APH(3')-Ie, an aminoglycoside-modifying enzyme discovered in a rabbit-derived Citrobacter gillenii isolate.

Background: Aminoglycoside-modifying enzymes (AMEs) play an essential role in bacterial resistance to aminoglycoside antimicrobials. With the development of sequencing techniques, more bacterial genomes have been sequenced, which has aided in the discovery of an increasing number of novel resistance mechanisms. Methods: The bacterial species was identified by 16S rRNA gene homology and average nucleotide identity (ANI) analyses. The minimum inhibitory concentration (MIC) of each antimicrobial was determined by the agar dilution method. The protein was expressed with the pCold I vector in E. coli BL21, and enzyme kinetic parameters were examined. The whole-genome sequence of the bacterium was obtained via the Illumina and PacBio sequencing platforms. Reconstruction of the phylogenetic tree, identification of conserved functional residues, and gene context analysis were performed using the corresponding bioinformatic techniques. Results: A novel aminoglycoside resistance gene, designated aph(3')-Ie, which confers resistance to ribostamycin, kanamycin, sisomicin and paromomycin, was identified in the chromosome of the animal bacterium Citrobacter gillenii DW61, which exhibited a multidrug resistance phenotype. APH(3')-Ie showed the highest amino acid identity of 74.90% with the functionally characterized enzyme APH(3')-Ia. Enzyme kinetics analysis demonstrated that it had phosphorylation activity toward four aminoglycoside substrates, exhibiting the highest affinity (K m, 4.22 ± 0.88 µM) and the highest catalytic efficiency [k cat/K m, (32.27 ± 8.14) × 104] for ribomycin. Similar to the other APH(3') proteins, APH(3')-Ie contained all the conserved functional sites of the APH family. The aph(3')-Ie homologous genes were present in C. gillenii isolates from different sources, including some of clinical significance. Conclusion: In this work, a novel chromosomal aminoglycoside resistance gene, designated aph(3')-Ie, conferring resistance to aminoglycoside antimicrobials, was identified in a rabbit isolate C. gillenii DW61. The elucidation of the novel resistance mechanism will aid in the effective treatment of infections caused by pathogens carrying such resistance genes.

Coexistence of a novel NDM-1-encoding MDR plasmid and an IMP-4-encoding IncN-IncU hybrid plasmid in a clinical isolate of Citrobacter freundii BC73.

Objectives: To investigate the genetic characteristics and transmission mechanism of the NDM-1-, IMP-4-, and SHV-12-producing multidrug-resistant (MDR) clinical isolate, Citrobacter freundii BC73. Methods: C. freundii BC73 was isolated from a urine specimen of a urological patient diagnosed with bladder cancer at a Chinese teaching hospital. Antimicrobial susceptibility testing was carried out using DL-120E susceptibility cards and DL-96A system. Whole genome sequencing (WGS) of the isolate was performed using the Illumina and Oxford Nanopore platforms to analyze the genetic context of drug resistance genes and plasmid characteristics. The phylogenetic tree was constructed and visualized by KSNP3.0 software and iTOL5.0 online database. Results: C. freundii isolate BC73 co-carrying bla NDM-1, bla IMP-4 and bla SHV-12 were multidrug-resistant. bla NDM-1 and bla IMP-4 were located on a novel IncFIB-like plasmid, pCFBC1, and an IncN-IncU hybrid plasmid, pCFBC2, respectively. The transferability of bla NDM-1 and bla IMP-4 from C. freundii BC73 to E. coli J53 was successfully demonstrated. The genetic context of the bla NDM-1 and bla IMP-4 genes were ISCR27-groEL-∆groES-cutA-dsbD-trpF-ble MBL-bla NDM-1-∆ISAba125-IS3000 and intI1-bla IMP-4-Kl.pn.13-mobC-IS6100, respectively. Additionally, two extensive transposition units (MGE1 in pCFBC1, MGE2 in pCFBC2) were identified and numerous antimicrobial resistance genes were discovered on it. Conclusion: To our knowledge, our study represents the first characterization of a ST22 C. freundii isolate co-harboring bla NDM-1, bla IMP-4, and bla SHV-12, obtained from a urine sample. The dissemination of this MDR isolate should be of close concern in future clinical surveillance.

Citrobacter spp. and Enterobacter spp. as reservoirs of carbapenemase blaNDM and blaKPC resistance genes in hospital wastewater.

Antibiotic resistance has emerged as a global threat to public health, generating a growing interest in investigating the presence of antibiotic-resistant bacteria in environments influenced by anthropogenic activities. Wastewater treatment plants in hospital serve as significant reservoirs of antimicrobial-resistant bacteria, where a favorable environment is established, promoting the proliferation and transfer of resistance genes among different bacterial species. In our study, we isolated a total of 243 strains from 5 hospital wastewater sites in Mexico, belonging to 21 distinct Gram-negative bacterial species. The presence of ß-lactamase was detected in 46.9% (114/243) of the isolates, which belonging to the Enterobacteriaceae family. We identified a total of 169 ß-lactamase genes; blaTEM in 33.1%, blaCTX-M in 25.4%, blaKPC in 25.4%, blaNDM 8.8%, blaSHV in 5.3%, and blaOXA-48 in 1.1% distributed in 12 different bacteria species. Among the 114 of the isolates, 50.8% were found to harbor at least one carbapenemase and were discharged into the environment. The carbapenemase blaKPC was found in six Citrobacter spp. and E. coli, while blaNDM was detected in two distinct Enterobacter spp. and E. coli. Notably, blaNDM-1 was identified in a 110 Kb IncFII conjugative plasmid in E. cloacae, E. xiangfangensis, and E. coli within the same hospital wastewater. In conclusion, hospital wastewater showed the presence of Enterobacteriaceae carrying a high frequency of carbapenemase blaKPC and blaNDM. We propose that hospital wastewater serves as reservoirs for resistance mechanism within bacterial communities and creates an optimal environment for the exchange of this resistance mechanism among different bacterial strains. IMPORTANCE: The significance of this study lies in its findings regarding the prevalence and diversity of antibiotic-resistant bacteria and genes identified in hospital wastewater in Mexico. The research underscores the urgent need for enhanced surveillance and prevention strategies to tackle the escalating challenge of antibiotic resistance, particularly evident through the elevated frequencies of carbapenemase genes such as blaKPC and blaNDM within the Enterobacteriaceae family. Moreover, the identification of these resistance genes on conjugative plasmids highlights the potential for widespread transmission via horizontal gene transfer. Understanding the mechanisms of antibiotic resistance in hospital wastewater is crucial for developing targeted interventions aimed at reducing transmission, thereby safeguarding public health and preserving the efficacy of antimicrobial therapies.

Substance P receptor signaling contributes to host maladaptive responses during enteric bacterial infection.

Immune responses in the intestine are intricately balanced to prevent pathogen entry without inducing immunopathology. The nervous system is well-established to interface with the immune system to fine-tune immunity in various organ systems including the gastrointestinal tract. Specialized sensory neurons can detect bacteria, bacterial products, and the resulting inflammation, to coordinate the immune response in the gastrointestinal tract. These sensory neurons release peptide neurotransmitters such as Substance P (SP), to induce both neuronal signaling and localized responses in non-neuronal cells. With this in mind, we assessed the immunoregulatory roles of SP receptor signaling during enteric bacterial infection with the non-invasive pathogen Citrobacter rodentium. Pharmacological antagonism of the SP receptor significantly reduced bacterial burden and prevented colonic crypt hyperplasia. Mice with SP receptor signaling blockade had significantly reduced inflammation and recruitment of T-cells in the colon. Reduced colonic T-cell recruitment is due to reduced expression of adhesion molecules on colonic endothelial cells in SP receptor antagonist-treated mice. Using SP receptor T-cell conditional knockout mice, we further confirmed SP receptor signaling enhanced select aspects of T-cell responses. Our data demonstrates that SP receptor signaling can significantly reduce inflammation and prevent host-maladaptive responses without impinging upon host protection.

Isolation and characterization of mercury and multidrug-resistant Citrobacter freundii strains from tannery effluents in Kolkata, India.

Mercury (Hg) is one of the most potent toxic heavy metals that distresses livestock, humans, and ecological health. Owing to uncontrolled exposure to untreated tannery industrial effluents, metals such as Hg are increasing in nature and are, therefore, becoming a global concern. As a result, understanding the thriving microflora in that severe condition and their characteristics becomes immensely important. During the course of this study, two Hg-resistant bacteria were isolated from tannery wastewater effluents from leather factories in Kolkata, India, which were able to tolerate 2.211 × 10- 3 M (600 µg/ml) Hg. 16 S rDNA analysis revealed strong sequence homology with Citrobacter freundii, were named as BNC22A and BNC22C for this study. In addition they showed high tolerance to nickel (Ni) and Chromium (Cr) at 6.31 × 10- 3 M (1500 µg/ml) and 6.792 × 10- 3 M (2000 µg/ml) respectively. However, both the isolates were sensitive to arsenic (As) and cadmium (Cd). Furthermore, their antibiotic sensitivity profiles reveal a concerning trend towards resistance to multiple drugs. Overuse and misuse of antibiotics in healthcare systems and agriculture has been identified as two of the main reasons for the decline in efficacy of antibiotics. Though their ability to produce lipase makes them industrially potent organisms, their competence to resist several antibiotics and metals that are toxic makes this study immensely relevant. In addition, their ability to negate heavy metal toxicity makes them potential candidates for bioremediation. Finally, the green mung bean seed germination test showed a significant favourable effect of BNC22A and BNC22C against Hg-stimulated toxicity.

The Unusual Suspect: Citrobacter Infection as a Rare Cause of Renal Abscess.

Citrobacter infections have emerged as now a common nosocomial pathogen. Most of the infections manifest in patients with underlying medical conditions. The features of infection can range from infections in the lower urinary tract, respiratory tract, gastrointestinal disease, or bacteremia, however renal abscesses remain uncommon. Here we present the case of a 48-year-old female with medical history of diabetes that presented with a right renal abscess secondary to Citrobacter koseri infection managed with drainage and antimicrobial treatment.

Epidemiology of Citrobacter spp. infections among hospitalized patients: a systematic review and meta-analysis.

BACKGROUND: Infections due to Citrobacter species are increasingly observed in hospitalized patients and are often multidrug-resistant. Yet, the magnitude and burden of Citrobacter spp. resistance in the hospital setting have not been reported. We aimed to evaluate the epidemiology of Citrobacter spp. infections among hospitalized patients, their main resistance patterns and Citrobacter spp. involvement in hospital outbreaks. METHODS: We conducted a systematic review and meta-analysis of published literature (PROSPERO registration Jan-2023, CRD42023390084). We searched Embase, Medline and grey literature for studies on hospitalized patients diagnosed with Citrobacter spp. infections, and nosocomial outbreaks due to Citrobacter spp. published during the years 2000-2022. We included observational, interventional, surveillance studies and outbreak reports. Outcomes of interest were the frequency of Citrobacter spp. infections among hospitalized patients and 3rd generation cephalosporin and/or carbapenem resistance percentages in these infections. We used random-effects models to generate pooled outcome estimates and evaluated risk of bias and quality of reporting of outbreaks. RESULTS: We screened 1609 deduplicated publications, assessed 148 full-texts, and included 41 studies (15 observational, 13 surveillance and 13 outbreak studies). Citrobacter spp. urinary tract- and bloodstream infections were most frequently reported, with Citrobacter freundii being the main causative species. Hospital-acquired infection occurred in 85% (838/990) of hospitalized patients with Citrobacter infection. After 2010, an increasing number of patients with Citrobacter spp. infections was reported in observational studies. Pooled frequency estimates for Citrobacter spp. infections could not be generated due to lack of data. The pooled prevalence of ESBL and carbapenemase producers among Citrobacter isolates were 22% (95%CI 4-50%, 7 studies) and 18% (95%CI 0-63%, 4 studies), respectively. An increased frequency of reported Citrobacter outbreaks was observed after 2016, with an infection/colonization ratio of 1:3 and a case-fatality ratio of 7% (6/89 patients). Common outbreak sources were sinks, toilets, contaminated food and injection material. Implemented preventive measures included environmental cleaning, isolation of positive patients and reinforcement of hand hygiene. Only seven out of 13 outbreaks (54%) were definitively controlled. CONCLUSION: This review highlights the clinical importance of endemic and epidemic Citrobacter spp. in healthcare settings. As an emerging, multidrug­resistant nosocomial pathogen it requires heightened awareness and further dedicated surveillance efforts.

Purulent Pericarditis in End-Stage Renal Disease: A Rare Case of Citrobacter freundii Infection.

BACKGROUND: Purulent pericarditis is a rare but life-threatening condition, particularly challenging when it occurs in immunocompromised individuals. CASE REPORT: We present the case of a 68-year-old man with end-stage renal disease who developed purulent pericarditis secondary to Citrobacter freundii infection. Despite initial challenges in diagnosis and management, the patient showed a favorable response to antibiotic therapy. CONCLUSIONS: This case highlights the importance of prompt recognition and treatment of purulent pericarditis, especially in patients with underlying immunosuppression and comorbidities.

Osteomyelitis Caused by Citrobacter koseri in a Young Immunocompetent Man.

Citrobacter koseri is a non-sporulating, motile, gram-negative, facultative anaerobic bacteria found in various environmental sources, including the human intestine. It is considered an opportunistic infection as it typically causes infection in newborns, elderly, and immunocompromised patients. Common sites of infection are the urinary tract, gastrointestinal system, and respiratory tract in immunocompromised adults, as well as the bloodstream and meninges in newborns. However, osteomyelitis secondary to C. koseri is very rare. We present an unusual case of Citrobacter osteomyelitis in a healthy young man with no identifiable risk factors.

Impaired neutrophil migration underpins host susceptibility to infectious colitis.

Citrobacter rodentium models infection with enteropathogenic Escherichia coli and ulcerative colitis (UC). While C57BL/6 (C57) mice recover, C3H/HeN (C3H) mice succumb to infection, partially due to increased colonic neutrophil elastase activity, also seen in UC patients; however, the underlying cause was unknown. Here, we found that bone marrow, blood, and colonic C57 neutrophils expressed (CD)11bHi and reached the infected colonic lumen, where they underwent productive NETosis. In contrast, while the number of C3H neutrophils increased in the bone marrow, blood, and colon, they remained CD11bLo and got trapped in the submucosa, away from C. rodentium, where they underwent harmful NETosis. CD11bLo neutrophils in C3H mice infected with CRi9, which triggers expression of neutrophil chemoattractants, reached the colonization site, resulting in host survival. UC patient neutrophils also displayed decreased levels of the activation/differentiation markers CD16/CXCR4. These results, suggesting that neutrophil malfunction contributes to exacerbated colitis, provide insight for future therapeutic prospects.

Emergence of an Extensive Drug Resistant Citrobacter portucalensis Clinical Strain Harboring bla SFO-1, bla KPC-2, and bla NDM-1.

Background: To explore the plasmid characteristics and transfer mechanisms of an extensive drug resistant (XDR) clinical isolate, Citrobacter portucalensis L2724hy, co-producing bla SFO-1, bla NDM-1, and bla KPC-2. Methods: Species confirmation of L2724hy was achieved through 16S rRNA sequencing and Average Nucleotide Identity (ANI) analysis. Antimicrobial susceptibility testing (AST) employed the agar dilution and micro broth dilution methods. Identification of resistance genes was carried out by PCR and whole-genome sequencing (WGS). Essential resistance gene locations were verified by S1 nuclease pulsed-field gel electrophoresis (S1-PFGE) and southern hybridization experiments. Subsequent WGS data analysis delved into drug resistance genes and plasmids. Results: The confirmation of the strain L2724hy as an extensive drug-resistant Citrobacter portucalensis, resistant to almost all antibiotics tested except polymyxin B and tigecycline, was achieved through 16S rRNA sequencing, ANI analysis and AST results. WGS and subsequent analysis revealed L2724hy carrying bla SFO-1, bla NDM-1, and bla KPC-2 on plasmids of various sizes. The uncommon ESBL gene bla SFO-1 coexists with the fosA3 gene on an IncFII plasmid, featuring the genetic environment IS26-fosA3-IS26-ampR-bla SFO-1-IS26. The bla NDM-1 was found on an IncX3 plasmid, coexisting with bla SHV-12, displaying the sequence IS5-IS3000-IS3000-Tn2-bla NDM-1-ble-trpF-dsbD-cutA-gros-groL, lacking ISAa125. The bla KPC-2 is located on an unclassified plasmid, exhibiting the sequence Tn2-tnpR-ISKpn27-bla KPC-2-ISKpn6-korC. Conjugation assays confirmed the transferability of both bla NDM-1 and bla KPC-2. Conclusion: We discovered the coexistence of bla SFO-1, bla NDM-1, and bla KPC-2 in C. portucalensis for the first time, delving into plasmid characteristics and transfer mechanisms. Our finding highlights the importance of vigilant monitoring of drug-resistance genes and insertion elements in uncommon strains.

Citrobacter rodentium infection impairs dopamine metabolism and exacerbates the pathology of Parkinson's disease in mice.

Parkinson's disease (PD) is a prevalent neurodegenerative disorder with indistinct etiology and ill-defined pathophysiology. Intestinal inflammation involved in the pathogenesis of PD, but the underlying mechanism is not fully understood. Citrobacter rodentium (C.R) is a gram-negative bacterium that can be used to induce human inflammatory bowel disease in mice. Here, we investigated whether the proinflammatory effects caused by C.R infection initiate PD-like injury and/or exacerbate PD pathology and extensively studied the underlying mechanism. Mice were gavaged once with C.R and monitored for several pathological features at 9 days post infection. The results showed that C.R delivery in mice induced IBD-like symptoms, including significant weight loss, increased fecal water content, an impaired intestinal barrier, intestinal hyperpermeability and inflammation, and intestinal microbiota disturbances. Notably, C.R infection modified dopamine (DA) metabolism in the brains of both male and female mice. Subsequently, a single high dose of MPTP or normal saline was administered at 6 days post infection. At 3 days after MPTP administration, the feces were collected for 16 S rRNA analysis, and PD-like phenotypes and mechanisms were systemically analyzed. Compared with C.R or MPTP injection alone, the injection of C.R and MPTP combined worsened behavioral performance. Moreover, such combination triggered more severe dopaminergic degeneration and glial cell overactivation in the nigrostriatal pathway of mice. Mechanistically, the combination of C.R and MPTP increased the expression of TLR4 and NF-κB p65 in the colon and striatum and upregulated proinflammatory cytokine expression. Therefore, C.R infection-induced intestinal inflammation can impair dopamine metabolism and exacerbate PD pathological processes.

Evaluation of the protective effect of the intranasal vaccines adjuvanted with bacterium-like particles against intestinal infection.

Mucosal vaccination presents a promising complement to parenteral vaccination. Bacterium-like particles (BLPs), peptidoglycan structures prepared from lactic acid bacteria, are explored as potential nasal vaccine adjuvants for respiratory infections. To date, studies on BLP-adjuvanted nasal vaccines against intestinal infections have remained limited. In this study, we demonstrated the efficacy of intranasal BLP-adjuvanted vaccination in controlling intestinal infections using the Citrobacter rodentium (C. rodentium) model in C57BL/6 mice. Intranasal vaccination of Intimin, an adhesin critical for intimate bacterial adhesion to colonic epithelial cells, combined with BLP (BLP+I) elicited robust Intimin-specific intestinal secretory IgA production, reduced bacterial load in feces and almost completely inhibited colonic hyperplasia, a characteristic symptom of C. rodentium infection in mice. Conversely, parenteral vaccination with Alhydrogel-adjuvanted Intimin failed to induce intestinal Intimin-specific IgA production, resulting in poor protection against C. rodentium infection. This underscores the pivotal role of mucosal IgA responses elicited by intranasal immunization in its protective efficacy. As this study did not delineate the precise protective mechanism conferred by BLP+I intranasal immunization against C. rodentium infection, further elucidation of the mechanisms underlying intranasal BLP+I immunization is required.

Genomic Characterization of Carbapenemase-Producing Enterobacter hormaechei, Serratia marcescens, Citrobacter freundii, Providencia stuartii, and Morganella morganii Clinical Isolates from Bulgaria.

Carbapenemase-producing Enterobacter spp. Serratia marcescens, Citrobacter freundii, Providencia spp., and Morganella morganii (CP-ESCPM) are increasingly identified as causative agents of nosocomial infections but are still not under systematic genomic surveillance. In this study, using a combination of whole-genome sequencing and conjugation experiments, we sought to elucidate the genomic characteristics and transferability of resistance genes in clinical CP-ESCPM isolates from Bulgaria. Among the 36 sequenced isolates, NDM-1 (12/36), VIM-4 (11/36), VIM-86 (8/36), and OXA-48 (7/36) carbapenemases were identified; two isolates carried both NDM-1 and VIM-86. The majority of carbapenemase genes were found on self-conjugative plasmids. IncL plasmids were responsible for the spread of OXA-48 among E. hormaechei, C. freundii, and S. marcescens. IncM2 plasmids were generally associated with the spread of NDM-1 in C. freundii and S. marcescens, and also of VIM-4 in C. freundii. IncC plasmids were involved in the spread of the recently described VIM-86 in P. stuartii isolates. IncC plasmids carrying blaNDM-1 and blaVIM-86 were observed too. blaNDM-1 was also detected on IncX3 in S. marcescens and on IncT plasmid in M. morganii. The significant resistance transfer rates we observed highlight the role of the ESCPM group as a reservoir of resistance determinants and stress the need for strengthening infection control measures.

Further Evaluation of Enterohemorrhagic Escherichia coli Gold Nanoparticle Vaccines Utilizing Citrobacter rodentium as the Model Organism.

Enterohemorrhagic E. coli (EHEC) is a group of pathogenic bacteria that is associated with worldwide human foodborne diarrheal illnesses and the development of hemolytic uremic syndrome, a potentially deadly condition associated with Shiga toxins (Stxs). Currently, approved vaccines for human prophylaxis against infection do not exist, and one barrier preventing the successful creation of EHEC vaccines is the absence of dependable animal models, including mice, which are naturally resistant to EHEC infection and do not manifest the characteristic signs of the illness. Our lab previously developed gold nanoparticle (AuNP)-based EHEC vaccines, and assessed their efficacy using Citrobacter rodentium, which is the mouse pathogen counterpart of EHEC, along with an Stx2d-producing strain that leads to more consistent disease kinetics in mice, including lethality. The purpose of this study was to continue evaluating these vaccines to increase protection. Here, we demonstrated that subcutaneous immunization of mice with AuNPs linked to the EHEC antigens EscC and intimin (Eae), either alone or simultaneously, elicits functional robust systemic humoral responses. Additionally, vaccination with both antigens together showed some efficacy against Stx2d-producing C. rodentium while AuNP-EscC successfully limited infection with non-Stx2d-producing C. rodentium. Overall, the collected results indicate that our AuNP vaccines have promising potential for preventing disease with EHEC, and that evaluation of novel vaccines using an appropriate animal model, like C. rodentium described here, could be the key to finally developing an effective EHEC vaccine that can progress into human clinical trials.

Diet-driven differential response of Akkermansia muciniphila modulates pathogen susceptibility.

The erosion of the colonic mucus layer by a dietary fiber-deprived gut microbiota results in heightened susceptibility to an attaching and effacing pathogen, Citrobacter rodentium. Nevertheless, the questions of whether and how specific mucolytic bacteria aid in the increased pathogen susceptibility remain unexplored. Here, we leverage a functionally characterized, 14-member synthetic human microbiota in gnotobiotic mice to deduce which bacteria and functions are responsible for the pathogen susceptibility. Using strain dropouts of mucolytic bacteria from the community, we show that Akkermansia muciniphila renders the host more vulnerable to the mucosal pathogen during fiber deprivation. However, the presence of A. muciniphila reduces pathogen load on a fiber-sufficient diet, highlighting the context-dependent beneficial effects of this mucin specialist. The enhanced pathogen susceptibility is not owing to altered host immune or pathogen responses, but is driven by a combination of increased mucus penetrability and altered activities of A. muciniphila and other community members. Our study provides novel insights into the mechanisms of how discrete functional responses of the same mucolytic bacterium either resist or enhance enteric pathogen susceptibility.

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.

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.

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 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.