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.

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.

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.

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.

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.

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.

Citrobacter sp. Y3 harbouring novel gene HBCD-hd-1 mineralizes hexabromocyclododecane via new metabolic pathways according to multi-omics characterization.

Hexabromocyclododecane (HBCD) is a typical persistent organic pollutant that is widely detected in the environment. Despite the significant efforts put into its mineralisation, there is still a lack of microorganism resources that can completely mineralise HBCD. Stable isotope analysis revealed that the Citrobacter sp. Y3 can use [13C]HBCD as its sole carbon source and degrade or even mineralise it into 13CO2, with a maximum conversion rate of 100% in approximately 14 days. Strain Y3 could completely mineralise HBCD, which it used as its only carbon source, and six debromination enzymes related to HBCD degradation were found in Y3, including haloalkane dehalogenase (DhaA), haloacid dehalogenase (HAD), etc. A functional gene named HBCD-hd-1, encoding a HAD, was found to be upregulated during HBCD degradation and heterologously expressed in Escherichia coli. Recombinant E. coli with the HBCD-hd-1 gene transformed the typical intermediate 4-bromobutyric acid to 4-hydroxybutanoic acid and showed excellent degradation performance on HBCD, accompanied by nearly 100% bromine (Br) ion generation. The expression of HBCD-hd-1 in Y3 rapidly accelerated the biodegradation of HBCD. With HBCD as its sole carbon source, strain Y3 could potentially degrade HBCD, especially in a low-nutrient environment.

Epidemiology and Molecular Characteristics of mcr-9 in Citrobacter spp. from Healthy Individuals and Patients in China.

With the globally prevailing carbapenemase-producing (CP) Citrobacter spp., polymyxin antibiotics have been reconsidered as one of the last-resort treatment options. Our study was conducted to investigate the prevalence of mcr-9 in Citrobacter species. From October to November 2021, 650 fecal samples and 215 Citrobacter isolates were collected from healthy individuals and infected patients, respectively. Isolates were screened for the presence of the mcr-9 gene by the PCR method. mcr-9-carrying strains were identified by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry. Due to the susceptibility to colistin, Citrobacter spp. isolates were first induced to increase the expression of mcr-9 on China blue agar plates containing colistin and were then subjected to conjugation experiments. Whole-genome sequencing was performed on the Illumina NovaSeq PE150 system. The prevalence of mcr-9 in the Citrobacter genus from healthy guts and infected patients was 0.62% and 1.86%, respectively. In all mcr-9-positive strains, MICs of polymyxin B were observed at ≤2 µg/mL, displaying a nonresistant phenotype. As for conjugation experiments, only one isolate successfully transferred the mcr-9 gene to Escherichia coli C600. Whole-genome sequencing showed that eight mcr-9-positive Citrobacter isolates carried mcr-9 and genes encoding resistance to beta-lactam antibiotics, including blaCMY, blaDHA, blaSHV, blaTEM, and blaCTX-M. We also discovered that mcr-9 could be located on the pKPC-CAV1321 plasmid. Our study investigated the prevalence of mcr-9 in Citrobacter spp. in both healthy individuals and infected patients and described the carriage of mcr-9 on the pKPC-CAV1321 plasmid for the first time. IMPORTANCE The emergence of mcr homologues posed a serious threat to the therapeutic efficiency of polymyxin antibiotics. Citrobacter freundii is generally regarded as an opportunistic pathogen associated with a variety of nosocomial infections. In this study, we investigated the prevalence of mcr-9 in Citrobacter spp. isolates from healthy individuals and infected patients and highlighted the importance of the rational use of antibiotics. In addition, this epidemiological investigation is the first to describe the carriage of mcr-9 on plasmid pKPC-CAV1321 and confirms the horizontal transfer of this plasmid. Our research may shed new light on further studies of mcr-9 dissemination in humans.

Cross-reaction of naturally-produced ß-lactamases from Citrobacter farmeri and Citrobacter amalonaticus with immunological detection of CTX-M enzymes.

The NG-Test CTX-M MULTI immunochromatographic assay has been developed to identify CTX-M-type ß-lactamases in Enterobacterales, being the most widespread extended-spectrum ß-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.

Identification and Genome Analysis of an Arsenic-Metabolizing Strain of Citrobacter youngae IITK SM2 in Middle Indo-Gangetic Plain Groundwater.

Whole-genome sequencing (WGS) data of a bacterial strain IITK SM2 isolated from an aquifer located in the middle Indo-Gangetic plain is reported here, along with its physiological, morphological, biochemical, and redox-transformation characteristics in the presence of dissolved arsenic (As). The aquifer exhibits oxidizing conditions relative to As speciation. Analyses based on 16S rRNA and recN sequences indicate that IITK SM2 was clustered with C. youngae NCTC 13708T and C. pasteuri NCTC UMH17T. However, WGS analyses using the digital DNA-DNA hybridization and Rapid Annotations using Subsystems Technology suggest that IITK SM2 belongs to a strain of C. youngae. This strain can effectively reduce As(V) to As(III) but cannot oxidize As(III) to As(V). It exhibited high resistance to As(V) [32,000 mg L-1] and As(III) [1,100 mg L-1], along with certain other heavy metals typically found in contaminated groundwater. WGS analysis also indicates the presence of As-metabolizing genes such as arsC, arsB, arsA, arsD, arsR, and arsH in this strain. Although these genes have been identified in several As(V)-reducers, the clustering of these genes in the forms of arsACBADR, arsCBRH, and an independent arsC gene has not been observed in any other Citrobacter species or other selected As(V)-reducing strains of Enterobacteriaceae family. Moreover, there were differences in the number of genes corresponding to membrane transporters, virulence and defense, motility, protein metabolism, phages, prophages, and transposable elements in IITK SM2 when compared to other strains. This genomic dataset will facilitate subsequent molecular and biochemical analyses of strain IITK SM2 to identify the reasons for high arsenic resistance in Citrobacter youngae and understand its role in As mobilization in middle Indo-Gangetic plain aquifers.

Phylogeographical Landscape of Citrobacter portucalensis Carrying Clinically Relevant Resistomes.

During a surveillance study conducted to assess the occurrence and genomic landscape of critical priority pathogens circulating at the human-animal-environment interface in Brazil, as part of the Grand Challenges Explorations-New Approaches to Characterize the Global Burden of Antimicrobial Resistance program, two multidrug-resistant (MDR) Citrobacter portucalensis carrying blaCTX-M-15 extended-spectrum ß-lactamase (ESBL) genes, isolated from green sea turtles, were characterized. Genomic and phylogeographical analysis of C. portucalensis genomes available in public databases revealed the intercontinental dissemination of clades carrying different arrays of clinically relevant genes conferring resistance to carbapenems, broad-spectrum cephalosporins, cephamycins, aminoglycosides and fluoroquinolones, disinfectants, and heavy metals. Our observations suggest that C. portucalensis could be emerging as critical priority bacteria of both public and One Health importance worldwide. IMPORTANCE The global spread of antibiotic-resistant priority pathogens beyond the hospital setting is a critical issue within a One Health context that integrates the human-animal-environment interfaces. On the other hand, next-generation sequencing technologies along with user-friendly and high-quality bioinformatics tools have improved the identification of bacterial species, and bacterial resistance surveillance. The novel Citrobacter portucalensis species was proposed in 2017 after taxonomic reclassification and definition of the strain A60T isolated in 2008. Here, we presented genomic data showing the occurrence of multidrug-resistant C. portucalensis isolates carrying blaCTX-M-15 ESBL genes in South America. Additionally, we observed the intercontinental dissemination of clades harboring a broad resistome to clinically relevant antibiotics. Therefore, these findings highlight that C. portucalensis is a global MDR bacteria that carries intrinsic blaCMY- and qnrB-type genes and has become a critical priority pathogen due to the acquisition of clinically relevant resistance determinants, such as ESBL and carbapenemase-encoding genes.

Molecular Cloning, Expression and Characterisation of a Bacterial Myrosinase from Citrobacter Wye1.

Glucosinolates are plant natural products which on degradation by myrosinases give rise to the beneficial bioactive isothiocyanates. Recently, a myrosinase activity was detected in a Citrobacter strain isolated from soil. This enzyme was purified enabling its amino acid sequence and gene sequence (cmyr) to be determined. In order to study this myrosinase it was necessary to establish an expression system that would enable future work such as a structural determination of the protein to be carried out. The myrosinase gene was amplified, cloned and expressed in Escherichia coli with a 6XHis-tag. The heterologous expression of cmyr enabled relatively large amounts of myrosinase to be produced (3.4 mg cmyr/100 ml culture). Myrosinase activity was determined by mixing substrate and enzyme and determining glucose release. Optimum pH and temperature were determined to be pH 6.0 and 25 °C for the Ni-NTA purified protein. The kinetic parameters of the purified myrosinase were determined using sinigrin as a substrate. Km and Vmax were estimated as 0.18 mM and 0.033 mmol/min/mg respectively for sinigrin under optimum conditions and compared to other kinetic data for myrosinases. The substrate specificity of myrosinase was determined having the highest affinity for sinigrin followed by glucoiberin, progoitrin, glucoerucin, glucoraphanin and glucotropaeolin.

Emergence of IncX3 Plasmid-Harboring blaNDM-5 in a Citrobacter sedlakii Isolated from Outdoor Aerosol in Wastewater Treatment Plant.

A carbapenem-resistant Citrobacter sedlakii strain AA2CS carrying blaNDM-5 was detected in outdoor aerosols of a wastewater treatment plant (WWTP) in China and the whole genome was sequenced subsequently. AA2CS was captured in an aerobic tank with aerosol particles of sizes ranging from 4.7 to 7.0 µm. Besides blaNDM-5, AA2CS also harbored 21 other antibiotic resistance genes and displayed a high level of resistance to ampicillin, cefotaxime, ceftazidime, tetracycline, and meropenem. BlaNDM-5 was located on the IncX3 plasmid (pCSNDM-5) with an IS3000-IS5-blaNDM-5-bleMBL-trpF-dsbD-IS26 structure. pCSNDM-5 was highly homologous to other blaNDM-5-carrying IncX3 plasmids in China and can be transferred to the Escherichia coli recipient J53. To our knowledge, this is the first report of carbapenem-resistant Enterobacteriaceae in outdoor aerosols in WWTPs.