Citrobacter freundii is a Gram-negative, facultative anaerobic, rod-shaped bacterium belonging to the family Enterobacteriaceae, typically measuring 1-5 μm in length and exhibiting motility via peritrichous flagella.[1] It utilizes citrate as a primary carbon source and ferments glucose and other carbohydrates, producing acid and gas.[2] First described in 1958, it represents one of 19 genomospecies within the Citrobacter genus and is phylogenetically related to genera such as Salmonella and Escherichia.[2]This bacterium is ubiquitous in the environment, commonly isolated from soil, sewage, water, food, and sludge, as well as serving as a commensal in the intestinal tracts of humans and animals.[2][1] Although generally harmless in healthy individuals, C. freundii can transition to an opportunistic pathogen, particularly in nosocomial settings.[3]Clinically, C. freundii is associated with a range of infections, including urinary tract infections, bacteremia, pneumonia, and sepsis, with higher incidence in immunocompromised patients, neonates, and those with underlying conditions like diabetes or cancer.[2][3] It is a notable cause of neonatal meningitis and gastroenteritis, often linked to foodborne transmission or healthcare-associated outbreaks.[3] Virulence factors such as Shiga-like toxins, heat-stable toxins, and a type VI secretionsystem contribute to its pathogenicity, enabling adhesion, cytotoxicity, and competition with host microbiota.[3]A major concern with C. freundii is its propensity for multidrug resistance, frequently harboring genes like ampC for beta-lactam resistance and emerging mechanisms against carbapenems and colistin (e.g., mcr-1). As of 2025, recent genomic studies have identified the global spread of multidrug-resistant clones, such as ST22, carrying carbapenemase genes like bla_{NDM}.[2][3][4] Studies report resistance rates exceeding 30% to multiple antibiotic classes (as of 2023), complicating treatment and contributing to high case-fatality rates, up to 30% in severe cases.[2] Its genetic diversity, with numerous sequence types identified globally, underscores its adaptability and potential as a public health threat.[3]
Taxonomy and Etymology
Discovery and Nomenclature
Citrobacter freundii was originally described in 1928 by C. Braak as "Bacterium freundii," based on bacterial strains that demonstrated the ability to ferment glycerol anaerobically to trimethylene glycol.[5] In 1932, C. H. Werkman and A. L. Gillen proposed the genusCitrobacter to accommodate Gram-negative, rod-shaped bacteria capable of utilizing citrate as a sole carbon source, which were biochemically intermediate between Escherichia and Aerobacter (now Klebsiella); they transferred "Bacterium freundii" to this new genus as the type species, with cultures isolated from soil extracts during studies on fermentation pathways.[6]The etymology of the genus name "Citrobacter" combines "citro-" (from citrate, reflecting its utilization) with "bacter" (Latin for rod or staff, denoting its morphology). The species epithet "freundii" honors the Czech bacteriologist August Freund, who in 1881 first reported the microbial conversion of glycerol to trimethylene glycol.[5]Initially grouped with Escherichia coli due to shared coliform characteristics, C. freundii was reclassified into the distinct Citrobacter genus based on differential biochemical traits, including positive citrate utilization, variable lactose fermentation, and production of 2,3-butylene glycol, as detailed in early taxonomic studies by C. A. Stuart and colleagues. The reference type strain is ATCC 8090, derived from an original soil isolate and maintained as the neotype for the species within the Enterobacteriaceae family.[7][5]
Phylogenetic Classification
Citrobacter freundii belongs to the domain Bacteria, phylum Pseudomonadota, class Gammaproteobacteria, order Enterobacterales, family Enterobacteriaceae, genus Citrobacter, and speciesC. freundii. This taxonomic placement reflects its position within the core group of enteric bacteria, characterized by Gram-negative rods that are facultative anaerobes. The species was originally described in the 1930s as part of early studies on citrate-utilizing bacteria isolated from soil and water.[8][9]Within the Enterobacteriaceae family, C. freundii is closely related to other Citrobacter species such as C. koseri, as well as Escherichia coli and Salmonella enterica. These relationships are supported by phylogenomic analyses showing shared evolutionary histories, particularly with Salmonella lineages, and frequent misidentifications due to phenotypic similarities. C. freundii is distinguished from these relatives by its ability to utilize citrate as a sole carbon source and its lack of lysine decarboxylase activity, traits that aid in biochemical differentiation.[10][11][12][13]Phylogenetic delineation of C. freundii is further reinforced by 16S rRNA gene sequence analysis, which reveals 98-99% similarity to other Citrobacterspecies, confirming its species status while highlighting intra-genus clustering. Although no formal subspecies are recognized, the species exhibits biochemical variability, including strains that are ornithine decarboxylation positive and negative, as identified in numerical taxonomy studies.[14][15]
Morphology and Physiology
Cellular Structure
Citrobacter freundii is a Gram-negative bacterium characterized by its rod-shaped bacilli morphology.[2] The cells typically measure 1.0 μm in width and 2.0–6.0 μm in length, appearing singly or in pairs under microscopic examination.[16] This bacillus structure is consistent with other members of the Enterobacteriaceae family.[9]The bacterium exhibits motility through peritrichous flagella distributed around the cell surface, which facilitate swimming in liquid environments.[9] These flagella enable active movement, contributing to the organism's ability to navigate aqueous habitats.[17]The cell wall of C. freundii features a typical Gram-negative envelope, including an inner cytoplasmic membrane, a thin peptidoglycan layer, and an outer membrane embedded with lipopolysaccharide (LPS).[2] The LPS, a key component of the outer membrane, consists of lipid A, core polysaccharide, and O-antigen chains, providing structural integrity and contributing to endotoxin properties.[18]Some strains of C. freundii possess a polysaccharide capsule, which forms an additional extracellular layer surrounding the cell.[19] This capsular material varies among isolates and can influence surface interactions, though it is not universally present.[20]
Growth and Cultural Characteristics
Citrobacter freundii is a facultative anaerobe capable of growth under aerobic, microaerophilic, or anaerobic conditions, with optimal proliferation observed at 37°C and a pH range of 6.5 to 7.5.[21][22] This bacterium exhibits mesophilic characteristics, with a broader temperature tolerance spanning 10°C to 45°C, allowing slow growth at lower temperatures relevant to refrigerated food storage.[23] Certain strains demonstrate psychrotrophic potential, enabling persistence and potential spoilage in contaminated chilled foods such as dairy products.[24]On selective media like MacConkey agar, C. freundii typically forms lactose-fermenting colonies that appear pink, round, and moist, measuring 2-3 mm in diameter after 24-48 hours of incubation at 37°C.[1][25] While most strains produce these characteristic pink colonies due to acid production from lactosefermentation, some variants may exhibit non-lactose-fermenting growth, resulting in colorless or pale colonies.[26]Regarding oxygen-related metabolism, C. freundii reduces nitrate to nitrite under anaerobic conditions but does not further reduce it to gaseous nitrogen products, distinguishing it from true denitrifying bacteria.[13] This nitrate reduction capability supports its survival in oxygen-limited environments, such as sediments or biofilms.[27]
Genomics and Evolution
Genome Organization
The genome of Citrobacter freundii is organized as a single circular chromosome with a size typically ranging from 5.0 to 5.4 Mb across strains.[28] For example, the type strain ATCC 8090 has a chromosome of 4,957,773 bp, while strain CFNIH1 measures 5,099,034 bp.[28][29] The G+C content is consistently around 52 mol%, aligning with the genomic signature of the Enterobacteriaceae family.[28]Annotation of sequenced genomes reveals approximately 4,600 to 5,300 protein-coding genes (CDSs), depending on the strain, alongside 70-80 RNA genes including rRNAs and tRNAs.[28][30] A notable feature is the presence of a significant number of hypothetical proteins, often comprising 20-40% of the predicted CDSs, reflecting gaps in functional annotation common to bacterial genomes in this family.[31] One of the earliest complete genome sequences, that of environmental isolate CFNIH1 (deposited in 2014 but isolated in 2012), spans 5.1 Mb and includes CRISPR-Cas arrays that provide adaptive immunity against bacteriophages.[29]In addition to the chromosome, C. freundiistrains frequently harbor extrachromosomal plasmids, typically 50-100 kb in length, which contribute to genetic plasticity.[32][33] These plasmids, such as those of the IncF incompatibility group, often carry genes conferring antibiotic resistance (e.g., carbapenemases like blaIMP-4) or virulence traits, facilitating horizontal gene transfer within bacterial communities.[34][32] For instance, strain B38 contains a ~127 kb IncFII plasmid with resistance determinants.[32]
Evolutionary History
Citrobacter freundii belongs to the family Enterobacteriaceae, which diversified from an ancestral lineage approximately 300-500 million years ago.[35] Within this family, the genus Citrobacter separated from closely related genera such as Escherichia and Salmonella around 100-140 million years ago, reflecting a deep phylogenetic split that underscores its evolutionary independence while maintaining shared core traits typical of gut-associated microbes.[36] This divergence likely occurred in environmental niches involving soil, water, and animal hosts, allowing C. freundii to adapt metabolic versatility for survival in diverse ecosystems.Horizontal gene transfer (HGT) has played a pivotal role in shaping the pathogenic capabilities of C. freundii, particularly through the acquisition of pathogenicity islands from related Enterobacteriaceae like Escherichia coli and Salmonella species. For instance, integrative conjugative elements similar to those in Klebsiella pneumoniae have mediated the transfer of high-pathogenicity islands originally from Yersinia species into C. freundii strains, enhancing their ability to cause extraintestinal infections.[37] Such HGT events highlight how C. freundii has evolved by borrowing genetic toolkits from co-occurring bacteria, promoting its transition from commensal to opportunistic pathogen.Genome plasticity in C. freundii is evident from structural variations including inversions, insertions, and rearrangements driven by transposons and insertion sequences, which contribute to niche adaptation and phenotypic diversity. Transposon-mediated insertions, such as those involving IS26 and IS5 elements, have been observed to alter plasmid and chromosomal architecture, enabling rapid responses to selective pressures like antimicrobial exposure.[38] These mechanisms of genomic rearrangement, combined with the bacterium's typical genome size of around 4.9-5.1 Mb, facilitate evolutionary flexibility without fundamentally altering core housekeeping genes.[28]In the 2020s, genomic and epidemiological studies have revealed clonal expansions of C. freundii lineages in hospital settings, driven by the evolution of multidrug resistance through accumulated mutations and HGT of resistance cassettes. As of 2025, phylogenetic analyses of over 700 global carbapenemase-producing C. freundii genomes have identified sequence type 22 (ST22) as the predominant multidrug-resistant clone, comprising about 31% of isolates.[4] Analyses of global isolates indicate that certain clones, often associated with carbapenemase genes like NDM-1, have proliferated via plasmidcointegration and vertical transmission, underscoring ongoing adaptive evolution in clinical environments.[39]
Metabolism and Biochemistry
Nutritional Requirements
Citrobacter freundii utilizes a range of carbon sources to support its growth, including citrate, glucose, galactose, and glycerol. The capacity to employ citrate as a sole carbon source is a key feature that differentiates the genus Citrobacter from related Enterobacteriaceae.[2][40][41]For nitrogen acquisition, C. freundii assimilates ammonia or amino acids as primary sources. The bacterium lacks urease activity but exhibits ornithine decarboxylase in certain strains, facilitating nitrogenmetabolism.[42][15]Essential minerals for C. freundii include magnesium and iron, which are critical for enzymatic functions. Magnesium supports metabolic processes, often in conjunction with other trace elements like cobalt, while iron is acquired via siderophore production to chelate the metal under limiting conditions.[42][43]Unlike some related bacteria that depend on external supplies, C. freundii is independent of exogenous B vitamins, as it synthesizes vitamin B12 internally.[44]C. freundii exhibits facultative anaerobic growth, enabling adaptation to diverse environmental oxygen levels.[45]
Key Metabolic Pathways
Citrobacter freundii performs mixed acid fermentation under anaerobic conditions, converting glucose into a mixture of organic acids and alcohols, including ethanol, lactate, and acetate, while also producing gas (CO₂ and H₂). This pathway allows the bacterium to generate energy without external electron acceptors, with typical product yields showing acetate at approximately 18% of glucose carbon, ethanol at 17%, lactate at 10%, and succinate at 13%, alongside formate and pyruvate.[46] The presence of nitrate shifts the fermentation profile, reducing ethanol and lactate production while increasing acetate, reflecting adaptive regulation of fermentative enzymes.[46]A hallmark of C. freundiimetabolism is its ability to utilize citrate as a sole carbon source via the citrate lyase pathway, which cleaves citrate into oxaloacetate and acetyl-CoA under aerobic conditions. This process is facilitated by a gene cluster encoding the citrate lyase subunits in Enterobacteriaceae, enabling efficient breakdown and integration into central carbon metabolism.[47] The regulation of this pathway involves repressors that ensure expression in response to citrate availability.[47]C. freundii produces hydrogen sulfide (H₂S) from cysteine through the action of cysteine desulfhydrase, an enzyme that decomposes L-cysteine into pyruvate, ammonia, and H₂S. This reaction is pyridoxal phosphate-dependent and results in a positive H₂S indicator in triple sugar iron agar, distinguishing it from related species like Escherichia coli. The enzyme's presence supports sulfur assimilation and is conserved across Enterobacteriaceae.As a facultative anaerobe, C. freundii employs a respiratory chain involving quinone and cytochrome b for electron transfer.[48] Under anaerobic conditions, it can utilize alternative electron acceptors such as nitrate. Nitrate reductase activity is oxygen-sensitive and supports energy conservation when oxygen is limited.
Ecology and Distribution
Natural Habitats
Citrobacter freundii is a ubiquitous bacterium commonly found in various environmental reservoirs, including soil, freshwater bodies, and sewage systems. It has been isolated from soil samples across diverse ecosystems, where it contributes to the natural microbial community.[2] In aquatic environments, particularly freshwater sources such as rivers and lakes, C. freundii persists as part of the coliform population, often indicating fecal contamination.[45]Sewage represents a major reservoir, with C. freundii comprising a notable proportion of coliform bacteria in untreated wastewater, where it demonstrates resilience against disinfection processes like chlorination due to its association with protective protozoa or inherent resistance traits.[49] Additionally, the bacterium is frequently detected in food sources contaminated by environmental or fecal matter, such as vegetables like basil and sprouts, as well as animal feces from various species.[50] Although less commonly reported, isolations from fruits and vegetables underscore its presence in agricultural products exposed to soil or water.[51]As a commensal organism, C. freundii inhabits the gastrointestinal tract of humans and animals at low levels. In humans, it colonizes the intestines asymptomatically in a small percentage of individuals, serving as part of the normal gut flora without causing disease in most cases.[52] Similar low-level colonization occurs in animals, including birds and reptiles, where it is detected in fecal samples. This commensal association facilitates its transmission through fecal-oral routes, linking environmental and host reservoirs.The global distribution of C. freundii is cosmopolitan, with isolations reported across continents, including Europe, North America, and Southeast Asia.[2] Its motility, enabled by peritrichous flagella, aids in dispersal within these habitats, enhancing its adaptability to varied conditions.[53]
Interactions in Ecosystems
Citrobacter freundii plays a symbiotic role in soil microbiomes by facilitating nitrogen cycling through anaerobicnitratereduction, often coupled with iron oxidation, which aids in nutrient transformation and potential remediation of contaminated environments. Strains such as PXL1 demonstrate the ability to reduce nitrate while oxidizing Fe(II), contributing to denitrification processes that mitigate nitrate accumulation in groundwater and agricultural soils. This activity supports broader microbial consortia in maintaining soil fertility by converting nitrates to less mobile forms, enhancing nitrogen availability for plants in anaerobic conditions.[54][27]In competitive interactions, C. freundii produces bacteriocins that inhibit other Enterobacteriaceae, providing a defensive advantage in polymicrobial environments like the gut or soil. For instance, a heat-stable bacteriocin isolated from C. freundii exhibits broad-spectrum activity against Gram-negative bacteria, including pathogens such as Escherichia coli.[55] Additionally, C. freundii employs quorum sensing mediated by N-acyl homoserine lactones (AHLs), enabling coordinated behaviors like virulence regulation and biofilm development in response to population density. Strains like ST2 produce multiple AHL variants, influencing interspecies competition within biofilms.[56]Biofilm formation by C. freundii occurs in diverse settings, including water distribution pipes where it contributes to biocorrosion, and plant roots where it promotes growth. In seawater environments, biofilms of C. freundii accelerate corrosion of aluminum alloys through metabolic byproducts like hydrogen sulfide from sulfate reduction. Conversely, in the rhizosphere, C. freundii forms beneficial biofilms that enhance plant growth promotion via nutrient solubilization and stress tolerance; for example, strain N51 isolated from wheat rhizosphere improves phosphorus uptake in host plants.[57][58]The zoonotic potential of C. freundii involves transmission from poultry to humans, often via contaminated water sources linked to animal husbandry. Poultry products, including eggs and meat, serve as reservoirs, with multidrug-resistant strains spreading through fecal contamination of water supplies during farming or processing. This pathway underscores the bacterium's role in bridging animal and human ecosystems, posing risks in regions with poor sanitation.[59][60]
Pathogenicity and Medical Importance
Associated Infections
Citrobacter freundii is an opportunistic pathogen that primarily causes infections in immunocompromised hosts, including neonates, the elderly, diabetics, and patients with underlying malignancies or hepatobiliary diseases. Approximately 85% of C. freundii infections are hospital-acquired (nosocomial), highlighting its role in healthcare-associated infections. These infections often occur in settings involving invasive procedures, such as catheterization or mechanical ventilation, and are uncommon overall, accounting for 0.8% of Gram-negative infections and 3–6% of Enterobacteriaceae isolates.Urinary tract infections (UTIs) represent the most frequent manifestation of C. freundiiinfection, comprising the majority of reported cases among hospitalized patients and particularly prevalent in catheter-associated settings. UTIs caused by Citrobacter species have been documented in 5–12% of bacterial urine isolates in adults, with increasing prevalence noted over time due to healthcare exposures. Systemic spread can lead to bacteremia, which is often secondary to urinary or intra-abdominal sources and carries a case fatality rate of up to 34% in nosocomial bloodstream infections.In neonates, Citrobacter species, including C. freundii and particularly C. koseri, are associated with severe systemic infections, including sepsis and meningitis, which account for about 1.3% of neonatal meningitis cases and are linked to high mortality rates of 25–50%. Meningitis in this population frequently progresses to brain abscesses.[61] Pneumonia caused by C. freundii occurs predominantly in mechanically ventilated patients, contributing to ventilator-associated pneumonia in intensive care units.Outbreaks of C. freundii infections have been documented in neonatal intensive care units, including clusters of meningitis and sepsis, often linked to contaminated hospital environments or equipment. The bacterium is also part of normal gastrointestinal carriage in humans, serving as a potential reservoir for opportunistic infections.
Virulence Factors and Mechanisms
Citrobacter freundii employs several virulence factors that facilitate its adherence to host tissues, invasion of epithelial cells, toxin-mediated damage, and evasion of immune responses, contributing to its opportunistic pathogenicity in humans and animals. These factors include adhesins for initial colonization, toxins that disrupt host cell function, mechanisms for cellular invasion, and structures that shield the bacterium from immune detection. While the bacterium is often commensal in the gut, expression of these factors in extraintestinal sites enables infections such as urinary tract infections and sepsis.Adhesins play a crucial role in the initial attachment of C. freundii to host epithelial surfaces. Type 1 fimbriae, encoded by the fimoperon, mediate mannose-sensitive adhesion to epithelial cells, promoting colonization in the urinary tract and other mucosal sites.[62] These fimbriae are assembled via the chaperone-usher pathway and are homologous to those in other Enterobacteriaceae, facilitating binding to mannosylated glycoproteins on host cells. Additionally, curli fimbriae, produced by the csgoperon, contribute to adherence and biofilm formation on abiotic surfaces like medical devices.[63]Toxins produced by C. freundii enhance its virulence by causing direct cytotoxicity and systemic inflammation. Shiga-like toxins and heat-stable enterotoxins are key determinants in diarrhea-associated strains, disrupting intestinal epithelial integrity and fluid secretion.[3]Lipopolysaccharide (LPS), the major component of the outer membrane, acts as an endotoxin that triggers severe inflammatory responses, including cytokine release leading to sepsis in susceptible hosts.[63]The bacterium's invasive capabilities allow it to penetrate host barriers, particularly in the urinary tract. C. freundii invades bladder epithelial cells through mechanisms involving fimbrial-mediated entry and host cytoskeletal rearrangements, enabling intracellular replication and persistence.[64] A Salmonella fim homologue in C. freundii directs this invasion into human bladder and gut epithelial cell lines, mimicking enteropathogenic strategies.[62]C. freundii also utilizes a type VI secretion system (T6SS) to deliver effector proteins into host cells and competing microbes, promoting cytotoxicity, activation of the NLRP3inflammasome, and pyroptosis in macrophages, which aids in immune evasion and tissue damage during infection.[65]Immune evasion strategies further bolster C. freundii's survival during infection. Capsular polysaccharides, including the Vi antigen, form a protective layer that masks surface antigens and inhibits phagocytosis by host immune cells.[66]Biofilm formation, facilitated by curli fimbriae and poly-β-1,6-N-acetyl-D-glucosamine (encoded by pgaABCD), allows persistence on indwelling devices and resistance to antimicrobial agents and immune clearance.[67] As a motile, rod-shaped bacterium, C. freundii also uses flagella to navigate host environments and reach infection sites.[68]
Antibiotic Resistance and Treatment
Resistance Profiles
Citrobacter freundii exhibits intrinsic resistance to ampicillin and first-generation cephalosporins primarily due to the production of a chromosomal AmpC β-lactamase, which hydrolyzes these β-lactam antibiotics and is inducible under exposure to certain β-lactams.[69] This mechanism is characteristic of several Enterobacterales, including C. freundii, and contributes to baseline resistance without the need for plasmid acquisition.[70]Acquired resistance in C. freundii has escalated, particularly through the production of extended-spectrum β-lactamases (ESBLs) such as CTX-M variants, with pooled prevalence around 22% (range 4-50%) in clinical isolates per recent meta-analysis.[71] Multidrug-resistant (MDR) strains often carry carbapenemases like NDM-1, reported since 2010 with increasing prevalence in subsequent years, rendering them resistant to carbapenems and complicating therapy in severe infections.[72] Resistance to aminoglycosides is mediated by enzymes such as AAC(6')-Ib-cr, while fluoroquinolone resistance frequently involves mutations in the gyrA gene, such as substitutions in the quinolone resistance-determining region.[72][73]A significant proportion of C. freundii isolates in hospital settings exhibit MDR profiles, with rates around 30% in recent studies and higher in Asia, where NDM-1-producing strains predominate in regions like China and India.[74][72] As of 2025, extremely drug-resistant strains, such as ST257 isolates producing multiple carbapenemases, have been reported in China, highlighting ongoing global dissemination.[75] These patterns underscore the role of C. freundii in nosocomial infections, where resistance mechanisms amplify clinical challenges.[71]
Clinical Management Strategies
The clinical management of Citrobacter freundii infections begins with empirical antibiotic therapy selected based on the site of infection, patient risk factors, and local resistance patterns, typically involving third-generation cephalosporins such as ceftazidime or cefotaxime for mild to moderate cases, or carbapenems like meropenem for severe infections such as bacteremia.[76][77] However, due to the organism's potential to induce AmpC beta-lactamase, leading to common resistance to beta-lactams, therapy must be promptly adjusted according to susceptibility testing results to avoid treatment failure.[78][79]Challenges in management include recurrence in urinary tract infections, with biofilm formation by C. freundii contributing to antibioticresistance and persistence on indwelling devices.[80] For bacteremia, combination therapy—such as a beta-lactam paired with an aminoglycoside like gentamicin—may be employed in critically ill patients to enhance efficacy and cover potential resistance, though monotherapy with a carbapenem is often sufficient once susceptibility is confirmed.[76][81]Prevention strategies emphasize infection control measures in healthcare settings, including rigorous hand hygiene with alcohol-based sanitizers or soap and water before and after patient contact, as well as meticulous catheter care protocols such as aseptic insertion, daily review of necessity, and prompt removal to minimize device-related colonization.[82][83] Additionally, probiotics such as Lactobacillus species have shown promise in reducing gut carriage of certain Enterobacterales in animal models.[84]The Infectious Diseases Society of America (IDSA) guidelines, updated in 2024, recommend de-escalation from broad-spectrum agents like carbapenems to narrower options such as trimethoprim-sulfamethoxazole or fluoroquinolones once susceptibility is established and the patient is clinically stable, aiming to curb resistance development while ensuring effective source control.[85][86]
Identification and Applications
Diagnostic Methods
The identification of Citrobacter freundii in clinical and environmental samples primarily relies on a combination of traditional biochemical tests, culture-based methods, molecular techniques, and serological approaches to ensure accurate differentiation from closely related Enterobacteriaceae such as Escherichia coli and Salmonella spp.[87]Biochemical profiling is a cornerstone of C. freundii diagnosis, with the IMViC pattern typically showing negative indole production, positive methyl red reaction, negative Voges-Proskauer test, and positive citrate utilization (--++), though indole and citrate results can vary across strains.[87]Hydrogen sulfide (H₂S) production is also variable, observed in approximately 63% of strains on triple sugar iron agar.[87] Commercial systems like the API 20E biochemical strip provide confirmatory identification by assessing 20 enzymatic and metabolic reactions, achieving approximately 90% accuracy for C. freundii among Gram-negative bacilli when combined with motility and ornithine decarboxylase tests.[88]Culture-based isolation exploits C. freundii's growth characteristics on selective media; the bacterium forms pink to colorless colonies on MacConkey agar due to delayed lactose fermentation and can be further differentiated on eosin methylene blue (EMB) agar, where it produces flat, dark colonies without the metallic sheen typical of E. coli.[89] These media, often supplemented with bile salts, inhibit Gram-positive organisms while supporting C. freundii proliferation at 37°C under aerobic conditions.[90]Molecular methods offer rapid and specific detection, with 16S rRNA gene sequencing serving as a gold standard for phylogenetic confirmation, enabling species-level identification through comparison to reference databases like NCBI GenBank.[20]Polymerase chain reaction (PCR) assays targeting genus-specific regions, such as those developed for Citrobacter detection and quantification, provide sensitivity down to 10³ CFU/mL in complex samples.[91] Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has emerged as a frontline tool in clinical labs, correctly identifying 95% of C. freundii isolates within minutes by comparing protein spectra to libraries like Bruker or VITEK MS.[92]Serotyping targets the lipopolysaccharide O-antigen for epidemiological tracking, with C. freundii encompassing multiple serogroups within the broader Citrobacter scheme of 43 O-groups, though reclassification has reassigned many to related species like C. youngae and C. braakii.[93] Slide agglutination with O-specific antisera remains the traditional method, aiding in outbreak investigations despite cross-reactivity challenges.[94]
Biotechnological and Research Uses
Citrobacter freundii has demonstrated significant potential in bioremediation, particularly through its ability to sequester heavy metals from wastewater via metabolic processes involving phosphate precipitation. The bacterium's alkaline phosphatase enzyme facilitates the hydrolysis of organic phosphates, leading to the formation of insoluble uranium phosphate complexes that immobilize the metal, with studies showing up to 90% uranium removal efficiency under optimized conditions.[95] This mechanism has been explored in recent investigations, including a 2022 review highlighting its application in treating uranium-contaminated effluents from mining and nuclear sites.[96] Additionally, C. freundii strains have been effective in bioaccumulating other heavy metals like copper.[97]In biotechnology, engineered Citrobacter freundii strains have been developed for biohydrogen production through dark fermentation of organic substrates such as glycerol and glucose. Optimization of culture conditions, including pH control around 6.5 and supplementation with iron, has yielded hydrogen production rates of up to 1.2 mol H₂/mol glucose, making it a promising candidate for renewable energy applications.[98] Furthermore, certain C. freundii isolates exhibit probiotic-like antagonistic activity against pathogens, producing bacteriocins and hydrogen peroxide that inhibit growth of bacteria like Aeromonas hydrophila in vitro, suggesting potential in competitive exclusion strategies for aquaculture or gut health modulation.[99]As a research model, C. freundii is utilized in quorum sensing studies due to its production of N-acyl homoserine lactone (AHL) signal molecules, such as C4-HSL and C8-HSL, which regulate biofilm formation and virulencegene expression.[100]Genome editing with CRISPR/Cas9 has enabled precise modifications in C. freundii, including gene knockouts to investigate antibiotic resistance evolution, with successful editing efficiencies reported in the type strain ATCC 8090, facilitating studies on horizontal gene transfer and mutation rates in resistance pathways.[101]Industrially, C. freundii serves as a source for enzyme production, particularly overexpressing strains that yield high levels of AmpC β-lactamases for research purposes, such as in vitro assays for antibiotic development and resistance mechanism elucidation. These enzymes, when constitutively expressed via mutations in regulatory genes like ampR, provide a model for studying β-lactam hydrolysis, with production levels enhanced up to 100-fold in derepressed mutants.[102]