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The symbiosis between Vibrio fischeri and the Hawaiian bobtail squid, Euprymna scolopes, is a tractable and well-studied model of bacteria-animal mutualism. Here, we developed a method to transiently colonize E. scolopes using D-alanine (D-ala) auxotrophy of the symbiont, controlling the persistence of viable infection by supplying or withholding D-ala. We generated alanine racemase (alr) mutants of V. fischeri that lack avenues for mutational suppression of auxotrophy or reversion to prototrophy. Surprisingly, an alr mutant did not require D-ala to grow in a minimal medium, a phenomenon requiring metC, which encodes cystathionine ß-lyase. Likewise, overexpression of metC suppressed D-ala auxotrophy in a rich medium. To block potential mechanisms of suppression, we combined the alr mutation with deletions of metC and/or bsrF, which encodes a broad-spectrum racemase and investigated the suppression rates of four D-ala auxotrophic strains. We then focused on alr bsrF mutant MC13, which has a suppression rate of <10-9. When D-ala was removed from a growing culture of MC13, cells rounded and lysed within 40 minutes. Transient colonization of E. scolopes was achieved by inoculating squid in seawater containing MC13 and D-ala, and then transferring the squid into water lacking D-ala, which resulted in loss of viable symbionts within hours. Interestingly, the symbionts within crypt 3 persisted longer than those of crypt 1, suggesting a difference in bacterial growth rate in distinct crypt environments. Our study highlights a new approach for inducing transient colonization and provides insight into the biogeography of the E. scolopes light organ. IMPORTANCE The importance of this study is multi-faceted, providing a valuable methodological tool and insight into the biology of the symbiosis between Vibrio fischeri and Euprymna scolopes. First, the study sheds light on the critical role of D-ala for bacterial growth, and the underpinnings of D-ala synthesis. Our observations that metC obviates the need for D-ala supplementation of an alr mutant in minimal medium and that MetC-dependent growth correlates with D-ala in peptidoglycan, corroborate and extend previous findings in Escherichia coli regarding a role of MetC in D-ala production. Second, our isolation of robust D-ala auxotrophs led us to a novel method for studying the squid-Vibrio symbiosis, allowing for transient colonization without the use of antibiotics, and revealed intriguing differences in symbiont growth parameters in distinct light organ crypts. This work and the methodology developed will contribute to our understanding of the persistence and dynamics of V. fischeri within its host. [ABSTRACT FROM AUTHOR]

In the U.S., baby spinach is mostly produced in Arizona (AZ) and California (CA). Characterizing the impact of growing region on the bacterial quality of baby spinach can inform quality management practices in industry. Between December 2021 and December 2022, baby spinach was sampled after harvest and packaging for microbiological testing, including shelf-life testing of packaged samples that were stored at 4°C. Samples were tested to (i) determine bacterial concentration, and (ii) obtain and identify bacterial isolates. Packaged samples from the Salinas, CA, area (n = 13), compared to those from the Yuma, AZ, area (n = 9), had a significantly higher bacterial concentration, on average, by 0.78 log10 CFU/g (P < 0.01, based on aerobic, mesophilic plate count data) or 0.67 log10 CFU/g (P < 0.01, based on psychrotolerant plate count data); the bacterial concentrations of harvest samples from the Yuma and Salinas areas were not significantly different. Our data also support that an increase in preharvest temperature is significantly associated with an increase in the bacterial concentration on harvested and packaged spinach. A Fisher's exact test and linear discriminant analysis (effect size), respectively, demonstrated that (i) the genera of 2,186 bacterial isolates were associated (P < 0.01) with growing region and (ii) Pseudomonas spp. and Exiguobacterium spp. were enriched in spinach from the Yuma and Salinas areas, respectively. Our findings provide preliminary evidence that growing region and preharvest temperature may impact the bacterial quality of spinach and thus could inform more targeted strategies to manage produce quality. [ABSTRACT FROM AUTHOR]

Biofilm formation is a common adaptation enabling bacteria to thrive in various environments and withstand external pressures. In the context of host-microbe interactions, biofilms play vital roles in establishing microbiomes associated with animals and plants and are used by opportunistic microbes to facilitate survival within hosts. Investigating biofilm dynamics, composition, and responses to environmental stressors is crucial for understanding microbial community assembly and biofilm regulation in health and disease. In this study, we explore in vivo colonization and in vitro biofilm formation abilities of core members of the honey bee (Apis mellifera) gut microbiota. Additionally, we assess the impact of glyphosate, a widely used herbicide with antimicrobial properties, and a glyphosate-based herbicide formulation on growth and biofilm formation in bee gut symbionts as well as in other biofilm-forming bacteria associated with diverse animals and plants. Our results demonstrate that several strains of core bee gut bacterial species can colonize the bee gut, which probably depends on their ability to form biofilms. Furthermore, glyphosate exposure elicits variable effects on bacterial growth and biofilm formation. In some instances, the effects correlate with the bacteria's ability to encode a susceptible or tolerant version of the enzyme inhibited by glyphosate in the shikimate pathway. However, in other instances, no such correlation is observed. Testing the herbicide formulation further complicates comparisons, as results often diverge from glyphosate exposure alone, suggesting that co-formulants influence bacterial growth and biofilm formation. These findings highlight the nuanced impacts of environmental stressors on microbial biofilms, with both ecological and host health-related implications. [ABSTRACT FROM AUTHOR]

Saccharomyces boulardii has been a subject of growing interest due to its potential as a probiotic microorganism with applications in gastrointestinal health, but the molecular cause for its probiotic potency has remained elusive. The recent discovery that S. boulardii contains unique mutations causing high acetic acid accumulation and inhibition of bacterial growth provides a possible clue. The natural S. boulardii isolates Sb.P and Sb.A are homozygous for the recessive mutation whi2S270* and accumulate unusually high amounts of acetic acid, which strongly inhibit bacterial growth. However, the homozygous whi2S270* mutation also leads to acetic acid sensitivity and acid sensitivity in general. In the present study, we have constructed a new S. boulardii strain, derived from the widely therapeutically used CMCN I-745 strain (isolated from the pharmaceutical product Enterol), producing even higher levels of acetic acid while keeping the same tolerance toward low pH as the parent Enterol (ENT) strain. This newly engineered strain, named ENT3, has a homozygous deletion of ACH1 and strong overexpression of ALD4. It is also able to accumulate much higher acetic acid concentrations when growing on low glucose levels, in contrast to the ENT wild-type and Sb.P strains. Moreover, we show the antimicrobial capacity of ENT3 against gut pathogens in vitro and observed that higher acetic acid production might correlate with better persistence in the gut in healthy mice. These findings underscore the possible role of the unique acetic acid production and its potential for improvement of the probiotic action of S. boulardii. [ABSTRACT FROM AUTHOR]

ABSTRACT Conventional Mycoplasma spp. diagnostics involve culture, often considered the gold standard in diagnostic test evaluation. However, culture protocols lack empirical derivation and primarily adhere to National Mastitis Council recommendations, tracing back to initial cultivation of Mycoplasma bovis. Despite a wide range of carbon dioxide (CO2) supplementation reported in literature, specific impacts of CO2 on Mycoplasma spp. growth remain unexplored. Our objective was to assess the effect of CO2 concentration on growth detection rates of 24 Mycoplasma spp. isolates from dairy cows. These isolates, mainly M. bovis, were incubated at 37°C in triplicate and three dilution ranges under three CO2 conditions: ambient air or 5% CO2 or 10% CO2. Bacterial growth was evaluated on incubation days 3, 5, 7, and 10. When cultured using ambient air, log10 cfu/mL was lower on days 3, 5, and 7 of incubation compared with isolates incubated in the recommended 5% or 10% CO2, with less variation observed in ambient air compared with 5% or 10% CO2. However, by 10 days of incubation, no differences in the detection of observable growth were noted among isolates incubated in ambient air, 5% CO2, or 10% CO2. Consequently, Mycoplasma spp. isolated from dairy cattle demonstrated growth after the recommended 7–10 days of culture, even in the absence of supplemental CO2. Given the expected concentration of M. bovis in (sub)clinical samples had similar concentrations to those used in our study, with the majority of isolates being M. bovis, we recommend expanding CO2 concentration ranges in M. bovis culture from 10% CO2 to ambient air when incubating for 10 days. However, the turnaround time could be shortened when incubating with supplemental CO2.IMPORTANCECurrent Mycoplasma spp. culture protocols lack empirical derivation concerning carbon dioxide (CO2) supplementation and are primarily based on the initial cultivation of Mycoplasma bovis. This study indicates that the suitable range for CO2 supplementation is broader than what is currently recommended by the National Mastitis Council for culturing within the specified 7–10 days. No differences in bacterial growth detection rates were observed among ambient air, 5% CO2, or 10% CO2 supplementation during the 7- and 10-day incubation intervals. These new insights provide evidence supporting the possibility of culturing Mycoplasma spp. under ambient air conditions in a laboratory setting.

Increases in Arctic temperatures have thawed permafrost and accelerated tundra soil microbial activity, releasing greenhouse gases that amplify climate warming. Warming over time has also accelerated shrub encroachment in the tundra, altering plant input abundance and quality, and causing further changes to soil microbial processes. To better understand the effects of increased temperature and the accumulated effects of climate change on soil bacterial activity, we quantified the growth responses of individual bacterial taxa to short-term warming (3 months) and long-term warming (29 years) in moist acidic tussock tundra. Intact soil was assayed in the field for 30 days using 18O-labeled water, from which taxon-specific rates of 18O incorporation into DNA were estimated as a proxy for growth. Experimental treatments warmed the soil by approximately 1.5°C. Short-term warming increased average relative growth rates across the assemblage by 36%, and this increase was attributable to emergent growing taxa not detected in other treatments that doubled the diversity of growing bacteria. However, long-term warming increased average relative growth rates by 151%, and this was largely attributable to taxa that co-occurred in the ambient temperature controls. There was also coherence in relative growth rates within broad taxonomic levels with orders tending to have similar growth rates in all treatments. Growth responses tended to be neutral in short-term warming and positive in long-term warming for most taxa and phylogenetic groups co-occurring across treatments regardless of phylogeny. Taken together, growing bacteria responded distinctly to short-term and long-term warming, and taxa growing in each treatment exhibited deep phylogenetic organization. IMPORTANCE Soil carbon stocks in the tundra and underlying permafrost have become increasingly vulnerable to microbial decomposition due to climate change. The microbial responses to Arctic warming must be understood in order to predict the effects of future microbial activity on carbon balance in a warming Arctic. In response to our warming treatments, tundra soil bacteria grew faster, consistent with increased rates of decomposition and carbon flux to the atmosphere. Our findings suggest that bacterial growth rates may continue to increase in the coming decades as faster growth is driven by the accumulated effects of long-term warming. Observed phylogenetic organization of bacterial growth rates may also permit taxonomy-based predictions of bacterial responses to climate change and inclusion into ecosystem models. [ABSTRACT FROM AUTHOR]

Traditional antibiotics target essential cellular components or metabolic pathways conserved in both pathogenic and nonpathogenic bacteria. Unfortunately, long-term antibiotic use often leads to antibiotic resistance and disruption of the overall microbiota. In this work, we identified a phenylamino acetamide compound, named 187R, that strongly inhibited the expression of the type III secretion system (T3SS) encoding genes and the secretion of the T3SS effector proteins in Pseudomonas aeruginosa. T3SS is an important virulence factor, as T3SS-deficient strains of P. aeruginosa are greatly attenuated in virulence. We further showed that 187R had no effect on bacterial growth, implying a reduced selective pressure for the development of resistance. 187R-mediated repression of T3SS was dependent on ExsA, the master regulator of T3SS in P. aeruginosa. The impact of 187R on the host-associated microbial community was also tested using the Arabidopsis thaliana phyllosphere as a model. Both culture-independent (Illumina sequencing) and culture-dependent (Biolog) methods showed that the application of 187R had little impact on the composition and function of microbial community compared to the antibiotic streptomycin. Together, these results suggested that compounds that target virulence factors could serve as an alternative strategy for disease management caused by bacterial pathogens. [ABSTRACT FROM AUTHOR]

ABSTRACT The growth and activity of bacteria have been extensively studied in nearly every environment on Earth, but there have been limited studies focusing on the air. Suspended bacteria (outside of water droplets) may stay in the atmosphere for time frames that could allow for growth on volatile compounds, including the potent greenhouse gas methane. We investigated the ability of aerosolized methanotrophic bacteria to grow on methane in the airborne state in rotating gas-phase bioreactors. The physical half-life of the aerial bacterium-sized particles was 3 days. To assess the potential for airborne growth, gas-phase bioreactors containing the aerosolized cultures were amended with 1,500 ppmv 13CH4 or 12CH4. Three of seven experiments demonstrated 13C incorporation into DNA, indicating growth in air. Bacteria associated with the genera Methylocystis and Methylocaldum were detected in 13C-DNA fractions, thus indicating that they were synthesizing new DNA, suggesting growth in air. We conclude that methanotrophs outside of water droplets in the air can potentially grow under certain conditions. Based on our data, humidity seems to be a major limitation to bacterial growth in air. Furthermore, low biomass levels can pose problems for detecting 13C-DNA synthesis in our experimental system. IMPORTANCE Currently, the cellular activities of bacteria in the airborne state outside of water droplets have not been heavily studied. Evidence suggests that these airborne bacteria produce ribosomes and metabolize gaseous compounds. Despite having a potentially important impact on atmospheric chemistry, the ability of bacteria in the air to metabolize substrates such as methane is not well understood. Demonstrating that bacteria in the air can metabolize and grow on substrates will expand knowledge about the potential activities and functions of the atmospheric microbiome. This study provides evidence for DNA synthesis and, ultimately, growth of airborne methanotrophs.

ABSTRACT Urinary tract infections (UTIs), mostly caused by uropathogenic E. coli (UPEC), affect most women, and often recur. Genomic and transcriptomic analyses have not identified a common set of virulence genes, which has suggested complex host-pathogen interactions and multiple virulence mechanisms. One aspect of the host-pathogen interaction is rapid UPEC growth in urine in vivo. When bacterial growth in urine is studied in vitro, urine is pooled, which is assumed to diminish individual variation. We grew one nonpathogenic and two pathogenic E. coli strains in urine from individuals who never had a UTI, had a UTI history but no current infection, and had a UTI history with a current infection. Bacterial growth showed large variations in individual urine samples, and pooled urine often supported significantly more growth than the average growth from individual urine samples. Total nutrient content tended to be higher in current group urine samples than the never and history grouped samples urine. We propose that pooling optimizes a nutrient mixture in the never and history group urine samples, which are often studied, whereas urine from current group individuals may have a more optimal nutrient mixture because of additional nutrient sources. We conclude that a pooled urine is not “an average urine sample,” and that the best comparisons of results between labs using pooled urine would also include results with a standardized synthetic urine. IMPORTANCE Urinary tract infections (UTIs) will affect most women, can recur especially in postmenopausal women, and can become antibiotic recalcitrant. Escherichia coli causes most community-acquired UTIs and recurrent UTIs. Current theories of virulence, based on studies of UTI-associated E. coli, propose multiple virulence mechanisms and complex host-pathogen interactions. Studies of bacterial growth in urine samples—one aspect of the host-pathogen interaction—invariably involve pooled urine that are assumed to eliminate variations between individuals. Our results show that a pooled urine is not necessarily an average urine sample, and we suggest that quantitative and qualitative variations in nutrient content are the basis for this discrepancy. Knowledge of growth-promoting urinary components is important for understanding host-pathogen interactions during UTIs and could contribute to developing nonantibiotic-based therapies.

ABSTRACT The life-threatening disease tularemia is caused by Francisella tularensis, an intracellular Gram-negative bacterial pathogen. Due to the high mortality rates of the disease, as well as the low respiratory infectious dose, F. tularensis is categorized as a Tier 1 bioterror agent. The identification and isolation from clinical blood cultures of F. tularensis are complicated by its slow growth. Iron was shown to be one of the limiting nutrients required for F. tularensis metabolism and growth. Bacterial growth was shown to be restricted or enhanced in the absence or addition of iron. In this study, we tested the beneficial effect of enhanced iron concentrations on expediting F. tularensis blood culture diagnostics. Accordingly, bacterial growth rates in blood cultures with or without Fe2+ supplementation were evaluated. Growth quantification by direct CFU counts demonstrated significant improvement of growth rates of up to 6 orders of magnitude in Fe2+-supplemented media compared to the corresponding nonmodified cultures. Fe2+ supplementation significantly shortened incubation periods for successful diagnosis and isolation of F. tularensis by up to 92 h. This was achieved in a variety of blood culture types in spite of a low initial bacterial inoculum representative of low levels of bacteremia. These improvements were demonstrated with culture of either Francisella tularensis subsp. tularensis or subsp. holarctica in all examined commercial blood culture types routinely used in a clinical setup. Finally, essential downstream identification assays, such as matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF-MS), immunofluorescence, or antibiotic susceptibility tests, were not affected in the presence of Fe2+. To conclude, supplementing blood cultures with Fe2+ enables a significant shortening of incubation times for F. tularensis diagnosis, without affecting subsequent identification or isolation assays. IMPORTANCE In this study, we evaluated bacterial growth rates of Francisella tularensis strains in iron (Fe)-enriched blood cultures as a means of improving and accelerating bacterial growth. The shortening of the culturing time should facilitate rapid pathogen detection and isolation, positively impacting clinical diagnosis and enabling prompt onset of efficient therapy.

ABSTRACT The nasopharyngeal commensal Streptococcus pneumoniae can become invasive and cause metastatic infection. This requires the pneumococcus to have the ability to adapt, grow, and reside in diverse host environments. Therefore, we studied whether the likelihood of severe disease manifestations was related to pneumococcal growth kinetics. For 383 S. pneumoniae blood isolates and 25 experimental mutants, we observed highly reproducible growth curves in nutrient-rich medium. The derived growth features were lag time, maximum growth rate, maximum density, and stationary-phase time before lysis. First, the pathogenicity of each growth feature was probed by comparing isolates from patients with and without marked preexisting comorbidity. Then, growth features were related to the propensity of causing severe manifestations of invasive pneumococcal disease (IPD). A high maximum bacterial density was the most pronounced pathogenic growth feature, which was also an independent predictor of 30-day mortality (P = 0.03). Serotypes with an epidemiologically higher propensity for causing meningitis displayed a relatively high maximum density (P

Iron is crucial for bacterial growth and virulence. Under iron-deficiency bacteria produce siderophores, iron chelators that facilitate the iron uptake into the cell via specific receptors. Erwinia amylovora, the causative agent of fire blight, produces hydroxamate-type desferrioxamine siderophores (DFO). The presented study reassesses the impact of DFO as a virulence factor of E. amylovora during its epiphytic phase on the apple flower. When inoculated in semisterile Golden Delicious flowers no difference in replication and induction of calyx necrosis could be observed between E. amylovora CFBP1430 siderophore synthesis (DfoA) or uptake (FoxR receptor) mutants and the parental strain. In addition, mutant strains only weakly induced a foxR promoter-gfpmut2 reporter construct in the flowers. When analyzing the replication of the receptor mutant in apple flowers harboring an established microbiome, either naturally, in case of orchard flowers, or by pre-inoculation of semisterile greenhouse flowers, it became evident that the mutant strain had a significantly reduced replication compared to the parental strain. The results suggest that apple flowers per se are not an iron-limiting environment for E. amylovora and that DFO is an important competition factor for the pathogen in precolonized flowers. [ABSTRACT FROM AUTHOR]

Legionella species are facultative intracellular pathogens that cause a lifethreatening pneumonia termed Legionnaires’ disease. Legionella pneumophila employs the Lqs-LvbR (Legionella quorum sensing-Legionella virulence and biofilm regulator) network to regulate virulence and motility, but its role for growth in media is illdefined. Here, we report that compared to the L. pneumophila reference strain JR32, a DlqsR mutant showed a reduced lag phase at 30°C and reached a higher cell density at 45°C, while the DlqsA, DlqsS, and DlqsT mutants showed a longer lag phase and reached a lower cell density. A DlvbR mutant resumed growth like the parental strain at 30°C but exhibited a substantially reduced cell density at 45°C. Thus, LvbR is an important cell density regulator at elevated temperatures. Environmental and clinical L. pneumophila strains grew in N-(2-acetamido)-2-aminoethanesulfonic acid (ACES)-buffered yeast extract (AYE) medium after distinct lag phases with similar rates at 30°C, reached different cell densities at the optimal growth temperature of 40°C, and no longer grew at 50°C. Legionella longbeachae reached a rather low cell density at 40°C and did not grow at and beyond 45°C. Genes encoding components of the Lqs-LvbR network were present in the genomes of the environmental and clinical L. pneumophila isolates, and upon growth at 30°C or 45°C, the PlqsR, PlqsA, PlqsS, and PlvbR promoters from strain JR32 were expressed in these strains with distinct patterns. Taken together, our results indicate that the Lqs-LvbR network governs the temperature- dependent growth onset and cell density of the L. pneumophila reference strain JR32 and possibly also of environmental and clinical L. pneumophila isolates. IMPORTANCE Environmental bacteria of the genus Legionella are the causative agents of the severe pneumonia Legionnaires’ disease, the incidence of which is on the rise worldwide. Legionella pneumophila and Legionella longbeachae are the clinically most relevant species. The opportunistic pathogens are inhaled through contaminated aerosols and replicate in human lung macrophages with a mechanism similar to that in their natural hosts, free-living amoebae. Given their prevalence in natural and technical water systems, an efficient control of Legionella spp. by physical, chemical, or biological means will reduce the incidence of Legionnaires’ disease. Here, we show that the Legionella quorum sensing (Lqs) system and the pleiotropic transcription factor LvbR govern the temperature-dependent growth onset and cell density of bacterial cultures. Hence, the growth of L. pneumophila in water systems is determined not only by the temperature and nutrient availability but also by quorum sensing, i.e., density- and signaling molecule-dependent gene regulation. [ABSTRACT FROM AUTHOR]

In this paper, a simple numerical procedure is presented to monitor the growth of Streptococcus sanguinis over time in the absence and presence of propolis, a natural antimicrobial. In particular, it is shown that the real-time decomposition of growth curves obtained through optical density measurements into growth rate and acceleration can be a powerful tool to precisely assess a large range of key parameters (i.e., lag time [t0], starting growth rate [g0], initial acceleration of the growth [a0], maximum growth rate [gmax], maximum acceleration [amax], and deceleration [amin] of the growth and the total number of cells at the beginning of the saturation phase [Ns]) that can be readily used to fully describe growth over time. Consequently, the procedure presented provides precise data of the time course of the different growth phases and features, which is expected to be relevant, for instance, to thoroughly evaluate the effect of new antimicrobial agents. It further provides insight into predictive microbiology, likely having important implications for assumptions adopted in mathematical models to predict the progress of bacterial growth. IMPORTANCE The new and simple numerical procedure presented in this paper to analyze bacterial growth will possibly allow the identification of true differences in efficacy among antimicrobial drugs for their applications in human health, food security, and the environment, among others. It further provides insight into predictive microbiology, likely helping in the development of proper mathematical models to predict the course of bacterial growth under diverse circumstances. [ABSTRACT FROM AUTHOR]

ABSTRACT Tranexamic acid (TXA) is extensively used in orthopedic surgery and traumatology as an antifibrinolytic agent to control intra- and postoperative bleeding and, therefore, indirectly, to reduce postsurgery infection rates. The hypothesis of an additional antibiotic effect against microorganisms associated with periprosthetic joint infection needs to be further evaluated. We aimed to assess whether TXA could reduce bacterial growth using an in vitro model. ATCC and clinical strains of staphylococci and Cutibacterium acnes were tested against TXA in both planktonic and sessile forms. We recorded the percent reduction in the following variables: log CFU/mL by microbiological culture, percentage of live cells by confocal laser scanning microscopy, and, additionally in sessile cells, metabolic activity by the 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide salt (XTT) assay. Variables were compared between groups using the Kruskal-Wallis test, and the results were reported as median (interquartile range [IQR]). Statistical significance was set at a P value of

The aim of this study was to investigate the temporal stability of microbial contamination during cheddar cheese production by examining patterns of nonstarter bacteria in 60-day aged cheddar collected from the start and end of 30 consecutive production days. Further, we explored the source of these temporal microbial variations by comparing microbial communities in the aged cheese to those on food contact surfaces from a piece of cheesemaking equipment previously identified as a major source of nonstarter bacteria in the same processing environment. 16S rRNA metabarcoding and culture-based sequencing methods identified two Streptococcus sequence variants significantly associated with the end of the production day in both the aged cheese and the cheese processing environment. Closer inspection of these sequence variants in the aged cheese over the 40-day sampling period revealed sinusoidal-like fluctuations in their relative ratios, which appeared to coincide with the Lactococcus starter rotation schedule. These results demonstrate that the microbial composition of finished cheese can vary according to the timing of processing within a production day. Further, our results demonstrate that time-of-day microbial differences in cheese can result from bacterial growth on food contact surfaces and that the composition of these microbial differences is subject to change day-to-day and may be linked to routine changes in the Lactococcus starter culture. [ABSTRACT FROM AUTHOR]

Cupriavidus necator H16 is an ideal strain for polyhydroxybutyrate (PHB) production from CO2. Low-oxygen stress can induce PHB synthesis in C. necator H16 while reducing bacterial growth under chemoautotrophic culture. The optimum growth and PHB synthesis of C. necator H16 cannot be achieved simultaneously, which restricts PHB production. The present study was initiated to address the issue through comparative transcriptome and gene function analysis. First, the comparative transcriptome of C. necator H16 chemoautotrophically cultured under low-oxygen stress and nonstress conditions was studied. Three types of genes were discovered to have differential levels of transcription: those involving PHB enzymatic synthesis, PHB granulation, and regulators. Under low-oxygen stress conditions, acetoacetyl-coenzyme A (CoA) reductase gene phaB2, PHB synthase gene phaC2, phasins genes phaP1 and phaP2, and regulator genes uspA and rpoN were upregulated 3.0-, 2.5-, 1.8-, 2.7-, 3.5-, and 1.6-fold, respectively. Second, the functions of upregulated genes and their applications in PHB synthesis were further studied. It was found that the overexpression of phaP1, phaP2, uspA, and rpoN can induce PHB synthesis under nonstress conditions, while phaB2 and phaC2 have no significant effect. Under the optimum conditions, the PHB percentage content in C. necator H16 was increased by 37.2%, 28.4%, 15.8%, and 41.0%, respectively, with overexpression of phaP1, phaP2, uspA, and rpoN, and the corresponding PHB production increased by 49.8%, 42.9%, 47.0%, and 77.5%, respectively, under nonstress chemoautotrophic conditions. Similar promotion by phaP1, phaP2, uspA, and rpoN was observed in heterotrophically cultured C. necator H16. The PHB percentage content and PHB production were increased by 54.4% and 103.1%, respectively, with the overexpression of rpoN under nonstress heterotrophic conditions. [ABSTRACT FROM AUTHOR]

The mechanisms controlling entry into and exit from the death phase in the bacterial life cycle remain unclear. Although bacterial growth studies in batch cultures traditionally focus on the first three phases during incubation, two additional phases, the death phase and the long-term stationary phase, are less understood. Although there are a number of stressors that arise during long-term batch culture, including nutrient depletion and the accumulation of metabolic toxins such as reactive oxidative species, their roles in cell death are not well-defined. By manipulating the environmental conditions of Escherichia coli incubated in long-term batch culture through chemical and mechanical means, we investigated the role of volatile metabolic toxins in modulating the onset of the death phase. Here, we demonstrate that with the introduction of substrates with high binding affinities for volatile compounds, toxic by-products of normal cell metabolism, into the headspace of batch cultures, cells display a prolonged stationary phase and delayed entry into the death phase. The addition of these substrates allows cultures to maintain a high cell density for hours to days longer than cultures incubated under standard growth conditions. A similar effect is observed when the gaseous headspace in culture flasks is continuously replaced with sterile air, mechanically preventing the accumulation of metabolic by-products in batch cultures. We establish that toxic compound(s) are produced during the exponential phase, demonstrate that buildup of toxic by-products influence entry into the death phase, and present a novel tool for improving high-density growth in batch culture that may be used in future research or industrial or biotechnology applications. [ABSTRACT FROM AUTHOR]

The suitability of stable isotope probing (SIP) and Raman microspectroscopy to measure growth rates of heterotrophic bacteria at the single-cell level was evaluated. Label assimilation into Escherichia coli biomass during growth on a complex 13C-labeled carbon source was monitored in time course experiments. 13C incorporation into various biomolecules was measured by spectral "red shifts" of Raman-scattered emissions. The 13C- and 12C-isotopologues of the amino acid phenylalanine (Phe) proved to be quantitatively accurate reporter molecules of cellular isotopic fractional abundances (fcell). Values of fcell determined by Raman microspectroscopy and independently by isotope ratio mass spectrometry (IRMS) over a range of isotopic enrichments were statistically indistinguishable. Progressive labeling of Phe in E. coli cells among a range of 13C/12C organic substrate admixtures occurred predictably through time. The relative isotopologue abundances of Phe determined by Raman spectral analysis enabled the accurate calculation of bacterial growth rates as confirmed independently by optical density (OD) measurements. The results demonstrate that combining SIP and Raman microspectroscopy can be a powerful tool for studying bacterial growth at the single-cell level on defined or complex organic 13C carbon sources, even in mixed microbial assemblages. [ABSTRACT FROM AUTHOR]

Nutritional dependencies, especially those regarding nitrogen sources, govern numerous microbial positive interactions. As for lactic acid bacteria (LAB), responsible for the sanitary, organoleptic, and health properties of most fermented products, such positive interactions have previously been studied between yogurt bacteria. However, they have never been exploited to create artificial cocultures of LAB that would not necessarily coexist naturally, i.e., from different origins. The objective of this study was to promote LAB positive interactions, based on nitrogen dependencies in cocultures, and to investigate how these interactions affect some functional outputs, e.g., acidification rates, carbohydrate consumption, and volatile-compound production. The strategy was to exploit both proteolytic activities and amino acid auxotrophies of LAB. A chemically defined medium was thus developed to specifically allow the growth of six strains used, three proteolytic and three nonproteolytic. Each of the proteolytic strains, Enterococcus faecalis CIRM-BIA2412, Lactococcus lactis NCDO2125, and CIRM-BIA244, was cocultured with each one of the nonproteolytic LAB strains, L. lactis NCDO2111 and Lactiplantibacillus plantarum CIRM-BIA465 and CIRM-BIA1524. Bacterial growth was monitored using compartmented chambers to compare growth in mono- and cocultures. Acidification, carbohydrate consumption, and volatile-compound production were evaluated in direct cocultures. Each proteolytic strain induced different types of interactions: strongly positive interactions, weakly positive interactions, and no interactions were seen with E. faecalis CIRM-BIA2412, L. lactis NCDO2125, and L. lactis CIRM-BIA244, respectively. Strong interactions were associated with higher concentrations of tryptophan, valine, phenylalanine, leucine, isoleucine, and peptides. They led to higher acidification rates, lower pH, higher raffinose utilization, and higher concentrations of five volatile compounds. [ABSTRACT FROM AUTHOR]

The common cooccurrence of antibiotics and phages in both natural and engineered environments underscores the need to understand their interactions and implications for bacterial control and antibiotic resistance propagation. Here, aminoglycoside antibiotics that inhibit protein synthesis (e.g., kanamycin and neomycin) impeded the replication of coliphage T3 and Bacillus phage BSP, reducing their infection efficiency and mitigating their hindrance of bacterial growth, biofilm formation, and tolerance to antibiotics. For example, treatment with phage T3 reduced subsequent biofilm formation by Escherichia coli liquid cultures to 53%65% of that of the no-phage control, but a smaller reduction of biofilm formation (89%610%) was observed for combined exposure to phage T3 and kanamycin. Despite sharing a similar mode of action with aminoglycosides (i.e., inhibiting protein synthesis) and antagonizing phage replication, albeit to a lesser degree, tetracyclines did not inhibit bacterial control by phages. Phage T3 combined with tetracycline showed higher suppression of biofilm formation than when combined with aminoglycosides (25%66% of the no-phage control). The addition of phage T3 to E. coli suspensions with tetracycline also suppressed the development of tolerance to tetracycline. However, this suppression of antibiotic tolerance development disappeared when tetracycline was replaced with 3 mg/liter kanamycin, corroborating the greater antagonism with aminoglycosides. Overall, this study highlights this overlooked antagonistic effect on phage proliferation, which may attenuate phage suppression of bacterial growth, biofilm formation, antibiotic tolerance, and maintenance of antibiotic resistance genes. IMPORTANCE The coexistence of residual antibiotics and phages is common in many environments, which underscores the need to understand their interactive effects on bacteria and the implications for antibiotic resistance propagation. Here, aminoglycosides acting as bacterial protein synthesis inhibitors impeded the replication of various phages. This alleviated the suppressive effects of phages against bacterial growth and biofilm formation and diminished bacterial fitness costs that suppress the emergence of tolerance to antibiotics. We show that changes in bacteria caused by environmentally relevant concentrations of sublethal antibiotics can affect phage-host dynamics that are commonly overlooked in vitro but can result in unexpected environmental consequences. [ABSTRACT FROM AUTHOR]

Inoculation studies are important when assessing microbial survival and growth in food products. These studies typically involve the pregrowth of multiple strains of a target pathogen under a single condition; this emphasizes strain diversity. To gain a better understanding of the impacts of strain diversity ("nature") and pregrowth conditions ("nurture") on subsequent bacterial growth in foods, we assessed the growth and survival of Salmonella enterica (n = 5), Escherichia coli (n = 6), and Listeria (n = 5) inoculated onto tomatoes, precut lettuce, and cantaloupe rind, respectively. Pregrowth conditions included (i) 37°C to stationary phase (baseline), (ii) low pH, (iii) high salt, (iv) reduced water activity, (v) log phase, (vi) minimal medium, and (vii) 21°C. Inoculated tomatoes were incubated at 21°C; lettuce and cantaloupe were incubated at 7°C. Bacterial counts were assessed over three phases, including initial reduction (phase 1), change in bacterial numbers over the first 24 h of incubation (phase 2), and change over the 7-day incubation (phase 3). E. coli showed overall decline in counts (,1 log) over the 7-day period, except for a, 1-log increase after pregrowth in high salt and to mid-log phase. In contrast, S. enterica and Listeria showed regrowth after an initial reduction. Pregrowth conditions had a substantial and significant effect on all three phases of S. enterica and E. coli population dynamics on inoculated produce, whereas strain did not show a significant effect. For Listeria, both pregrowth conditions and strain affected changes in phase 2 but not phases 1 and 3. IMPORTANCE Our findings suggest that inclusion of multiple pregrowth conditions in inoculation studies can best capture the range of growth and survival patterns expected for Salmonella enterica and Escherichia coli present on produce. This is particularly important for fresh and fresh-cut produce, where stress conditions encountered by pathogens prior to contamination can vary widely, making selection of a typical pregrowth condition virtually impossible. Pathogen growth and survival data generated using multiple pregrowth conditions will allow for more robust microbial risk assessments that account more accurately for uncertainty. [ABSTRACT FROM AUTHOR]

Listeria monocytogenes causes the severe foodborne illness listeriosis and survives in food-associated environments due to its high stress tolerance. A data assembly and analysis protocol for microbial growth experiments was compiled to elucidate the strain variability of L. monocytogenes stress tolerance. The protocol includes measurement of growth ability under stress (step 1), selection of a suitable method for growth parameter calculation (step 2), comparison of growth patterns between strains (step 3), and biological interpretation of the discovered differences (step 4). In step 1, L. monocytogenes strains (n=388) of various serovars and origins grown on media with 9.0% NaCl were measured using a Bioscreen C microbiology reader. Technical variability of the growth measurements was assessed and eliminated. In step 2, the growth parameters determined by Gompertz, modified- Gompertz, logistic, and Richards models and model-free splines were compared, illustrating differences in the suitability of these methods to describe the experimental data. In step 3, hierarchical clustering was used to describe the NaCl tolerance of L. monocytogenes measured by strain-specific variation in growth ability; tolerant strains had higher growth rates and maximum optical densities and shorter lag phases than susceptible strains. The spline parameter area under the curve best classified "poor," "average," and "good" growers. In step 4, the tested L. monocytogenes lineage I strains (serovars 4b and 1/2b) proved to be significantly more tolerant toward 9.0% NaCl than lineage II strains (serovars 1/2a, 1/2c, and 3a). Our protocol provides systematic tools to gain comparable data for investigating strain-specific variation of bacterial growth under stress. [ABSTRACT FROM AUTHOR]

The biosynthesis of histidine, a proteinogenic amino acid, has been extensively studied due to its importance in bacterial growth and survival. Histidinol-phosphate phosphatase (Hol-Pase), which is responsible for the penultimate step of histidine biosynthesis, is generally the last enzyme to be characterized in many bacteria because its origin and evolution are more complex compared to other enzymes in histidine biosynthesis. However, none of the enzymes in histidine biosynthesis, including Hol-Pase, have been characterized in Pseudomonas aeruginosa, which is an important opportunistic gram-negative pathogen that can cause serious human infections. In our previous work, a transposon mutant of P. aeruginosa was found to display a growth defect on glucose-containing minimal solid medium. In this study, we found that the growth defect was due to incomplete histidine auxotrophy caused by PA0335 inactivation. Subsequently, PA0335 was shown to encode Hol-Pase, and its function and enzymatic activity were investigated using genetic and biochemical methods. In addition to PA0335, the roles of 12 other predicted genes involved in histidine biosynthesis in P. aeruginosa were examined. Among them, hisC2(PA3165), hisH2(PA3152), and hisF2(PA3151) were found to be dispensable for histidine synthesis, whereas hisG(PA4449), hisE(PA5067), hisF1(PA5140), hisB(PA5143), hisI(PA5066), hisC1(PA4447), and hisA(PA5141) were essential because deletion of each resulted in complete histidine auxotrophy; similar to PA0335, hisH1(PA5142) or hisD(PA4448) deletion caused incomplete histidine auxotrophy. Taken together, our results outline the histidine synthesis pathway of P. aeruginosa. [ABSTRACT FROM AUTHOR]

ABSTRACT The global regulatory protein CsrA coordinates gene expression in response to physiological cues reflecting cellular stress and nutrition. CsrA binding to the 5′ segments of mRNA targets affects their translation, RNA stability, and/or transcript elongation. Recent studies identified probable mRNA targets of CsrA that are involved in iron uptake and storage in Escherichia coli, suggesting an unexplored role for CsrA in regulating iron homeostasis. Here, we assessed the impact of CsrA on iron-related gene expression, cellular iron, and growth under various iron levels. We investigated five new targets of CsrA regulation, including the genes for 4 ferritin or ferritin-like iron storage proteins (ISPs) and the stress-inducible Fe-S repair protein, SufA. CsrA bound with high affinity and specificity to ftnB, bfr, and dps mRNAs and inhibited their translation, while it modestly activated ftnA expression. Furthermore, CsrA was found to regulate cellular iron levels and support growth by repressing the expression of genes for ISPs, most importantly, ferritin B (FtnB) and bacterioferritin (Bfr). Iron starvation did not substantially affect cellular levels of CsrA or its small RNA (sRNA) antagonists, CsrB and CsrC. csrA disruption led to increased resistance to the lethal effects of H2O2 during exponential growth, consistent with a regulatory role in oxidative stress resistance. We propose that during exponential growth and under minimal stress, CsrA represses the deleterious expression of the ISPs that function under oxidative stress and stationary-phase conditions (FtnB, Bfr, and Dps), thus ensuring that cellular iron is available to processes that are required for growth. IMPORTANCE Iron is an essential micronutrient for nearly all living organisms but is toxic in excess. Consequently, the maintenance of iron homeostasis is a critical biological process, and the genes involved in this function are tightly regulated. Here, we explored a new role for the bacterial RNA binding protein CsrA in the regulation of iron homeostasis. CsrA was shown to be a key regulator of iron storage genes in Escherichia coli, with consequential effects on cellular iron levels and growth. Our findings establish a model in which robust CsrA activity during the exponential phase of growth leads to repression of genes whose products sequester iron or divert it to unnecessary stress response processes. In so doing, CsrA supports E. coli growth under iron-limiting laboratory conditions and may promote fitness in the competitive iron-limited environment of the host large intestine.

Listeria monocytogenes, the causative agent of the serious food-borne disease listeriosis, can rapidly adapt to a wide-range of environmental stresses, including visible light. This study shows that exposure of the L. monocytogenes EGDe strain to low-intensity broad spectrum visible light inhibited bacterial growth and caused altered multicellular behavior during growth on semi-solid agar as compared to when the bacteria were grown in complete darkness. These light-dependent changes were observed regardless of the presence of the blue light receptor (Lmo0799) and the stressosome regulator Sigma B (SigB), which have been suggested to be important for the ability of L. monocytogenes to respond to blue light. A genome wide transcriptional analysis revealed that exposure of L. monocytogenes EGDe to broad- spectrum visible light caused altered expression of 2409 genes belonging to 18 metabolic pathways, as compared to bacteria grown in darkness. The light-dependent differentially expressed genes are involved in functions such as glycan metabolism, cell wall synthesis, chemotaxis, flagellar synthesis and resistance to oxidative stress. Exposure to light conferred reduced bacterial motility in semi-solid agar, which correlates well with the light-dependent reduction in transcript levels of flagellar and chemotaxis genes. Similar light-induced reduction in growth and motility was also observed in two different L. monocytogenes food isolates, suggesting that these responses are typical for L. monocytogenes. Together, the results show that even relatively small doses of broad-spectrum visible light causes genome-wide transcriptional changes, reduced growth and motility in L. monocytogenes. [ABSTRACT FROM AUTHOR]

Differentiating between contamination and the genuine presence of 16S rRNA genes in gestational tissue samples is the gold standard for supporting the in utero colonization hypothesis. During gestation, the fetus undergoes signifi- cant physiological changes that may be directly affected by maternal colonization of key bacterial genera. In this study, lab benches, necropsy tables, and air ducts were swabbed at the same time as clinical sampling. The relative and absolute abundance of bacteria present in sheep samples was determined by culture-independent and culture-dependent means. Of 14 healthy pregnant ewes, there was no evidence of any bacteria in the fetal liver, spleen, or brain cortex using culture-independent techniques despite evidence of the presence of bacteria in various locations of the necropsy room used for 11 of these 14 sheep. Of the 336 bacterial genera found in the room swabs, only 12 (5%) were also found in the saliva and vaginal swabs among the three ewes for which bacteria were detected. These 12 taxa represent 1.32% of the relative abundance and approximately 393 16S rRNA copies/swab in these three ewes. Using careful necropsy protocols, bacterial contamination of sheep tissues was avoided. Contamination of saliva and vaginal samples was limited to less than 2% of the bacterial population. [ABSTRACT FROM AUTHOR]

In this work, we shed light on the metabolism of dihydroxyacetone (DHA), a versatile, ubiquitous and important intermediate for various chemicals in industry, by analysing its metabolism at the system level in E. coli. Using constraints based modelling, we show that the growth of E. coli on DHA is suboptimal and we identify the potential causes. NMR analysis shows that DHA is degraded non- enzymatically into substrates known to be unfavourable to high growth rates. Transcriptomic analysis reveals that DHA promotes genes involved in biofilm formation, which may reduce the bacterial growth rate. Functional analysis of the genes involved in DHA metabolism proves that under the aerobic conditions used in this study, DHA is mainly assimilated via the dihydroxyacetone kinase pathway. In addition, these results show that the alternative routes of DHA assimilation (i.e. the glycerol and fructose-6-phosphate aldolase pathways) are not fully activated under our conditions because of anaerobic mediated hierarchical control. These pathways are therefore certainly unable to sustain fluxes as high as the ones predicted in silico for optimal aerobic growth on DHA. Overexpressing some of the genes in these pathways releases these constraints and restores the predicted optimal growth on DHA. IMPORTANCE: Dihydroxyacetone (DHA) is an attractive triose molecule with a wide range of applications, notably in cosmetics and the food and pharmaceutical industries. DHA is found in many species from microorganisms to humans and can be used by Escherichia coli as growth substrate. However, knowledge about the mechanisms and regulation of this process is currently lacking, motivating our investigation of DHA metabolism in E. coli. We show that in aerobic conditions, E. coli growth on DHA is far from optimal and is hindered by chemical, hierarchical and possibly allosteric constraints. We show that optimal growth on DHA can be restored by releasing the hierarchical constraint. These results improve our understanding of DHA metabolism and are likely to help unlock biotechnological applications involving DHA as an intermediate, such as the bioconversion of glycerol or C1 substrates into value added chemicals. [ABSTRACT FROM AUTHOR]

Adaptation to osmotic stress is crucial for bacterial growth and survival in changing environments. Although a large number of osmotic stress response genes have been identified in various bacterial species, how osmotic changes affect bacterial motility, biofilm formation, and colonization of host niches remains largely unknown. In this study, we report that the LrhA regulator is an osmoregulated transcription factor that directly binds to the promoters of the flhDC, eps, and opgGH operons and differentially regulates their expression, thus inhibiting motility and promoting exopolysaccharide (EPS) production, synthesis of osmoregulated periplasmic glucans (OPGs), biofilm formation, and root colonization of the plant growth-promoting bacterium Pantoea alhagi LTYR-11Z. Further, we observed that the LrhA-regulated OPGs control RcsCD-RcsB activation in a concentration-dependent manner, and a high concentration of OPGs induced by increased medium osmolarity is maintained to achieve the high level of activation of the Rcs phosphorelay, which results in enhanced EPS synthesis and decreased motility in P. alhagi. Moreover, we showed that the osmosensing regulator OmpR directly binds to the promoter of lrhA and promotes its expression, while lrhA expression is feedback inhibited by the activated Rcs phosphorelay system. Overall, our data support a model whereby P. alhagi senses environmental osmolarity changes through the EnvZ-OmpR two-component system and LrhA to regulate the synthesis of OPGs, EPS production, and flagellum-dependent motility, thereby employing a hierarchical signaling cascade to control the transition between a motile lifestyle and a biofilm lifestyle. IMPORTANCE Many motile bacterial populations form surface-attached biofilms in response to specific environmental cues, including osmotic stress in a range of natural and host-related systems. However, cross talk between bacterial osmosensing, swimming, and biofilm formation regulatory networks is not fully understood. Here, we report that the pleiotropic regulator LrhA in Pantoea alhagi is involved in the regulation of flagellar motility, biofilm formation, and host colonization and responds to osmotic upshift. We further show that this sensing relies on the EnvZ-OmpR two-component system that was known to detect changes in external osmotic stress. The EnvZ-OmpR-LrhA osmosensing signal transduction cascade is proposed to increase bacterial fitness under hyperosmotic conditions inside the host. Our work proposes a novel regulatory mechanism that links osmosensing and motile-sessile lifestyle transitions, which may provide new approaches to prevent or promote the formation of biofilms and host colonization in P. alhagi and other bacteria possessing a similar osmoregulatory mechanism. [ABSTRACT FROM AUTHOR]

Rhodococcus equi is a leading cause of severe pneumonia in foals. Standard treatment is dual antimicrobial therapy with a macrolide and rifampin, but the emergence of macrolide- and rifampin-resistant R. equi isolates is an increasing problem. The objective of this study was to determine the effect of macrolide and/or rifampin resistance on fitness of R. equi. Three unique isogenic sets were created, each consisting of four R. equi strains: a susceptible parent isolate, strains resistant to macrolides or rifampin, and a dual macrolide- and rifampin resistant strain. Each isogenic set's bacterial growth curve was generated in enriched medium, minimal medium (MM) and minimal medium without iron (MM-I). Bacterial survival in soil was analyzed over 12 months at -20°C, 4°C, 25°C, and 37°C, and the ability of these strains to retain antimicrobial resistance during sequential subculturing was determined. Insertion of the mobile element conferring macrolide resistance had minimal effect on in vitro growth. However, two of three rpoB mutations conferring rifampin resistance resulted in a decreased growth rate in MM. In soil, macrolide or rifampin resistant R. equi strains exhibited limited growth compared to the susceptible R. equi isolate at all temperatures except -20°C. During subculturing, macrolide resistance was lost over time, and two of three rpoB mutations reverted to the wild type form. The growth of rifampin-resistant R. equi is delayed under nutrient restriction. In soil, possession of rifampin or macrolide resistance results in decreased fitness. Both macrolide and rifampin resistance can be lost after repeated subculturing. [ABSTRACT FROM AUTHOR]

Bacterial pathogens that carry antibiotic resistance alleles sometimes pay a cost in the form of impaired growth in antibiotic-free conditions. This cost of resistance is expected to be a key parameter for understanding how resistance spreads and persists in pathogen populations. Analysis of individual resistance alleles from laboratory evolution and natural isolates has shown they are typically costly, but these costs are highly variable and influenced by genetic variation at other loci. It therefore remains unclear how strongly resistance is linked to impaired antibioticfree growth in bacteria from natural and clinical scenarios, where resistance alleles are likely to coincide with other types of genetic variation. To investigate this, we measured the growth of 92 natural and clinical Escherichia coli isolates across three antibiotic-free environments. We then tested whether variation of antibiotic-free growth among isolates was predicted by their resistance to 10 antibiotics, while accounting for the phylogenetic structure of the data. We found that isolates with similar resistance profiles had similar antibiotic-free growth profiles, but it was not simply that higher average resistance was associated with impaired growth. Next, we used whole-genome sequences to identify antibiotic resistance genes and found that isolates carrying a greater number of resistance gene types grew relatively poorly in antibiotic-free conditions, even when the resistance genes they carried were different. This suggests that the resistance of bacterial pathogens is linked to growth costs in nature, but it is the total genetic burden and multivariate resistance phenotype that predict these costs, rather than individual alleles or mean resistance across antibiotics. IMPORTANCE Managing the spread of antibiotic resistance in bacterial pathogens is a major challenge for global public health. Central to this challenge is understanding whether resistance is linked to impaired bacterial growth in the absence of antibiotics, because this determines whether resistance declines when bacteria are no longer exposed to antibiotics. We studied 92 isolates of the key bacterial pathogen Escherichia coli; these isolates varied in both their antibiotic resistance genes and other parts of the genome. Taking this approach, rather than focusing on individual genetic changes associated with resistance as in much previous work, revealed that growth without antibiotics was linked to the number of specialized resistance genes carried and the combination of antibiotics to which isolates were resistant but was not linked to average antibiotic resistance. This approach provides new insights into the genetic factors driving the long-term persistence of antibiotic-resistant bacteria, which is important for future efforts to predict and manage resistance. [ABSTRACT FROM AUTHOR]

Aeromonas salmonicida subsp. salmonicida is a major pathogen affecting fisheries worldwide and is a well-known pigmented member of the Aeromonas genus. This subspecies produces melanin at 22°C. However, melanogenesis decreases as the culture temperature increases and is completely suppressed at 30°C to 35°C, while bacterial growth is unaffected. The mechanism and biological significance of this temperature-dependent melanogenesis remain unclear. Heterologous expression of an A. salmonicida subsp. salmonicida 4-hydroxyphenylpyruvate dioxygenase (HppD), the most critical enzyme in the homogentisic acid (HGA)-melanin synthesis pathway, results in thermosensitive pigmentation in Escherichia coli, suggesting that HppD plays a key role in this process. In this study, we demonstrated that the thermolability of HppD is responsible for the temperature-dependent melanization of A. salmonicida subsp. salmonicida. Substitutions of three residues, S18T, P103Q, and L119P, in A. salmonicida subsp. salmonicida HppD increased the thermostability of this enzyme and resulted in temperature-independent melanogenesis. Moreover, the replacement of the corresponding residues in HppD from Aeromonas media strain WS, which forms pigment independent of temperature, with those of A. salmonicida subsp. salmonicida HppD resulted in thermosensitive melanogenesis. A structural analysis suggested that mutations at these sites, especially at position P103, strengthen the secondary structure of HppD and greatly improve its thermal stability. Additionally, we found that the HppD sequences of all A. salmonicida subsp. salmonicida isolates were identical and that two of the three residues were clearly distinct from those of other Aeromonas strains. IMPORTANCE Aeromonas salmonicida subsp. salmonicida is the causative agent of furunculosis, a bacterial septicemia of cold-water fish of the Salmonidae family. Although other Aeromonas species can produce melanin, A. salmonicida subsp. salmonicida is the only member of this genus that has been reported to exhibit temperature-dependent melanization. Here, we demonstrated that thermosensitive melanogenesis in A. salmonicida subsp. salmonicida strains is due to the thermolability of 4-hydroxyphenylpyruvate dioxygenase (HppD). Additionally, we confirmed that this thermolabile HppD exhibited higher activity at low temperatures than its mesophilic homologues, suggesting this as an adaptive strategy of this enzyme to the psychrophilic lifestyle of A. salmonicida subsp. salmonicida. The strictly conserved hppD sequences among A. salmonicida subsp. salmonicida isolates and the specific possession of P103 and L119 residues could be used as a reference for the identification of A. salmonicida subsp. salmonicida isolates. [ABSTRACT FROM AUTHOR]

Vibrio vulnificus and Vibrio parahaemolyticus are important human pathogens that are frequently transmitted via consumption of contaminated raw oysters. A small amount of D-tryptophan (D-Trp) inhibits some foodborne pathogenic bacteria under high-salt environments. In this study, we aimed to evaluate the antibacterial effect of D-Trp on V. vulnificus and V. parahaemolyticus inoculated in culture media, artificial seawater, shucked and live oysters. The effectiveness of D-Trp in growth inhibition of Vibrio spp. was highly dependent on environmental NaCl concentrations. Higher levels of NaCl (>4.0%) with D-Trp (>20 mM) resulted in higher and consistent growth inhibition of both Vibrio spp. Treatment with 40 mM D-Trp significantly (P < 0.05) reduced viable V. parahaemolyticus cell counts in tryptic soy broth (TSB) with >4.0% NaCl at 25°C. In contrast, compared to V. parahaemolyticus, V. vulnificus was more sensitive to D-Trp (20 mM). D-Trp (40 mM) treatment with NaCl (>4.5%) significantly (P < 0.05) inhibited the growth of V. parahaemolyticus and V. vulnificus in shucked oysters immersed in peptone water at 25°C throughout a 48 h incubation period. In artificial seawater, D-Trp exhibited stronger growth inhibitory effect on V. vulnificus and V. parahaemolyticus at 25°C than in TSB at same level of salinity and inhibited the growth of both V. parahaemolyticus and V. vulnificus in live oysters at 25°C for 48 h. Furthermore, we tested the synergistic effect D-Trp and salinity on the inhibition of total viable bacterial counts (TVC) at refrigeration temperature. 40 mM D-Trp inhibited the growth of TVC in shucked oysters immersed in artificial seawater at 4°C. Therefore, these results revealed that D-Trp will serve as a novel and an alternative food preservative to control Vibrio spp. in live oysters at ambient temperature and extending the shelf-life of shucked oysters at refrigeration temperature. [ABSTRACT FROM AUTHOR]

Only a small fraction of bacteria can autonomously initiate growth on agar plates. Nongrowing bacteria typically enter a metabolically inactive dormant state and require specific chemical trigger factors or signals to exit this state and to resume growth. Micrococcus luteus has become a model organism for this important yet poorly understood phenomenon. Only a few resuscitation signals have been described to date, and all of them are produced endogenously by bacterial species. We report the discovery of a novel type of resuscitation signal that allows M. luteus to grow on agar but not agarose plates. Fractionation of the agar polysaccharide complex and sulfation of agarose allowed us to identify the signal as highly sulfated saccharides found in agar or carrageenans. Purification of hydrolyzed κ-carrageenan ultimately led to the identification of the signal as a small fragment of a large linear polysaccharide, i.e., an oligosaccharide of five or more sugars with a repeating disaccharide motif containing D-galactose-4-sulfate (G4S) 1,4-linked to 3,6-anhydro-α-Dgalactose (DA), G4S-(DA-G4S)n≥2. IMPORTANCE Most environmental bacteria cannot initiate growth on agar plates, but they can flourish on the same plates once growth is initiated. While there are a number of names for and manifestations of this phenomenon, the underlying cause appears to be the requirement for a molecular signal indicating safe growing conditions. Micrococcus luteus has become a model organism for studying this growth initiation process, often called resuscitation, because of its apparent connection with the persistent or dormant form of Mycobacterium tuberculosis, an important human pathogen. In this report, we identify a highly sulfated saccharide from agar or carrageenans that robustly resuscitates dormant M. luteus on agarose plates. We identified and characterized the signal as a small repeating disaccharide motif. Our results indicate that signals inherent in or absent from the polysaccharide composition of solid growth media can have major effects on bacterial growth. [ABSTRACT FROM AUTHOR]

Microgreens, like sprouts, are relatively fast-growing products and are generally consumed raw. Moreover, as observed for sprouts, microbial contamination from preharvest sources may also be present in the production of microgreens. In this study, two Salmonella enterica serovars (Hartford and Cubana), applied at multiple inoculation levels, were evaluated for survival and growth on alfalfa sprouts and Swiss chard microgreens by using the most-probable-number (MPN) method. Various abiotic factors were also examined for their effects on Salmonella survival and growth on sprouts and microgreens. Community-level physiological profiles (CLPPs) of sprout/microgreen rhizospheres with different levels of S. enterica inoculation at different growth stages were characterized by use of Biolog EcoPlates. In the seed contamination group, the ability of S. enterica to grow on sprouting alfalfa seeds was affected by both seed storage time and inoculation level but not by serovar. However, the growth of S. enterica on Swiss chard microgreens was affected by serovar and inoculation level. Seed storage time had little effect on the average level of Salmonella populations in microgreens. In the irrigation water contamination group, the growth of Salmonella on both alfalfa sprouts and microgreens was largely affected by inoculation level. Surprisingly, the growth medium was found to play an important role in Salmonella survival and growth on microgreens. CLPP analysis showed significant changes in the microbial community metabolic diversity during sprouting for alfalfa sprouts, but few temporal changes were seen with microgreens. The data suggest that the change in rhizosphere bacterial functional diversity was dependent on the host but independent of Salmonella contamination. [ABSTRACT FROM AUTHOR]

Shigella flexneri and Shigella sonnei bacteria cause the majority of all shigellosis cases worldwide. However, their distributions differ, with S. sonnei predominating in middle- and high-income countries and S. flexneri predominating in low-income countries. One proposed explanation for the continued range expansion of S. sonnei is that it can survive in amoebae, which could provide a protective environment for the bacteria. In this study, we demonstrate that while both S. sonnei and S. flexneri can survive coculture with the free-living amoebae Acanthamoebae castellanii, bacterial growth is predominantly extracellular. All isolates of Shigella were degraded following phagocytosis by A. castellanii, unlike those of Legionella pneumophila, which can replicate intracellularly. Our data suggest that S. sonnei is not able to use amoebae as a protective host to enhance environmental survival. Therefore, alternative explanations for S. sonnei emergence need to be considered. [ABSTRACT FROM AUTHOR]

Most isolated nitrate-reducing Fe(II)-oxidizing microorganisms are mixotrophic, meaning that Fe(II) is chemically oxidized by nitrite that forms during heterotrophic denitrification, and it is debated to which extent Fe(II) is enzymatically oxidized. One exception is the chemolithoautotrophic enrichment culture KS, a consortium consisting of a dominant Fe(II) oxidizer, Gallionellaceae sp., and less abundant heterotrophic strains (e.g., Bradyrhizobium sp., Nocardioides sp.). Currently, this is the only nitrate-reducing Fe(II)-oxidizing culture for which autotrophic growth has been demonstrated convincingly for many transfers over more than 2 decades. We used 16S rRNA gene amplicon sequencing and physiological growth experiments to analyze the community composition and dynamics of culture KS with various electron donors and acceptors. Under autotrophic conditions, an operational taxonomic unit (OTU) related to known microaerophilic Fe(II) oxidizers within the family Gallionellaceae dominated culture KS. With acetate as an electron donor, most 16S rRNA gene sequences were affiliated with Bradyrhizobium sp. Gallionellaceae sp. not only was able to oxidize Fe(II) under autotrophic and mixotrophic conditions but also survived over several transfers of the culture on only acetate, although it then lost the ability to oxidize Fe(II). Bradyrhizobium spp. became and remained dominant when culture KS was cultivated for only one transfer under heterotrophic conditions, even when conditions were reverted back to autotrophic in the next transfer. This study showed a dynamic microbial community in culture KS that responded to changing substrate conditions, opening up questions regarding carbon cross-feeding, metabolic flexibility of the individual strains in KS, and the mechanism of Fe(II) oxidation by a microaerophile in the absence of O2. [ABSTRACT FROM AUTHOR]

Tannerella forsythia and Fusobacterium nucleatum are dental plaque bacteria implicated in the development of periodontitis. These two species have been shown to form synergistic biofilms and have been found to be closely associated in dental plaque biofilms. A number of genetic loci for TonB-dependent membrane receptors (TDR) for glycan acquisition, with many existing in association with genes coding for enzymes involved in the breakdown of complex glycans, have been identified in T. forsythia. In this study, we focused on a locus, BFO_0186-BFO_0188, that codes for a predicted TDR-SusD transporter along with a putative β-glucan hydrolyzing enzyme (BFO_0186). This operon is located immediately downstream of a 2-gene operon that codes for a putative stress-responsive extracytoplasmic function (ECF) sigma factor and an anti-sigma factor. Here, we show that BFO_0186 expresses a β-glucanase that cleaves glucans with β-1,6 and β-1,3 linkages. Furthermore, the BFO_0186-BFO_0188 locus is upregulated, with an induction of β-glucanase activity, in cobiofilms of T. forsythia and F. nucleatum. The β-glucanase activity in mixed biofilms in turn leads to an enhanced hydrolysis of β-glucans and release of glucose monomers and oligomers as nutrients for F. nucleatum. In summary, our study highlights the role of T. forsythia β-glucanase expressed by the asaccharolytic oral bacterium T. forsythia in the development of T. forsythia-F. nucleatum mixed species biofilms, and suggest that dietary β-glucans might contribute in plaque development and periodontal disease pathogenesis IMPORTANCE The development of dental plaque biofilm is a complex process in which metabolic, chemical and physical interactions between bacteria take a central role. Previous studies have shown that the dental pathogens T. forsythia and F. nucleatum form synergistic biofilms and are closely associated in human dental plaque. In this study, we show that β-glucanase from the periodontal pathogen T. forsythia plays a role in the formation of T. forsythia-F. nucleatum cobiofilms by hydrolyzing β-glucans to glucose as a nutrient. We also unveiled that the expression of T. forsythia β-glucanase is induced in response to F. nucleatum sensing. This study highlights the involvement ofβ-glucanase activity in the development of T. forsythia-F. nucleatum biofilms and suggests that intake of dietaryβ-glucans might be a contributing risk factor in plaque development and periodontal disease pathogenesis. [ABSTRACT FROM AUTHOR]

Valle d'Aosta Lard d'Arnad is a protected designation of origin (PDO) product produced from fat of the shoulder and back of heavy pigs. Its manufacturing process can be very diverse, especially regarding the maturation temperature and the NaCl concentration used for the brine; thereby, the main goal of this study was to investigate the impact of those parameters on the microbiota developed during curing and ripening. Three farms producing Lard d'Arnad were selected. Two plants, reflecting the industrial process characterized either by low maturation temperature (plant A [10% NaCl, 2°C]) or by using a low NaCl concentration (plant B [2.5% NaCl, 4°C]), were selected, while the third was characterized by an artisanal process (plant C [30% NaCl, 8°C]). Lard samples were obtained at time 0 and after 7, 15, 30, 60, and 90 days of maturation. From each plant, 3 independent lots were analyzed. The diversity of live microbiota was evaluated by using classical plate counts and amplicon target sequencing of small subunit (SSU) rRNA. The main taxa identified by sequencing were Acinetobacter johnsonii, Psychrobacter, Staphylococcus equorum, Staphylococcus sciuri, Pseudomonas fragi, Brochothrix, Halomonas, and Vibrio, and differences in their relative abundances distinguished samples from the individual plants. The composition of the microbiota was more similar among plants A and B, and it was characterized by the higher presence of taxa recognized as undesired bacteria in food-processing environments. Oligotype analysis of Halomonas and Acinetobacter revealed the presence of several characteristic oligotypes associated with A and B samples. IMPORTANCE Changes in the food production process can drastically affect the microbial community structure, with a possible impact on the final characteristics of the products. The industrial processes of Lard d'Arnad production are characterized by a reduction in the salt concentration in the brines to address a consumer demand for less salty products; this can negatively affect the dynamics and development of the live microbiota and, as a consequence, can negatively impact the quality of the final product due to the higher abundance of spoilage bacteria. This study is an overview of the live microbiota that develop during lard manufacturing, and it highlights the importance of the use of traditional process to produce PDO from a spoilage perspective. [ABSTRACT FROM AUTHOR]

Alginate beads represent a simple and highly reproducible in vitro model system for diffusion-limited bacterial growth. In this study, alginate beads were inoculated with Pseudomonas aeruginosa and followed for up to 72 h. Confocal microscopy revealed that P. aeruginosa formed dense clusters similar in size to in vivo aggregates observed ex vivo in cystic fibrosis lungs and chronic wounds. Bacterial aggregates primarily grew in the bead periphery and decreased in size and abundance toward the center of the bead. Microsensor measurements showed that the O2 concentration decreased rapidly and reached anoxia ~100 μm below the alginate bead surface. This gradient was relieved in beads supplemented with NO3- as an alternative electron acceptor allowing for deeper growth into the beads. A comparison of gene expression profiles between planktonic and alginate-encapsulated P. aeruginosa confirmed that the bacteria experienced hypoxic and anoxic growth conditions. Furthermore, alginate-encapsulated P. aeruginosa exhibited a lower respiration rate than the planktonic counterpart and showed a high tolerance toward antibiotics. The inoculation and growth of P. aeruginosa in alginate beads represent a simple and flexible in vivo-like biofilm model system, wherein bacterial growth exhibits central features of in vivo biofilms. This was observed by the formation of small cell aggregates in a secondary matrix with O2-limited growth, which was alleviated by the addition of NO3- as an alternative electron acceptor, and by reduced respiration rates, as well as an enhanced tolerance to antibiotic treatment. IMPORTANCE Pseudomonas aeruginosa has been studied intensively for decades due to its involvement in chronic infections, such as cystic fibrosis and chronic wounds, where it forms biofilms. Much research has been dedicated to biofilm formation on surfaces; however, in chronic infections, most biofilms form small aggregates of cells not attached to a surface, but embedded in host material. In this study, bacteria were encapsulated in small alginate beads and formed aggregates similar to what is observed in chronic bacterial infections. Our findings show that aggregates are exposed to steep oxygen gradients, with zones of oxygen depletion, and that nitrate may serve as an alternative to oxygen, enabling growth in oxygendepleted zones. This is important, as slow growth under low-oxygen conditions may render the bacteria tolerant toward antibiotics. This model provides an alternative to surface biofilm models and adds to the comprehension that biofilms do not depend on a surface for formation. [ABSTRACT FROM AUTHOR]

Complex media are routinely used to cultivate diverse bacteria. However, this complexity can obscure the factors that govern cell growth. While studying protein acetylation in buffered tryptone broth supplemented with glucose (TB7-glucose), we observed that Escherichia coli did not fully consume glucose prior to stationary phase. However, when we supplemented this medium with magnesium, the glucose was completely consumed during exponential growth, with concomitant increases in cell number and biomass but reduced cell size. Similar results were observed with other sugars and other peptide-based media, including lysogeny broth. Magnesium also limited cell growth for Vibrio fischeri and Bacillus subtilis in TB7-glucose. Finally, magnesium supplementation reduced protein acetylation. Based on these results, we conclude that growth in peptide-based media is magnesium limited. We further conclude that magnesium supplementation can be used to tune protein acetylation without genetic manipulation. These results have the potential to reduce potentially deleterious acetylated isoforms of recombinant proteins without negatively affecting cell growth. IMPORTANCE: Bacteria are often grown in complex media. These media are thought to provide the nutrients necessary to grow bacteria to high cell densities. In this work, we found that peptide-based media containing a sugar are magnesium limited for bacterial growth. In particular, magnesium supplementation is necessary for the bacteria to use the sugar for cell growth. Interestingly, in the absence of magnesium supplementation, the bacteria still consume the sugar. However, rather than use it for cell growth, the bacteria instead use the sugar to acetylate lysines on proteins. As lysine acetylation may alter the activity of proteins, this work demonstrates how lysine acetylation can be tuned through magnesium supplementation. These findings may be useful for recombinant protein production, when acetylated isoforms are to be avoided. They also demonstrate how to increase bacterial growth in complex media. [ABSTRACT FROM AUTHOR]

Fungal blue-light photoreceptors have been proposed as integrators of light and oxidative stress. However, additional elements participating in the integrative pathway remain to be identified. In Trichoderma atroviride, the blue-light regulator (BLR) proteins BLR-1 and -2 are known to regulate gene transcription, mycelial growth, and asexual development upon illumination, and recent global transcriptional analysis revealed that the histone deacetylase-encoding gene hda-2 is induced by light. Here, by assessing responses to stimuli in wild-type and hda-2 backgrounds, we evaluate the role of HDA-2 in the regulation of genes responsive to light and oxidative stress. Δhda-2 strains present reduced growth, misregulation of the con-1 gene, and absence of conidia in response to light and mechanical injury. We found that the expression of hda-2 is BLR-1 dependent and HDA-2 in turn is essential for the transcription of early and late light-responsive genes that include blr-1, indicating a regulatory feedback loop. When subjected to reactive oxygen species (ROS), Δhda-2 mutants display high sensitivity whereas Δblr strains exhibit the opposite phenotype. Consistently, in the presence of ROS, ROS-related genes show high transcription levels in wild-type and Δblr strains but misregulation in Δhda-2 mutants. Finally, chromatin immunoprecipitations of histone H3 acetylated at Lys9/ Lys14 on cat-3 and gst-1 promoters display low accumulation of H3K9K14ac in Δblr and Δhda-2 strains, suggesting indirect regulation of ROS-related genes by HDA-2. Our results point to a mutual dependence between HDA-2 and BLR proteins and reveal the role of these proteins in an intricate gene regulation landscape in response to blue light and ROS. [ABSTRACT FROM AUTHOR]

Pervasive environmental stressors on coral reefs are attributed with shifting the competitive balance in favor of alternative dominants, such as macroalgae. Previous studies have demonstrated that macroalgae compete with corals via a number of mechanisms, including the production of potent primary and secondary metabolites that can influence coral-associated microbial communities. The present study investigates the effects of the Pacific brown macroalga Lobophora sp. (due to the shifting nature of the Lobophora species complex, it will be referred to here as Lobophora sp.) on coral bacterial isolates, coral larvae, and the microbiome associated with the coral Porites cylindrica. Crude aqueous and organic macroalgal extracts were found to inhibit the growth of coral-associated bacteria. Extracts and fractions were also shown to inhibit coral larval settlement and cause mortality at concentrations lower (<0.3 mg · ml-1) than calculated natural concentrations (4.4 mg · ml-1). Microbial communities associated with coral tissues exposed to aqueous (e.g., hydrophilic) crude extracts demonstrated a significant shift to Vibrio dominance and a loss of sequences related to the putative coral bacterial symbiont, Endozoicomonas sp., based on 16S rRNA amplicon sequencing. This study contributes to growing evidence that macroalgal allelochemicals, dissolved organic material, and native macroalgal microbial assemblages all play a role in shifting the microbial equilibrium of the coral holobiont away from a beneficial state, contributing to a decline in coral fitness and a shift in ecosystem structure. [ABSTRACT FROM AUTHOR]

We show in this report that traces of juices released from salad leaves as they become damaged can significantly enhance colonization of salad leaves by Salmonella enterica. Salad juices in water increased Salmonella growth by 110% over the level seen with the unsupplemented control and in host-like serum-based media by more than 2,400-fold over control levels. In serum-based media, salad juices induced growth of Salmonella via provision of Fe from transferrin, and siderophore production was found to be integral to the growth induction process. Other aspects relevant to salad leaf colonization and retention were enhanced, such as motility and biofilm formation, which were increased over control levels by >220% and 250%, respectively; direct attachment to salad leaves increased by >350% when a salad leaf juice was present. In terms of growth and biofilm formation, the endogenous salad leaf microbiota was largely unresponsive to leaf juice, suggesting that Salmonella gains a marked growth advantage from fluids released by salad leaf damage. Salad leaf juices also enhanced pathogen attachment to the salad bag plastic. Over 5 days of refrigeration (a typical storage time for bagged salad leaves), even traces of juice within the salad bag fluids increased Salmonella growth in water by up to 280-fold over control cultures, as well as enhancing salad bag colonization, which could be an unappreciated factor in retention of pathogens in fresh produce. Collectively, the study data show that exposure to salad leaf juice may contribute to the persistence of Salmonella on salad leaves and strongly emphasize the importance of ensuring the microbiological safety of fresh produce. [ABSTRACT FROM AUTHOR]