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The emergence of multidrug-resistant Citrobacter freundii poses a significant threat to public health. C. freundii isolates were collected from clinical patients in a Chinese hospital during 2020-2022. An unusual strain, GMU8049, was not susceptible to any of the antibiotics tested, including the novel β-lactam/β-lactamase inhibitor combination ceftazidime-avibactam. Whole-genome sequencing (WGS) revealed that GMU8049 harbors a circular chromosome belonging to the rare ST257 and an IncX3 resistance plasmid. Genomic analysis revealed the coexistence of two β-lactamase genes, including plasmid-mediated bla NDM-1 and chromosomal bla CMY encoding a novel CMY variant, combined with an outer membrane porin deficiency, which may account for the extreme resistance to β-lactams. Conjugation experiment confirmed that the bla NDM-1 resistance gene located on pGMU8049 could be successfully transferred to Escherichia coli EC600. The novel CMY variant had an amino acid substitution at position 106 (N106S) compared to the closely related CMY-51. Additionally, a GMU8049-specific truncation in an OmpK37 variant that produces a premature stop codon. Moreover, a variety of chromosome-located efflux pump coding genes and virulence-related genes were also identified. Analysis of strain GMU8049 in the context of other C. freundii strains reveals an open pan-genome and the presence of mobile genetic elements that can mediate horizontal gene transfer of antimicrobial resistance and virulence genes. Our work provides comprehensive insights into the genetic mechanisms of highly resistant C. freundii , highlighting the importance of genomic surveillance of this opportunistic pathogen as a high-risk population for emerging resistance and pathogenicity.IMPORTANCEEmerging pathogens exhibiting multi-, extremely, and pan-drug resistance are a major concern for hospitalized patients and the healthcare community due to limited antimicrobial treatment options and the potential for spread. Genomic technologies have enabled clinical surveillance of emerging pathogens and modeling of the evolution and transmission of antimicrobial resistance and virulence. Here, we report the genomic characterization of an extremely drug-resistant ST257 Citrobacter freundii clinical isolate. Genomic analysis of GMU8049 with a rare ST type and unusual phenotypes can provide information on how this extremely resistant clinical isolate has evolved, including the acquisition of bla NDM-1 via the IncX3 plasmid and accumulation through chromosomal mutations leading to a novel CMY variant and deficiency of the outer membrane porin OmpK37. Our work highlights that the emergence of extremely resistant C. freundii poses a significant challenge to the treatment of clinical infections. Therefore, great efforts must be made to specifically monitor this opportunistic pathogen., Competing Interests: The authors declare no conflict of interest.

Objective: To investigate the distribution of carbapenem-resistant Enterobacterales (CRE) in the community and to describe the genomic characteristics., Methods: CRE screened from fecal samples in healthy people at the health examination center of a tertiary hospital in China underwent Whole genome sequencing (WGS) to analyze genotypic characteristics of CRE. The flanking DNA sequence of blaNDM-5 and mcr1.1 genes were analyzed by Gcluster software., Results: A total of 7187 fecal samples were screened, and CRE carriage was detected in 0.4 % of the sampled population. In total, 30 Escherichia coli, one Citrobacter freundii and one Klebsiella aerogene were screened. The 30 carbapenem-resistant Escherichia coli (CREC) isolates displayed slight resistance to amikacin (13.3 %) and aztreonam (20.0 %). All the CRE isolates contained blaNDM, and blaNDM-5 (84.4 %) was the most common one. B1 (n = 11) and A (n = 7) were predominant phylogroups. Furthermore, 34 distinct plasmid replicons, 67 different VFs, 22 distinct STs, 17 different FimH types, 26 O:H serotypes as well as 74 MGEs including 61 insertion sequences and 13 transposons were identified. The flanking DNA sequence analysis of blaNDM-5 and mcr1.1 genes indicates the key role of horizontal transfer of blaNDM-5 in the CRE development evidenced by diverse STs and phylogenetic tree., Conclusion: E. coli was the most predominant CRE isolates in community setting, and blaNDM (blaNDM-5) was the main CHβL encoding genes. The high prevalence of ARGs was associated with high resistance to commonly used antimicrobials. Besides, the genetic diversity of these isolates suggested the key role of blaNDM horizontal transfer in the CRE development. Thus, active screening of blaNDM in communities is particularly important for the prevention and control of CRE., Competing Interests: Declaration of competing interest The authors declare that they have no competing interest., (Copyright © 2024. Published by Elsevier Ltd.)

Factors involved in the susceptibility of third-generation cephalosporins (3GCs) to bacteremia caused by Citrobacter freundii complex, Enterobacter cloacae complex, and Klebsiella aerogenes were investigated based on a case-case-control design. Antimicrobial therapy administered 30 days prior to bacteremia and hospitalization within 90 days were common risk factors for the 3GC susceptible and 3GC non-susceptible groups, while hospitalization from an institution or another hospital was a specific risk factor for the 3GC non-susceptible group. We also attempted to examine the factors affecting the clinical outcome of bacteremia. Hospitalization more than 14 days before the onset of bacteremia was an independent factor indicating poor clinical outcome. In contrast, the implementation of source control was an independent predictor of successful treatment. Although a longer hospital stay before the onset of bacteremia was associated with worse clinical outcomes, implementation of source control may have contributed to improved treatment outcomes for bacteremia.

Introduction: Multi-carbapenemase-producing Enterobacterales (M-CPE) are increasingly described. We characterized the M-CPE isolates prospectively recovered in our hospital (Madrid, Spain) over two years (2021-2022)., Methods: We collected 796 carbapenem resistant Enterobacterales (CRE) from clinical and surveillance samples. Carbapenemase production was confirmed with phenotypic (immunochromatographic, disk diffusion) and molecular (PCR, WGS) techniques. Antimicrobial susceptibility was evaluated by a standard broth microdilution method. Clinical and demographic data were collected., Results: Overall, 23 M-CPE (10 Klebsiella pneumoniae, 6 Citrobacter freundii complex, 3 Escherichia coli, 2 Klebsiella oxytoca, and 2 Enterobacter hormaechei) isolates were recovered from 17 patients (3% with CPE, 0.26-0.28 cases per 1000 admissions). OXA-48 + KPC-3 (7/23) and KPC-3 + VIM-1 (5/23) were the most frequent carbapenemase combinations. All patients had prior antibiotics exposure, including carbapenems (8/17). High resistance rates to ceftazidime/avibactam (14/23), imipenem/relebactam (16/23) and meropenem/vaborbactam (7/23) were found. Ceftazidime/avibactam + aztreonam combination was synergistic in all metallo-β-lactamase producers. Clonal and non-clonal related isolates were found, particularly in K. pneumoniae (5 ST29, 3 ST147, 3 ST307) and C. freundii (3 ST8, 2 ST125, 1 ST563). NDM-1 + OXA-48 was introduced with the ST147-K. pneumoniae high-risk clone linked to the transfer of a Ukrainian patient. We identified four possible nosocomial clonal transmission events between patients of the same clone with the same combination of carbapenemases (KPC-3 + VIM-1-ST29-K. pneumoniae, NDM-1 + OXA-48-ST147-K. pneumoniae and KPC-2 + VIM-1-ST145-K. oxytoca). Carbapenemase-encoding genes were located on different plasmids, except for VIM-1 + KPC-2-ST145-K. oxytoca. Cross-species transmission and a possible acquisition overtime was found, particularly between K. pneumoniae and E. coli producing OXA-48 + KPC-3., Conclusion: M-CPE is an emerging threat in our hospital. Co-production of different carbapenemases, including metallo-β-lactamases, limits therapeutic options and depicts the need to reinforce infection control measures., Competing Interests: Declaration of competing interest None., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Fosfomycin (FOS) is an effective antibiotic against multidrug-resistant Enterobacterales , but its effectiveness is reducing. Little is known on the current prevalence of FosA enzymes in low-risk pathogens, such as Citrobacter freundii . The aim of the study was the molecular characterization of a carbapenemase- and FosA-producing C. freundii collected in Italy. AK867, collected in 2023, showed an XDR profile, retaining susceptibility only to colistin. AK867 showed a FOS MIC >128 mg/L by ADM. Based on WGS, AK867 belonged to ST116 and owned a wide resistome, including fosA3 , bla KPC-2, and bla VIM-1. fosA3 was carried by a conjugative pKPC-CAV1312 plasmid of 320,480 bp, on a novel composite transposon (12,907 bp). FosA3 transposon shared similarities with other fosA3 -harboring pKPC-CAV1312 plasmids among Citrobacter spp. We report the first case of FosA3 production in clinical carbapenemase-producing C. freundii ST116. The incidence of FosA3 enzymes is increasing among Enterobacterales , affecting even low-virulence pathogens, as C. freundii ., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Mattioni Marchetti, Venturelli, Cassetti, Meschiari, Migliavacca and Bitar.)

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., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

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., Competing Interests: Declaration of competing interest The authors declare no competing interest., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Objectives: KHM-1-metallo-β-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 bla KHM-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 bla KHM-1 on their chromosomes and plasmids. In all four isolates, bla KHM-1 was located on 137 kbp to 213 kbp plasmids of IncC replicon type. Although there were common resistance genes such as bla KHM-1 -ISEc68, class I integron cassette, and fosG, the four bla KHM-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 bla KHM-1 -encoding IncC plasmids might have spread horizontally to multiple bacterial species through repeated recombination and insertion., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)