Earthworm gut bacteria facilitate cadmium immobilization through the formation of CdS nanoparticles.

Gut bacteria of earthworm Amynthas hupeiensis exhibit significant potential for the in-situ remediation of cadmium (Cd)-contaminated soil. However, the mechanisms by which these gut bacteria immobilize and tolerate Cd remain elusive. The composition of the gut bacterial community was characterized by high-throughput sequencing. Cd-tolerant bacteria were isolated from the gut, and their roles in Cd immobilization, as well as their tolerance mechanisms, were explored through chemical characterization and transcriptome analysis. The predominant taxa in the gut bacterial community included unclassified Enterobacteriaceae, Citrobacter, and Bacillus, which were distinctly different from those in the surrounding soil. Notably, the most Cd-tolerant gut bacterium, Citrobacter freundii DS strain, immobilized 63.61% of Cd2+ within 96 h through extracellular biosorption and intracellular bioaccumulation of biosynthetic CdS nanoparticles, and modulation of solution pH and NH4+ concentration. Moreover, the characteristic signals of CdS were also observed in the gut content of A. hupeiensis when the sterilized Cd-contaminated soil was inoculated with C. freundii. The primary pathways involved in the response of C. freundii to Cd stress included the regulation of ABC transporters, bacterial chemotaxis, cell motility, oxidative phosphorylation, and two-component system. In conclusion, C. freundii facilitates Cd immobilization both in vitro and in vivo, thereby enhancing the host earthworm's adaptation to Cd-contaminated soil.

Effects of Citrobacter freundii on sturgeon: Insights from skin mucosal immunology and microbiota.

Skin mucus analysis has recently been used as a non-invasive method to evaluate for fish welfare. The present research study was conducted to examine the skin mucosal immunity and skin microbiota profiles of sturgeons infected with Citrobacter freundii. Our histology results showed that the thickness of the epidermal layer of skin remained thinner, and the number of mucous cells was significantly decreased in sturgeons after infection (p < 0.05). Total protein, alanine aminotransferase, aspartate aminotransferase, superoxide dismutase, and creatine kinase levels in the mucus showed biphasic pattern (decrease and then increase). Lactate dehydrogenase, lysozyme, and acid phosphatase activities in the mucus showed an increasing trend after infection. Furthermore, 16S rRNA sequencing also revealed that C. freundii infection also affected the diversity and community structure of the skin mucus microbiota. An increase in microbial diversity (p > 0.05) and a decrease in microbial abundance (p < 0.05) after infection were noted. The predominant bacterial phyla in the skin mucus were Proteobacteria, Fusobacteria, Bacteroidetes, Firmicutes, and Actinobacteria. Specifically, the relative abundance of Fusobacteria increased after infection. The predominant bacterial genera in the skin mucus were Cetobacterium, Pelomonas, Bradyrhizobium, Flavobacterium, and Pseudomonas. The relative abundance of Cetobacterium, Pseudomonas, and Flavobacterium increased after infection. Our current research findings will provide new insights into the theoretical basis for future research studies exploring the mechanism of sturgeon infection with C. freundii.

A sophisticated virulence repertoire and colistin resistance of Citrobacter freundii ST150 from a patient with sepsis admitted to ICU in a tertiary care hospital in Uganda, East Africa: Insight from genomic and molecular docking analyses.

Sepsis and multidrug resistance comprise a complex of factors attributable to mortality among intensive care unit (ICU) patients globally. Pathogens implicated in sepsis are diverse, and their virulence and drug resistance remain elusive. From a tertiary care hospital ICU in Uganda, we isolated a Citrobacter freundii strain RSM030 from a patient with sepsis and phenotypically tested it against a panel of 16 antibiotics including imipenem levofloxacin, cotrimoxazole and colistin, among others. We sequenced the organism's genome and integrated multilocus sequencing (MLST), PathogenFinder with Virulence Factor analyzer (VFanalyzer) to establish its pathogenic relevance. Thereafter, we combined antiSMASH and PRISM genome mining with molecular docking to predict biosynthetic gene clusters (BGCs), pathways, toxin structures and their potential targets in-silico. Finally, we coupled ResFinder with comprehensive antibiotic resistance database (CARD) to scrutinize the genomic antimicrobial resistance profile of the isolate. From PathogenFinder and MLST, this organism was confirmed to be a human pathogen (p = 0.843), sequence type (ST)150, whose virulence is determined by chromosomal type III secretion system (T3SS) (the injectosome) and plasmid-encoded type IV secretion system (T4SS), the enterobactin biosynthetic gene cluster and biofilm formation through the pgaABCD operon. Pathway and molecular docking analyses revealed that the shikimate pathway can generate a toxin targeting multiple host proteins including spectrin, detector of cytokinesis protein 2 (Dock2) and plasmalemma vesicle-associated protein (PLVAP), potentially distorting the host cell integrity. From phenotypic antibiotic testing, we found indeterminate results for amoxicillin/clavulanate and levofloxacin, with resistance to cotrimoxazole and colistin. Detailed genome analysis revealed chromosomal beta lactam resistance genes, i.e. blaCMY-79, blaCMY-116 and blaTEM-1B, along with multiple mutations of the lipopolysaccharide modifying operon genes PmrA/PmrB, pmrD, mgrA/mgrB and PhoP/PhoQ, conferring colistin resistance. From these findings, we infer that Citrobacter freundii strain RSM030 is implicated in sepsis and resistance to standard antibiotics, including colistin, the last resort.

Assessment of the Microbiological Quality and Effect of Public Health of Ready-to-Eat Salad Samples in Isparta.

Salmonella spp. and Citrobacter spp. are among the microorganisms causing important foodborne outbreaks. In this study, it was tried to determine the presence and rate of Salmonella spp. and Citrobacter spp. in salad samples collected from certain regions of province of Isparta in Türkiye. A total of 50 salad samples were analyzed. Classical culture technique was used for microbiological analysis of salad samples. Suspected isolates obtained were identified using the VITEK-2 system. Although no negative visual changes were observed in the salad samples used in the study, it was determined that the number of Gram-negative microorganisms was very high and six salad samples were not suitable for public health. In 50 salad samples, 2% Salmonella and 4% Citrobacter freundii were detected. In addition, it was determined that the Salmonella strain isolated from the salad sample was resistant to three different antibiotics and Citrobacter was resistant to two different antibiotics. Salmonella spp. and Citrobacter spp. are considered very dangerous to public health because they are associated with foodborne outbreaks and can develop antibiotic resistance very quickly. Salad producers should try to reduce the possibility of microbial contamination by using different technologies.

The effect of triclosan on intergeneric horizontal transmission of plasmid-mediated tigecycline resistance gene tet(X4) from Citrobacter freundii isolated from grass carp gut.

The transmission of antibiotic resistance genes (ARGs) in pathogenic bacteria affects culture animal health, endangers food safety, and thus gravely threatens public health. However, information about the effect of disinfectants - triclosan (TCS) on ARGs dissemination of bacterial pathogens in aquatic animals is still limited. One Citrobacter freundii (C. freundii) strain harboring tet(X4)-resistant plasmid was isolated from farmed grass carp guts, and subsequently conjugative transfer frequency from C. freundii to Escherichia coli C600 (E. coli C600) was analyzed under different mating time, temperature, and ratio. The effect of different concentrations of TCS (0.02, 0.2, 2, 20, 200 and 2000 µg/L) on the conjugative transfer was detected. The optimum conditions for conjugative transfer were at 37 °C for 8h with mating ratio of 2:1 or 1:1 (C. freundii: E. coli C600). The conjugative transfer frequency was significantly promoted under TCS treatment and reached the maximum value under 2.00 µg/L TCS with 18.39 times that of the control group. Reactive oxygen species (ROS), superoxide dismutase (SOD) and catalase (CAT) activities, cell membrane permeability of C. freundii and E. coli C600 were obviously increased under TCS stress. Scanning electron microscope showed that the cell membrane surface of the conjugative strains was wrinkled and pitted, even broken at 2.00 µg/L TCS, while lysed or even ruptured at 200.00 µg/L TCS. In addition, TCS up-regulated expression levels of oxidative stress genes (katE, hemF, bcp, hemA, katG, ahpF, and ahpC) and cell membrane-related genes (fimC, bamE and ompA) of donor and recipient bacteria. Gene Ontology (GO) enrichment demonstrated significant changes in categories relevant to pilus, porin activity, transmembrane transporter activity, transferase activity, hydrolase activity, material transport and metabolism. Taken together, a tet(X4)-resistant plasmid could horizontal transmission among different pathogens, while TCS can promote the propagation of the resistant plasmid.

Genetic characterization of KHM-1 metallo-ß-lactamase-producing Enterobacterales isolates from inpatient sources in Osaka, Japan.

OBJECTIVES: KHM-1-metallo-ß-lactamase-producing Enterobacterales strains, of which only a few have been found, were isolated from four inpatients in Osaka, Japan during 2016 to 2020. We compared whole genomes of the four KHM-1-producing isolates, including one Enterobacter hormaechei subsp. hoffmannii, one Escherichia coli, and two Citrobacter freundii. METHODS: These isolates were characterized by whole-genome sequencing, comparative analysis of blaKHM-1-encoding plasmids with earlier reported plasmids, and antimicrobial susceptibility tests. RESULTS: Multilocus sequence typing classified the E. hormaechei subsp. hoffmannii isolate to ST78, the E. coli isolate to ST354, and the two C. freundii isolates to ST95. These isolates harboured various antimicrobial resistance genes aside from blaKHM-1 on their chromosomes and plasmids. In all four isolates, blaKHM-1 was located on 137 kbp to 213 kbp plasmids of IncC replicon type. Although there were common resistance genes such as blaKHM-1-ISEc68, class I integron cassette, and fosG, the four blaKHM-1-encoding plasmids were distinguishable into two lineages based on differences of the resistance gene components and their surrounding regions. CONCLUSION: Because no epidemiological contact was observed among the inpatients, the blaKHM-1-encoding IncC plasmids might have spread horizontally to multiple bacterial species through repeated recombination and insertion.

Colistin heteroresistance in Citrobacter freundii clinical isolates from Republic of Korea.

We investigated colistin heteroresistance in Citrobacter freundii isolates from Korean hospitals. Using population analysis profiling (PAP), we detected colistin heteroresistance in 31.3% of isolates. Among these, ST217 was the most prevalent clone (58.5%), particularly within colistin-heteroresistant isolates (80.0%). Interestingly, the second most common clone, ST248, was not found in heteroresistant isolates. We identified amino acid changes in PhoQ, PmrA, and PmrB, along with mRNA overexpression in pmrB and arnD. Colistin monotherapy showed no efficacy, but a combination of colistin and ciprofloxacin successfully eradicated all five isolates, even at 0.5 × minimum inhibitory concentrations. This study underscores the high prevalence of colistin heteroresistance in C. freundii isolates, limiting the effectiveness of colistin monotherapy. Combining colistin with ciprofloxacin may offer a viable treatment option for C. freundii infections.

Genomic analysis of a cAmpC (CMY-41)-producing Citrobacter freundii ST64 isolated from patient.

Antibiotic resistance in Citrobacter freundii is a public health concern. This study evaluated the closed genome of a C. freundii isolated from the stool of a hospitalized patient initially related to a Salmonella outbreak. Confirmation of the isolate was determined by whole-genome sequencing. Nanopore sequencing was performed using a MinION with a Flongle flow cell. Assembly using SPAdes and Unicycler yielded a closed genome annotated by National Center for Biotechnology Information Prokaryotic Genome Annotation Pipeline. Genomic analyses employed MLST 2.0, ResFinder4.1, PlasmidFinder2.1, and VFanalyzer. Phylogenetic comparison utilized the Center for Food Safety and Applied Nutrition (CFSAN)-single nucleotide polymorphism pipeline and Genetic Algorithm for Rapid Likelihood Inference. Antimicrobial susceptibility was tested by broth microdilution following Clinical and Laboratory Standards Institute criteria. Multi-locus sequence type in silico analysis assigned the C. freundii as sequence type 64 and the blaCMY-41 gene was detected in resistome investigation. The susceptibility to antibiotics, determined using Sensititre® plates, revealed resistance to aztreonam, colistin, cefoxitin, amoxicillin/clavulanic acid, sulfisoxazole, ampicillin, and streptomycin. The genetic relatedness of the C. freundii CFSAN077772 with publicly available C. freundii genomes revealed a close relationship to a C. freundii SRR1186659, isolated in 2009 from human stool in Tanzania. In addition, C. freundii CFSAN077772 is nested in the same cluster with C. freundii clinical strains isolated in Denmark, Mexico, Myanmar, and Canada, suggesting a successful intercontinental spread.

Genomic scan of a healthcare-associated NDM-1-producing Citrobacter freundii ST18 isolated from a green sea turtle impacted by plastic pollution.

BACKGROUND: Carbapenemase-producing Citrobacter freundii has been reported as a leading cause of healthcare-associated infections. Particularly, C. freundii belonging to the sequence type (ST) 18 is considered to be an emerging nosocomial clone. OBJECTIVES: To report the genomic background and phylogenomic analysis of a multidrug-resistant NDM-1-producing C. freundii ST18 (strain CF135931) isolated from an endangered green sea turtle affected by plastic pollution in Brazil. METHODS: Genomic DNA was extracted and sequenced using the Illumina NextSeq platform. De novo assembly was performed by CLC Workbench, and in silico analysis accomplished by bioinformatics tools. For phylogenomic analysis, publicly available C. freundii (txid:546) genome assemblies were retrieved from the NCBI database. RESULTS: The genome size was calculated at 5 290 351 bp, comprising 5263 total genes, 4 rRNAs, 77 tRNAs, 11ncRNAs, and 176 pseudogenes. The strain belonged to C. freundii ST18, whereas resistome analysis predicted genes encoding resistance to ß-lactams (blaNDM-1, blaOXA-1, blaCMY-117, and blaTEM-1C), aminoglycosides (aph(3'')-Ib, aadA16, aph(3')-VI, aac(6')-Ib-cr, and aph(6)-Id), quinolones (aac(6')-Ib-cr), macrolides (mph(A) and erm(B)), sulphonamides (sul1 and sul2), tetracyclines (tetA and tetD), and trimethoprim (dfrA27). The phylogenomic analysis revealed that CF135931 strain is closely related to international human-associated ST18 clones producing NDM-1. CONCLUSION: Genomic surveillance efforts are necessary for robust monitoring of the emergence of drug-resistant strains and WHO critical priority pathogens within a One Health framework. In this regard, this draft genome and associated data can improve understanding of dissemination dynamics of nosocomial clones of carbapenemase-producing C. freundii beyond hospital walls. In fact, the emergence of NDM-1-producing C. freundii of global ST18 in wildlife deserves considerable attention.

Genomic characterization of a carbapenem-resistant Citrobacter freundii clinical isolate from China carrying blaNDM-5 on a novel IncC-IncFIB-IncX3 plasmid.

OBJECTIVES: Citrobacter freundii is one of the important pathogens that can cause nosocomial infections. The advent of carbapenem-resistant C. freundii complicates clinical treatment. Here, we reported the genome sequence of a carbapenem-resistant C. freundii strain carrying a novel IncC-IncFIB-IncX3 plasmid in China. METHODS: The genome sequence of C. freundii CRNMS1 was obtained using the Illumina NovaSeq 6000 platform and the long-read Nanopore sequencer. Multilocus sequence typing was identified using MLST (v.2.23.0). The identification of antimicrobial resistance genes (ARGs) and plasmid replicons was performed using the resfinder and plasmidfinder of ABRicate (v.1.0.1). Circular comparisons of plasmids were performed using the BLAST Ring Image Generator (BRIG). RESULTS: CRNMS1 belongs to ST116 in the C. freundii MLST scheme. Thirteen ARGs were predicted in all, including blaNDM-5, which was located in a plasmid. The plasmid pblaNDM5-S1, which carried the blaNDM-5 gene, was discovered to be a novel plasmid including three plasmid replicons (IncC, IncFIB, and IncX3) as well as seven ARGs (sul1, sul2, floR, dfrA17, aadA5, qnrA1, and blaNDM-5). A total of 38 blaNDM-5-bearing C. freundii strains can be retrieved from the NCBI database. Phylogenetic analysis revealed a worldwide distribution of C. freundii strains carrying the blaNDM-5 gene, with China having the highest prevalence (39%, 15/38). However, they were distantly related to CRNMS1 with SNP differences >2545. CONCLUSION: In summary, we reported a novel IncC-IncFIB-IncX3 plasmid carrying blaNDM-5 in a carbapenem-resistant C. freundii strain in China. The development of such hybrid plasmids facilitates the transmission of ARGs.

Characterization of a blaNDM-1-positive Citrobacter freundii strain isolated from earthworms.

OBJECTIVES: Earthworms are one of the key components of soil, and they play a crucial role in the transformation of various nutrients and pollutants in the soil. The purpose of this study is to characterize the NDM-1-producing C. freundii isolated from soil-dwelling earthworms near a hospital, exploring their potential role as carriers of carbapenem-resistant genes. METHODS: Isolates were isolated from the intestines of earthworms and identified by MALDI-TOF MS. The presence of NDM enzyme was verified through the CARBA-5 Assay. Whole genome sequencing was conducted using the Illumina NovaSeq PE150 platform. Antimicrobial susceptibility testing and conjugation experiment were performed for phenotypic analysis. RESULTS: This isolate exhibited a multidrug-resistant profile, including resistance to imipenem, meropenem, and ertapenem and successfully transferred blaNDM-1 gene to Escherichia coli. Whole genomic sequencing showed that blaNDM-1 gene was located on an IncFIIY-type plasmid. Phylogenetic analysis revealed a close relationship between the QY221001 strain obtained from earthworms and the human isolate F2021 in the NCBI database, both of which were collected in Hangzhou, China. CONCLUSION: To our knowledge, this is the first report of an NDM-1-producing bacteria isolated from the intestine of an earthworm. Our finding suggested that earthworms could be a potential reservoir of carbapenem resistance genes, emphasizing the importance of enhanced environmental monitoring of antimicrobial resistance.

Citrobacter freundii-associated lesions in stranded loggerhead sea turtles (Caretta caretta).

Citrobacter freundii, an opportunistic bacterial pathogen belonging to the family Enterobacteriaceae, has been sporadically reported in sea turtles. Here, the authors describe 3 unusual lesions associated with C. freundii infection in 3 loggerhead sea turtles stranded on the coast of Gran Canaria Island, Spain. It is possible that these 3 distinct lesions played a major role in the death of these turtles. The first turtle had caseous cholecystitis, a lesion not previously described in sea turtles. The second turtle had large intestinal diverticulitis, a rare condition in loggerheads. The third turtle had bilateral caseous salt gland adenitis. Histologically, numerous gram-negative bacilli were observed at the deepest edge of inflammation in all cases. Pure cultures of C. freundii were obtained from these 3 lesions. Molecular detection of C. freundii DNA in formalin-fixed paraffin-embedded samples from the lesions of the 3 turtles confirmed the microbiological isolation. These cases, in addition to expanding the limited body of knowledge on bacterial infections in sea turtles, highlight the potential pathogenic role of C. freundii in loggerhead turtles.

Emergence and plasmid cointegration-based evolution of NDM-1-producing ST107 Citrobacter freundii high-risk resistant clone in China.

Carbapenem-resistant Citrobacter freundii (CRCF) poses an enormous challenge in the health care setting. However, the epidemiology and plasmid dynamic evolution of this species have not been well studied, especially for the novel high-risk resistant clones in the intensive care units (ICUs). Here, we characterised the cointegration-based plasmid dynamic evolution of the emerging ST107 CRCF clone in China. Twenty CRCF strains were identified, including ST22 (30%), ST107 (25%), ST396 (10%) and ST116 (10%). Interestingly, the tigecycline (TGC) resistance gene cluster tmexCD2-toprJ2 and blaNDM-1 and blaKPC-2 were simultaneously found in one ST107 strain. Epidemiological analysis showed that ST107 clone contained human- and environment-derived strains from five countries. Notably, 93.75% (15/16) of the isolates harboured blaNDM-1 or blaKPC-2. Plasmid fusion among various ST107 strains of two patients occurred in the same ICU, mediated by Tn5403 and IS26-based insertion and deletion events. pCF1807-2 carried blaNDM-1 while pCF1807-3 carried both tmexCD2-toprJ2 and blaKPC-2 in the CF1807 strain. Importantly, the cointegrate plasmid pCF1807-2 exhibited higher transfer efficiency and could remain stable after serial passage. Notably, no fitness cost was observed for the host. In conclusion, ST107 CRCF is a high-risk resistant clone due to its ability to integrate resistant plasmids. Our findings elucidated the potential threat and global transmission of the ST107 lineage, and reasonable monitoring should be performed to prevent its further spread in hospitals.

Development and evaluation of a colorimetric LAMP-based biosensor for rapid detection of a nosocomial infection agent, Citrobacter freundii.

Simple and fast diagnosis of Citrobacter freundii which is an important cause of nosocomial infection in human is crucial to achieve early treatment. We have developed and evaluated an optical LAMP-based biosensor for the visual detection of C. freundii for the first time. The efficiency of the assay was investigated and compared to PCR method. The selectivity and specificity of the biosensor were analyzed using Morganella morganii, Enterobacter aerogenes, Pseudomonas aeruginosa, Yersinia enterocolitica, Shigella sonnei, Serratia marcescens, Burkholderia cepacia and Klebsiella pneumoniae and a mixed-culture medium. Endpoint analysis using hydroxy naphthol blue was applied, and the color change to sky blue and no color change from violet indicated positive and negative results, respectively. The absorption at 650 nm was measured 0.39 for the positive sample, while the mean absorption of the test samples, including water, was 0.23. The specificity of the method was equal to that of PCR. However, the sensitivity was determined as 12.24 fg/µL of the genomic content of C. freundii, higher than PCR assay. The developed LAMP-based method provided a rapid and accurate technique for molecular diagnostics of C. freundii, making it a suitable technique for point-of-care diagnostics in cases of urgent situations.

Identification and characterization of a highly virulent Citrobacter freundii isolate and its activation on immune responses in largemouth bass (Micropterus salmoides).

Citrobacter freundii, a common pathogen of freshwater fish, causes significant commercial losses to the global fish farming industry. In the present study, a highly pathogenic C. freundii strain was isolated and identified from largemouth bass (Micropterus salmoides). The pathogenicity and antibiotic sensitivity of the C. freundii strain were evaluated, and the histopathology and host immune response of largemouth bass infected with C. freundii were investigated. The results showed that C. freundii was the pathogen causing disease outbreaks in largemouth bass, and the infected fish showed typical signs of acute hemorrhages and visceral enlargement. Antimicrobial susceptibility testing showed that the C. freundii strain was resistant to Kanamycin, Medimycin, Clindamycin, Penicillin, Oxacillin, Ampicillin, Cephalexin, Cefazolin, Cefradine and Vancomycin. Histopathological analysis showed different pathological changes in major tissues of diseased fish. In addition, humoral immune factors such as superoxide dismutase (SOD), catalase (CAT) and lysozyme (LZM) were used as serum indicators to evaluate the immune response of largemouth bass after infection. Quantitative real-time PCR (qRT-PCR) was performed to investigate the expression pattern of immune-related genes (CXCR1, IL-8, IRF7, IgM, CD40, IFN-γ, IL-1ß, Hep1, and Hep2) in liver, spleen, and head kidney tissues, which demonstrated a strong immune response induced by C. freundii infection in largemouth bass. The present study provides insights into the pathogenic mechanism of C. freundii and immune response in largemouth bass, promoting the prevention and treatment of diseases caused by C. freundii infection.

Complete-genome sequencing and comparative genomic characterization of blaNDM-5 carrying Citrobacter freundii isolates from a patient with multiple infections.

BACKGROUND: The emergence and wide spread of carbapenemase-producing Enterobacteriaceae (CPE) poses a growing threat to global public health. However, clinically derived carbapenemase-producing Citrobacter causing multiple infections has rarely been investigated. Here we first report the isolation and comparative genomics of two blaNDM-5 carrying Citrobacter freundii (C. freundii) isolates from a patient with bloodstream and urinary tract infections. RESULTS: Antimicrobial susceptibility testing showed that both blaNDM-5 carrying C. freundii isolates were multidrug-resistant. Positive modified carbapenem inactivation method (mCIM) and EDTA-carbapenem inactivation method (eCIM) results suggested metallo-carbapenemase production. PCR and sequencing confirmed that both metallo-carbapenemase producers were blaNDM-5 positive. Genotyping and comparative genomics analyses revealed that both isolates exhibited a high level of genetic similarity. Plasmid analysis confirmed that the blaNDM-5 resistance gene is located on IncX3 plasmid with a length of 46,161 bp, and could successfully be transferred to the recipient Escherichia coli EC600 strain. A conserved structure sequence (ISAba125-IS5-blaNDM-5-trpF-IS26-umuD-ISKox3) was found in the upstream and downstream of the blaNDM-5 gene. CONCLUSIONS: The data presented in this study showed that the conjugative blaNDM-5 plasmid possesses a certain ability to horizontal transfer. The dissemination of NDM-5-producing C. freundii isolates should be of close concern in future clinical surveillance. To our knowledge, this is the first study to characterize C. freundii strains carrying the blaNDM-5 gene from one single patient with multiple infections.

FDH/Hases-S-chain mediated electron redistributing in Citrobacter freundii JH@FeS during degradation of sulfamethoxazole and nitrate.

Considering the negligent degradation of sulfamethoxazole (SMX) by Citrobacter freundii JH, the incorporation of bio-FeS could initiate the SMX biodegradation to 0.0444 (S-FeS), and further to 0.0564 mg L-1 mg-1 protein d-1 (SN-FeS) when coexisted with nitrate. Electrochemical (LSV, I-t, DPV, EIS and EDC) and respiratory inhibition experiments clarified that the bio-FeS could greatly switch/redistribute electron transmembrane-transfer from intracellular to extracellular mainly via FDH/Hases-S-chain, as revealed by the significant increase of ipa-FDH/Hases/ipa-FC-Cyts and ipc-FDH/Hases/ipc-FC-Cyts (from 1.09 and 1.07 (SN-native) to 1.50 and 3.58 (SN-FeS)), while nitrate (linear fitting with NADH (R2 = 0.9903)) mainly intensified CoQ-L-chain related INET from Complex I to CoQ to compensate for the electronic competition with SMX. SN-FeS system detoxified the SMX on microbial metabolism (such as membrane rupture and oxidative stress induction) with high SOD activity (737.93 U gFW-1). Structural equation modeling indicated that bio-FeS up-regulated PMF-mediated ATP synthesis (PPMF-ATPs from 0.12 (SN-native) to 0.74 (SN-FeS)) and PMF-mediated NADH (PPMF-NADH from -0.72 (SN-native) to 0.63 (SN-FeS)), and the nitrate addition intensified this positive feedback. Overall, this study provides a new perspective for bionanoparticles via electron transfer/redistribution to detoxify and launch the antibiotics biodegradation in ecological environment.

Sanitary installations and wastewater plumbing as reservoir for the long-term circulation and transmission of carbapenemase producing Citrobacter freundii clones in a hospital setting.

BACKGROUND: Accumulating evidence shows a role of the hospital wastewater system in the spread of multidrug-resistant organisms, such as carbapenemase producing Enterobacterales (CPE). Several sequential outbreaks of CPE on the geriatric ward of the Ghent University hospital have led to an outbreak investigation. Focusing on OXA-48 producing Citrobacter freundii, the most prevalent species, we aimed to track clonal relatedness using whole genome sequencing (WGS). By exploring transmission routes we wanted to improve understanding and (re)introduce targeted preventive measures. METHODS: Environmental screening (toilet water, sink and shower drains) was performed between 2017 and 2021. A retrospective selection was made of 53 Citrobacter freundii screening isolates (30 patients and 23 environmental samples). DNA from frozen bacterial isolates was extracted and prepped for shotgun WGS. Core genome multilocus sequence typing was performed with an in-house developed scheme using 3,004 loci. RESULTS: The CPE positivity rate of environmental screening samples was 19.0% (73/385). Highest percentages were found in the shower drain samples (38.2%) and the toilet water samples (25.0%). Sink drain samples showed least CPE positivity (3.3%). The WGS data revealed long-term co-existence of three patient sample derived C. freundii clusters. The biggest cluster (ST22) connects 12 patients and 8 environmental isolates taken between 2018 and 2021 spread across the ward. In an overlapping period, another cluster (ST170) links eight patients and four toilet water isolates connected to the same room. The third C. freundii cluster (ST421) connects two patients hospitalised in the same room but over a period of one and a half year. Additional sampling in 2022 revealed clonal isolates linked to the two largest clusters (ST22, ST170) in the wastewater collection pipes connecting the rooms. CONCLUSIONS: Our findings suggest long-term circulation and transmission of carbapenemase producing C. freundii clones in hospital sanitary installations despite surveillance, daily cleaning and intermittent disinfection protocols. We propose a role for the wastewater drainage system in the spread within and between rooms and for the sanitary installations in the indirect transmission via bioaerosol plumes. To tackle this problem, a multidisciplinary approach is necessary including careful design and maintenance of the plumbing system.

Citrobacter freundii resistant to novel ß-lactamase inhibitor combinations and cefiderocol, co-producing class A, B and D carbapenemases encoded by transferable plasmids.

OBJECTIVES: To characterize a carbapenem-resistant Citrobacter freundii (Cf-Emp) co-producing class A, B and D carbapenemases, resistant to novel ß-lactamase inhibitor combinations (BLICs) and cefiderocol. METHODS: Carbapenemase production was tested by an immunochromatography assay. Antibiotic susceptibility testing (AST) was performed by broth microdilution. WGS was performed using short- and long-read sequencing. Transfer of carbapenemase-encoding plasmids was assessed by conjugation experiments. RESULTS: Cf-Emp was isolated on selective medium for carbapenem-resistant Enterobacterales from the surveillance rectal swab taken at hospital admission from a patient of Moroccan origin. Cf-Emp produced three different carbapenemases, including KPC-2, OXA-181 and VIM-1, and was resistant to all ß-lactams including carbapenems, novel BLICs (ceftazidime/avibactam, meropenem/vaborbactam and imipenem/relebactam) and cefiderocol. MIC of aztreonam/avibactam was 0.25 mg/L. The strain belonged to ST22, one of the C. freundii lineages of global diffusion, known to be associated with carbapenemase production. Each carbapenemase gene was located aboard a different plasmid (named pCf-KPC, pCf-OXA and pCf-VIM, respectively), which also carried other clinically relevant resistance genes, such as armA (pCf-KPC), blaSHV-12 (pCf-VIM) and qnrS1 (pCf-OXA). Transferability to Escherichia coli J53 by conjugation was observed for all plasmids. CONCLUSIONS: The finding of enterobacterial strains carrying multiple carbapenemase genes on transferable plasmids is alarming, because similar strains could provide an important reservoir for disseminating these clinically relevant resistance determinants.