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MBio Oct 2021The gut microbiota plays a crucial role in susceptibility to enteric pathogens, including Citrobacter rodentium, a model extracellular mouse pathogen that colonizes the...
The gut microbiota plays a crucial role in susceptibility to enteric pathogens, including Citrobacter rodentium, a model extracellular mouse pathogen that colonizes the colonic mucosa. C. rodentium infection outcomes vary between mouse strains, with C57BL/6 and C3H/HeN mice clearing and succumbing to the infection, respectively. Kanamycin (Kan) treatment at the peak of C57BL/6 mouse infection with Kan-resistant C. rodentium resulted in relocalization of the pathogen from the colonic mucosa and cecum to solely the cecal luminal contents; cessation of the Kan treatment resulted in rapid clearance of the pathogen. We now show that in C3H/HeN mice, following Kan-induced displacement of C. rodentium to the cecum, the pathogen stably colonizes the cecal lumens of 65% of the mice in the absence of continued antibiotic treatment, a phenomenon that we term antibiotic-induced bacterial commensalization (AIBC). AIBC C. rodentium was well tolerated by the host, which showed few signs of inflammation; passaged AIBC C. rodentium robustly infected naive C3H/HeN mice, suggesting that the AIBC state is transient and did not select for genetically avirulent C. rodentium mutants. Following withdrawal of antibiotic treatment, 35% of C3H/HeN mice were able to prevent C. rodentium commensalization in the gut lumen. These mice presented a bloom of a commensal species, Citrobacter amalonaticus, which inhibited the growth of C. rodentium in a contact-dependent manner and the luminal growth of AIBC C. rodentium . Overall, our data suggest that commensal species can confer colonization resistance to closely related pathogenic species. Gut bacterial infections involve three-way interactions between virulence factors, the host immune responses, and the microbiome. While the microbiome erects colonization resistance barriers, pathogens employ virulence factors to overcome them. Treating mice infected with kanamycin-resistant Citrobacter rodentium with kanamycin caused displacement of the pathogen from the colonic mucosa to the cecal lumen. Following withdrawal of the kanamycin treatment, 65% of the mice were persistently colonized by C. rodentium, which seemed to downregulate virulence factor expression. In this model of luminal gut colonization, 35% of mice were refractory to stable C. rodentium colonization, suggesting that their microbiotas were able to confer colonization resistance. We identify a commensal bacterium of the genus, , which inhibits C. rodentium growth and . These results show that the line separating commensal and pathogenic lifestyles is thin and multifactorial and that commensals may play a major role in combating enteric infection.
Topics: Animals; Citrobacter; Citrobacter rodentium; Colon; Enterobacteriaceae Infections; Female; Gastrointestinal Microbiome; Humans; Intestinal Mucosa; Mice; Mice, Inbred C3H; Mice, Inbred C57BL
PubMed: 34609899
DOI: 10.1128/mBio.02410-21 -
Frontiers in Microbiology 2021species often occur in sewage, food, soil, wastewater, and in the intestinal tract of animals and humans. spp. cause urinary tract infections (UTIs) and infantile...
species often occur in sewage, food, soil, wastewater, and in the intestinal tract of animals and humans. spp. cause urinary tract infections (UTIs) and infantile meningitis in humans. Due to the presence of plasmid-encoded resistance genes, spp. are often resistant to many antibiotics. In this study, virus HCF1, a novel virulent bacteriophage capable of killing and , was isolated from the sewage water. The isolated bacteriophage was characterized with respect to transmission electron microscopy, one-step growth curve, host range, efficacy, storage stability, and environmental stress tolerance. The one-step growth curve analysis revealed that the latent period of HCF1 was 30 min and the estimated burst size was 121 plaque-forming units (PFU) per bacterial cell. Host range testing indicated that the HCF1 was specific to the genus. efficacy assay in the effluent of an anaerobic biodigester showed that the HCF1 completely eliminated the host within 4 and 5 h at MOI:100 and MOI:10, respectively, thereby indicating its potential for combating infections. The isolated bacteriophage is considerably stable and tolerant to environmental stress. Furthermore, the complete genome of HCF1 was sequenced using Oxford Nanopore sequencing and the data were subjected to detailed bioinformatic analyses. NCBI-BLASTn analysis revealed that the HCF1 genome had a query coverage of 15-21% and a maximum similarity of 77.27-78.49% with 11 bacteriophages of the family. Detailed bioinformatic analysis of the genome profile suggests that HCF1 is a novel belonging to the subfamily of the family.
PubMed: 33569047
DOI: 10.3389/fmicb.2021.644013 -
Frontiers in Cellular and Infection... 2022spp. are Gram-negative bacteria commonly found in environments and intestinal tracts of humans and animals. They are generally susceptible to third-generation...
spp. are Gram-negative bacteria commonly found in environments and intestinal tracts of humans and animals. They are generally susceptible to third-generation cephalosporins, carbapenems and colistin. However, several antibiotic resistant genes have been increasingly reported in spp., which leads to the postulation that spp. could potentially be a reservoir for spreading of antimicrobial resistant genes. In this study, we characterized two colistin-resistant spp. isolated from the feces of a healthy individual in Thailand. Based on MALDI-TOF and ribosomal multilocus sequence typing, both strains were identified as and . Genomic analysis and S1-nuclease pulsed field gel electrophoresis/DNA hybridization revealed that and harbored gene on pSY_CS01 and pSY_CA01 plasmids, respectively. Both plasmids belonged to IncFII(pCoo) replicon type, contained the same genetic context (TnISΔTnAsIS) and exhibited high transferring frequencies ranging from 1.03×10 - 4.6×10 CFU/recipient cell J53. Colistin-MICs of transconjugants increased ≥ 16-fold suggesting that on these plasmids can be expressed in other species. However, beside , other major antimicrobial resistant determinants in multidrug resistant Enterobacterales were not found in these two isolates. These findings indicate that gene continued to evolve in the absence of antibiotics selective pressure. Our results also support the hypothesis that could be a reservoir for spreading of antimicrobial resistant genes. To the best of our knowledge, this is the first report that discovered human-derived spp. that harbored but no other major antimicrobial resistant determinants. Also, this is the first report that described the presence of gene in and in .
Topics: Animals; Humans; Anti-Bacterial Agents; Citrobacter; Colistin; Drug Resistance, Bacterial; Escherichia coli; Escherichia coli Proteins; Plasmids; Thailand
PubMed: 36683683
DOI: 10.3389/fcimb.2022.1067572 -
Current Research in Microbial Sciences 2022Gut microbiota metabolism can have profound effects on human health. Choline, a quaternary amine (QA) highly abundant in our diet, is canonically cleaved by a glycyl...
Gut microbiota metabolism can have profound effects on human health. Choline, a quaternary amine (QA) highly abundant in our diet, is canonically cleaved by a glycyl radical enzyme, choline trimethylamine lyase (CutC), and its SAM-dependent radical activator, CutD. CutC cleaves choline to form trimethylamine (TMA) and acetaldehyde. TMA is oxidized to TMAO by FMO3 in the liver, which plays a role in causing atherosclerosis. We hypothesized that alternative pathways for choline degradation occur within gut microbes and that certain gut microbiota can anaerobically respire or ferment QAs, such as choline. Based on this prediction we established QA-supplemented enrichment cultures using fecal material from healthy volunteers as the inocula. We have isolated, from a choline-supplemented enrichment of a human fecal sample, a strain of , that we have designated CJ25. This strain is capable of anaerobically utilizing choline as its sole carbon and energy source. Its genome does not contain the genes or genes encoding any COG5598 methyltransferases. We have confirmed the degradation of choline and production of acetate by the organism during growth of the strain. However, we used multiple analytical methods to confirm that no TMA accumulated in the medium during growth. Hence, strain CJ25 is a unique bacterium that degrades choline without the production of the proatherogenic metabolite TMA.
PubMed: 36518168
DOI: 10.1016/j.crmicr.2022.100157 -
Diagnostic Microbiology and Infectious... Oct 2022The NG-Test CTX-M MULTI immunochromatographic assay has been developed to identify CTX-M-type β-lactamases in Enterobacterales, being the most widespread...
The NG-Test CTX-M MULTI immunochromatographic assay has been developed to identify CTX-M-type β-lactamases in Enterobacterales, being the most widespread extended-spectrum β-lactamases. We showed here that the chromosomally-encoded ß-lactamases from Citrobacter farmeri and Citrobacter amalonaticus generated false-positive NG-Test CTX-M MULTI results, compromising the specificity of the test.
Topics: Citrobacter; Microbial Sensitivity Tests; beta-Lactamases
PubMed: 35940102
DOI: 10.1016/j.diagmicrobio.2022.115760 -
ACS Infectious Diseases Jan 2022Non-antibiotic alternative treatments to combat the increasing number of infections caused by multidrug resistant bacteria are urgently needed. In recent years,...
Non-antibiotic alternative treatments to combat the increasing number of infections caused by multidrug resistant bacteria are urgently needed. In recent years, bacteriophages have reemerged to potentially replace or complement the role of antibiotics, as bacterial viruses have the ability to inactivate pathogens. This study aimed to evaluate the synergy of phage-antibiotic combinations. A isolate was used in this study together with the phage MRM57. Eight different antibiotics with different mechanisms of action were used in combination with the phage to study the impact of the combination treatment on the minimal inhibitory concentrations. We found that antibiotic concentration dependent synergism exists, albeit at different extents, with very low numbers of phages. This demonstrates the use of phages as an adjuvant with a sublethal concentration of antibiotics as an effective therapeutic strategy.
Topics: Anti-Bacterial Agents; Bacteriophages; Citrobacter; Microbial Sensitivity Tests
PubMed: 34979073
DOI: 10.1021/acsinfecdis.1c00117 -
Biotechnology For Biofuels 2017Y19 is a good biocatalyst for production of hydrogen (H) from oxidation of carbon monoxide (CO) via the so-called water-gas-shift reaction (WGSR). It has a high...
BACKGROUND
Y19 is a good biocatalyst for production of hydrogen (H) from oxidation of carbon monoxide (CO) via the so-called water-gas-shift reaction (WGSR). It has a high H-production activity (23.83 mmol H g cell h) from CO, and can grow well to a high density on various sugars. However, its H-production activity is expressed only when CO is present as an inducer and in the absence of glucose.
RESULTS
In order to avoid dependency on CO and glucose, in the present study, the native CO-inducible promoters of WGSR operons (CO dehydrogenase, CODH, and CODH-dependent hydrogenase, CO-) in Y19 were carefully analyzed and replaced with strong and constitutive promoters screened from Y19. One engineered strain (Y19-PR1), selected from three positive ones after screening ~10,000 colonies, showed a similar CO-dependent H-production activity to that of wild-type Y19, without being affected by glucose and/or CO. Compared with wild-type Y19, transcription of the CODH operon in Y19-PR1 increased 1.5-fold, although that of the CO- operon remained at a similar level. To enhance the activity of CO-Hyd in Y19-PR1, further modifications, including an increase in gene copy number and engineering of the 5' untranslated region, were attempted, but without success.
CONCLUSIONS
Convenient recombinant Y19-PR1 that expresses CO-dependent H-production activity without being limited by CO and glucose was obtained.
PubMed: 28360938
DOI: 10.1186/s13068-017-0770-8 -
Combined strategies for improving expression of Citrobacter amalonaticus phytase in Pichia pastoris.BMC Biotechnology Sep 2015Phytase is used as an animal feed additive that degrades phytic acid and reduces feeding costs and pollution caused by fecal excretion of phosphorus. Some phytases have...
BACKGROUND
Phytase is used as an animal feed additive that degrades phytic acid and reduces feeding costs and pollution caused by fecal excretion of phosphorus. Some phytases have been expressed in Pichia pastoris, among which the phytase from Citrobacter amalonaticus CGMCC 1696 had high specific activity (3548 U/mg). Improvement of the phytase expression level will contribute to facilitate its industrial applications.
METHODS
To improve the phytase expression, we use modification of P AOX1 and the α-factor signal peptide, increasing the gene copy number, and overexpressing HAC1 (i) to enhance folding and secretion of the protein in the endoplasmic reticulum. The genetic stability and fermentation in 10-L scaled-up fed-batch fermenter was performed to prepare for the industrial production.
RESULTS
The phytase gene from C. amalonaticus CGMCC 1696 was cloned under the control of the AOX1 promoter (P AOX1 ) and expressed in P. pastoris. The phytase activity achieved was 414 U/mL. Modifications of P AOX1 and the α-factor signal peptide increased the phytase yield by 35 and 12%, respectively. Next, on increasing the copy number of the Phy gene to six, the phytase yield was 141% higher than in the strain containing only a single gene copy. Furthermore, on overexpression of HAC1 (i) (i indicating induced), a gene encoding Hac1p that regulates the unfolded protein response, the phytase yield achieved was 0.75 g/L with an activity of 2119 U/mL, 412% higher than for the original strain. The plasmids in this high-phytase expression strain were stable during incubation at 30 °C in Yeast Extract Peptone Dextrose (YPD) Medium. In a 10-L scaled-up fed-batch fermenter, the phytase yield achieved was 9.58 g/L with an activity of 35,032 U/mL.
DISCUSSION
The production of a secreted protein will reach its limit at a specific gene copy number where further increases in transcription and translation due to the higher abundance of gene copies will not enhance the secretion process any further. Enhancement of protein folding in the ER can alleviate bottlenecks in the folding and secretion pathways during the overexpression of heterologous proteins in P. pastoris.
CONCLUSIONS
Using modification of P AOX1 and the α-factor signal peptide, increasing the gene copy number, and overexpressing HAC1 (i) to enhance folding and secretion of the protein in the endoplasmic reticulum, we have successfully increased the phytase yield 412% relative to the original strain. In a 10-L fed-batch fermenter, the phytase yield achieved was 9.58 g/L with an activity of 35,032 U/mL. Large-scale production of phytase can be applied towards different biocatalytic and feed additive applications.
Topics: 6-Phytase; Animal Husbandry; Citrobacter; Cloning, Molecular; Food Additives; Gene Expression Regulation, Enzymologic; Industrial Microbiology; Pichia
PubMed: 26410558
DOI: 10.1186/s12896-015-0204-2 -
Scientific Reports Feb 2016Cell-cell communication is also known as quorum sensing (QS) that happens in the bacterial cells with the aim to regulate their genes expression in response to increased...
Cell-cell communication is also known as quorum sensing (QS) that happens in the bacterial cells with the aim to regulate their genes expression in response to increased cell density. In this study, a bacterium (L8A) isolated from dental plaque biofilm was identified as Citrobacter amalonaticus by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS). Its N-acylhomoserine-lactone (AHL) production was screened by using two types of AHL biosensors namely Chromobacterium violaceum CV026 and Escherichia coli [pSB401]. Citrobacter amalonaticus strain L8A was identified and confirmed producing numerous types of AHL namely N-butyryl-L-homoserine lactone (C4-HSL), N-hexanoyl-L-homoserine lactone (C6-HSL), N-octanoyl-L-homoserine lactone (C8-HSL) and N-hexadecanoyl-L-homoserine lactone (C16-HSL). We performed the whole genome sequence analysis of this oral isolate where its genome sequence reveals the presence of QS signal synthase gene and our work will pave the ways to study the function of the related QS genes in this bacterium.
Topics: 4-Butyrolactone; Bacterial Proteins; Citrobacter; DNA, Bacterial; Dental Plaque; High-Throughput Nucleotide Sequencing; Homoserine; Humans; Lactones; Phylogeny; Quorum Sensing; Real-Time Polymerase Chain Reaction; Sequence Analysis, DNA; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
PubMed: 26860259
DOI: 10.1038/srep20702 -
New Microbes and New Infections May 2016Citrobacter amalonaticus is a bacterium that has rarely been reported as a human pathogen. Here we report four cases of C. amalonaticus infections occurring in patients...
Citrobacter amalonaticus is a bacterium that has rarely been reported as a human pathogen. Here we report four cases of C. amalonaticus infections occurring in patients hospitalized in Marseille, France, and review all cases described in the published literature.
PubMed: 26958347
DOI: 10.1016/j.nmni.2016.01.003