Multiple resistance factors collectively promote inoculum-dependent dynamic survival during antimicrobial peptide exposure in Enterobacter cloacae.

Antimicrobial peptides (AMPs) are a promising tool with which to fight rising antibiotic resistance. However, pathogenic bacteria are equipped with several AMP defense mechanisms, whose contributions to AMP resistance are often poorly defined. Here, we evaluate the genetic determinants of resistance to an insect AMP, cecropin B, in the opportunistic pathogen Enterobacter cloacae. Single-cell analysis of E. cloacae's response to cecropin revealed marked heterogeneity in cell survival, phenotypically reminiscent of heteroresistance (the ability of a subpopulation to grow in the presence of supra-MIC concentration of antimicrobial). The magnitude of this response was highly dependent on initial E. cloacae inoculum. We identified 3 genetic factors which collectively contribute to E. cloacae resistance in response to the AMP cecropin: The PhoPQ-two-component system, OmpT-mediated proteolytic cleavage of cecropin, and Rcs-mediated membrane stress response. Altogether, our data suggest that multiple, independent mechanisms contribute to AMP resistance in E. cloacae.

Molecular Mechanism of Ciprofloxacin Translocation Through the Major Diffusion Channels of the ESKAPE Pathogens Klebsiella pneumoniae and Enterobacter cloacae.

Experimental studies on the translocation and accumulation of antibiotics in Gram-negative bacteria have revealed details of the properties that allow efficient permeation through bacterial outer membrane porins. Among the major outer membrane diffusion channels, OmpF has been extensively studied to understand the antibiotic translocation process. In a few cases, this knowledge has also helped to improve the efficacy of existing antibacterial molecules. However, the extension of these strategies to enhance the efficacy of other existing and novel drugs require comprehensive molecular insight into the permeation process and an understanding of how antibiotic and channel properties influence the effective permeation rates. Previous studies have investigated how differences in antibiotic charge distribution can influence the observed permeation pathways through the OmpF channel, and have shown that the dynamics of the L3 loop can play a dominant role in the permeation process. Here, we perform all-atom simulations of the OmpF orthologs, OmpE35 from Enterobacter cloacae and OmpK35 from Klebsiella pneumoniae. Unbiased simulations of the porins and biased simulations of the ciprofloxacin permeation processes through these channels provide insight into the differences in the permeation pathway and energetics. In addition, we show that similar to the OmpF channel, antibiotic-induced dynamics of the L3 loop are also operative in the orthologs. However, the sequence and structural differences, influence the extent of the L3 loop fluctuations with OmpK35 showing greater stability in unbiased runs and subdued fluctuations in simulations with ciprofloxacin.

The nucleoid protein HU positively regulates the expression of type VI secretion systems in Enterobacter cloacae.

Enterobacter cloacae is an emerging pathogen isolated in healthcare-associated infections. A major virulence factor of this bacterium is the type VI secretion system (T6SS). The genome of E. cloacae harbors two T6SS gene clusters (T6SS-1 and T6SS-2), and the functional characterization of both systems showed that these two T6SSs are not expressed under the same conditions. Here, we report that the major histone-like protein HU positively regulates the expression of both T6SSs and, therefore, the function that each T6SS exerts in E. cloacae. Single deletions of the genes encoding the HU subunits (hupA and hupB) decreased mRNA levels of both T6SS. In contrast, the hupA hupB double mutant dramatically affected the T6SS expression, diminishing its transcription. The direct binding of HU to the promoter regions of T6SS-1 and T6SS-2 was confirmed by electrophoretic mobility shift assay. In addition, single and double mutations in the hup genes affected the ability of inter-bacterial killing, biofilm formation, adherence to epithelial cells, and intestinal colonization, but these phenotypes were restored when such mutants were trans-complemented. Our data broaden our understanding of the regulation of HU-mediated T6SS in these pathogenic bacteria. IMPORTANCE: T6SS is a nanomachine that functions as a weapon of bacterial destruction crucial for successful colonization in a specific niche. Enterobacter cloacae expresses two T6SSs required for bacterial competition, adherence, biofilm formation, and intestinal colonization. Expression of T6SS genes in pathogenic bacteria is controlled by multiple regulatory systems, including two-component systems, global regulators, and nucleoid proteins. Here, we reported that the HU nucleoid protein directly activates both T6SSs in E. cloacae, affecting the T6SS-related phenotypes. Our data describe HU as a new regulator involved in the transcriptional regulation of T6SS and its impact on E. cloacae pathogenesis.

Improved application of immobilized Enterobacter cloacae into a bio-based polymer for Reactive Blue 19 removal, an eco-friendly advancement in potential decolorizing systems.

The widespread use of highly complex synthetic dyes like reactive dyes in the textile industry has some adverse environmental impacts and deserves close attention. Biological treatment of these effluents utilizing various species of bacteria with remarkable efficiency in dye removal is still considered promising. Our current study deals with immobilizing an isolated bacterial strain into calcium alginate (Ca/Alg) gel beads and using it to treat pernicious pollutants like synthetic dyes. A potential Reactive Blue 19 (RB19)-degrading Enterobacter cloacae strain A1 was isolated from the Kashan textile industry and was characterized by 16S rDNA gene sequencing. The decolorization ability of strain A1 was assessed by time-based studies using free bacterial cells/immobilized in Ca/Alg. Based on the results of the 16S rDNA gene sequencing, it appears that strain A1 belonged to E. cloacae, with a 99.74% similarity. The findings suggest that immobilized strain A1 accomplished maximum decolorization activity compared with the free cells. The immobilized strain could utterly decompose and decolorize 0.05 mg/mL of RB19 within 48 h, while the free bacterial strain decolorized RB19 within 5 days. Moreover, Ca/Alg gel beads can maintain their efficiency for over three decolorization cycles. Further infrared spectroscopy (FTIR) and gas chromatograph mass spectrometer (GC/MS) investigation declared complete RB19 decomposition on reaction products. Artemia salina was used to investigate the toxicity of dye and its degraded metabolites. The LC50 values signified the pure dye as very toxic with 0.01 mg/mL concentration, while after-treatment products showed no toxic effect on larvae. This immobilization technique increased the applicability of bacterial strain for dye removal. It was beneficial for the decolorization of RB19 from textile wastewater due to a remarkable reduction in time. Notably, strain A1-immobilized beads can maintain their activity for three consecutive decolorization cycles without a considerable decrease in efficiency. PRACTITIONER POINTS: The remarkable capacity of immobilized Enterobacter cloacae strain A1 for Reactive Blue 19 (RB19) removal Immobilized A1 strain showed two-fold higher removal than free one over 48 h A promising method for enhancing RB19 decolorization Decolorization was due to degradation based on UV-Vis, FTIR, and GC/MS analysis Non-toxic posttreatment products for Artemia.

Study on the mechanism of salt relief and growth promotion of Enterobacter cloacae on cotton.

AIMS: In-depth studies on plant ion uptake and plant growth-promoting rhizobacteria (PGPR) at the molecular level will help to further reveal the effects of PGPR on plants and their interaction mechanisms under salt stress. METHODS: Cotton was inoculated with a PGPR-Enterobacter cloacae Rs-35, and the ion uptake capacity, membrane transporter protein activity, and expression of key genes were determined under salt stress. Changes in the endogenous hormone content of cotton were also determined. Further, the genome-wide metabolic pathway annotation of E. cloacae Rs-35 and its differential enrichment pathway analysis of multi-omics under salinity environments were performed. RESULTS: In a pot experiment of saline-alkali soil, E. cloacae Rs-35-treated cotton significantly increased its uptake of K+ and Ca2+ and decreased uptake of Na+, elevated the activity of the H+-ATPase, and increased the sensitivity of the Na+/H+ reverse transporter protein on the vesicle membrane. Meanwhile, inoculation with E. cloacae Rs-35 could promote cotton to maintain the indole-3-acetic acid (IAA) content under salt stress. Genome-wide annotation showed that E. cloacae Rs-35 was respectively annotated to 31, 38, and 130 related genes in osmotic stress, phytohormone and organic acid metabolism, and ion uptake metabolic pathway. Multi-omics differences analysis showed that E. cloacae Rs-35 were enriched to tryptophan metabolism, multiple amino acid biosynthesis, carbon and glucose synthesis, and oxidative phosphorylation metabolic pathways at the transcriptome, proteome, and metabolome. CONCLUSION: E. cloacae Rs-35 can promote cotton balance cell ion concentration, stabilize intracellular IAA changes, stimulate induction of systemic tolerance, and promote the growth of cotton plants under salt stress.

X-ray structures of Enterobacter cloacae allose-binding protein in complexes with monosaccharides demonstrate its unique recognition mechanism for high affinity to allose.

d-Allose is an aldohexose of the C3-epimer of d-glucose, existing in very small amounts in nature, called a rare sugar. The operon responsible for d-allose metabolism, the allose operon, was found in several bacteria, which consists of seven genes: alsR, alsB, alsA, alsC, alsE, alsK, and rpiB. To understand the biological implication of the allose operon utilizing a rare sugar of d-allose as a carbon source, it is important to clarify whether the allose operon functions specifically for d-allose or also functions for other ligands. It was proposed that the allose operon can function for d-ribose, which is essential as a component of nucleotides and abundant in nature. Allose-binding protein, AlsB, coded in the allose operon, is thought to capture a ligand outside the cell, and is expected to show high affinity for the specific ligand. X-ray structure determinations of Enterobacter cloacae AlsB (EtcAlsB) in ligand-free form, and in complexes with d-allose, d-ribose, and d-allulose, and measurements of the thermal parameters of the complex formation using an isothermal titration calorimeter were performed. The results demonstrated that EtcAlsB has a unique recognition mechanism for high affinity to d-allose by changing its conformation from an open to a closed form depending on d-allose-binding, and that the binding of d-ribose to EtcAlsB could not induce a completely closed form but an intermediate form, explaining the low affinity for d-ribose.

Biological treatment of triclosan using a novel strain of Enterobacter cloacae and introducing naphthalene dioxygenase as an effective enzyme.

In recent years, triclosan (TCS) has been widely used as an antibacterial agent in personal care products due to the spread of the Coronavirus. TSC is an emerging contaminant, and due to its stability and toxicity, it cannot be completely degraded through traditional wastewater treatment methods. In this study, a novel strain of Enterobacter cloacae was isolated and identified that can grow in high TCS concentrations. Also, we introduced naphthalene dioxygenase as an effective enzyme in TCS biodegradation, and its role during the removal process was investigated along with the laccase enzyme. The change of cell surface hydrophobicity during TCS removal revealed that a glycolipid biosurfactant called rhamnolipid was involved in TCS removal, leading to enhanced biodegradation of TCS. The independent variables, such as initial TCS concentration, pH, removal duration, and temperature, were optimized using the response surface method (RSM). As a result, the maximum TCS removal (97%) was detected at a pH value of 7 and a temperature of 32 °C after 9 days and 12 h of treatment. Gas chromatography-mass spectrometry (GC/MS) analysis showed five intermediate products and a newly proposed pathway for TCS degradation. Finally, the phytotoxicity experiment conducted on Cucumis sativus and Lens culinaris seeds demonstrated an increase in germination power and growth of stems and roots in comparison to untreated water. These results indicate that the final treated water was less toxic.

Temocillin Resistance in the Enterobacter cloacae Complex Is Conferred by a Single Point Mutation in BaeS, Leading to Overexpression of the AcrD Efflux Pump.

The Enterobacter cloacae complex (ECC) has become a major opportunistic pathogen with antimicrobial resistance issues. Temocillin, an "old" carboxypenicillin that is remarkably stable toward ß-lactamases, has been used as an alternative for the treatment of multidrug-resistant ECC infections. Here, we aimed at deciphering the never-investigated mechanisms of temocillin resistance acquisition in Enterobacterales. By comparative genomic analysis of two clonally related ECC clinical isolates, one susceptible (Temo_S [MIC of 4 mg/L]) and the other resistant (Temo_R [MIC of 32 mg/L]), we found that they differed by only 14 single-nucleotide polymorphisms, including one nonsynonymous mutation (Thr175Pro) in the two-component system (TCS) sensor histidine kinase BaeS. By site-directed mutagenesis in Escherichia coli CFT073, we demonstrated that this unique change in BaeS was responsible for a significant (16-fold) increase in temocillin MIC. Since the BaeSR TCS regulates the expression of two resistance-nodulation-cell division (RND)-type efflux pumps (namely, AcrD and MdtABCD) in E. coli and Salmonella, we demonstrated by quantitative reverse transcription-PCR that mdtB, baeS, and acrD genes were significantly overexpressed (15-, 11-, and 3-fold, respectively) in Temo_R. To confirm the role of each efflux pump in this mechanism, multicopy plasmids harboring mdtABCD or acrD were introduced into either Temo_S or the reference strain E. cloacae subsp. cloacae ATCC 13047. Interestingly, only the overexpression of acrD conferred a significant increase (from 8- to 16-fold) of the temocillin MIC. Altogether, we have shown that temocillin resistance in the ECC can result from a single BaeS alteration, likely resulting in the permanent phosphorylation of BaeR and leading to AcrD overexpression and temocillin resistance through enhanced active efflux.

LY2183240 regioisomers act as competitive and selective inhibitors of class C ß-lactamases.

The regioisomers of the anandamide-acting drug LY2183240 exhibited specific potent and competitive inhibitory activities against class C ß-lactamases. More explicitly, the 1,5- and 2,5-regioisomers inhibited AmpC from Enterobacter hormaechei (formerly Enterobacter cloacae) with inhibitor binding affinity values of 1.8 µM and 2.45 µM, respectively. Structural molecular modelling studies revealed the interaction of the regioisomers with the relevant residues of the catalytic site of cephalosporinase from E. hormaechei P99, which included Tyr150, Lys315 and Thr316.

Intracellular-to-extracellular localization switch of acidic lipase in Enterobacter cloacae: evaluation of production kinetics and enantioselective esterification potential for pharmaceutical applications.

Downstream processing is a significant part of a production process and accounts for 50-90% of the production cost of biotechnological products. Post-fermentation localization of a microbial metabolite contributes significantly to the recovery cost of the product. Enterobacter cloacae produced naturally, acidic lipase with a 0.023:1 extracellular localization ratio. This research aimed to re-direct the localization of lipase to the extracellular milieu to reduce recovery costs using multi-objective response surface optimization (MO-RSM). The approach resulted in a 1:0.32 extracellular: intracellular lipase ratio, with product formation kinetics of Luedeking-Piret function showing a significant switch from a completely growth-associated intracellular production to a predominantly non-growth-associated extracellular localization. The enzyme was purified by an aqueous two-phase system which extracted 95.22% lipase with 72.36 purity. Characterization of the enzyme showed a molecular weight of 55.7 kDa, kcat of 68.59 s-1, and a Km of 0.63 mmol. Lipase activity occurred optimally at pH 2.5-3.5 and 50 °C, and was stable in most organic solvents tested. The acidic lipase demonstrated pH-dependent enantioselective esterification in resolving (R, S)-ibuprofen (E = 14, pH 4.5) and (R, S)-Naproxen (E = 13, pH 2.5), with an enantioselective preference for (S)-enantiomer in both drugs thus underpinning its potential for pharmaceutical applications.

In Vitro Selection of Enterobacter cloacae with Cefepime, Meropenem, and Ceftazidime-Avibactam Generates Diverse Resistance Mechanisms.

Five Enterobacter cloacae isolates were subjected to 10-day serial passage in broth microdilution with cefepime, meropenem, or ceftazidime-avibactam to evaluate increases in minimum inhibitory concentration (MIC) and resistance mechanisms after exposure. Post-exposure isolates displaying >2-fold changes from the parent isolate were analysed alongside the parent isolate. Increases in MIC were 4- to 256-fold (median: 16-fold) after cefepime exposure, 16- to 128-fold (64-fold) after meropenem, and 2- to 32-fold (8-fold) after ceftazidime-avibactam. Post-exposure isolates had diverse mechanisms, identified using a combination of short and long whole-genome sequencing. All agents selected for AmpC alterations in one isolate set. OmpC and TetA/AcrR regulator alterations were noted in meropenem and ceftazidime-avibactam post-exposure isolates of the same set. Other mutations in AmpC were noted when isolates were exposed to cefepime or ceftazidime-avibactam. A premature stop codon in the cell division inhibitor protein, MioC was observed when one parent isolate was exposed to any of the agents, indicating a cell persistence mechanism. Mutations in less common transporter systems and protein synthesis components were also noted. All agents showed cross-resistance to other ß-lactams and resistance mechanisms were diverse, with some not usually associated with ß-lactam resistance in Enterobacterales. This initial evaluation indicates that cefepime and meropenem select for isolates with higher MIC values compared to ceftazidime-avibactam. Further studies evaluating these findings should be performed for other species for which the primary ß-lactam resistance mechanism is not gene acquisition. These studies should evaluate these observations in vivo to assess their translation into patient treatment policies.

Comparative genome analyses uncovered the cadmium resistance mechanism of enterobacter cloacae.

Cadmium (Cd) can be transported into plants from polluted soils and may cause animal and human diseases through food chains, which requires the development of highly efficient methods for soil Cd remediation. Although we isolated an Enterobacter cloacae strain Cu6 with Cd resistance, this strain cannot be used for soil Cd remediation due to its lower resistance. Here, we domesticated Cu6 and obtained a highly Cd-resistant strain, LPY6, and found that this strain can attenuate the toxic effects of Cd on wheat seedling growth. We deciphered the high Cd-resistance mechanism of LPY6 by genome comparative and genetic analysis. Compared with Cu6, 75 genes were mutated in LPY6. Thirty-four of these genes were deleted, and 41 had single nucleotide polymorphisms (SNPs). Most of these mutated proteins are involved in basic metabolism, substrate transport, stress response and formate and hydrogen metabolism. RNA quantitative analysis and promoter activity assays showed that the transcription or mRNA levels of two operons (cadA and norVW) in these mutated genes were regulated by Cd, zinc (Zn) or lead (Pb) ions, suggesting that these two operons might be required for Cd, Zn or Pb resistance. Expression of cadA and norVW operons in LPY6 partially recovered Cd susceptibility, demonstrating that CadA and NorVW are involved in Cd resistance in E. cloacae. Our findings illustrate that E. cloacae acquires Cd resistance through different pathways and lay a foundation for developing highly efficient methods for soil Cd remediation.

Intracellular-to-extracellular localization switch of acidic lipase in Enterobacter cloacae through multi-objective medium optimization: aqueous two-phase purification and activity kinetics.

As physiological impairments that require replacement therapy continue to increase, so also does the need for improved production of acidic lipase from new microbial sources. Enterobacter cloacae strain UCCM 00116 produced a novel acidic lipase in kernel oil-processing waste-basal broth with 0.023:1 extracellular: intracellular localization ratio. This research re-directed enzyme localization to the extracellular milieu to reduce recovery cost using multi-objective response surface optimization of medium parameters. Results revealed a 1:0.32 extracellular:intracellular lipase ratio. Product formation kinetics, modeled by the Luedeking-Piret function, showed a significant switch from a completely growth-associated intracellular production to a predominantly non-growth-associated extracellular localization through medium optimization. Aqueous two-phase system purification conditions extracted 95.22% lipase with 72.36 purity, a Vmax of 370.37 µmolmin-1, and a Km of 0.63 mmol. Enzyme activity was enhanced by K+ and Ca2+ ions, stable in many organic solvents, except acetone, and had pH and temperature optima at 2.5-3.5 and 50 °C, respectively.

Newly isolated Enterobacter cloacae sp. HN01 and Klebsiella pneumoniae sp. HN02 collaborate with self-secreted biosurfactant to improve solubility and bioavailability for the biodegradation of hydrophobic and toxic gaseous para-xylene.

The gas-liquid mass transfer rate of hydrophobic volatile organic compounds (VOCs) is the limiting step in a biological treatment system. The present study aimed to utilize self-producing biosurfactants to enhance the bioavailability of hydrophobic gaseous VOCs. Two novel gram-negative rod-shaped bacteria, Enterobacter cloacae strain HN01 and Klebsiella pneumoniae strain HN02 were successfully isolated from sewage sludge by using blood agar and methylene blue agar plates. The two strains can use para-xylene (PX), a hydrophobic VOC model, as the only carbon source for biosurfactant production. Both strains can produce glycolipid biosurfactants, as confirmed by the emulsification index, Nuclear magnetic resonance, and Fourier transform infrared spectroscopy. Results indicated that PX can be completely decomposed at an initial concentration of 15.50 mg L-1, pH value of 7.0, and temperature of 30 °C within 36 h. The Yano model is suitable for the prediction of the growth kinetics of strains over the entire PX concentration range. Gas chromatography/mass spectrometry analysis indicated that PX was converted into four and four intermediates in the presence of the strains HN01 and HN02, respectively, and the possible mechanisms were proposed. The results can be used in purifying industrial hydrophobic gaseous VOCs and improving the bioavailability of VOCs with self-produced biosurfactants.

High-level carbapenem tolerance requires antibiotic-induced outer membrane modifications.

Antibiotic tolerance is an understudied potential contributor to antibiotic treatment failure and the emergence of multidrug-resistant bacteria. The molecular mechanisms governing tolerance remain poorly understood. A prominent type of ß-lactam tolerance relies on the formation of cell wall-deficient spheroplasts, which maintain structural integrity via their outer membrane (OM), an asymmetric lipid bilayer consisting of phospholipids on the inner leaflet and a lipid-linked polysaccharide (lipopolysaccharide, LPS) enriched in the outer monolayer on the cell surface. How a membrane structure like LPS, with its reliance on mere electrostatic interactions to maintain stability, is capable of countering internal turgor pressure is unknown. Here, we have uncovered a novel role for the PhoPQ two-component system in tolerance to the ß-lactam antibiotic meropenem in Enterobacterales. We found that PhoPQ is induced by meropenem treatment and promotes an increase in 4-amino-4-deoxy-L-aminoarabinose [L-Ara4N] modification of lipid A, the membrane anchor of LPS. L-Ara4N modifications likely enhance structural integrity, and consequently tolerance to meropenem, in several Enterobacterales species. Importantly, mutational inactivation of the negative PhoPQ regulator mgrB (commonly selected for during clinical therapy with the last-resort antibiotic colistin, an antimicrobial peptide [AMP]) results in dramatically enhanced tolerance, suggesting that AMPs can collaterally select for meropenem tolerance via stable overactivation of PhoPQ. Lastly, we identify histidine kinase inhibitors (including an FDA-approved drug) that inhibit PhoPQ-dependent LPS modifications and consequently potentiate meropenem to enhance lysis of tolerant cells. In summary, our results suggest that PhoPQ-mediated LPS modifications play a significant role in stabilizing the OM, promoting survival when the primary integrity maintenance structure, the cell wall, is removed.

Enterobacter cloacae: a villain in CaOx stone disease?

To explore the roles microbiome of urinary tract played in calcium oxalate stones (CaOx) formation, we collected two sides' pelvis urine of patients with unilateral CaOx stones to set self-control to diminish the influence of systemic factors. Patients with unilateral CaOx stones were recruited in our study according to strict criteria. 16S rRNA gene sequencing was applied to every pair of pelvis urine. Bacterial genome sequencing of Enterobacter cloacae was conducted and bioinformatic analysis was applied to explore the possible pathways of Enterobacter cloacae inducing CaOx stones formation. In vivo experiments were conducted to validate our claims. Von Kossa staining, TUNEL assay and Western Blot were applied to SD rats exploring the mechanism of stone formation. We found 26 significantly different bacteria between stone sides and non-stone sides' pelvis urine, among which Enterobacter cloacae ranked the most different. Bacterial genome sequencing of Enterobacter cloacae revealed that its virulence factors included Flagellin, LPS and Fimbrial. GO and KEGG analysis revealed it probably induced CaOx stone formation via ion binging and signaling transduction pathways. The results of animal experiments indicated that Glyoxylic Acid could promote apoptosis and crystal depositions of kidney comparing with control group while pre-injected with Enterobacter cloacae could apparently compound the effects. While Western Blot demonstrated that Glyoxylic Acid or Enterobacter cloacae could increase the expression of IL-6, Mcp-1, BMP2 and OPN in rats' kidney, Glyoxylic Acid and Enterobacter cloacae together could aggravate these increases. These findings indicated that Enterobacter cloacae might play important roles in CaOx stones formation. However, this study is just a preliminary exploration; further studies still need to be conducted.

Interaction with mammalian enteric viruses alters outer membrane vesicle production and content by commensal bacteria.

Intestinal commensal bacteria contribute to maintaining gut homeostasis. Disruptions to the commensal flora are linked to the development and persistence of disease. The importance of these organisms is further demonstrated by the widespread ability of enteric viruses to exploit commensal bacteria to enhance viral infection. These viruses interact directly with commensal bacteria, and while the impact of this interaction on viral infection is well described for several viruses, the impact on the commensal bacteria has yet to be explored. In this article, we demonstrate, for the first time, that enteric viruses alter the gene expression and phenotype of individual commensal bacteria. Human and murine norovirus interaction with bacteria resulted in genome-wide differential gene expression and marked changes in the surface architecture of the bacterial cells. Furthermore, the interaction of the virus with bacteria led to increased production of smaller outer membrane vesicles (OMVs). Enhanced production of smaller vesicles was also observed when noroviruses were incubated with other commensal bacteria, indicating a potentially broad impact of norovirus interaction. The vesicle production observed in the in vivo model followed a similar trend where an increased quantity of smaller bacterial vesicles was observed in stool collected from virus-infected mice compared to mock-infected mice. Furthermore, changes in vesicle size were linked to changes in protein content and abundance, indicating that viral binding induced a shift in the mechanism of the OMV biogenesis. Collectively, these data demonstrate that enteric viruses induce specific changes in bacterial gene expression, leading to changes in bacterial extracellular vesicle production that can potentially impact host responses to infection.

Genomic Investigation and Successful Containment of an Intermittent Common Source Outbreak of OXA-48-Producing Enterobacter cloacae Related to Hospital Shower Drains.

The hospital environment has been reported as a source of transmission events and outbreaks of carbapenemase-producing Enterobacterales. Interconnected plumbing systems and the microbial diversity in these reservoirs pose a challenge for outbreak investigation and control. A total of 133 clinical and environmental OXA-48-producing Enterobacter cloacae isolates collected between 2015 and 2021 were characterized by whole-genome sequencing (WGS) to investigate a prolonged intermittent outbreak involving 41 patients in the hematological unit. A mock-shower experiment was performed to investigate the possible acquisition route. WGS indicated the hospital water environmental reservoir as the most likely source of the outbreak. The lack of diversity of the blaOXA-48-like harbouring plasmids was a challenge for data interpretation. The detection of blaOXA-48-like-harboring E. cloacae strains in the shower area after the mock-shower experiment provided strong evidence that showering is the most likely route of acquisition. Initially, in 20 out of 38 patient rooms, wastewater traps and drains were contaminated with OXA-48-positive E. cloacae. Continuous decontamination using 25% acetic acid three times weekly was effective in reducing the trap/drain positivity in monthly environmental screening but not in reducing new acquisitions. However, the installation of removable custom-made shower tubs did prevent new acquisitions over a subsequent 12-month observation period. In the present study, continuous decontamination was effective in reducing the bacterial burden in the nosocomial reservoirs but was not sufficient to prevent environment-to-patient transmission in the long term. Construction interventions may be necessary for successful infection prevention and control. IMPORTANCE The hospital water environment can be a reservoir for a multiward outbreak, leading to acquisitions or transmissions of multidrug-resistant organisms in a hospital setting. The majority of Gram-negative bacteria are able to build biofilms and persist in the hospital plumbing system over a long period of time. The elimination of the reservoir is essential to prevent further transmission and spread, but proposed decontamination regimens, e.g., using acetic acid, can only suppress but not fully eliminate the environmental reservoir. In this study, we demonstrated that colonization with multidrug-resistant organisms can be acquired by showering in showers with contaminated water traps and drains. A construction intervention by installing removable and autoclavable shower inserts to avoid sink contact during showering was effective in containing this outbreak and may be a viable alternative infection prevention and control measure in outbreak situations involving contaminated shower drains and water traps.

Human commensal gut Proteobacteria withstand type VI secretion attacks through immunity protein-independent mechanisms.

While the major virulence factors for Vibrio cholerae, the cause of the devastating diarrheal disease cholera, have been extensively studied, the initial intestinal colonization of the bacterium is not well understood because non-human adult animals are refractory to its colonization. Recent studies suggest the involvement of an interbacterial killing device known as the type VI secretion system (T6SS). Here, we tested the T6SS-dependent interaction of V. cholerae with a selection of human gut commensal isolates. We show that the pathogen efficiently depleted representative genera of the Proteobacteria in vitro, while members of the Enterobacter cloacae complex and several Klebsiella species remained unaffected. We demonstrate that this resistance against T6SS assaults was mediated by the production of superior T6SS machinery or a barrier exerted by group I capsules. Collectively, our data provide new insights into immunity protein-independent T6SS resistance employed by the human microbiota and colonization resistance in general.

Norovirus diarrhea is significantly associated with higher counts of fecal histo-blood group antigen expressing Enterobacter cloacae among black South African infants.

The study tested the hypothesis that harboring high levels of histo-blood group antigen-expressing Enerobactero cloacae is a risk factor for norovirus diarrhea. The fecal E. cloacae abundance in diarrheic norovirus positive (DNP), non-diarrheic norovirus negative (NDNN), diarrhea norovirus negative (DNN), and non-diarrhea norovirus positive (NDNP) infants was determined by qPCR, and the risk of norovirus diarrhea was assessed by logistical regression. DNP infants contained significantly higher counts of E. cloacae than NDNN and DNN infants, p = .0294, and 0.0001, respectively. The risk of norovirus diarrhea was significantly high in infants with higher counts of E. cloacae than those with lower counts, p = .009. Harboring higher counts of E. cloacae is a risk factor for norovirus diarrhea.