration history of this strain between Eastern European countries. We infer that MDR Ural/4.2 likely evolved (via acquisition of rpoB S450L, which confers resistance to rifampin) in the early 1990s, during a period of social turmoil following Moldovan independence from the Soviet Union. This strain subsequently underwent substantial population size expansion in the early 2000s, at a time when national guidelines encouraged inpatient treatment of TB patients. We infer exportation of this strain and its isoniazid-resistant ancestral precursor from Moldova to neighbouring countries starting as early as 1985. Our findings suggest temporal and ecological associations between specific public health practices, including inpatient hospitalization of drug-resistant TB cases from the early 2000s until 2013, and the evolution of drug-resistant M. tuberculosis in Moldova. These findings underscore the need for regional coordination in TB control and expanded genomic surveillance efforts across Eastern Europe.

Changes in Bacterial Communities During Treatment of Municipal Wastewater in Arctic Wastewater Stabilization Ponds

Wastewater stabilization ponds (WSPs) are commonly used to treat municipal wastewater in the Canadian Arctic. Bacterial community structure and functionality remain mostly uncharacterized for arctic WSPs, yet are presumed important for treatment outcomes during the 3-month summer treatment season with open water in the WSPs. The objective of this study was to investigate treatment performance and related temporal and spatial changes in the structure and putative function of bacterial communities during treatment of municipal wastewater in the WSPs of Pond Inlet and Clyde River, Nunavut over two consecutive summer treatment seasons. Influent raw wastewater contained a high organic load and large bacterial communities (~9 log 16S rRNA copies/mL) belonging mainly to Proteobacteria. Although designed to be facultative ponds, both WSPs remained anaerobic with neutral pH values (7.5–7.8) throughout the summer treatment season. Water quality data showed that nutrients [measured as carbonaceous biological oxygen demand (CBOD5)], total suspended solids, and total ammonia nitrogen were progressively reduced during treatment in the ponds as the summer progressed. The pond bacterial population size and species richness depended on the pond temperature (2–18°C), with 8.5 log 16S rRNA copies/mL and the largest alpha diversities (Shannon-Wiener index of 4-4.5) observed mid-season (late July). While the phylogenetic beta diversity in raw wastewater from the two locations remained similar, pond bacterial communities underwent significant (p < 0.05) changes to dominance of Comamonadaceae, Geobacteracea, and Porphyromonadaceae. Multivariate distance based redundancy analysis and predicted gene functionalities in the microbiota agreed with water quality results that microbial removal of nutrients (e.g., CBOD5) peaked in the middle of the summer coinciding with the treatment period with the highest pond temperatures. Information from this study will be useful for further development of models to predict biological treatment outcomes, which could be used to size and assess the feasibility of WSPs in extreme climates. Higher pond temperatures resulted in optimal biological processes and nutrient removal in the middle of the summer. While it is challenging to control environmental factors in a passive wastewater treatment system there are some design considerations that could be used to optimize temperature regimes, such as the depth of the pond.

Bottleneck size and selection level reproducibly impact evolution of antibiotic resistance

During antibiotic treatment, the evolution of bacterial pathogens is fundamentally affected by bottlenecks and varying selection levels imposed by the drugs. Bottlenecks—that is, reductions in bacterial population size—lead to an increased influence of random effects (genetic drift) during bacterial evolution, and varying antibiotic concentrations during treatment may favour distinct resistance variants. Both aspects influence the process of bacterial evolution during antibiotic therapy and thereby treatment outcome. Surprisingly, the joint influence of these interconnected factors on the evolution of antibiotic resistance remains largely unexplored. Here we combine evolution experiments with genomic and genetic analyses to demonstrate that bottleneck size and antibiotic-induced selection reproducibly impact the evolutionary path to resistance in pathogenic Pseudomonas aeruginosa, one of the most problematic opportunistic human pathogens. Resistance is favoured—expectedly—under high antibiotic selection and weak bottlenecks, but—unexpectedly—also under low antibiotic selection and severe bottlenecks. The latter is likely to result from a reduced probability of losing favourable variants through drift under weak selection. Moreover, the absence of high resistance under low selection and weak bottlenecks is caused by the spread of low-resistance variants with high competitive fitness under these conditions. We conclude that bottlenecks, in combination with drug-induced selection, are currently neglected key determinants of pathogen evolution and outcome of antibiotic treatment.