Mejos, Al Jay T.

Bio 120 Synthesis Paper

Citrobacter

The Citrobacter genus is classified under the family Enterobacteriaceae. They typically utilize

citrate as their sole carbon source. They are Gram-negative, nonsporeforming, facultative anaerobic and

motile bacilli employing peritrichous flagella for locomotion. They do not have oxidase but have catalase

and are methyl red positive. They produce a double positive result in the TSI biochemical test due to the

production of acids and gas from the fermentation of glucose and other carbohydrates. They are

frequently isolated from urine, blood, fecal samples and cerebrospinal fluid and have been found in the

urinary tract causing urinary tract infections and human nose and are believed to be natural commensal

organisms in the human digestive tract. They are most related to the Salmonella and Escherichia genus

groups. Species in the genus have been referred to by numerous names such as Bacterium freundii (Braak,

1928), “Citrobacter freundii” (Werkman and Gillen, 1932), Escherichia freundii, Colobactrum freundii,

Paracolobactrum freundii, Salmonella ballerup, Salmonella hormaechei, the Ballerup group, the

Bethesda group, and the Bethesda-Ballerup group. Only through genetic analyses did the Judicial

Commission of the International Committee on Systematic Bacteriology identified eleven species

included in the group and these genomospecies are Citrobacter freundii, Citrobacter koseri, Citrobacter

amalonaticus, Citrobacter farmeri, Citrobacter youngae, Citrobacter braakii, Citrobacter werkmanii,

Citrobacter sedlakii (Brenner et al., 1993), Citrobacter rodentium (Schauer et al., 1995), Citrobacter

gillenii and Citrobacter murliniae (Brenner et al., 1999). The assignment of binomial nomenclature to the

genomospecies number is ordered (i.e. species 1 to species 11 corresponding to C. freundii to C.

murliniae) as in the list below.

C. freundii

The type species of the genus is C. freundii. It is regarded as an opportunistic pathogen but has

been found to have relatively rare cases involving neonatal infections documented so far (Kaufman &

Fairchild, 2004). It produces abundant amounts of Hydrogen sulfide in Triple Sugar Iron (TSI) agar but

does not produce indole (Sedlak, 1973). It is a rather big group compared to others and is divided into
seven biotypes (Brenner et al., 1999). They have been observed in some hospitals to have the most

number of infection cases among the members of the genus (Samonis, et al. 2008). Moreover, recent

studies have shown that C. freundii infects and replicates in the human brain microvascular endothelial

cells (Badger et al., 1999).

C. koseri

Like C. freundii, C. koseri is also being regarded as an opportunistic pathogen. It accounts for

most of the medical cases involving Citrobacter species in human diseases such as meningitis and

neonatal infections (Kaufman & Fairchild, 2004). Unlike C. freundii it does not produce Hydrogen sulfide

in TSI but instead it is most differentiated from other Citrobacter species due to its capability to produce

acids from adonitol and D-arabitol. The species was also once referred to using different names such as

Colobactrum freundii, Paracolobactrum freundii and Colobactrum diversus. It is not a normal inhabitant

of the maternal urinary tract though also sometimes found; as such it was found to be the cause of

chorioamnionitis, maternal UTI and is highly involved in neonate infection affecting the central nervous

system and lodging in the cerebrospinal fluid in most cases and even sepsis after premature birth

(Kaufman & Fairchild, 2004).

C. amalonaticus

Once named Levinea malonatica, C. amalonaticus is distinguished from others in the genus by its

ability to utilize citrate but not malonate, incapacity of Hydrogen sulfide production in TSI, indole

production and ability to grow in KCN.

C. farmeri

Named after John Farmer III (Brenner at al., 1999), it is identified by its ability to produce indole.

Also, it has the ability to produce indole, arginine dehydrolase and ornithine decarboxylase. It is capable

of producing acids from certain sugars such as α-methyl-D-glucoside, melibiose, raffinose, and sucrose. It

digests and utilize benzoate, 4-hydroxybenzoate, malitol, D-melibiose, 1-O-methyl-α-galactoside,

palatinose, protocatechuate, D-raffinose, and sucrose as sole carbon sources but incapable of utilizing m-

coumarate, dulcitol and malonate. It is also able to, while sometimes delayed, utilize citrate.
C. youngae

Named after Viola Young (Brenner at al., 1999), it is identified through being negative in indole

and ornithine decarboxylase. It is positive, sometimes delayed, for citrate and arginine dihydrolase. It

utilizes dulcitol, 3-phenylpropionate, and L-sorbose, but not gentisate, 3-hydroxybenzoate, malonate, D-

melibiose, 1-O-methyl- α-galactoside, 3-Omethyl- D-glucose, or tricarballylate as sole carbon sources.

C. braakii

Named after Hendrik Braak (Brenner at al., 1999), it has two biotypes differentiated in their

abilities to utilize 4-aminobutyrate, lactose and lactulose as sole carbon sources. The species is capable of

utilizing m-coumarate, 1-O-methyl- α-galactoside, 3-phenylpropionate, and L-tyrosine (delayed), but not

L-sorbose as sole carbon sources. It is also adentified by variable indole production, ornithine

decarboxylase, arginine dehydrolase, and positive, sometimes delayed, in citrate utilization

C. werkmanii

Named after Chester Werkman by Brenner (Brenner at al., 1999), it has citrate and arginine

dehydrolase but does not produce indole and has no ornithine decarboxylase. As its carbon source it

utilizes m-coumarate, D-lyxose, malonate, 3-phenylpropionate, L-sorbose and Dtartrate, but not dulcitol,

4-hydroxybenzoate, Dmelibiose and 1-O-methyl-α-galactoside.

C. sedlakii

Named after Jiri Sedlak (Brenner et al., 1993), it uses it utilizes benzoate, dulcitol, 4-

hydroxybenzoate, myo-inositol, lactulose, malonate, 1-O-methyl-α-galactoside and protocatechuate as its

sole carbon sources but not 5-ketogluconate or L-sorbose. It also produces acid from dulcitol and

melibiose but does not utilize sucrose. Similar to C. braakii, it is positive for arginine dehydrolase,

ornithine decarboxylase and indole production and citrate (sometimes delayed).

C. rodentium

It is the one among Citrobacter species found to have infected animals and man so far, another is

C. freundii (Deng et al., 2003; Hartland et al. 2000) but it was maintained by Luperchio and Schauer

(2001) to be have only been isolated from mice and was found to be the cause of transmissible murine
colonic hyperplasia. It grows in KCN and is negative for Hydrogen sulfide production in TSI and indole.

Citrate utilization has been observed to have an extension of two days delay in producing a positive

reaction. It is positive for arginine dihydrolase and ornithine decarboxylase. C. rodentium is typically

non-motile but Brenner et al. (1999) have observed motility ater four days. It utilizes malonate and

produces acids from melibiose, sucrose, dulcitol and glycerol. It causes bacterial lesions as a means of

attachment and infection and appears as an effacing bacterial pathogen in mice.

C. gillenii

Named after George Francis Gillen (Brenner et al., 1999), it is positive for malonate utilization

and delayed positive for citrate growth and arginine dihydrolase production. It is negative for the

production of indole, urease production, dulcitol fermentation and ornithine decarboxylase production. It

is also incapable of utilizing gentisate, 3-hydroxybenzoate, 3- O-methyl-D-glucose, L-sorbose, or

tricarballylate as sole sole carbon sources.

C. murlinae

Named after Alma McWhorter-Murlin (Brenner et al., 1999), It was found positive (sometimes

delayed) for indole production and citrate utilization and delayed positive for arginine dehydrolase. It is

negative for ornithine decarboxylase. It produces acid from dulcitol and esculin and grows on sodium

acetate (usually delayed). It utilizes malonate, Dulcitol, D-lyxose, 1-O-methyl-α- galactoside (delayed),

and L-tyrosine as sole carbon sources but not malonate or protocatechuate.

Identification

A diagram lifted from O’Harra et al. (1995) [below] is a schematic diagram useful in identifying

the colonies of Citrobacter species.

Habitat and Pathology

Citrobacter species have been found to inhabit the nasal passages, cerebrospinal fluid, brain,

urinary tract, and is believed to be a commensal in the human and animal intestines, occasionally shed in

the bowel hence isolation of samples in feces, water, sewage and soil.

Many diseases have been found to be commonly associated by Citrobacter including urinary tract

infections, wound infections, pneumonia, abscesses, septicemia, meningitis, and endocarditis in adults, as

well as septicemia, meningitis, and brain abscesses in neonates (Graham and Band, 1981; Shamir et al.,

1990; Tang et al., 1994; Shih et al., 1996, Samonis et al. 2008)

Townsend and colleagues (2003) have observed that C. koseri survive and even replicate within

human macrophages, intracellular.

In a very rare case (the study specifically mentioned that the case was second recorded in the

world and first case in the United States), neonatal diffuse pneumocephaly has been observed instead of

the usual abcess production (Alviedo et al., 2009).

 Despite the advances in medicine and recently developed techniques in Citrobacter infection

treatments, a third of the patients die and most of those that survive have sequelae or deformities.

Diagnostics

Citrobacter infections are usually treated a variety of formulations (with corresponding

concentrations) such as colistin (100%), fosfomycin (100%), imipenem (97.4%), gentamicin (89.5%),

nitrofurantoin (89.5%), ciprofloxacin (80.6%), and cefepime (73.7%), (Samonis et al., 2008). However,

due to the increasing rate of nosocomial infections, Citrobacter has also been observed to accumulate

certain resistance to drugs such as sulfonamides, trimethoprim, aminoglycosides, chloramphenicol,

tetracycline, nalidixic acid, fluoroquinolones, nitrofurantoin, polymyxins and fosfomycin (Pepperell et al.,

2002). Being enterobacteria, it is worth noting that Citrobacter are resistant to erythromycin, other

macrolides, lincosamides, fusidic acid, and vancomycin.

Microbiological and Genetic Applications

Studies have been conducted as to the mechanism of lesion formation as a method of bacterial

introduction leading to infections. Shiga-like Toxin II-Related Cytotoxins have been observed by Schmidt

and colleagues (1993) as responsible for the high pathogenicity of C. freundii. These toxin complexes

have two subunits identified as slt-IIcA and slt-IIcB. The cytotoxins are most closely related to the E. coli

slt-II family causative of diarrhea, vomiting and food poisoning. Their study have also found that some

strains hold permanent levels of toxicity while others exhibit loss of number of toxin complexes resulting

to corresponding loss of toxicity.

In 2000, Hartland and colleagues have discovered five types of carboxy-terminal 280 amino acids

(Int280) present in C. rodentium, designated ά, β, γ, δ and ε. They have found that intimin γ binds to the

translocated intimin receptor (Tir) suggesting that intimin may contribute to host specificity of C.

rodentium and has the ability to produce attaching and effacing lesions on HEp-2 cells.

Levy and colleagues (1973) was able to extract a heat-stable ribonuclease from Citrobacter

species that catalyzes the conversion of polyuridylic acid to cyclic-2’, 3’-uridylic or polyadenylic acids.

Due to the high degree of sensitivity and selectivity of the found ribonuclease, they have speculated its
possible usefulness in the elucidation of various RNA types and postulated a possible role of polyamines

in the regulation and mediation capacities in RNA degradation.

References:

Alviedo, J. N. MD, B. G. Sood, MD, MS, FAAPa, J. V. Aranda, MD, PhD, C. Becker, MD. 2009.
Diffuse Pneumocephalus in Neonatal Citrobacter Meningitis. Pediatrics 2006;118;e1576-e1579.
American Academy of Pediatrics.

Badger, J. L., M. F. Stins, and K. S. Kim. 1999. Citrobacter freundii Invades and Replicates in Human
Brain Microvascular Endothelial Cells. Infection and Immunity, Aug. 1999, p. 4208–4215 Vol.
67, No. 8

Borenshtein, D. and David B. Schauer. The Genus Citrobacter. Prokaryotes (2006) 6:90–98.
_________.

Braak, H. R. 1928. Onderzoekingen over Vergisting van Glycerine [thesis]. W. D. Meinema-

Utitgeurerm. Delft, The Netherlands.

Brenner, D. J., P. A., Grimont, A. G. Steigerwalt, G. R. Fanning, E. Ageron, and C. F. Riddle.

1993. Classification of Citrobacteria by DNA hybridization: designation of Citrobacter
farmeri sp. nov., Citrobacter youngae sp. nov., Citrobacter braakii sp. nov., Citrobacter
werkmanii sp. nov., Citrobacter sedlakii sp. nov., and three unnamed Fig. 3. Rep-PCR patterns
amplified from DNA from Citrobacter species type strains, taken from Luperchio et al. (2000).
The Genus Citrobacter 97 Citrobacter genomospecies. International Journal of Systemic
Bacteriology. 43: 645–658.

Brenner, D. J., C. M. O’Hara, P. A. Grimont, J. M. Janda, E. Falsen, E. Aldova, E. Ageron, J.

Schindler, S. L. Abbott, and A. G. Steigerwalt. 1999. Biochemical identification of Citrobacter
species defined by DNA hybridization and description of Citrobacter gillenii sp. nov. (formerly
Citrobacter genomospecies 10) and Citrobacter murliniae sp. nov. (formerly Citrobacter
genomospecies 11). J. Clin. Microbiol. 37:2619–2624.

Deng, W., B. A. Vallance, Y. Li, J. L. Puente and B. B. Finlay. 2003. Citrobacter rodentium translocated
intimin receptor (Tir) is an essential virulence factor needed for actin condensation, intestinal
colonization and colonic hyperplasia in mice. Molecular Microbiology (2003) 48 (1), 95–115.
Blackwell Publishing Ltd.

Graham, D. R., and J. D. Band. 1981. Citrobacter diversus Brain Abscess and Meningitis in Neonates.
JAMA 245:1923–1925.

Hartland, E. L., E. Huter, L. M. Higgins, N. S. Goncalves, G. Dougan, L. A. Phillips,

T. T. Macdonald, and G. Frankel. 2000. Expression of Intimin g from Enterohemorrhagic
Escherichia coli in Citrobacter rodentium. Infection and Immunity. p. 4637–4646 Vol. 68, No. 8
American Society for Microbiology.

Kaufman, D. and K. D. Fairchild. 2004. Clinical Microbiology of Bacterial and Fungal Sepsis in Very-
Low-Birth-Weight Infants. Clinical Microbiology Reviews, July 2004, p. 638–680 Vol. 17, No. 3

Levy, C. C., W. E. Mitch and M. Schmucker. 1973. Effect of Polyamiues on a Ribonuclease Which
 Hydrolyzes Ribonucleic Acid at Uridylic Acid Residues. The Journal of Biological Chemistry
Vol. 248, No. 16, Issue of August 25, pp. 5712-5719, 1973

Luperchio, S. A., J. V. Newman, C. A. Dangler, M. D. Schrenzel, D. J. Brenner, A. G. Steigerwalt, and D.

B. Schauer. 2000. Citrobacter rodentium, the causative agent of transmissible murine colonic
hyperplasia, exhibits clonality: Synonymy of C. rodentium and mouse-pathogenic Escherichia
coli. J. Clin. Microbiol. 38:4343–4350.

O’Hara, C. M., S. B. Roman, and J. M. Miller. 1995. Ability of commercial identification systems to
identify newly recognized species of Citrobacter. J. Clin. Microbiol. 33:242–245.

Pepperell, C., J. V. Kus, M. A. Gardam, A. Humar, and L. L. Burrows. 2002. Low-virulence Citrobacter
species encode resistance to multiple antimicrobials. Antimicrob. Agents Chemother. 46:3555–
3560.

Samonis, G., D. E. Karageorgopoulos, D. P. Kofteridis, D. K. Matthaiou, V. Sidiropoulou, S.

Maraki and M. E. Falagas. 2008. Citrobacter in a general hospital: characteristics and outcomes.
Springer-Verlag 2008.European Journal of Clinical Microbiology of Infectious Diseases (2009)
28:61–68

Schauer, D. B., B. A. Zabel, I. F. Pedraza, C. M. O’Hara, A. G. Steigerwalt, and D. J. Brenner.

1995. Genetic and biochemical characterization of Citrobacter rodentium sp. nov. J. Clin.
Microbiol. 33:2064–2068.

Schmidt H., M. Montag, J. Bockemuhl, J. Heesemann and H. Karch1. 1993. Shiga-Like Toxin II-Related
Cytotoxins in Citrobacter J. freundii Strains from Humans and Beef Samples. Infection and
Immunity, Feb. 1993, p. 534-543 Vol. 61, No. 2 American Society for Microbiology

Sedlak, J. 1973. Present knowledge and aspects of Citrobacter. Current Topics in Microbiology and
Immunology. 62:41–59.

Shamir, R., G. Horev, P. Merlob, and J. Nutman. 1990. Citrobacter diversus lung abscess in a preterm
infant. Pediatr. Infect. Dis. J. 9:221–222.

Shih, C. C., Y. C. Chen, S. C. Chang, K. T. Luh, and W. C. Hsieh. 1996. Bacteremia due to Citrobacter
species: Significance of primary intraabdominal infection. Clin. Infect. Dis. 23:543–549.

Tang, L. M., S. T. Chen, and T. N. Lui. 1994. Citrobacter meningitis in adults. Clin. Neurol. Neurosurg.
96:52–57.

Townsend, S. M., H. A. Pollack, I. Gonzalez-Gomez, H. Shimada, and J. L. Badger. 2003. Citrobacter

koseri brain abscess in the neonatal rat: Survival and replication within human and rat
macrophages. Infect. Immun. 71:5871–5880.

Werkman, C. H., and C. F. Gillen. 1932. Bacteria producing trimethylene glycol. Journal of Bacteriology.
23:167–182.