Genetically dissecting the electron transport chain of a soil bacterium reveals a generalizable mechanism for biological phenazine-1-carboxylic acid oxidation.

The capacity for bacterial extracellular electron transfer via secreted metabolites is widespread in natural, clinical, and industrial environments. Recently, we discovered the biological oxidation of phenazine-1-carboxylic acid (PCA), the first example of biological regeneration of a naturally produced extracellular electron shuttle. However, it remained unclear how PCA oxidation was catalyzed. Here, we report the mechanism, which we uncovered by genetically perturbing the branched electron transport chain (ETC) of the soil isolate Citrobacter portucalensis MBL. Biological PCA oxidation is coupled to anaerobic respiration with nitrate, fumarate, dimethyl sulfoxide, or trimethylamine-N-oxide as terminal electron acceptors. Genetically inactivating the catalytic subunits for all redundant complexes for a given terminal electron acceptor abolishes PCA oxidation. In the absence of quinones, PCA can still donate electrons to certain terminal reductases, albeit much less efficiently. In C. portucalensis MBL, PCA oxidation is largely driven by flux through the ETC, which suggests a generalizable mechanism that may be employed by any anaerobically respiring bacterium with an accessible cytoplasmic membrane. This model is supported by analogous genetic experiments during nitrate respiration by Pseudomonas aeruginosa.

Three-component systems represent a common pathway for extracytoplasmic addition of pentofuranose sugars into bacterial glycans.

Cell surface glycans are major drivers of antigenic diversity in bacteria. The biochemistry and molecular biology underpinning their synthesis are important in understanding host-pathogen interactions and for vaccine development with emerging chemoenzymatic and glycoengineering approaches. Structural diversity in glycostructures arises from the action of glycosyltransferases (GTs) that use an immense catalog of activated sugar donors to build the repeating unit and modifying enzymes that add further heterogeneity. Classical Leloir GTs incorporate α- or ß-linked sugars by inverting or retaining mechanisms, depending on the nucleotide sugar donor. In contrast, the mechanism of known ribofuranosyltransferases is confined to ß-linkages, so the existence of α-linked ribofuranose in some glycans dictates an alternative strategy. Here, we use Citrobacter youngae O1 and O2 lipopolysaccharide O antigens as prototypes to describe a widespread, versatile pathway for incorporating side-chain α-linked pentofuranoses by extracytoplasmic postpolymerization glycosylation. The pathway requires a polyprenyl phosphoribose synthase to generate a lipid-linked donor, a MATE-family flippase to transport the donor to the periplasm, and a GT-C type GT (founding the GT136 family) that performs the final glycosylation reaction. The characterized system shares similarities, but also fundamental differences, with both cell wall arabinan biosynthesis in mycobacteria, and periplasmic glucosylation of O antigens first discovered in Salmonella and Shigella. The participation of auxiliary epimerases allows the diversification of incorporated pentofuranoses. The results offer insight into a broad concept in microbial glycobiology and provide prototype systems and bioinformatic guides that facilitate discovery of further examples from diverse species, some in currently unknown glycans.

Outbreak of carbapenemase-producing Citrobacter farmeri in an intensive care haematology department linked to a persistent wastewater reservoir in one hospital room, France, 2019 to 2022.

In 2019-2022, a prolonged outbreak of oxacillinase (OXA)-48-producing Citrobacter farmeri due to a persistent environmental contamination, occurred in our haematology intensive care unit. In April 2019, we isolated OXA-48-producing C. farmeri from rectal samples of two patients in weekly screenings. The cases had stayed in the same hospital room but 4 months apart. We screened five patients who had stayed in this room between the two cases and identified a third case. Over the following 3 years, five other cases were detected, the last case in September 2022. In total, eight cases were detected: seven colonised with the bacterium and one infected with a lethal outcome. All cases stayed in the same hospital room. We detected OXA-48-producing C. farmeri from a shower, washbasin drains and wastewater drainage of the bathroom of the hospital room. Molecular typing confirmed that all C. farmeri isolates from the environment and the cases were indistinguishable. Despite bundle measures to control the outbreak, the bacterium persisted in the system, which resulted in transmission to new patients. A design defect in the placement of wastewater drains contributed to the persistence and proliferation of the bacterium. The room was closed after the last case and the bathroom rebuilt.

Emergence of OXA-48-like producing Citrobacter species, Germany, 2011 to 2022.

BackgroundCarbapenemase-producing Enterobacterales are a public health threat worldwide and OXA-48 is the most prevalent carbapenemase in Germany and western Europe. However, the molecular epidemiology of OXA-48 in species other than Escherichia coli and Klebsiella pneumoniae remains poorly understood.AimTo analyse the molecular epidemiology of OXA-48 and OXA-48-like carbapenemases in Citrobacter species (spp.) in Germany between 2011 and 2022.MethodsData of 26,822 Enterobacterales isolates sent to the National Reference Centre (NRC) for Gram-negative bacteria were evaluated. Ninety-one Citrobacter isolates from 40 German hospitals harbouring bla OXA-48/OXA-48­like were analysed by whole genome sequencing and conjugation experiments.ResultsThe frequency of OXA-48 in Citrobacter freundii (CF) has increased steadily since 2011 and is now the most prevalent carbapenemase in this species in Germany. Among 91 in-depth analysed Citrobacter spp. isolates, CF (n = 73) and C. koseri (n = 8) were the most common species and OXA-48 was the most common variant (n = 77), followed by OXA-162 (n = 11) and OXA­181 (n = 3). Forty percent of the isolates belonged to only two sequence types (ST19 and ST22), while most other STs were singletons. The plasmids harbouring bla OXA­48 and bla OXA-162 belonged to the plasmid types IncL (n = 85) or IncF (n = 3), and plasmids harbouring bla OXA­181 to IncX3 (n = 3). Three IncL plasmid clusters (57/85 IncL plasmids) were identified, which were highly transferable in contrast to sporadic plasmids.ConclusionIn CF in Germany, OXA-48 is the predominant carbapenemase. Dissemination is likely due to distinct highly transmissible plasmids harbouring bla OXA­48 or bla OXA-48-like and the spread of the high-risk clonal lineages ST19 and ST22.

Whole-genome sequencing of clinical isolates of Citrobacter Europaeus in China carrying blaOXA-48 and blaNDM-1.

OBJECTIVE: To analyze the clinical infection characteristics and genetic environments of resistance genes in carbapenem-resistant Citrobacter europaeus using whole-genome sequencing. METHODS: The susceptibility of two clinical isolates of C. europaeus (WF0003 and WF1643) to 24 antimicrobial agents was assessed using the BD Phoenix™ M50 System and Kirby-Bauer (K-B) disk-diffusion method. Whole-genome sequencing was performed on the Illumina and Nanopore platforms, and ABRicate software was used to predict resistance and virulence genes of carbapenem-resistant C. europaeus. The characteristics of plasmids carrying carbapenem-resistance genes and their genetic environments were analyzed. Single nucleotide polymorphisms were used to construct a phylogenetic tree to analyze the homology of these two C. europaeus strains with ten strains of C. europaeus in the NCBI database. RESULTS: The two strains of carbapenem-resistant C. europaeus are resistant to various antimicrobial agents, particularly carbapenems and ß-lactams. WF0003 carries blaNDM- 1, which is located on an IncX3 plasmid that has high homology to the pNDM-HN380 plasmid. blaNDM- 1 is located on a truncated Tn125. It differs from Tn125 by the insertion of IS5 in the upstream ISAba125 and the deletion of the downstream ISAba125, which is replaced by IS26. WF1643 carries blaOXA- 48 in a Tn1999 transposon on the IncL/M plasmid, carrying only that single drug resistance gene. Homology analysis of these two strains of C. europaeus with ten C. europaeus strains in the NCBI database revealed that the 12 strains can be classified into three clades, with both WF0003 and WF1643 in the B clade. CONCLUSION: To the best of our knowledge, this is the first study to report an IncX3 plasmid carrying blaNDM- 1 in C. europaeus in China. C. europaeus strains harboring carbapenem-resistance genes are concerning in relation to the spread of antimicrobial resistance, and the presence of carbapenem-resistance genes in C. europaeus should be continuously monitored.

Characterization of cytotoxic Citrobacter braakii isolated from human stomach.

Citrobacter braakii (C. braakii) is an anaerobic, gram-negative bacterium that has been isolated from the environment, food, and humans. Infection by C. braakii has been associated with acute mucosal inflammation in the intestine, respiratory tract, and urinary tract. However, the pathogenesis of C. braakii in the gastric mucosa has not yet been clarified. In this study, the bacterium was detected in 35.5% (61/172) of patients with chronic gastritis (CG) and was closely associated with the severity of mucosal inflammation. Citrobacter braakii P1 isolated from a patient with CG exhibited urease activity and acid resistance. It contained multiple secretion systems, including a complete type I secretion system (T1SS), T5aSS and T6SS. We then predicted the potential pilus-related adhesins. Citrobacter braakii P1 diffusely adhered to AGS cells and significantly increased lactate dehydrogenase (LDH) release; the adhesion rate and LDH release were much lower in HEp-2 cells. Strain P1 also induced markedly increased mRNA and protein expression of IL-8 and TNF-α in AGS cells, and the fold increase was much higher than that in HEp-2 cells. Our results demonstrate proinflammatory and cytotoxic role of C. braakii in gastric epithelial cells, indicating the bacterium is potentially involved in inducing gastric mucosa inflammation.

Citrobacter enshiensis sp. nov., isolated from seleniferous soil.

Two Gram-stain-negative, facultative anaerobic, rod-shaped bacterial strains, S171T and S2-9, were isolated from seleniferous soil in China. Comprehensive phylogenetic analyses based on 16S rRNA genes, multilocus sequences and whole genome sequences indicated that the two strains belonged to the genus Citrobacter. The average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values of strains S171T and S2-9 with the closest relative Citrobacter koseri NCTC 10786T were 83.6-83.7% and 27.7-27.8 %, respectively, which were below the species cutoff values. The ANI and dDDH values between the two strains were 97.9% and 84.8 %, respectively. The biochemical characteristics revealed that selenite tolerance, H2S and indole production, arginine dihydrolase, ornithine decarboxylase, as well as acid production from carbon sources such as d-sorbitol and arbutin are distinctive features of the two strains. Based on these results, strain S171T and strain S2-9 represent a novel species of the genus Citrobacter, for which the name Citrobacter enshiensis sp. nov. is proposed, with strain S171T (=GDMCC 1.3637T=JCM 35851T) as the type strain. The genome size of strain S171T was 4.92 Mb with a G+C content of 52.6 mol%. The genome size of strain S2-9 was 4.89 Mb with a G+C content of 52.6 mol%.

A bacteriophage against Citrobacter braakii and its synergistic effect with antibiotics.

A bacteriophage BD49 specific for Citrobacter braakii was screened out and purified by double-layer plate method. It consists of a polyhedral head of 93.1 ± 1.2 nm long and 72.9 ± 4.2 nm wide, tail fibers, collar, sheath and baseplate. The bacteriophage was identified by morphology observed with transmission electron microscope (TEM), whole genome sequencing carried out by Illumina next generation sequencing (NGS) technique, and gene annotation based on Clusters of Orthologous Groups of proteins (COG) database. It was identified primarily as a member of Caudovirales by morphology and further determined as Caudovirales, Myoviridae, and Citrobacter bacteriophage by alignment of its whole genome sequence with the NCBI database and establishment of phylogenetic tree. The bacteriophage showed good environmental suitability with optimal multiplicity of infection (MOI) of 0.01, proliferation time of 80 min, optimum living temperature of 30-40 °C, and living pH of 5-10. In addition, it exhibited synergistic effect with ciprofloxacin against C. braakii in antibacterial tests.

The phylogenomic landscape of extended-spectrum ß-lactamase producing Citrobacter species isolated from surface water.

BACKGROUND: Citrobacter species are Gram-negative opportunistic pathogens commonly reported in nosocomial-acquired infections. This study characterised four Citrobacter species that were isolated from surface water in the North West Province, South Africa. RESULTS: Phenotypic antimicrobial susceptibility profiles of the isolates demonstrated their ability to produce the extended-spectrum ß-lactamase (ESBL). Whole genomes were sequenced to profile antibiotic resistance and virulence genes, as well as mobile genetic elements. In silico taxonomic identification was conducted by using multi-locus sequence typing and average nucleotide identity. A pangenome was used to determine the phylogenomic landscape of the Citrobacter species by using 109 publicly available genomes. The strains S21 and S23 were identified as C. braakii, while strains S24 and S25 were C. murliniae and C. portucalensis, respectively. Comparative genomics and sequenced genomes of the ESBL-producing isolates consisted of n = 91; 83% Citrobacter species in which bla-CMY-101 (n = 19; 32,2%) and bla-CMY-59 (n = 12; 38,7%) were prevalent in C. braakii, and C. portucalensis strains, respectively. Macrolide (acrAB-TolC, and mdtG) and aminoglycoside (acrD) efflux pumps genes were identified in the four sequenced Citrobacter spp. isolates. The quinolone resistance gene, qnrB13, was exclusive to the C. portucalensis S25 strain. In silico analysis detected plasmid replicon types IncHI1A, IncP, and Col(VCM04) in C. murliniae S24 and C. portucalensis S25, respectively. These potentially facilitate the T4SS secretion system in Citrobacter species. In this study, the C. braakii genomes could be distinguished from C. murliniae and C. portucalensis on the basis of gene encoding for cell surface localisation of the CPS (vexC) and identification of genes involved in capsule polymer synthesis (tviB and tviE). A cluster for the salmochelin siderophore system (iro-BCDEN) was found in C. murliniae S24. This is important when it comes to the pathogenicity pathway that confers an advantage in colonisation. CONCLUSIONS: The emerging and genomic landscapes of these ESBL-producing Citrobacter species are of significant concern due to their dissemination potential in freshwater systems. The presence of these ESBL and multidrug-resistant (MDR) pathogens in aquatic environments is of One Health importance, since they potentially impact the clinical domain, that is, in terms of human health and the agricultural domain, that is, in terms of animal health and food production as well as the environmental domain.

Nationwide molecular epidemiology of carbapenemase-producing Citrobacter spp. in France in 2019 and 2020.

IMPORTANCE: The emergence of carbapenemase producers in Enterobacterales mostly involves Escherichia coli, Klebsiella pneumoniae, and Enterobacter cloacae complex species. However, in France, we observed the emergence and the rapid dissemination of carbapenemase in Citrobacter spp. In this study, we demonstrated that a wide variety of carbapenemases is produced by many different species of Citrobacter spp. However, we clearly identify three high-risk clones of Citrobacter freundii, ST8, ST22, and ST91 that drive the spread of carbapenemase in France. This epidemiological study paves the way of further analysis that would aim to identify the virulence factors involved in this pellicular ability of these three clones to disseminate at the hospital.

Oral microbiome homogeneity across diverse human groups from southern Africa: first results from southwestern Angola and Zimbabwe.

BACKGROUND: While the human oral microbiome is known to play an important role in systemic health, its average composition and diversity patterns are still poorly understood. To gain better insights into the general composition of the microbiome on a global scale, the characterization of microbiomes from a broad range of populations, including non-industrialized societies, is needed. Here, we used the portion of non-human reads obtained through an expanded exome capture sequencing approach to characterize the saliva microbiomes of 52 individuals from eight ethnolinguistically diverse southern African populations from Angola (Kuvale, Kwepe, Himba, Tjimba, Kwisi, Twa, !Xun) and Zimbabwe (Tshwa), including foragers, food-producers, and peripatetic groups (low-status communities who provide services to their dominant neighbors). RESULTS: Our results indicate that neither host genetics nor livelihood seem to influence the oral microbiome profile, with Neisseria, Streptococcus, Prevotella, Rothia, and Porphyromonas being the five most frequent genera in southern African groups, in line with what has been shown for other human populations. However, we found that some Tshwa and Twa individuals display an enrichment of pathogenic genera from the Enterobacteriaceae family (i.e. Enterobacter, Citrobacter, Salmonella) of the Proteobacteria phylum, probably reflecting deficient sanitation and poor health conditions associated with social marginalization. CONCLUSIONS: Taken together, our results suggest that socio-economic status, rather than ethnolinguistic affiliation or subsistence mode, is a key factor in shaping the salivary microbial profiles of human populations in southern Africa.

Quality changes, potential spoilage organisms, and shelf-life prediction of brackish river prawn (Macrobrachium macrobrachion) at different storage temperatures.

The brackish river prawn (Macrobrachium macrobrachion) is a species of commercial importance in West Africa. However, like other fishery products, it is prone to deterioration due mainly to microbial activities. The present study aimed at evaluating the spoilage characteristics of M. macrobrachion and predicting the growth of the main spoilage bacteria as well as the shelf-life of the product as a function of storage temperature. Freshly caught brackish river prawn samples from Lake Aheme were aerobically stored at 0, 7, 15, and 28 °C and, at pre-determined times during storage, they were taken for microbiological, chemical, and sensory analysis. At sensory rejection times, the spoilage potential of 185 isolates from specific groups of organisms enumerated was assessed in prawn of which the endogenous microbiota was heat inactivated. Isolates capable of producing strong off-odor were identified using 16S rRNA sequencing. Models predicting the maximum growth rate of Pseudomonas spp. and H2S-producing bacteria in the brackish river prawn as well as the shelf-life of the product were developed. These models were validated using an independent experiment during which prawn was stored at 0, 4, 10, and 25 °C. Results showed that Pseudomonas spp. at 0 °C, Pseudomonas spp. and H2S-producing bacteria at 7 °C, and H2S-producing bacteria at 15 °C and 28 °C were the dominant groups of microorganisms during storage. As expected, total volatile basic nitrogen, trimethylamine, and pH with initial values of 21.2 ± 3.0 mg-N/100 g, 4.1 ± 0.8 mg-N/100 g, and 7.46 ± 0.15 increased during storage reaching approximately 35 mg-N/100 g, 10 mg/ 100 g and 8, respectively at sensory rejection times which were 7 h at 28 °C, 1.2 d at 15 °C, 4.6 d at 7 °C, and 11.7 d at 0 °C. The main spoilage organisms were Citrobacter braakii at 28 °C, Citrobacter braakii, Pseudomonas kurunegalensis, and Shewanella bicestrii at 15 °C, Shewanella putrefaciens, Shewanella baltica, and Pseudomonas bubulae at 7 °C, and Pseudomonas versuta at 0 °C. The validation of the developed models showed an adequate agreement between the predicted and observed values. This study highlights the specific spoilage characteristics of the brackish river prawn and reveals that Gram-negative rod bacteria are the main spoilage organisms even at high storage temperatures, contrary to many earlier reports on the spoilage of tropical fishery products.

Sialic acid plays a pivotal role in licensing Citrobacter rodentium's transition from the intestinal lumen to a mucosal adherent niche.

Enteric bacterial pathogens pose significant threats to human health; however, the mechanisms by which they infect the mammalian gut in the face of daunting host defenses and an established microbiota remain poorly defined. For the attaching and effacing (A/E) bacterial family member and murine pathogen Citrobacter rodentium, its virulence strategy likely involves metabolic adaptation to the host's intestinal luminal environment, as a necessary precursor to reach and infect the mucosal surface. Suspecting this adaptation involved the intestinal mucus layer, we found that C. rodentium was able to catabolize sialic acid, a monosaccharide derived from mucins, and utilize it as its sole carbon source for growth. Moreover, C. rodentium also sensed and displayed chemotactic activity toward sialic acid. These activities were abolished when the nanT gene, encoding a sialic acid transporter, was deleted (ΔnanT). Correspondingly, the ΔnanT C. rodentium strain was significantly impaired in its ability to colonize the murine intestine. Intriguingly, sialic acid was also found to induce the secretion of two autotransporter proteins, Pic and EspC, which possess mucinolytic and host-adherent properties. As a result, sialic acid enhanced the ability of C. rodentium to degrade intestinal mucus (through Pic), as well as to adhere to intestinal epithelial cells (through EspC). We thus demonstrate that sialic acid, a monosaccharide constituent of the intestinal mucus layer, functions as an important nutrient and a key signal for an A/E bacterial pathogen to escape the colonic lumen and directly infect its host's intestinal mucosa.

Pangenome-driven insights into nitrogen metabolic characteristics of Citrobacter portucalensis strain AAK_AS5 associated with wastewater nitrogen removal.

Nitrogen metabolism in the genus Citrobacter is very poorly studied despite its several implications in wastewater treatment. In the current study, Citrobacter portucalensis strain AAK_AS5 was assessed for remediation of simulated wastewater supplemented with different inorganic nitrogen sources. Combination of (NH4)2SO4 with KNO3 was the most preferred for achieving high growth density followed by (NH4)2SO4 and KNO3 alone. This was in agreement with highest ammonical nitrogen removal of 92.9% in the presence of combined nitrogen sources and the corresponding nitrate nitrogen removal of 93% in the presence of KNO3. Furthermore, these removal capacities were validated by investigating the uniqueness and the spread of metabolic features through pan-genomic approach that revealed the largest number of unique genes (2097) and accessory genes (705) in strain AAK_AS5. Of the total 44 different types of nitrogen metabolism-related genes, 39 genes were associated with the core genome, while 5 genes such as gltI, nasA, nasR, nrtA, and ntrC uniquely belonged to the accessory genome. Strain AAK_AS5 possessed three major nitrate removal pathways viz., assimilatory and dissimilatory nitrate reduction to ammonia (ANRA & DNRA), and denitrification; however, the absence of nitrification was compensated by ammonia assimilation catalyzed by gene products of the GDH and GS-GOGAT pathways. narGHIJ encoding the respiratory nitrate reductase was commonly identified in all the studied genomes, while genes such as nirK, norB, and nosZ were uniquely present in the strain AAK_AS5 only. A markedly different genetic content and metabolic diversity between the strains reflected their adaptive evolution in the environment thus highlighting the significance of C. portucalensis AAK_AS5 for potential application in nitrogen removal from wastewater.

Carbapenemase-producing Gram-negative bacteria in hospital wastewater, wastewater treatment plants and surface waters in a metropolitan area in Germany, 2020.

Carbapenemase-producing bacteria (CPB) such as Klebsiella pneumoniae and Escherichia coli are causing hospital outbreaks worldwide. An important transfer route into the aquatic environment is the urban water cycle. We aimed to determine the presence of CPB in hospital wastewater, wastewater treatment plants (WWTPs) and surface waters in a German metropolitan area and to characterise these bacteria by whole-genome comparisons. During two periods in 2020, 366 samples were collected and cultivated on chromogenic screening media. Bacterial colonies were selected for species identification and PCR-based carbapenemase gene screening. Genomes of all detected CPB were sequenced and analysed for resistance gene content, followed by multilocus sequence typing (MLST) and core genome MLST (cgMLST) for K. pneumoniae and E. coli isolates. Carbapenemase genes were detected in 243 isolates, most of which belonged to genera/species Citrobacter spp. (n = 70), Klebsiella spp. (n = 57), Enterobacter spp. (n = 52) and E. coli (n = 42). Genes encoding KPC-2 carbapenemase were detected in 124 of 243 isolates. K. pneumoniae produced mainly KPC-2 and OXA-232 whereas E. coli harboured various enzymes (KPC-2, VIM-1, OXA-48, NDM-5, KPC-2 + OXA-232, GES-5, GES-5 + VIM-1, IMP-8 + OXA-48). Eight and twelve sequence types (STs) were identified for K. pneumoniae and E. coli, respectively, forming different clusters. The detection of numerous CPB species in hospital wastewater, WWTPs and river water is of concern. Genome data highlight a hospital-specific presence of distinct carbapenemase-producing K. pneumoniae and E. coli strains belonging to "global epidemic clones" in wastewater samples representing local epidemiology. The various detected CPB species including E. coli ST635, which is not known to cause human infections, could serve as reservoirs/vectors for the spread of carbapenemase genes in the environment. Therefore, effective pretreatment of hospital wastewater prior to discharge into the municipal wastewater system may be required, although swimming lakes do not appear to be a relevant risk factor for CPB ingestion and infection.

Citrobacter meridianamericanus sp. nov., isolated from a soil sample.

A Gram-stain-negative strain, designated BR102T, isolated from a soil sample in Brazil was characterized by a polyphasic approach. Comparative 16S rRNA gene sequences indicated that strain BR102T belonged to the genus Citrobacter. The recN- and whole-genome-based phylogeny, and multilocus sequence analysis based on concatenated partial fusA, leuS, pyrG and rpoB sequences strongly supported a clade encompassing strain BR102T and a strain from public database that was distinct from currently recognized species of the genus Citrobacter. Average nucleotide identity and digital DNA-DNA hybridization values between strain BR102T and the closest relative Citrobacter freundii ATCC 8090T were 91.8 and 48.8 %, respectively. The ability to metabolize different compounds further discriminated strain BR102T from other closely related species of the genus Citrobacter. The novel variants bla CMY-179 and qnrB97, which encoded a CMY-2-like ß-lactamase and a QnrB-type protein, respectively, were identified in strain BR102T. BR102T was resistant to ampicillin, amoxicillin/clavulanate and cefoxitin. The DNA G+C content of strain BR102T is 51.3 mol%. Based on these results, strain BR102T represents a novel species of the genus Citrobacter, for which the name Citrobacter meridianamericanus sp. nov. is proposed. The type strain is BR102T (=MUM 22.55T=IMI 507229T).

Parametric and kinetic studies of activated sludge dewatering by cationic chitosan-like bioflocculant BF01314 produced from Citrobacter youngae.

Bacterial strains belonging to Citrobacter spp. were reported to produce polysaccharides consisting of N-acetylglucosamine and glucosamine like chitosan, with high flocculation activity. In this work, the flocculation dewatering performance of activated sludge conditioned by a novel cationic chitosan-like bioflocculant (BF) named BF01314, produced from Citrobacter youngae GTC 01314, was evaluated under the influences of flocculant dosage, pH, and temperature. At BF dosage as low as 0.5 kg/t DS, the sludge dewaterability was significantly enhanced in comparison to the raw (untreated) sludge, featuring well-flocculated characteristic (reduction in CST from 22.0 s to 9.4 s) and good sludge filterability with reduced resistance (reduction in SRF by one order from 7.42 × 1011 to 9.59 × 1010 m/kg) and increased compactness of sludge (increase in CSC from 15.2 to 23.2%). Besides, the BF demonstrated comparable high sludge dewatering performance within the pH range between 2 and 8, and temperature range between 25 °C and 80 °C. Comparison between the BF, the pristine chitosan and the commercial cationic copolymer MF 7861 demonstrated equivalent performance with enhanced dewaterability at the dosage between 2.0 and 3.0 kg/t DS. Besides, the BF demonstrated strong flocculation activity (>99%) when added to the sludge suspension using moderate to high flocculation speeds (100-200 rpm) with at least 3-min mixing time. The BF's reaction in sludge flocculation was best fitted with a pseudo first-order kinetic model. Electrostatic charge patching and polymer bridging mechanisms are believed to be the dominant mechanistic phenomena during the BF's sludge conditioning process (coagulation-flocculation).

ompX contribute to biofilm formation, osmotic response and swimming motility in Citrobacter werkmanii.

Citrobacter werkmanii, an aerobe and mesophilic Proteobacterium, is universal in industrial putrefaction, coastal water, and human blood. Our previous studies have discovered that outer membrane protein X (OmpX) of C. werkmanii is involved in calcium response, but the underlying mechanisms and its molecular characteristics remain elusive. To that end, the ompX gene was deleted from the genome of C. werkmanii and its phenotypic variations were thoroughly investigated in conjunction with the wild type (WT) and complementary strains using biochemical and molecular techniques such as RNA-Seq, respectively. The results demonstrated that deleting ompX reduces biofilm formation on polystyrene and glass surfaces. Meanwhile, ΔompX's swimming ability but not for its twitching or swarming abilities, was also reduced on semi-solid plates compared with WT, which was caused by inhibition of flagellar assembly genes, such as flgC, flhB, and fliE, etc. Furthermore, ompX inactivation altered susceptibility to various bactericide classes, as well as responses to Ca2+ and Mg2+ stress. In addition, when compared to WT, ΔompX captures a total of 1,357 deferentially expressed genes (DEGs), of which 465 were up-regulated and 892 were down-regulated, which can be enriched into various GO ontology and KEGG pathway terms. Furthermore, ompX, as well as ompD and ompW, can be modulated at the transcriptional levels by rbsR and tdcA. Overall, the ompX gene contributed to a variety of biological functions in C. werkmanii and could be served as a targeted site for controlling biofilm formation and developing new bactericides.

Phylogenomic analysis of Citrobacter sp. strain AAK_AS5 and its metabolic capabilities to support nitrogen removal behavior.

Despite the ubiquity of the genus Citrobacter in clinical, industrial, and environmental scenarios, a large number of Citrobacter strains have not been explored at the genome-scale level. In this study, accurate taxonomic assignment of strain AAK_AS5 isolated from activated sludge was achieved by in-silico genomic comparison using Overall Genome-based Relatedness Indices (ANI(OAT): 97.55%, ANIb:97.28%, and ANIm: 97.83%) that indicated its closest identity to the related strain Citrobacter portucalensis A60T . Results were consistent with a digital DNA-DNA hybridization value of 80% with C. portucalensis A60T which was greater than the species boundary value >70% for delineating closely related bacterial species. Gene mining through Kyoto Encyclopedia of Genes and Genomes (KEGG), and annotation using rapid annotation subsystem technology (RAST) revealed the notable gene contents for nitrogen metabolism and other pathways associated with nitrate/nitrite ammonification (28 genes), ammonia assimilation (22 genes), and denitrification pathways (14 genes). Furthermore, the strain AAK_AS5 also exhibited a high soluble chemical oxygen demand (sCOD), NH4 + -N, and NO3 - -N removal efficiency of 91.4%, 90%, and 93.6%, respectively thus validating its genetic capability for utilizing both (NH4 )2 SO4 and KNO3 as the nitrogen source. The study provided deeper insights into the phylogenomics and the genetic potential of Citrobacter, sp. strain AAK AS5 associated with nitrogen metabolism thus signifying the potential application of the isolate for treating nitrogen-rich wastewaters.

Citrobacter portucalensis Sb-2 contains a metalloid resistance determinant transmitted by Citrobacter phage Chris1.

Bacterial adaptation to extreme environments is often mediated by horizontal gene transfer (HGT) via genetic mobile elements. Nevertheless, phage-mediated HGT conferring bacterial arsenic resistance determinants has rarely been investigated. In this study, a highly arsenite and antimonite resistant bacterium, Citrobacter portucalensis strain Sb-2, was isolated, and genome analysis showed that several putative arsenite and antimonite resistance determinants were flanked or embedded in prophages. Furthermore, an active bacteriophage carrying one of the ars clusters (arsRDABC arsR-yraQ/arsP) was obtained and sequenced. These genes encoding putative arsenic resistance determinants were induced by arsenic and antimony as demonstrated by RT-qPCR, and one gene arsP/yraQ of the ars cluster was shown to give resistance to MAs(III) and Rox(III), thereby showing function. Here, we were able to directly show that these phage-mediated arsenic and antimony resistances play a significant role in adapting to As- and Sb-contaminated environments. In addition, we demonstrate that this phage is responsible for conferring arsenic and antimony resistances to C. portucalensis strain Sb-2.