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The misuse of antibiotics and the emergence of antimicrobial resistance (AMR) is a concern in the aquaculture industry because it contributes to global health risks and impacts the environment. This study analyzed the AMR of sentinel bacteria associated with striped catfish ( Pangasisanodon hypophthalmus ) and giant snakehead ( Channa micropeltes ), the two main fish species reared in the pond culture in Cambodia. Phenotypic and genotypic characterization of the recovered isolates from fish, water, and sediment samples revealed the presence of bacteria, such as 22 species belonging to families Aeromonadaceae , Enterobacteriaceae , and Pseudomonadaceae . Among 48 isolates, Aeromonas caviae (n = 2), Aeromonas hydrophila (n = 2), Aeromonas ichthiosmia (n = 1), Aeromonas salmonicida (n = 4) were detected. A. salmonicida and A. hydrophilla are known as fish pathogens that occur worldwide in both fresh and marine water aquaculture. Antibiotic susceptibility testing revealed antibiotic resistance patterns of 24 (50 %) isolates among 48 isolates with higher multiple antibiotic resistance index (> 0.2). All the isolates of Enterobacteriaceae were susceptible to ciprofloxacin. Ciprofloxacin is a frontline antibiotic that is not recommended to use in aquaculture. Therefore, its use has to be strictly controlled. This study expands our knowledge of the AMR status in aquaculture farms which is very limited in Cambodia., Competing Interests: The authors declare there is no conflict., (© 2024 The Authors This is an Open Access article distributed under the terms of the Creative Commons Attribution Licence (CC BY 4.0), which permits copying, adaptation and redistribution, provided the original work is properly cited (http://creativecommons.org/licenses/by/4.0/).)

Biofilms within drinking water distribution systems serve as a habitat for drinking water microorganisms. However, biofilms can negatively impact drinking water quality by causing water discoloration and deterioration and can be a reservoir for unwanted microorganisms. In this study, we investigated whether indicator organisms for drinking water quality, such as coliforms, can settle in mature drinking water biofilms. Therefore, a biofilm monitor consisting of glass rings was used to grow and sample drinking water biofilms. Two mature drinking water biofilms were characterized by flow cytometry, ATP measurements, confocal laser scanning microscopy, and 16S rRNA sequencing. Biofilms developed under treated chlorinated surface water supply exhibited lower cell densities in comparison with biofilms resulting from treated groundwater. Overall, the phenotypic as well as the genotypic characteristics were significantly different between both biofilms. In addition, the response of the biofilm microbiome and possible biofilm detachment after minor water quality changes were investigated. Limited changes in pH and free chlorine addition, to simulate operational changes that are relevant for practice, were evaluated. It was shown that both biofilms remained resilient. Finally, mature biofilms were prone to invasion of the coliform, Serratia fonticola . After spiking low concentrations (i.e., ±100 cells/100 mL) of the coliform to the corresponding bulk water samples, the coliforms were able to attach and get established within the mature biofilms. These outcomes emphasize the need for continued research on biofilm detachment and its implications for water contamination in distribution networks., Importance: The revelation that even low concentrations of coliforms can infiltrate into mature drinking water biofilms highlights a potential public health concern. Nowadays, the measurement of coliform bacteria is used as an indicator for fecal contamination and to control the effectiveness of disinfection processes and the cleanliness and integrity of distribution systems. In Flanders (Belgium), 533 out of 18,840 measurements exceeded the established norm for the coliform indicator parameter in 2021; however, the source of microbial contamination is mostly unknown. Here, we showed that mature biofilms, are susceptible to invasion of Serratia fonticola . These findings emphasize the importance of understanding and managing biofilms in drinking water distribution systems, not only for their potential to influence water quality, but also for their role in harboring and potentially disseminating pathogens. Further research into biofilm detachment, long-term responses to operational changes, and pathogen persistence within biofilms is crucial to inform strategies for safeguarding drinking water quality., Competing Interests: The authors declare no conflict of interest.

Facultative vertically transmitted symbionts are a common feature of insects that determine many aspects of their hosts' phenotype. Our capacity to understand and exploit these symbioses is commonly compromised by the microbes unculturability and consequent lack of genetic tools, an impediment of particular significance for symbioses of pest and vector species. Previous work had established that insecticide susceptibility of the economically important pest of rice, the brown planthopper Nilaparvata lugens, was higher in field-collected lineages that carry Ca. Arsenophonus nilaparvatae. We established Ca. A. nilaparvatae into cell-free culture and used this to establish the complete closed genome of the symbiont. We transformed the strain to express GFP and reintroduced it to N. lugens to track infection in vivo. The symbiont established vertical transmission, generating a discrete infection focus towards the posterior pole of each N. lugens oocyte. This infection focus was retained in early embryogenesis before transition to a diffuse somatic infection in late N. lugens embryos and nymphs. We additionally generated somatic infection in novel host species, but these did not establish vertical transmission. Transinfected planthopper lines acquired the insecticide sensitivity trait, with associated downregulation of the P450 xenobiotic detoxification system of the host. Our results causally establish the role of the symbiont in increasing host insecticide sensitivity with implications for insecticide use and stewardship. Furthermore, the culturability and transformation of this intracellular symbiont, combined with its ease of reintroduction to planthopper hosts, enables novel approaches both for research into symbiosis and into control of insect pest species., (© The Author(s) 2024. Published by Oxford University Press on behalf of the International Society for Microbial Ecology.)

Bacteriophages are viruses that infect bacteria and thus threaten industrial processes relying on the production executed by bacterial cells. Industries bear huge economic losses due to such recurring and resilient infections. Depending on the specificity of the process, there is a need for appropriate methods of bacteriophage inactivation, with an emphasis on being inexpensive and high efficiency. In this review, we summarize the reports on antiphagents, i.e., antibacteriophage agents on inactivation of bacteriophages. We focused on bacteriophages targeting the representatives of the Enterobacteriaceae family, as its representative, Escherichia coli , is most commonly used in the bio-industry. The review is divided into sections dealing with bacteriophage inactivation by physical factors, chemical factors, and nanotechnology-based solutions.

Macrophages clear infections by engulfing and digesting pathogens within phagolysosomes. Pathogens escape this fate by engaging in a molecular arms race; they use WxxxE motif-containing "effector" proteins to subvert the host cells they invade and seek refuge within protective vacuoles. Here, we define the host component of the molecular arms race as an evolutionarily conserved polar "hot spot" on the PH domain of ELMO1 (Engulfment and Cell Motility protein 1), which is targeted by diverse WxxxE effectors. Using homology modeling and site-directed mutagenesis, we show that a lysine triad within the "patch" directly binds all WxxxE effectors tested: SifA (Salmonella), IpgB1 and IpgB2 (Shigella), and Map (enteropathogenic Escherichia coli). Using an integrated SifA-host protein-protein interaction network, in silico network perturbation, and functional studies, we show that the major consequences of preventing SifA-ELMO1 interaction are reduced Rac1 activity and microbial invasion. That multiple effectors of diverse structure, function, and sequence bind the same hot spot on ELMO1 suggests that the WxxxE effector(s)-ELMO1 interface is a convergence point of intrusion detection and/or host vulnerability. We conclude that the interface may represent the fault line in coevolved molecular adaptations between pathogens and the host, and its disruption may serve as a therapeutic strategy., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

The black carpenter ant (Camponotus pennsylvanicus) is a pest species found widely throughout North America. From a single individual I used long-read nanopore sequencing to assemble a phased diploid genome of 306 Mb and 60X coverage, with quality assessed by a 97.0% BUSCO score, improving upon other ant assemblies. The mitochondrial genome reveals minor rearrangements from other ants. The reads also allowed assembly of parasitic and symbiont genomes. I include a complete Wolbachia bacterial assembly with a size of 1.2 Mb, as well as a commensal symbiont Blochmannia pennsylvanicus, at 791 kb. DNA methylation and hydroxymethylation were measured at base-pair resolution level from the same reads and confirmed extremely low levels seen in the Formicidae family. There was moderate heterozygosity, with 0.16% of bases being biallelic from the parental haplotypes. Protein prediction yielded 14 415 amino acid sequences with 95.8% BUSCO score and 86% matching to previously known proteins. All assemblies were derived from a single MinION flow cell generating 20 Gb of sequence for a cost of $1047 including consumable reagents. Adding fixed costs for equipment brings the total for an ant-sized genome to less than $5000. All analyses were performed in 1 week on a single desktop computer., (© The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The risk assessment of Bacillus thuringiensis (Bt) crops on nontarget pests has received much attention. Despite the knowledge of various beneficial bacterial symbionts in pests, whether Bt proteins affect these symbionts and subsequently alter the pest's ecology remains largely unknown. The whitefly Bemisia tabaci is one of the most serious nontarget pests in Bt cotton. Here, we explored the Bt Cry1Ac protein-induced changes in whitefly symbiont abundance and the subsequent effects on whitefly response against a naturally prevalent entomopathogenic fungus Cordyceps javanica. The obligate symbiont 'Candidatus Portiera aleyrodidarum' (hereafter P. aleyrodidarum) as well as facultative symbionts 'Candidatus Hamiltonella defensa' (hereafter H. defensa), 'Candidatus Cardinium hertigii' (hereafter C. hertigii) and 'Candidatus Rickettsia bellii' (hereafter R. bellii) dominate the microbial community of whiteflies. The Bt exposure had no effects on H. defensa infected (H) and H. defensa-C. hertigii doubly infected (HC) whiteflies, but decreased the total copy number of symbionts as well as the R. bellii proportion in H. defensa-C. hertigii- R. bellii triply infected whiteflies (HCR). C. javanica caused whitefly adults 100 % mortality within 8 days. Without Bt protein exposure, HCR whiteflies survived significantly longer than H and HC whiteflies sprayed by C. javanica, suggesting that R. bellii confers protection. However, in Bt-exposed groups, C. javanica generated synchronous death of H, HC and HCR whiteflies. Specifically, in H and HC whiteflies, Bt protein-exposure showed no significant difference in progress of death caused by C. javanica. But in HCR whiteflies, Bt exposure hastened death induced by C. javanica, suppressing the R. bellii-conferred protection. This is the first report revealing that Bt protein altered symbiont community conferred adverse effects on nontarget pests, providing a new perspective for Bt risk assessment and biocontrol strategies of nontarget pests., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Children in low-resource settings carry enteric pathogens asymptomatically and are frequently treated with antibiotics, resulting in opportunities for pathogens to be exposed to antibiotics when not the target of treatment (i.e., bystander exposure). We quantified the frequency of bystander antibiotic exposures for enteric pathogens and estimated associations with resistance among children in eight low-resource settings. We analyzed 15,697 antibiotic courses from 1,715 children aged 0 to 2 y from the MAL-ED birth cohort. We calculated the incidence of bystander exposures and attributed exposures to respiratory and diarrheal illnesses. We associated bystander exposure with phenotypic susceptibility of E. coli isolates in the 30 d following exposure and at the level of the study site. There were 744.1 subclinical pathogen exposures to antibiotics per 100 child-years. Enteroaggregative Escherichia coli was the most frequently exposed pathogen, with 229.6 exposures per 100 child-years. Almost all antibiotic exposures for Campylobacter (98.8%), enterotoxigenic E. coli (95.6%), and typical enteropathogenic E. coli (99.4%), and the majority for Shigella (77.6%), occurred when the pathogens were not the target of treatment. Respiratory infections accounted for half (49.9%) and diarrheal illnesses accounted for one-fourth (24.6%) of subclinical enteric bacteria exposures to antibiotics. Bystander exposure of E. coli to class-specific antibiotics was associated with the prevalence of phenotypic resistance at the community level. Antimicrobial stewardship and illness-prevention interventions among children in low-resource settings would have a large ancillary benefit of reducing bystander selection that may contribute to antimicrobial resistance.

Salmonella enterica persist in the chicken gut by suppressing inflammatory responses via expansion of intestinal regulatory T cells (Tregs). In humans, T cell activation is controlled by neurochemical signaling in Tregs; however, whether similar neuroimmunological signaling occurs in chickens is currently unknown. In this study, we explore the role of the neuroimmunological axis in intestinal Salmonella resistance using the drug reserpine, which disrupts intracellular storage of catecholamines like norepinephrine. Following reserpine treatment, norepinephrine release was increased in both ceca explant media and Tregs. Similarly, Salmonella killing was greater in reserpine-treated explants, and oral reserpine treatment reduced the level of intestinal Salmonella Typhimurium and other Enterobacteriaceae in vivo. These antimicrobial responses were linked to an increase in antimicrobial peptide and IL-2 gene expression as well as a decrease in CTLA-4 gene expression. Globally, reserpine treatment led to phosphorylative changes in epidermal growth factor receptor (EGFR), mammalian target of rapamycin (mTOR), and the mitogen-associated protein kinase 2(MEK2). Exogenous norepinephrine treatment alone increased Salmonella resistance, and reserpine-induced antimicrobial responses were blocked using beta-adrenergic receptor inhibitors, suggesting norepinephrine signaling is crucial in this mechanism. Furthermore, EGF treatment reversed reserpine-induced antimicrobial responses, whereas mTOR inhibition increased antimicrobial activities, confirming the roles of metabolic signaling in these responses. Finally, MEK1/2 inhibition suppressed reserpine, norepinephrine, and mTOR-induced antimicrobial responses. Overall, this study demonstrates a central role for MEK1/2 activity in reserpine induced neuro-immunometabolic signaling and subsequent antimicrobial responses in the chicken intestine, providing a means of reducing bacterial colonization in chickens to improve food safety., (© 2021. The Author(s).)

In the present work, we evaluated the effects of a mixture of biocontrol agents against two toxigenic strains of Penicillium expansum isolated in Argentine Patagonia from pome fruits. The two strains, INTA-5 and INTA-10, were previusly selected among ten strains coming from the Alto Valle (Rio Negro-Argentina) for their high production of patulin. For the biocontrol, Kosakonia radicincitans, Cryptococcus laurentii, and Rhodosporidium fluviale were tested in vitro experiments on Potato Dextrose Agar (PDA) dishes against the INTA-5 and INTA-10 strains. The bacterium K. radicincitans and the yeast C. laurentii were selected to be used in a mixture due to their capacity to control the fungus and reduce the mycotoxin severely. In vitro assays with the mixture showed a high antagonism against P. expansum INTA-5 and INTA-10, at 21 d of incubation at 25 °C and a patulin reduction of 98%. The mixture of microorganisms was also effective in apples stored at 25 °C for 10 d and 4 °C for 30 d. At cold storage, the mixture controlled moderately the development of rot and decreased patulin concentration. At 25 °C, the pathogen's optimal growth temperature, the mixture of Biological Control Agent (BCAs) assured both the control of rot and decrease of patulin concentration. The combination of two microorganisms, with different requirements and abilities, resulted in a mix with a strong antagonism against P. expansum with the capability to decrease the patulin concentration. Treatment with the selected mixture could be a good option for controlling strains with different behaviours and in different environmental conditions., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

This study investigated the clinical characteristics and dynamic changes of intestinal bacterial community to evaluate the curative effect of fecal microbiota transplantation (FMT) on irritable bowel syndrome with predominant diarrhea (IBS-D) comorbid with anxiety and depression. Total two treatments were designed in randomize-controlled trial includes oral FMT capsules with 1 week (A1), 8 weeks (A2), and 12 weeks (A3), as well as oral empty capsules with 1 week (B1), 8 weeks (B2), and 12 weeks (B3) as control for comparison. The positive therapeutic effects occurred in FMT colonized patient with IBS-D comorbid psychological disorder, demonstrated at alleviated IBS-D severity (IBS-SSS score from 291.11 reduced to 144.44), altered stool type (from 6 changed to 4), reduced anxiety and depression scores (from 18.33 to 8.39 and from 22.33 to 17.78) after FMT-treated 12 weeks. The FMT therapy improved bacterial alpha diversity and the majority bacterial community predominant by Bacteroidetes and Firmicutes , and the relative abundance (RA) was higher after FMT-treated 12 weeks (50.61% and 45.52%) than control (47.62% and 38.96%). In short, FMT therapy has great potential for IBS-D patients combined with anxiety and depression by alleviated clinical symptoms and restore the intestinal micro-ecology.

Gastrointestinal infections cause significant morbidity and mortality worldwide. The complexity of human biology and limited insights into host-specific infection mechanisms are key barriers to current therapeutic development. Here, we demonstrate that two-dimensional epithelial monolayers derived from human intestinal organoids, combined with in vivo- like bacterial culturing conditions, provide significant advancements for the study of enteropathogens. Monolayers from the terminal ileum, cecum, and ascending colon recapitulated the composition of the gastrointestinal epithelium, in which several techniques were used to detect the presence of enterocytes, mucus-producing goblet cells, and other cell types following differentiation. Importantly, the addition of receptor activator of nuclear factor kappa-B ligand (RANKL) increased the presence of M cells, critical antigen-sampling cells often exploited by enteric pathogens. For infections, bacteria were grown under in vivo- like conditions known to induce virulence. Overall, interesting patterns of tissue tropism and clinical manifestations were observed. Shigella flexneri adhered efficiently to the cecum and colon; however, invasion in the colon was best following RANKL treatment. Both Salmonella enterica serovars Typhi and Typhimurium displayed different infection patterns, with S . Typhimurium causing more destruction of the terminal ileum and S . Typhi infecting the cecum more efficiently than the ileum, particularly with regard to adherence. Finally, various pathovars of Escherichia coli validated the model by confirming only adherence was observed with these strains. This work demonstrates that the combination of human-derived tissue with targeted bacterial growth conditions enables powerful analyses of human-specific infections that could lead to important insights into pathogenesis and accelerate future vaccine development. IMPORTANCE While traditional laboratory techniques and animal models have provided valuable knowledge in discerning virulence mechanisms of enteric pathogens, the complexity of the human gastrointestinal tract has hindered our understanding of physiologically relevant, human-specific interactions; and thus, has significantly delayed successful vaccine development. The human intestinal organoid-derived epithelial monolayer (HIODEM) model closely recapitulates the diverse cell populations of the intestine, allowing for the study of human-specific infections. Differentiation conditions permit the expansion of various cell populations, including M cells that are vital to immune recognition and the establishment of infection by some bacteria. We provide details of reproducible culture methods and infection conditions for the analyses of Shigella , Salmonella , and pathogenic Escherichia coli in which tissue tropism and pathogen-specific infection patterns were detected. This system will be vital for future studies that explore infection conditions, health status, or epigenetic differences and will serve as a novel screening platform for therapeutic development.

We performed a retrospective case-control cohort study following 146 preterm infants (≤32 weeks of gestation) who had been colonized with extended spectrum beta-lactamase producing Enterobacterales and compared them with 1:1 matched controls regarding rates of hospitalizations and outpatient visits because of infectious and gastrointestinal diseases and developmental impairment up to school age. Preterm infants with extended spectrum beta-lactamase producing Enterobacterales colonization did have neither higher rates of gastrointestinal or infectious diseases nor higher rates of developmental impairments up to the age of 6 years., Competing Interests: The authors have no funding or conflicts of interest to disclose., (Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.)

Many bacterial species employ systems for interference competition with other microorganisms. Some systems are effective without contact (e.g., through secretion of toxins), while other systems (e.g., type VI secretion system [T6SS]) require direct contact between cells. Here, we provide the initial characterization of a novel contact-dependent competition system for Proteus mirabilis. In neonatal mice, a commensal P. mirabilis strain apparently eliminated commensal Escherichia coli. We replicated the phenotype in vitro and showed that P. mirabilis efficiently reduced the viability of several Enterobacteriaceae species but not Gram-positive species or yeast cells. Importantly, P. mirabilis strains isolated from humans also killed E. coli. A reduction of viability occurred from early stationary phase to 24 h of culture and was observed in shaking liquid media as well as on solid media. Killing required contact but was independent of T6SS, which is the only contact-dependent killing system described for P. mirabilis. Expression of the killing system was regulated by osmolarity and components secreted into the supernatant. Stationary-phase P. mirabilis culture supernatant itself did not kill but was sufficient to induce killing in an exponentially growing coculture. In contrast, killing was largely prevented in media with low osmolarity. In summary, we provide the initial characterization of a potentially novel interbacterial competition system used by P. mirabilis. IMPORTANCE The study of bacterial competition systems has received significant attention in recent years. These systems are important in a multitude of polymicrobial environments and collectively shape the composition of complex ecosystems like the mammalian gut. They are also being explored as narrow-spectrum alternatives to specifically eliminate problematic pathogenic species. However, only a small fraction of the estimated number of interbacterial competition systems has been identified. We discovered a competition system that is novel for Proteus mirabilis. Inspired by an observation in infant mice, we confirmed in vitro that P. mirabilis was able to efficiently kill several Enterobacteriaceae species. This killing system might represent a new function of a known competition system or even a novel system, as the observed characteristics do not fit with described contact-dependent competition systems. Further characterization of this system might help understand how P. mirabilis competes with other Enterobacteriaceae in various niches.

Kosakonia cowanii (syn. Enterobacter cowanii ) is a highly competitive bacterium that lives with plant, insect, fish, bird, and human organisms. It is pathogenic on some plants and an opportunistic pathogen of human. Nine novel viruses that lyse plant pathogenic strains and/or human strains of K. cowanii were isolated, sequenced, and characterized. Kc166A is a novel kayfunavirus, Kc261 is a novel bonnellvirus, and Kc318 is a new cronosvirus (all Autographiviridae ). Kc237 is a new sortsnevirus, but Kc166B and Kc283 are members of new genera within Podoviridae . Kc304 is a new winklervirus, and Kc263 and Kc305 are new myoviruses. The viruses differ in host specificity, plaque phenotype, and lysis kinetics. Some of them should be suitable also as pathogen control agents.

The association of different species of endophytic bacteria with the rhizosphere of the host plants can stimulate growth, development and acclimatization, offering a greater quantity of seedlings, in addition to reducing the cycle, providing economic return to the producer. The objective of this study was to evaluate the effect of introduction four bacterial isolates through inoculation into the root system in three banana cultivars (Prata Anã, Grande Naine and BRS Princesa) in the acclimatization phase. The evaluated treatments were: control (nutrient broth without bacteria); Bacillus cereus strain 1 (BC1); Bacillus cereus strain 2 (BC2); Bacillus thuringiensis (BT); Buttiauxella agrestis (BA). The morphological characteristics related to the development of the plants (total height and pseudostem diameter) were evaluated throughout the acclimatization period. After 90 days of transplanting and acclimatization, root length, leaf number, dry root weight, pseudostem and leaf, leaf area, internal carbon concentration, stomatal conductance, photosynthesis rate, transpiration rate, leaf temperature and chlorophyll were evaluated. The bacteria showed different results in relation to the studied cultivars. Considering the morphological and physiological characteristics observed in this study, B. thuringiensis for the cultivars Prata Anã and Grande Naine and the B. agrestis for the cultivar BRS Princesa are recommended for the process of acclimatization of banana seedlings, as they stimulated growth of the plant, increasing the dry mass, besides promoting the growth of roots. In this way, they improved the physiological aspects of the plants and reduced the period of acclimatization of the banana., (Copyright © 2021 Elsevier GmbH. All rights reserved.)

The mutualistic relationship between alien plant species and microorganisms is proposed to facilitate or hinder invasive success, depending on whether plants can form novel associations with microorganisms in the introduced habitats. However, this hypothesis has not considered seed endophytes that would move together with plant propagules. Little information is available on the seed endophytic bacteria of invasive species and their effects on plant performance. We isolated the seed endophytic bacteria of a xerophytic invasive plant, Lactuca serriola, and examined their plant growth-promoting traits. In addition, we assessed whether these seed endophytes contributed to plant drought tolerance. Forty-two bacterial species were isolated from seeds, and all of them exhibited at least one plant growth-promoting trait. Kosakonia cowanii occurred in all four tested plant populations and produced a high concentration of exopolysaccharides in media with a highly negative water potential. Notably, applying K. cowanii GG1 to Arabidopsis thaliana stimulated plant growth under drought conditions. It also reduced soil water loss under drought conditions, suggesting bacterial production of exopolysaccharides might contribute to the maintenance of soil water content. These results imply that invasive plants can disperse along with beneficial bacterial symbionts, which potentially improve plant fitness and help to establish alien plant species.

Introduction: Minas fresh cheese (MFC), a Brazilian white cheese, is one of the most popular cheeses nationwide. Studies have shown that Listeria monocytogenes occurrence in this product is generally low, while high populations of coliforms can be found. This study aimed to evaluate the influence of coliforms in the behavior of L. monocytogenes in MFC., Methods: Pasteurized milk was inoculated with L. monocytogenes and coliforms, and the acidification was made by lactic acid or by the addition of a starter culture. The cheeses of each production were divided into 3 groups and stored at 5 ºC, 12 ºC and cycles of 5 ºC followed by 25 ºC. In predetermined days, samples were taken and L. monocytogenes, coliforms and lactic acid bacteria populations were evaluated, besides the pH, water activity (aw), titratable acidity and NaCl concentration., Results: The inhibition of L. monocytogenes in the presence of coliforms was observed (p < 0.05), except for those samples prepared with lactic acid and stored at temperature cycles. The values of pH and aw were not sufficiently low to cause inhibition; however, titratable acidity was higher in cheeses containing coliforms. In vitro tests containing lactic acid and L. monocytogenes showed that the bacterium is sensitive to concentration of lactic acid ≥ 0.3%, indicating that lactic acid produced by coliforms strongly influences the population of L. monocytogenes., Conclusions: Thus, it can be concluded that coliforms negatively impact populations of L. monocytogenes in MFC. We strongly recommend that producers of MFC adopt good hygiene practices to not only avoid contamination with L. monocytogenes, but also coliforms.

Background: International travel could facilitate the spread of antimicrobial-resistant bacteria including extended spectrum β-lactamase-producing Enterobacteriaceae (ESBL-E). Previous studies, which attempted to understand the role of gut microbiota in the acquisition of antimicrobial resistant bacteria during international travels, are limited to western travellers., Methods: We established a prospective cohort of 90 Hong Kong travellers to investigate gut microbiota determinants and associated risk factors for the acquisition of ESBL-E. Baseline characteristics and travel-associated risk factors were gathered through questionnaires. Faecal samples were collected in 3-4 days before and after travel. Antimicrobial susceptibility of ESBL-E isolates was tested, and gut microbiota were profiled by 16S rDNA amplicon sequencing. Non-parametric tests were used to detect potential associations, and logistic regression models were used to quantify the associations. Random forest models were constructed to identify microbial predictors for ESBL-E acquisition., Results: In total, 49 (54.4%) participants were tested negative for ESBL-E colonization before travel and were followed up after travel. A total of 60 ESBL-E isolates were cultured from 20 (40.8%) participants. Having low Actinobacteria richness and low abundance of short-chain fatty acid-producing bacteria in the gut microbiota before travel increased the risk of acquiring ESBL-E and the risk can be further exacerbated by eating raw seafood during travel. Besides, post-travel ESBL-E positive participants had increased abundances of several opportunistic pathogens such as Staphylococcus, Enterococcus, Escherichia/Shigella and Klebsiella. The random forest model integrating pre-travel microbiota and the identified travel-related risk factor could predict ESBL-E acquisition with an area under the curve of 75.4% (95% confidence interval: 57.9-93.0%)., Conclusions: In this study, we identified both travel-related risk factors and microbiota predictors for the risk of ESBL-E acquisition. Our results provide foundational knowledge for future developments of microbiota-based interventions to prevent ESBL-E acquisition during international travels., (© International Society of Travel Medicine 2021. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Introduction. Nitrofurantoin is one of the preferred antibiotics in the treatment of uropathogenic multidrug-resistant (MDR) infections. However, resistance to nitrofurantoin in extensively drug-resistant (XDR) bacteria has severely limited the treatment options. Gap statement. Information related to co-resistance or collateral sensitivity (CS) with reference to nitrofurantoin resistant bacteria is limited. Aim. To study the potential of nitrofurantoin resistance as an indicator of the XDR phenotype in Enterobacteriaceae . Methods. One hundred (45 nitrofurantoin-resistant, 21 intermediately resistant and 34 nitrofurantoin-susceptible) Enterobacteriaceae were analysed in this study. Antibiotic susceptibility testing (AST) against nitrofurantoin and 17 other antimicrobial agents across eight different classes was performed by using the Vitek 2.0 system. The isolates were screened for the prevalence of acquired antimicrobial resistance (AMR) and efflux pump genes by PCR. Results. In total, 51 % of nitrofurantoin-resistant and 28 % of intermediately nitrofurantoin resistant isolates exhibited XDR characteristics, while only 3 % of nitrofurantoin-sensitive isolates were XDR ( P =0.0001). Significant co-resistance was observed between nitrofurantoin and other tested antibiotics (β-lactam, cephalosporin, carbapenem, aminoglycoside and tetracycline). Further, the prevalence of AMR and efflux pump genes was higher in the nitrofurantoin-resistant strains compared to the susceptible isolates. A strong association was observed between nitrofurantoin resistance and the presence of bla PER-1 , bla NDM-1 , bla OXA-48 , ant(2) and oqxA-oqxB genes. Tigecycline (84 %) and colistin (95 %) were the only antibiotics to which the majority of the isolates were susceptible. Conclusion. Nitrofurantoin resistance could be an indicator of the XDR phenotype among Enterobacteriaceae , harbouring multiple AMR and efflux pump genes. Tigecycline and colistin are the only antibiotics that could be used in the treatment of such XDR infections. A deeper understanding of the co-resistance mechanisms in XDR pathogens and prescription of AST-based appropriate combination therapy may help mitigate this problem.

Developing mathematical models to accurately predict microbial growth dynamics remains a key challenge in ecology, evolution, biotechnology, and public health. To reproduce and grow, microbes need to take up essential nutrients from the environment, and mathematical models classically assume that the nutrient uptake rate is a saturating function of the nutrient concentration. In nature, microbes experience different levels of nutrient availability at all environmental scales, yet parameters shaping the nutrient uptake function are commonly estimated for a single initial nutrient concentration. This hampers the models from accurately capturing microbial dynamics when the environmental conditions change. To address this problem, we conduct growth experiments for a range of micro-organisms, including human fungal pathogens, baker's yeast, and common coliform bacteria, and uncover the following patterns. We observed that the maximal nutrient uptake rate and biomass yield were both decreasing functions of initial nutrient concentration. While a functional form for the relationship between biomass yield and initial nutrient concentration has been previously derived from first metabolic principles, here we also derive the form of the relationship between maximal nutrient uptake rate and initial nutrient concentration. Incorporating these two functions into a model of microbial growth allows for variable growth parameters and enables us to substantially improve predictions for microbial dynamics in a range of initial nutrient concentrations, compared to keeping growth parameters fixed., Competing Interests: The authors have declared that no competing interests exist.

A homogeneous polysaccharide named SHNP with apparent molecular weight of 8.4 kDa was purified from brown algae Sargassum henslowianum using ethanol precipitation, ion-exchange chromatography, and gel-filtration column chromatography. Structural analyses reveal that SHNP is completely composed of glucose, and its backbone consists of β-D-(1→3)-Glcp with side chains comprising t-β-D-Glcp attached at the O-6 position. Thus, SHNP is a laminarin-type polysaccharide. In vitro fermentation test results showed that SHNP was digested by gut microbiota; the pH value in the fecal culture of SHNP was significantly decreased; and total short-chain fatty acids, acetic, propionic and n-butyric acids were significantly increased. Furthermore, SHNP regulated the intestinal microbiota composition by stimulating the growth of species belonging to Enterobacteriaceae while depleting Haemophilus parainfluenzae and Gemmiger formicilis. Taken together, these results indicate that SHNP has the potential for regulating gut microbiota, but its specific role in the regulation requires to be further investigated., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Purpose: Tigecycline is one of few antibiotics active against multidrug-resistant bacteria; however, the assessment of dosing strategies to optimize its activity is needed. The purpose was to use Monte Carlo Simulation (MCS) to determine if safe tigecycline dosing options attaining breakpoints for pharmacokinetic/pharmacodynamic (PK-PD) targets in non-critically ill adults could be identified., Methods: Publications that evaluated tigecycline dosing regimens and provided mean PK variables of interest (minimum 2 of: elimination rate constant or half-life and volume of distribution or clearance), with SDs, were included. Weighted mean (±SDs) for each PK parameter were determined. Food and Drug Administration minimum inhibitory concentration (MIC) tigecycline breakpoints for susceptible (MIC ≤ 2 μg/mL), intermediate (MIC 4 μg/mL), and resistant (MIC ≥ 8 μg/mL) Enterobacteriaceae were used. MCS probability distributions for PK-PD target attainment of AUC for total tigecycline plasma concentration from 0 to 24 h following an intravenous dose (AUC total, 0-24h ) to MIC ratios of ≥ 18, 7, and 4.5 were generated, with success defined as ≥ 80% probability of target attainment at a given MIC., Results: Ten studies (n = 442) were eligible. Tigecycline 150 mg IV q12h for ward patients with resistant bacteria up to a MIC of 0.48, 1, and 2 μg/mL for an AUC total, 0-24h /MIC target attainment of 18, 7, and 4.5, respectively, may be appropriate., Conclusion: Bacterial infections with tigecycline MICs ≥ 0.48-2 μg/mL, depending on AUC total, 0-24h /MIC target, may require treatment with alternate antibiotics due to target attainment failure.

The aim of the research was to estimate the effect of selected lactic acid bacterial strains on the technological quality and microbiological stability of mechanically separated poultry meat (MSPM) cured with a reduced amount of sodium nitrite. The 5 different treatments of MSPM batters were prepared: C150 - control cured with sodium nitrite at 150 mg/kg, C50 - control cured with sodium nitrite at 50 mg/kg, PL1 - cured with sodium nitrite at 50 mg/kg and inoculated Lactobacillus plantarum SCH1 at about 10 7  cfu/g, PL2 - cured with sodium nitrite at 50 mg/kg and inoculated Lactobacillus brevis KL5 at about 10 7  cfu/g, and PL3 - cured sodium nitrite at 50 mg/kg and inoculated L. plantarum S21 at about 10 7  cfu/g. The MSPM batters were tested at 1, 4 and 7 d of being in refrigerated storage. The scope of the research was as follows - physicochemical determinations: pH and redox, nitrates and nitrites as well as nitrosyl pigments levels, color estimation with a Comission Internationale de l'Eclairage Lab system and microbiological determinations: the total viable counts, the mesophilic lactic acid bacteria counts, Escherichia coli and Enterobacteriaceae counts. The inhibitory effect of L. plantarum SCH1 isolated from the ecological raw fermented meat product on E. coli in cured MSPM batters during refrigerated storage was proved (P < 0.05). The use of lactic acid bacterial strains in cured batters that were prepared and based on mechanically separated poultry meat did not have a negative effect on their technological quality. The positive effect of L. brevis KL5 on the level of nitrosyl pigments in the cured MSPM batters was observed (P < 0.05). The conducted research suggested the possibility of using the selected bacterial strains of the Lactobacillus genus to improve the microbiological quality of MSPM cured with a reduced amount of sodium nitrite., (Copyright © 2020. Published by Elsevier Inc.)

Obligate symbioses involving intracellular bacteria have transformed eukaryotic life, from providing aerobic respiration and photosynthesis to enabling colonization of previously inaccessible niches, such as feeding on xylem and phloem, and surviving in deep-sea hydrothermal vents. A major challenge in the study of obligate symbioses is to understand how they arise. Because the best studied obligate symbioses are ancient, it is especially challenging to identify early or intermediate stages. Here we report the discovery of a nascent obligate symbiosis in Howardula aoronymphium , a well-studied nematode parasite of Drosophila flies. We have found that H aoronymphium and its sister species harbor a maternally inherited intracellular bacterial symbiont. We never find the symbiont in nematode-free flies, and virtually all nematodes in the field and the laboratory are infected. Treating nematodes with antibiotics causes a severe reduction in fly infection success. The association is recent, as more distantly related insect-parasitic tylenchid nematodes do not host these endosymbionts. We also report that the Howardula nematode symbiont is a member of a widespread monophyletic group of invertebrate host-associated microbes that has independently given rise to at least four obligate symbioses, one in nematodes and three in insects, and that is sister to Pectobacterium , a lineage of plant pathogenic bacteria. Comparative genomic analysis of this group, which we name Candidatus Symbiopectobacterium, shows signatures of genome erosion characteristic of early stages of symbiosis, with the Howardula symbiont's genome containing over a thousand predicted pseudogenes, comprising a third of its genome., Competing Interests: The authors declare no competing interest.

The nitrated compounds 2,4-dinitrotoluene (2,4-DNT), 2,4,6-trinitrotoluene (TNT), and pentaerythritol tetranitrate (PETN) are toxic xenobiotics widely used in various industries. They often coexist as environmental contaminants. The aims of this study were to evaluate the transformation of 100 mg L -1 of TNT, 2,4-DNT, and PETN by Raoultella planticola M30b and Rhizobium radiobacter M109c and identify enzymes that may participate in the transformation. These strains were selected from 34 TNT transforming bacteria. Cupriavidus metallidurans DNT was used as a reference strain for comparison purposes. Strains DNT, M30b and M109c transformed 2,4-DNT (100%), TNT (100, 94.7 and 63.6%, respectively), and PETN (72.7, 69.3 and 90.7%, respectively). However, the presence of TNT negatively affects 2,4-DNT and PETN transformation (inhibition > 40%) in strains DNT and M109c and fully inhibited (100% inhibition) 2,4-DNT transformation in R. planticola M30b.Genomes of R. planticola M30b and R. radiobacter M109c were sequenced to identify genes related with 2,4-DNT, TNT or PETN transformation. None of the tested strains presented DNT oxygenase, which has been previously reported in the transformation of 2,4-DNT. Thus, unidentified novel enzymes in these strains are involved in 2,4-DNT transformation. Genes encoding enzymes homologous to the previously reported TNT and PETN-transforming enzymes were identified in both genomes. R. planticola M30b have homologous genes of PETN reductase and xenobiotic reductase B, while R. radiobacter M109c have homologous genes to GTN reductase and PnrA nitroreductase. The ability of these strains to transform explosive mixtures has a potentially biotechnological application in the bioremediation of contaminated environments.

Urinary tract infection (UTI) is a common complication in kidney transplant recipients and can lead to significant morbidity and mortality. Recent evidence supports a role for the gut as a source for UTIs but little is known about the relationship between gut commensal bacteria and UTI development. We hypothesized that the abundance of gut commensal bacteria is associated with a lower risk of developing bacteriuria and UTIs. We performed gut microbiome profiling using 16S rRNA gene sequencing of the V4-V5 hypervariable region on 510 fecal specimens in 168 kidney transplant recipients. Fifty-one kidney transplant recipients (30%) developed Enterobacteriaceae bacteriuria within the first 6 months after transplantation ( Enterobacteriaceae Bacteriuria Group) and 117 did not (No Enterobacteriaceae Bacteriuria Group). The relative abundances of Faecalibacterium and Romboutsia were significantly higher in the fecal specimens from the No Enterobacteriaceae Bacteriuria Group than those from the Enterobacteriaceae Bacteriuria Group (Adjusted P value<.01). The combined relative abundance of Faecalibacterium and Romboutsia was inversely correlated with the relative abundance of Enterobacteriaceae (r = -0.13, P = .003). In a multivariable Cox Regression, a top tercile cutoff of the combined relative abundance of Faecalibacterium and Romboutsia of ≥13.7% was independently associated with a decreased risk for Enterobacteriaceae bacteriuria (hazard ratio 0.3, P = .02) and Enterobacteriaceae UTI (hazard ratio 0.4, P = .09). In conclusion, we identify bacterial taxa associated with decreased risk for Enterobacteriaceae bacteriuria and Enterobacteriaceae UTI in kidney transplant recipients, which supports future studies on modulating the gut microbiota as a novel treatment for preventing UTIs.

This study aimed to analyse the frequency of occurrence of spontaneous decolonization in intensive care unit patients colonized by extended-spectrum beta-lactamase-producing Enterobacterales (ESBL-E) in order to assess the added value of continuing weekly ESBL-E rectal carriage screening in these patients. In total, 49,468 weekly rectal screening samples taken from 20,846 patients over 12 years were included. Among the 4280 ESBL-E carriers, only 109 patients (2.5%) could be considered decolonized at the end of their hospitalization with at least three consecutive negative samples. Overall, 7957 samples (16.1%) were requested for patients already identified as ESBL-E carriers. Avoiding unnecessary weekly screening following positive ESBL-E colonization results could decrease nursing and laboratory work loads., (Copyright © 2020 The Healthcare Infection Society. Published by Elsevier Ltd. All rights reserved.)

Plasmids are autonomously replicating sequences that help cells adapt to diverse stresses. Theta plasmids are the most frequent plasmid class in enterobacteria. They co-opt two host replication mechanisms: replication at oriC , a DnaA-dependent pathway leading to replisome assembly (theta class A), and replication fork restart, a PriA-dependent pathway leading to primosome assembly through primer extension and D-loop formation (theta classes B, C, and D). To ensure autonomy from the host's replication and to facilitate copy number regulation, theta plasmids have unique mechanisms of replication initiation at the plasmid origin of replication ( ori ). Tight plasmid copy number regulation is essential because of the major and direct impact plasmid gene dosage has on gene expression. The timing of plasmid replication and segregation are also critical for optimizing plasmid gene expression. Therefore, we propose that plasmid replication needs to be understood in its biological context, where complex origins of replication (redundant origins, mosaic and cointegrated replicons), plasmid segregation, and toxin-antitoxin systems are often present. Highlighting their tight functional integration with ori function, we show that both partition and toxin-antitoxin systems tend to be encoded in close physical proximity to the ori in a large collection of Escherichia coli plasmids. We also propose that adaptation of plasmids to their host optimizes their contribution to the host's fitness while restricting access to broad genetic diversity, and we argue that this trade-off between adaptation to host and access to genetic diversity is likely a determinant factor shaping the distribution of replicons in populations of enterobacteria.

'Filth flies' facilitate the dispersal of pathogens between animals and humans. The objective was to study the intestinal colonization with antimicrobial resistant and enteropathogenic bacteria in 'filth flies' from Nigeria. Flies from Southern Nigeria were screened for extended-spectrum β-lactamase producing Enterobacterales (ESBL-E), Staphylococcus aureus, Salmonella sp., Shigella sp., Campylobacter sp. and Yersinia enterocolitica by culture. ESBL-E were tested for bla SHV , bla CTX-M and bla TEM ; S. aureus was screened for enterotoxins. Spa typing and multilocus sequence typing (MLST) was done for S. aureus and MLST for Escherichia coli. Of 2,000 flies, 400 were randomly collected for species identification. The most common species were Musca domestica (44.8%, 179/400), Chrysomya putoria (21.6%, 85/400) and Musca sorbens (18.8%, 75/400). Flies were colonized with S. aureus (13.8%, 275/2,000) and ESBL-E (0.8%, 16/2,000). No other enteropathogenic bacteria were detected. The enterotoxin sei was most common (26%, 70/275) in S. aureus, followed by sea (12%, n = 32/275). Four S. aureus isolates were methicillin resistant (mecA positive, t674 and t5305, ST15). The bla CTX-M (n = 16) was the most prevalent ESBL subtype, followed by bla TEM (n = 8). 'Filth flies' can carry antimicrobial resistant bacteria in Nigeria. Enterotoxin-positive S. aureus might be the main reason for food poisoning by 'filth flies' in the study area.

Outer membrane vesicles (OMVs) are naturally released from Gram-negative bacteria and play important roles in various biological functions. Released vesicles are not uniform in shape, size, or characteristics, and little is known about this diversity of OMVs. Here, we show that deletion of tolB , which encodes a part of the Tol-Pal system, leads to the production of multiple types of vesicles and increases overall vesicle production in the high-vesicle-forming Buttiauxella agrestis type strain JCM 1090. The Δ tolB mutant produced small OMVs and multilamellar/multivesicular OMVs (M-OMVs) as well as vesicles with a striking similarity to the wild type. M-OMVs, previously undescribed, contained triple-lamellar membrane vesicles and multiple vesicle-incorporating vesicles. Ultracentrifugation enabled the separation and purification of each type of OMV released from the Δ tolB mutant, and visualization by quick-freeze deep-etch and replica electron microscopy indicated that M-OMVs are composed of several lamellar membranes. Visualization of intracellular compartments of Δ tolB mutant cells showed that vesicles were accumulated in the broad periplasm, which is probably due to the low linkage between the outer and inner membranes attributed to the Tol-Pal defect. The outer membrane was invaginating inward by wrapping a vesicle, and the precursor of M-OMVs existed in the cell. Thus, we demonstrated a novel type of bacterial OMV and showed that unconventional processes enable the B. agrestis Δ tolB mutant to form unique vesicles. IMPORTANCE Membrane vesicle (MV) formation has been recognized as a common mechanism in prokaryotes, and MVs play critical roles in intercellular interaction. However, a broad range of MV types and their multiple production processes make it difficult to gain a comprehensive understanding of MVs. In this work, using vesicle separation and electron microscopic analyses, we demonstrated that diverse types of outer membrane vesicles (OMVs) were released from an engineered strain, Buttiauxella agrestis JCM 1090 T Δ tolB mutant. We also discovered a previously undiscovered type of vesicle, multilamellar/multivesicular outer membrane vesicles (M-OMVs), which were released by this mutant using unconventional processes. These findings have facilitated considerable progress in understanding MV diversity and expanding the utility of MVs in biotechnological applications., (Copyright © 2020 American Society for Microbiology.)

Bacterial intracellular symbiosis (endosymbiosis) is widespread in nature and impacts many biological processes. In holometabolous symbiotic insects, metamorphosis entails a complete and abrupt internal reorganization that creates a constraint for endosymbiont transmission from larvae to adults. To assess how endosymbiosis copes-and potentially evolves-throughout this major host-tissue reorganization, we used the association between the cereal weevil Sitophilus oryzae and the bacterium Sodalis pierantonius as a model system. S. pierantonius are contained inside specialized host cells, the bacteriocytes, that group into an organ, the bacteriome. Cereal weevils require metabolic inputs from their endosymbiont, particularly during adult cuticle synthesis, when endosymbiont load increases dramatically. By combining dual RNA-sequencing analyses and cell imaging, we show that the larval bacteriome dissociates at the onset of metamorphosis and releases bacteriocytes that undergo endosymbiosis-dependent transcriptomic changes affecting cell motility, cell adhesion, and cytoskeleton organization. Remarkably, bacteriocytes turn into spindle cells and migrate along the midgut epithelium, thereby conveying endosymbionts to midgut sites where future mesenteric caeca will develop. Concomitantly, endosymbiont genes encoding a type III secretion system and a flagellum apparatus are transiently up-regulated while endosymbionts infect putative stem cells and enter their nuclei. Infected cells then turn into new differentiated bacteriocytes and form multiple new bacteriomes in adults. These findings show that endosymbiosis reorganization in a holometabolous insect relies on a synchronized host-symbiont molecular and cellular "choreography" and illustrates an adaptive feature that promotes bacteriome multiplication to match increased metabolic requirements in emerging adults., Competing Interests: The authors declare no competing interest.

Numerous adaptations are gained in light of a symbiotic lifestyle. Here, we investigated the obligate partnership between tortoise leaf beetles (Chrysomelidae: Cassidinae) and their pectinolytic Stammera symbionts to detail how changes to the bacterium's streamlined metabolic range can shape the digestive physiology and ecological opportunity of its herbivorous host. Comparative genomics of 13 Stammera strains revealed high functional conservation, highlighted by the universal presence of polygalacturonase, a primary pectinase targeting nature's most abundant pectic class, homogalacturonan (HG). Despite this conservation, we unexpectedly discovered a disparate distribution for rhamnogalacturonan lyase, a secondary pectinase hydrolyzing the pectic heteropolymer, rhamnogalacturonan I (RG-I). Consistent with the annotation of rhamnogalacturonan lyase in Stammera, cassidines are able to depolymerize RG-I relative to beetles whose symbionts lack the gene. Given the omnipresence of HG and RG-I in foliage, Stammera that encode pectinases targeting both substrates allow their hosts to overcome a greater diversity of plant cell wall polysaccharides and maximize access to the nutritionally rich cytosol. Possibly facilitated by their symbionts' expanded digestive range, cassidines additionally endowed with rhamnogalacturonan lyase appear to utilize a broader diversity of angiosperms than those beetles whose symbionts solely supplement polygalacturonase. Our findings highlight how symbiont metabolic diversity, in concert with host adaptations, may serve as a potential source of evolutionary innovations for herbivorous lineages., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Insect-bacterial symbioses are ubiquitous, but there is still much to uncover about how these relationships establish, persist and evolve. The tsetse endosymbiont Sodalis glossinidius displays intriguing metabolic adaptations to its microenvironment, but the process by which this relationship evolved remains to be elucidated. The recent chance discovery of the free-living species of the genus Sodalis , Sodalis praecaptivus , provides a serendipitous starting point from which to investigate the evolution of this symbiosis. Here, we present a flux balance model for S. praecaptivus and empirically verify its predictions. Metabolic modelling is used in combination with a multi-objective evolutionary algorithm to explore the trajectories that S. glossinidius may have undertaken from this starting point after becoming internalized. The order in which key genes are lost is shown to influence the evolved populations, providing possible targets for future in vitro genetic manipulation. This method provides a detailed perspective on possible evolutionary trajectories for S. glossinidius in this fundamental process of evolutionary and ecological change.

Enteric pathogens rely on a variety of toxins, adhesins and other virulence factors to cause infections. Some of the best studied pathogens belong to the Enterobacterales order; these include enteropathogenic and enterohemorrhagic Escherichia coli , Shigella spp., and the enteropathogenic Yersiniae . The pathogenesis of these organisms involves two different secretion systems, a type 3 secretion system (T3SS) and type 5 secretion systems (T5SSs). The T3SS forms a syringe-like structure spanning both bacterial membranes and the host cell plasma membrane that translocates toxic effector proteins into the cytoplasm of the host cell. T5SSs are also known as autotransporters, and they export part of their own polypeptide to the bacterial cell surface where it exerts its function, such as adhesion to host cell receptors. During infection with these enteropathogens, the T3SS and T5SS act in concert to bring about rearrangements of the host cell cytoskeleton, either to invade the cell, confer intracellular motility, evade phagocytosis or produce novel structures to shelter the bacteria. Thus, in these bacteria, not only the T3SS effectors but also T5SS proteins could be considered "cytoskeletoxins" that bring about profound alterations in host cell cytoskeletal dynamics and lead to pathogenic outcomes., Competing Interests: The authors declare no conflict of interest.

Vacuole generation occurs frequently during the enlargement of bacterial protoplasts and spheroplasts. Gram-positive Enterococcus faecalis protoplasts and gram-negative Lelliottia amnigena spheroplasts had large and small vacuoles inside the cytoplasm, respectively. Although no vacuoles were found at the early stage of cell enlargement, all enlarged cells used in the microinjection procedures had vacuoles. The plasma membrane of L. amnigena was more flexible than that of E. faecalis. In addition, E. faecalis protoplasts had unique discoidal structures as well as spherical structures in the cytoplasm. Our findings showed that the number of vacuoles increased as the L. amnigena plasma membrane expanded and that the size of vacuoles increased as the E. faecalis plasma membrane expanded, suggesting that bacterial cell enlargement involved vacuole generation. Thus, biosynthesis of the plasma and vacuolar membranes was synchronous with the bacterial cell enlargement. Differences in the plasma membrane flexibility might influence the different types of vacuole generation.

The biofilms of Enterobacteriaceae are fortified by assembly of curli amyloid fibres on the cell surface. Curli not only provides structural reinforcement, but also facilitates surface adhesion. To prevent toxic intracellular accumulation of amyloid precipitate, secretion of the major curli subunit, CsgA, is tightly regulated. In this work, we have employed solution state NMR spectroscopy to characterise the structural ensemble of the pre-fibrillar state of CsgA within the bacterial periplasm, and upon recruitment to the curli pore, CsgG, and the secretion chaperone, CsgE. We show that the N-terminal targeting sequence (N) of CsgA binds specifically to CsgG and that its subsequent sequestration induces a marked transition in the conformational ensemble, which is coupled to a preference for CsgE binding. These observations lead us to suggest a sequential model for binding and structural rearrangement of CsgA at the periplasmic face of the secretion machinery.

There is limited data on the gut colonization rate of colistin resistant (Col-R) bacteria in patients and healthy volunteers in India. Aim of this study was to investigate the stool carriage rate of Col-R in hospitalized patients. Stool samples were inoculated in Eosin Methylene Blue agar plates supplemented with colistin. Colistin minimum inhibitory concentrations (MICs) were determined by the broth microdilution method. PCR for the mcr-1 was performed on Col-R Enterobacteriaceae isolates. Mutations in the mgrB gene were analyzed in K. pneumoniae isolates. Mcr-1 positive E. coli was subjected to whole-genome sequencing. Out of 65 stool samples screened, 33 (51%) samples carried Col-R bacteria. Majority (76.7%) of the isolates were sensitive to carbapenem., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Objectives: The objective of this study was to assess the prevalence of maternal recto-vaginal extended-spectrum β-lactamase producing Enterobacteriacea (ESBL-E) colonization, identify risk factors for maternal and neonatal ESBL-E colonization, and subsequent impact on neonatal mortality., Methods: A prospective, cross-sectional study was conducted at the University of Abuja Teaching Hospital from April 2016 to May 2017. Maternal-neonatal pairs were screened for ESBL-E exposure at time of delivery. Neonatal mortality was assessed at 28 days., Results: A total of 1161 singleton deliveries were evaluated. In total, 9.7% (113/1161) of mothers and 4.3% (50/1161) of infants had ESBL-E-positive cultures at delivery. Maternal antibiotic exposure was associated with ESBL-E recto-vaginal colonization (18.6% (21/113) vs. 8.4% (88/1048), p < 0.001)). Maternal ESBL-E colonization (adjusted odds ratio (AOR) 14.85; 95% CI 7.83-28.15) and vaginal delivery (AOR 6.35; 95% CI 2.63-17.1) were identified as a risk factor for positive ESBL-E neonatal surface cultures. Neonatal positive ESBL-E surface cultures were a risk factor for neonatal mortality (stillbirths included, AOR 4.84; 95% CI 1.44-16.31). The finding that maternal ESBL-E recto-vaginal colonization appeared protective in regards to neonatal mortality (AOR 0.22; 95% CI .06-0.75) requires further evaluation., Conclusions: Maternal ESBL-E recto-vaginal colonization is an independent risk factor for neonatal ESBL-E colonization and neonates with positive ESBL-E surface cultures were identified as having increased risk of neonatal mortality., (Copyright © 2019 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.)

The widespread use of pesticides has been one of the major anthropogenic sources of environmental pollution. Organophosphorus (OP) pesticides are predominantly used in agriculture due to their broad-spectrum insecticidal activity and chemical stability. The study was focused on the biodegradation of OP pesticides, Profenofos (PF) and Quinalphos (QP) in culture media using bacterium isolated from wetland paddy rhizosphere. The strain VITPSCQ3 showed higher pesticide tolerance, efficient biofilm formation and was capable of synthesizing organophosphate degrading enzymes. Based on the 16S rRNA gene sequencing the isolate exhibited maximum sequence similarity with Kosakinia oryzae (GenBank accession number: KR149275). Biodegradation assay with various concentrations of PF and QP (200, 400, 600 and 800 mg L -1 ) showed maximum degradation up to 82% and 92% within 48 h. The kinetic studies revealed the biodegradation rates (k) to be 0.0844 min -1 and 0.107 min -1 with half-lives (h) of 18 h and 14.8 h for PF and QP. The degradation products were identified by GCMS and possible degradation pathways were proposed using Insilico techniques. To the best of our knowledge, this is the first report on the biodegradation of PF and QP using Kosakonia oryzae. Bioremoval of PF and QP from aqueous solution was performed using the biofilm of VITPSCQ3 developed on selected substrates in a circulating Vertical-flow packed-bed biofilm (VFPBB) bioreactor. Charcoal, gravel and mushroom (Agaricus bisporus) were used as biofilm carriers. Mushroom showed strong biofilm formation with optimum biodegradation capacity of up to 96% for PF and 92% for QP within 120 min reaction time., Competing Interests: Declaration of competing interest The authors would like to declare that they have no conflict of interest., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Bacterial soft rot diseases caused by Pectobacterium spp. and Dickeya spp. affect a wide range of crops, including potatoes, a major food crop. As of today, farmers mostly rely on sanitary practices, water management, and plant nutrition for control. We tested the bacterial predators Bdellovibrio and like organisms (BALOs) to control potato soft rot. BALOs are small, motile predatory bacteria found in terrestrial and aquatic environments. They prey on a wide range of Gram-negative bacteria, including animal and plant pathogens. To this end, BALO strains HD100, 109J, and a Δ merRNA derivative of HD100 were shown to efficiently prey on various rot-causing strains of Pectobacterium and Dickeya solani BALO control of maceration caused by a highly virulent strain of Pectobacterium carotovorum subsp. brasilense was then tested in situ using a potato slice assay. All BALO strains were highly effective at reducing disease, up to complete prevention. Effectivity was concentration dependent, and BALOs applied before P. carotovorum subsp. brasilense inoculation performed significantly better than those applied after the disease-causing agent, maybe due to in situ consumption of glucose by the prey, as glucose metabolism by live prey bacteria was shown to prevent predation. Dead predators and the supernatant of BALO cultures did not significantly prevent maceration, indicating that predation was the major mechanism for the prevention of the disease. Finally, plastic resistance to predation was affected by prey and predator population parameters, suggesting that population dynamics affect prey response to predation. IMPORTANCE Bacterial soft rot diseases caused by Pectobacterium spp. and Dickeya spp. are among the most important plant diseases caused by bacteria. Among other crops, they inflict large-scale damage to potatoes. As of today, farmers have few options to control them. The bacteria Bdellovibrio and like organisms (BALOs) are obligate predators of bacteria. We tested their potential to prey on Pectobacterium spp. and Dickeya spp. and to protect potato. We show that different BALOs can prey on soft rot-causing bacteria and prevent their growth in situ , precluding tissue maceration. Dead predators and the supernatant of BALO cultures did not significantly prevent maceration, showing that the effect is due to predation. Soft rot control by the predators was concentration dependent and was higher when the predator was inoculated ahead of the prey. As residual prey remained, we investigated what determines their level and found that initial prey and predator population parameters affect prey response to predation., (Copyright © 2020 American Society for Microbiology.)

Objective: Extended-spectrum beta-lactamase (ESBL) is a rapidly evolving enzyme that cleaves beta-lactam-containing antibiotics, forming resistance to certain types of antibiotics, such as penicillin, cephalosporins and monobactams. Colonization with ESBL-producing bacteria during pregnancy is harmful, however this topic is currently underrepresented in the literature., Study Design: Using a retrospective design, we analyzed data of all consecutive pregnant women who were identified with a vaginal colonization of ESBL-producing bacteria from 2011 to 2016 at the Medical University of Vienna, Department of Obstetrics and Gynecology. Swabs were taken during pregnancy and/or at delivery, as well as from neonates. Demographic and clinical data were obtained from the central in-house alert system and patients' clinical records., Results: Of the 14,279 deliveries performed in our department during the study period, we identified 13 women with vaginal colonization of ESBL-producing bacteria during pregnancy. Of these cases, 6 born neonates were tested ESBL positive. The maternal-to-neonatal transmission rate was 43 %, associated with a 70 % rate of preterm premature rupture of the membranes (pPROM) and a preterm birth rate of 83 %. Of the 6 neonates with ESBL colonization, 4 neonates (67 %) were born to mothers who were still tested positive at the time of delivery., Conclusion: Maternal colonization of ESBL-producing bacteria is an important risk factor for transmission. The vaginal presence of ESBL-producing bacteria during pregnancy is associated with preterm birth and pPROM, which shows the need for clear diagnostic and therapeutic guidelines., Competing Interests: Declaration of Competing Interest All authors declare that they have no conflict of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Acute oak decline (AOD) affects native UK oak species causing rapid decline and mortality in as little as five years. A major symptom of AOD is black weeping stem lesions associated with bacterial phytopathogens, Brenneria goodwinii and Gibbsiella quercinecans. However, there is limited knowledge on the ecological and environmental reservoirs of these phytopathogens. Rainwater and soils are common reservoirs of plant pathogens in a forest environment; therefore, the aim of this study was to investigate the survival of B. goodwinii and G. quercinecans in vitro when inoculated into rainwater and forest soil using a combination of agar-based colony counts and gyrB gene-targeted quantitative PCR (qPCR). Brenneria goodwinii lost viability on inoculation into soil and rainwater, but was detectable at low abundance in soil for 28 days using qPCR, suggesting a limited ability to persist outside of the host, potentially in a viable but non-culturable (VBNC) state. Conversely, Gibbsiella quercinecans, was re-isolated from rainwater for the entire duration of the experiment (84 days) and was re-isolated from forest soil after 28 days, with qPCR analysis corroborating these trends. These data demonstrate that B. goodwinii is unable to survive in forest soils and rainwater, suggesting that it may be an endosymbiont of oak trees, whereas G. quercinecans remains viable in soil and rainwater biomes, suggesting a broad ecological distribution. These data advance understanding of the potential epidemiology of AOD-associated bacteria and their ecological reservoirs, thus increasing the overall knowledge of the pathology of AOD, which assists the development of future management strategies., (Copyright © 2020 Elsevier GmbH. All rights reserved.)

While genome erosion is extensively studied in intracellular symbionts, the metabolic implications of reductive evolution in microbes subsisting extracellularly remain poorly understood. Stammera capleta-an extracellular symbiont in leaf beetles-possesses an extremely reduced genome (0.27 Mb), enabling the study of drastic reductive evolution in the absence of intracellularity. Here, we outline the genomic and transcriptomic profiles of Stammera and its host to elucidate host-symbiont metabolic interactions. Given the symbiont's substantial demands for nutrients and membrane components, the host's symbiotic organ shows repurposing of internal resources by upregulating nutrient transporters and cuticle-processing genes targeting epithelial chitin. Facilitated by this supplementation and its localization, Stammera exhibits a highly streamlined gene expression profile and a fermentation pathway for energy conversion, sharply contrasting the respiratory metabolism retained by most intracellular symbionts. Our results provide insights into a tightly regulated and metabolically integrated extracellular symbiosis, expanding our understanding of the minimal metabolism required to sustain life outside of a host cell.

The testing of bacterial preservation should be included in preliminary studies to epidemiological studies. In the case of multidrug-resistant organism (MDRO) studies, quantifications of the bacteria make it possible to understand their emergence. The purpose of this preliminary study was to evaluate the performance of ESwab TM on survival of Escherichia coli, Klebsiella pneumoniae, and Enterococcus faecalis, based on the number of freezing and thawing (F/T) cycles at -80 °C and freezing time. A first experiment with 9 samples showed that multiple F/T cycles drastically affected Enterobacteriaceae viabilities and less E. faecalis one. A single freezing maintained the three species viabilities during three weeks. A second experiment showed that E. coli survival was maintained with a 3-month single freezing. This study which used a limited number of bacterial isolates is however a proof of concept establishing the utility of ESwab TM samples when frozen once in quantitative studies of bacteria., (Copyright © 2019 Elsevier Inc. All rights reserved.)

The effects of 3 ethanol levels (30, 50, and 70%) with and without thiamine dilaurylsulfate (TDS; 1,000 ppm) were evaluated for the reduction of natural mesophilic aerobic bacteria (MAB), coliforms, and inoculated Salmonella Typhimurium (S. Typhimurium) in chicken skin. The chicken skin was inoculated with a 7 log cfu/mL suspension of S. Typhimurium. Loosely, intermediately, and tightly attached cells were recovered from chicken skin through shaking at 200 rpm for 5 min, stomaching for 1 min, and blending for 1 min, respectively. Increasing the ethanol concentration reduced the number of MAB, coliforms, and S. Typhimurium on the chicken skin, whereas TDS treatment without ethanol was not effective. Intermediately and tightly attached microorganisms (total MAB, coliforms, and S. Typhimurium) were more resistant to chemical disinfectants than loosely attached microorganisms. The combination of 70% ethanol with TDS was most effective than the combination of TDS with lower concentrations of ethanol in reducing populations of loosely, intermediately, and tightly attached MAB (by 1.88 log cfu/g, 1.21 log cfu/g, and 0.84 log cfu/g, respectively), coliforms (by 1.14 log cfu/g, 1.04 log cfu/g, and 0.67 log cfu/g, respectively), and S. Typhimurium (by 1.62 log cfu/g, 1.72 log cfu/g, and 1.27 log cfu/g, respectively). However, the chicken skin treated with higher concentrations of ethanol was tougher (P < 0.05) and more yellow and less red (P < 0.05) than that treated with lower concentrations of ethanol or with water (control). On the other hand, a combination of 30% ethanol and TDS yielded the best results, showing the reduction greater than 0.5 log cfu/g in S. Typhimurium, with no negative effect on chicken skin color or texture. Thus, a combination of 30% ethanol and TDS appears to be the optimal treatment for reducing microbial contamination of skin-on chicken products to enhance poultry safety without decreasing food quality, and this treatment could be applied in the poultry industry., (Copyright © 2019. Published by Elsevier Inc.)

The emergence of the plasmid-mediated colistin resistance gene mcr-1 has led to serious multidrug-resistant (MDR) Enterobacteriaceae infections, which are a great threat to the clinic. This study aims to find an inhibitor of MCR-1 to reestablish the use of polymyxins against MDR Enterobacteriaceae infections. Here, we determined that the natural compound honokiol could enhance the efficacy of polymyxins against MDR Enterobacteriaceae infections by a checkerboard MIC assay, a time-kill assay, a combined disk test, Western blotting, molecular simulation dynamics, and mouse infection models. The MIC results indicated that honokiol can recover the sensitivity of polymyxins against MCR-1-positive Klebsiella pneumoniae and Escherichia coli (with a fractional inhibitory concentration index ranging from 0.09 ± 0.00 to 0.27 ± 0.06). Based on time-kill curve analysis, all of the tested bacteria were killed within 1 h following the combined therapy with honokiol and polymyxins. Molecular simulation dynamics results suggested that honokiol directly binds to the MCR-1 active region, reducing the biological activity of MCR-1. The combination of honokiol and polymyxins could increase the 40% protection rate and reduce the bacterial load on the thigh muscles of mice. Our study indicates that honokiol is a predominant natural compound whose combination therapy with polymyxins is very promising in future treatment options for MCR-1-positive Enterobacteriaceae infections. IMPORTANCE In the present study, honokiol could effectively inhibit the activity of MCR-1 and showed almost no cytotoxicity to MH-S cells. According to our results, the combination of honokiol and polymyxin had a clear synergistic effect against MCR-1-positive Enterobacteriaceae in vitro Combination therapy also showed a powerful therapeutic effect in vivo , which can significantly improve mouse livability, reduced the load of bacteria, and reduced pathological change. This combined therapy of small molecule compounds and antibiotics may not continue to induce new bacterial resistance, due to the fact that MCR-1 targeted by honokiol is not indispensable for the bacterial viability; on the other hand, it can reduce the dosage of combined antibiotics, and it is also a promising alternative therapy for the treatment of drug-resistant infections in the future., (Copyright © 2020 American Society for Microbiology.)

Animal-associated microbes are highly variable, contributing to a diverse set of symbiont-mediated phenotypes. Given that host and symbiont genotypes, and their interactions, can impact symbiont-based phenotypes across environments, there is potential for extensive variation in fitness outcomes. Pea aphids, Acyrthosiphon pisum , host a diverse assemblage of heritable facultative symbionts (HFS) with characterized roles in host defense. Protective phenotypes have been largely studied as single infections, but pea aphids often carry multiple HFS species, and particular combinations may be enriched or depleted compared to expectations based on chance. Here, we examined the consequences of single infection versus coinfection with two common HFS exhibiting variable enrichment, the antiparasitoid Hamiltonella defensa and the antipathogen Regiella insecticola , across three host genotypes and environments. As expected, single infections with either H. defensa or R. insecticola raised defenses against their respective targets. Single infections with protective H. defensa lowered aphid fitness in the absence of enemy challenge, while R. insecticola was comparatively benign. However, as a coinfection, R. insecticola ameliorated H. defensa infection costs. Coinfected aphids continued to receive antiparasitoid protection from H. defensa , but protection was weakened by R. insecticola in two clones. Notably, H. defensa eliminated survival benefits conferred after pathogen exposure by coinfecting R. insecticola Since pathogen sporulation was suppressed by R. insecticola in coinfected aphids, the poor performance likely stemmed from H. defensa -imposed costs rather than weakened defenses. Our results reveal a complex set of coinfection outcomes which may partially explain natural infection patterns and suggest that symbiont-based phenotypes may not be easily predicted based solely on infection status. IMPORTANCE The hyperdiverse arthropods often harbor maternally transmitted bacteria that protect against natural enemies. In many species, low-diversity communities of heritable symbionts are common, providing opportunities for cooperation and conflict among symbionts, which can impact the defensive services rendered. Using the pea aphid, a model for defensive symbiosis, we show that coinfections with two common defensive symbionts, the antipathogen Regiella and the antiparasite Hamiltonella , produce outcomes that are highly variable compared to single infections, which consistently protect against designated enemies. Compared to single infections, coinfections often reduced defensive services during enemy challenge yet improved aphid fitness in the absence of enemies. Thus, infection with multiple symbionts does not necessarily create generalist aphids with "Swiss army knife" defenses against numerous enemies. Instead, particular combinations of symbionts may be favored for a variety of reasons, including their abilities to lessen the costs of other defensive symbionts when enemies are not present., (Copyright © 2020 American Society for Microbiology.)

Introduction. Infections attributable to carbapenem-resistant Gram-negative bacilli are increasing globally. New antimicrobial agents are urgently needed to treat patients with these infections. Aim. To describe susceptibility to the novel carbapenem-β-lactamase inhibitor combination imipenem-relebactam and comparators of clinical isolates of non-Proteeae Enterobacteriaceae (NPE) and Pseudomonas aeruginosa from intraabdominal infections (IAIs) and urinary tract infections (UTIs). Methods. Broth microdilution MICs were determined for isolates collected in 22 European countries in 2015-2017 and interpreted using EUCAST breakpoints; imipenem-relebactam MICs were interpreted using imipenem breakpoints. Results. For NPE, 98.4 % of isolates from IAIs ( n =10,465) and 98.5 % of UTI isolates ( n =7,446) were susceptible to imipenem-relebactam, as were 42.4 % of imipenem-nonsusceptible ( n =474), 98.6 % of Klebsiella pneumoniae carbapenemase (KPC)-positive ( n =138), and 93.9 % of multidrug-resistant (MDR) isolates ( n =4,424) from IAIs and UTIs combined. Molecular analysis demonstrated that two-thirds of imipenem-nonsusceptible isolates rendered susceptible by relebactam carried KPCs; 96 % (261/271) of imipenem-nonsusceptible isolates of NPE that remained nonsusceptible in the presence of relebactam carried metallo-β-lactamase (MBL)-type and/or OXA-48-like carbapenemases. Among P. aeruginosa , 94.4 % of IAI ( n =1,245) and 93.0 % of UTI isolates ( n =714) were susceptible to imipenem-relebactam, as were 74.4 % of imipenem-nonsusceptible ( n =469) and 79.8 % of MDR isolates ( n =595) from IAIs and UTIs combined. Among the 120 isolates of P. aeruginosa that remained nonsusceptible to imipenem upon addition of relebactam, 72 % carried MBLs. The distribution of NPE and P. aeruginosa carrying carbapenemases varied substantially across Europe, as did resistance to imipenem and imipenem-relebactam. Conclusions. Continued surveillance of antimicrobial resistance and resistance mechanisms, including the study of imipenem-relebactam as it approaches regulatory approval, appears warranted.