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PubMed2004

CFE-1, a novel plasmid-encoded AmpC beta-lactamase with an ampR gene originating from Citrobacter freundii.

A clinical isolate of Escherichia coli from a patient in Japan, isolate KU6400, was found to produce a plasmid-encoded beta-lactamase that conferred resistance to extended-spectrum cephalosporins and cephamycins. Resistance arising from production of a beta-lactamase could be transferred by either conjugation or transformation with plasmid pKU601 into E. coli ML4947. The substrate and inhibition profiles of this enzyme resembled those of the AmpC beta-lactamase. The resistance gene of pKU601, which was cloned and expressed in E. coli, proved to contain an open reading frame showing 99.8% DNA sequence identity with the ampC gene of Citrobacter freundii GC3. DNA sequence analysis also identified a gene upstream of ampC whose sequence was 99.0% identical to the ampR gene from C. freundii GC3. In addition, a fumarate operon (frdABCD) and an outer membrane lipoprotein (blc) surrounding the ampR-ampC genes in C. freundii were identified, and insertion sequence (IS26) elements were observed on both sides of the sequences identified (forming an IS26 composite transposon); these results confirm the evidence of the translocation of a beta-lactamase-associated gene region from the chromosome to a plasmid. Finally, we describe a novel plasmid-encoded AmpC beta-lactamase, CFE-1, with an ampR gene derived from C. freundii.

Amino Acid Sequence↗
PubMed2005

Novel TEM-type extended-spectrum beta-lactamase, TEM-134, in a Citrobacter koseri clinical isolate.

A new natural TEM derivative with extended-spectrum beta-lactamase activity, TEM-134, was identified in a ceftazidime-resistant clinical isolate of Citrobacter koseri. Compared to TEM-1, TEM-134 contains the following mutations: Q39K, E104K, R164H, and G238S. The bla(TEM-134) gene was not transferable by conjugation and, apparently, was chromosomally encoded. Expression studies with Escherichia coli revealed efficient cefotaximase and ceftazidimase activity for TEM-134.

Anti-Bacterial Agents↗
PubMed1994

Vitamin B12 production by Citrobacter freundii or Klebsiella pneumoniae during tempeh fermentation and proof of enterotoxin absence by PCR.

The influence of some fermentation parameters on vitamin B12 formation by strains of Citrobacter freundii and Klebsiella pneumoniae isolated from Indonesian tempeh samples during tempeh fermentation was investigated. A decrease in fermentation temperature from 32 to 24 degrees C led to a decrease in vitamin B12 formation. Inoculation of soybeans with different numbers of cells of C. freundii at the beginning of solid-substrate fermentation showed that only the velocity of vitamin formation and not the final amount of vitamin formed depended on the number of cells. The addition of cobalt and 5,6-dimethylbenzimidazole increased the vitamin B12 content of tempeh. Nevertheless, levels of incorporation of the two precursors into the vitamin B12 molecule were very low. Neither C. freundii nor K. pneumoniae possessed the genes encoding the enterotoxins Shiga-like toxin SLT IIA, heat-labile enterotoxin LT Ih, and heat-stable enterotoxin ST Ih, as indicated by PCR. This result supports the suggested use of these two strains to form vitamin B12 during tempeh fermentation in Indonesia.

Base Sequence↗
PubMed1994

Comparison of the taxonomy, serology, drug resistance transfer, and virulence of Citrobacter freundii strains from mammals and poikilothermic hosts.

In this study, the phenotypic, antigenic, and virulence characteristics of 32 Citrobacter freundii strains of fish, human, and veterinary origin were comparatively analyzed. In addition, the spread of drug resistance factors by conjugation was investigated. Regardless of the source of isolation, the strains exhibited variable reactions mainly for arginine dihydrolase, ornithine decarboxylase, and fermentation of sucrose, melibiose, amygdalin, and salicin. Total fatty acid methyl ester analysis by gas chromatography proved to be useful for an intratypic differentiation within the C. freundii strains studied. In fact, although all of the isolates exhibited similar fatty acid methyl ester profiles, significant differences in the major fatty acids 16:1 and 16:0 and in the 17:0 delta region were observed between the isolates from salmonids and the remaining strains. Serological studies using agglutination tests, analysis of lipopolysaccharides (LPS), and the corresponding immunoblots with 13 antisera indicated a great antigenic diversity among the strains. Common LPS patterns were shared only by some isolates showing high cross-agglutination titers. In contrast, although all strains exhibited very similar surface protein patterns, only two common outer membrane proteins of 54 and 58 kDa were immunologically related. Infectivity trials performed in mice and rainbow trout indicated that all of the C. freundii strains were not pathogenic for mice (50% lethal dose of > 5 x 10(7)). Although the isolates displayed a low degree of virulence for trout, inoculated strains were always recovered from the survivors in pure culture.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed2005

Triphenylmethane reductase from Citrobacter sp. strain KCTC 18061P: purification, characterization, gene cloning, and overexpression of a functional protein in Escherichia coli.

We purified to homogeneity an enzyme from Citrobacter sp. strain KCTC 18061P capable of decolorizing triphenylmethane dyes. The native form of the enzyme was identified as a homodimer with a subunit molecular mass of about 31 kDa. It catalyzes the NADH-dependent reduction of triphenylmethane dyes, with remarkable substrate specificity related to dye structure. Maximal enzyme activity occurred at pH 9.0 and 60 degrees C. The enzymatic reaction product of the triphenylmethane dye crystal violet was identified as its leuco form by UV-visible spectral changes and thin-layer chromatography. A gene encoding this enzyme was isolated based on its N-terminal and internal amino acid sequences. The nucleotide sequence of the gene has a single open reading frame encoding 287 amino acids with a predicted molecular mass of 30,954 Da. Although the deduced amino acid sequence displays 99% identity to the hypothetical protein from Listeria monocytogenes strain 4b H7858, it shows no overall functional similarity to any known protein in the public databases. At the N terminus, the amino acid sequence has high homology to sequences of NAD(P)H-dependent enzymes containing the dinucleotide-binding motif GXXGXXG. The enzyme was heterologously expressed in Escherichia coli, and the purified recombinant enzyme showed characteristics similar to those of the native enzyme. This is the first report of a triphenylmethane reductase characterized from any organism.

Amino Acid Sequence↗
PubMed2005

Degradation of cyanophycin by Sedimentibacter hongkongensis strain KI and Citrobacter amalonaticus strain G Isolated from an anaerobic bacterial consortium.

Using a combination of various enrichment techniques, the strictly anaerobic, gram-positive, endospore-forming bacterium Sedimentibacter hongkongensis strain KI as revealed by 16S rRNA analysis and the gram-negative enterobacterium Citrobacter amalonaticus strain G as revealed by physiological tests were isolated from an anaerobic cyanophycin (CGP)-degrading bacterial consortium. S. hongkongensis strain KI is the first anaerobic bacterium with the ability to hydrolyze CGP to beta-Asp-Arg and beta-Asp-Lys dipeptides, as revealed by electrospray ionization-mass spectrometry and reversed-phase high-performance liquid chromatography analysis. However, these primary accumulated hydrolysis products were only partially used by S. hongkongensis strain KI, and significant growth on CGP did not occur. On the other hand, C. amalonaticus strain G did not degrade CGP but grew on the beta-linked iso-dipeptides formed in vitro by enzymatic CGP degradation or in vivo by metabolic activity of S. hongkongensis strain KI. Dipeptide utilization occurred at the highest rate if both strains were used in cocultivation experiments with CGP, indicating that cooperation between different bacteria occurs in anaerobic natural environments for complete CGP turnover. The amino acids obtained from the cleavage of dipeptides were fermented to ethanol, acetic acid, and succinic acid, as revealed by gas chromatographic analysis and by spectrophotometric enzyme assays.

Anaerobiosis↗
PubMed2006

In vivo bioluminescence imaging of the murine pathogen Citrobacter rodentium.

Citrobacter rodentium is a natural mouse pathogen related to enteropathogenic and enterohemorrhagic Escherichia coli. We have previously utilized bioluminescence imaging (BLI) to determine the in vivo colonization dynamics of C. rodentium. However, due to the oxygen requirement of the bioluminescence system and the colonic localization of C. rodentium, in vivo localization studies were performed using harvested organs. Here, we report the detection of bioluminescent C. rodentium and commensal E. coli during colonization of the gastrointestinal tract in intact living animals. Bioluminescence was dependent on intact blood circulation, suggesting that the colonic environment is not anaerobic but nanaerobic. In addition, BLI revealed that C. rodentium colonizes the rectum, a site previously unreported for this pathogen.

Animals↗
PubMed1989

Citrobacter freundii produces an 18-amino-acid heat-stable enterotoxin identical to the 18-amino-acid Escherichia coli heat-stable enterotoxin (ST Ia).

We purified and sequenced the heat-stable enterotoxin produced by Citrobacter freundii. The toxin was detected during purification by reaction with monoclonal antibody to Escherichia coli heat-stable enterotoxin. The C. freundii toxin amino acid sequence was identical to that of the 18-amino-acid heat-stable enterotoxin (STa) produced by toxigenic E. coli.

Amino Acid Sequence↗
PubMed1991

Pathophysiology of Citrobacter diversus neonatal meningitis: comparative studies in an infant mouse model.

Citrobacter diversus is a cause of devastating neonatal meningitis, with illness characterized by formation of multiple brain abscesses. We developed an infant mouse intracranial inoculation model to evaluate the pathophysiology of C. diversus neonatal infections. Eighteen of 26 strains inoculated intracranially at a dose of ca. 3.3 x 10(3) CFU caused greater than 50% mortality in 2-day-old mice. No correlation was seen between the epidemiologic characteristics of a strain and its rate of mortality. When seven C. diversus isolates (four isolates from patients with meningitis, three from non-central nervous system [CNS] sites) were further evaluated, mortality was significantly correlated with bacteremia. The initial lesion in the CNS was a suppurative ventriculitis beginning 1 to 2 days postinoculation. Subsequent ventriculomegaly was associated with ventriculitis and periventricular abscessation. Brain lesions were seen with all strains, although strains of low virulence (as measured by having no bacteremia and low mortality) caused less-severe damage. An age-related susceptibility to C. diversus brain lesions was demonstrated, with 5-day-old mice showing a significant reduction in, and 8-day-old mice being apparently resistant to, infection and CNS damage. Our data indicate that C. diversus has a propensity to cause abscess formation in the neonatal mouse brain, with characteristic pathologic findings; however, the factors that determine whether a strain will cause meningitis in a human infant remain to be identified.

Animals↗
PubMed1993

The eae gene of Citrobacter freundii biotype 4280 is necessary for colonization in transmissible murine colonic hyperplasia.

Transmissible murine colonic hyperplasia is characterized by proliferation of anchored stem cells in the mucosa of the descending colon of laboratory mice and is caused by Citrobacter freundii biotype 4280. This bacterium produces attaching and effacing lesions in the descending colon prior to the onset of gross hyperplasia. By mutational analysis, the chromosomal eae gene of C. freundii biotype 4280 was shown to be necessary for colonic colonization. Conversely, bacteria cured of a 65-kb plasmid, which was identified in C. freundii biotype 4280, were not attenuated for colonic colonization or for the induction of colonic hyperplasia.

Adhesins, Bacterial↗
PubMed1993

Shiga-like toxin II-related cytotoxins in Citrobacter freundii strains from humans and beef samples.

By hybridizing colonies grown from 928 individual stool samples of patients suffering from diarrhea with oligonucleotide probes 772 and 849 complementary to Shiga-like toxin I (SLT-I) and SLT-II gene sequences, respectively, we identified two strains that hybridized with probe 849, which biochemical identification revealed as Citrobacter freundii. An additional five slt-II probe-positive isolates were screened from 81 beef samples. Polymerase chain reaction analysis and restriction of amplified products provided evidence for slt-II-related genes in all seven strains. From C. freundii LM 76, the genes encoding the A and B subunits were cloned in pUC 18 vectors and sequenced. The gene encoding the A subunit differed from that of Escherichia coli slt-IIvhc in 4 bases, resulting in two amino acid residue differences. In 11, 13, and 11 nucleotides, differentiation of slt-IIA, slt-IIcA, and vtx2haA, respectively, was found. These differences affected the predicted amino acid sequence as follows: there were six amino acid differences with SLT-IIA, five with SLT-IIcA, and four with VTx2haA. The nucleotide sequence of the gene encoding the B subunit is identical to slt-IIvhcB and differed from slt-IIcB and vtx2haB by only a single nucleotide base, but this resulted in a predicted amino acid sequence identical to that reported for these toxins. We therefore termed the toxin genes C. freundii slt-IIcA and slt-IIcB. Culture filtrates inoculated with material from the colonies from primary cultures were cytotoxic to Vero cells. Neutralization assays with antisera to E. coli SLT-I, SLT-II, and SLT-IIvhc revealed that antibodies against SLT-IIvhc reduced the C. freundii cytotoxic activity specifically and to the same degree as with the E. coli SLT-IIvhc control strain. In five of the seven strains tested, subcultivation on both a liquid or solid medium resulted in loss of cytotoxic activity. With polymerase chain reaction, we demonstrated that loss of cytotoxic activity ran parallel with the loss of slt genes. These data demonstrate the intergeneric occurrence of SLT-II-related toxins, which may well be a new marker of enteropathogenicity in C. freundii. Our findings that the toxin genes belong to the slt-II family plus their evident instability in the majority of strains should help pave the way to a better understanding of their role in diarrhea or food poisoning.

Animals↗
PubMed1993

Attaching and effacing locus of a Citrobacter freundii biotype that causes transmissible murine colonic hyperplasia.

Citrobacter freundii biotype 4280 produces attaching and effacing (AE) lesions in the large intestine of laboratory mice and is the causative agent of transmissible murine colonic hyperplasia. AE lesions are also produced by enteropathogenic Escherichia coli in humans. Southern analysis revealed that biotype 4280, but not 20 other strains of C. freundii, contained DNA homologous to the eae (E. coli attaching and effacing) gene which is necessary for AE activity by enteropathogenic E. coli in vitro. We have cloned and determined the nucleotide sequence of the C. freundii eae homolog. Our findings suggest that the eae locus of C. freundii biotype 4280 is necessary for AE activity and has a role in the pathogenesis of transmissible murine colonic hyperplasia.

Adhesins, Bacterial↗
PubMed1994

Characterization of the C-terminal domains of intimin-like proteins of enteropathogenic and enterohemorrhagic Escherichia coli, Citrobacter freundii, and Hafnia alvei.

Surface proteins called intimins (Int), which are homologous to the invasin protein (Inv) of Yersinia spp., play a role in inducing brush border damage, termed attachment and effacement, which follows infection by enteropathogenic and enterohemorrhagic Escherichia coli, Citrobacter freundii biotype 4280, and Hafnia alvei. Maltose-binding protein (MBP) fusions containing the C-terminal 280 amino acids of Int-like proteins of strains of enteropathogenic E. coli, enterohemorrhagic E. coli, H. alvei, and C. freundii biotype 4280 and of Yersinia pseudotuberculosis Inv were constructed and purified. The 3' end of the gene for the H. alvei Int-like protein was sequenced and showed homology to corresponding regions of other Int-encoding genes. Binding of MBP-Int-like and MBP-Inv fusion proteins to HEp-2 cells was demonstrated by immunofluorescence microscopy and by fluorescence-activated cell sorting. MBP-Inv induced attachment and spreading of HEp-2 cells to plastic-coated wells, but MBP-Int-like fusion proteins did not. Preincubation of HEp-2 cells with MBP-Inv, but not with MBP-Int-like fusion proteins, inhibited MBP-Inv-induced cell attachment. Fixed staphylococci and fluorescent polymer microspheres coated with both MBP-Int-like and MBP-Inv fusion proteins showed enhanced adhesion to HEp-2 cells. These fusion proteins will facilitate studies of the role of intimin in the pathogenesis of diarrhea associated with members of the family Enterobacteriaeceae that induce attachment and effacement.

ATP-Binding Cassette Transporters↗
PubMed1996

Intimin from enteropathogenic Escherichia coli restores murine virulence to a Citrobacter rodentium eaeA mutant: induction of an immunoglobulin A response to intimin and EspB.

The formation of attaching and effacing (A/E) lesions is central to the pathogenesis of enteropathogenic Escherichia coli (EPEC)-mediated disease in humans and Citrobacter rodentium (formerly C. freundii biotype 4280)-mediated transmissible colonic hyperplasia in mice. Closely related outer membrane proteins, known as intimins, are required for formation of the A/E lesion by both EPEC (Int(EPEC)) and C. rodentium (Int(CR)). A secreted protein, EspB (formally EaeB), is also necessary for A/E-lesion formation. Here we report that expression of a cloned Int(EPEC), encoded by plasmid pCVD438, restores murine virulence to an intimin-deficient mutant of C. rodentium DBS255. Replacement of Cys937 with Ala abolished the ability of the cloned EPEC intimin to complement the deletion mutation in DBS255. Ultrastructural examination of tissues from wild-type C. rodentium and DBS255(pCVD438)-infected mice revealed multiple A/E lesion on infected cells and loss of contact between enterocytes and basement membrane. Histological investigation showed that although both wild-type C. rodentium and DBS255(pCVD438) colonized the descending colon and induced colonic hyperplasia in orally infected 21-day-old mice, the latter strain adhered to epithelial cells located deeper within crypts. Nonetheless, infection with the wild-type strain was consistently more virulent, as indicated by a higher mortality rate. All the surviving mice, challenged with either wild-type C. rodentium or DBS255(pCVD438), developed a mucosal immunoglobulin A response to intimin and EspB. These results show that C. rodentium infection provides a relevant, simple, and economic model to investigate the role of EPEC proteins in the formation of A/E lesions in vivo and in intestinal disease.

Adhesins, Bacterial↗
PubMed1999

Citrobacter rodentium espB is necessary for signal transduction and for infection of laboratory mice.

Citrobacter rodentium is the causative agent of transmissible murine colonic hyperplasia and contains a locus of enterocyte effacement (LEE) similar to that found in enteropathogenic Escherichia coli (EPEC). EPEC espB is necessary for intimate attachment and signal transduction between EPEC and cultured cell monolayers. Mice challenged with wild-type C. rodentium develop a mucosal immunoglobulin A response to EspB. In this study, C. rodentium espB has been cloned and its nucleotide sequence has been determined. C. rodentium espB was found to have 90% identity to EPEC espB. A nonpolar insertion mutation in C. rodentium espB was constructed and used to replace the chromosomal wild-type allele. The C. rodentium espB mutant exhibited reduced cell association and had no detectable fluorescent actin staining activity on cultured cell monolayers. The C. rodentium espB mutant also failed to colonize laboratory mice following experimental inoculation. The espB mutation could be complemented with a plasmid-encoded copy of the gene, which restored both cell association and fluorescent actin staining activity, as well as the ability to colonize laboratory mice. These studies indicate that espB is necessary for signal transduction and for colonization of laboratory mice by C. rodentium.

Actins↗
PubMed1999

Citrobacter rodentium infection in mice elicits a mucosal Th1 cytokine response and lesions similar to those in murine inflammatory bowel disease.

Citrobacter rodentium is a classically noninvasive pathogen of mice that is similar to enteropathogenic Escherichia coli (EPEC) in man. Following oral infection of young mice, the organism colonizes the distal colon, and within 1 week the colonic mucosa doubles in thickness and there is massive epithelial cell hyperplasia. Since T-cell responses in mouse models of inflammatory bowel disease (IBD) also cause epithelial hyperplasia, we have investigated the possibility that C. rodentium promotes similar T-cell responses in the mucosa, thereby increasing epithelial shedding, transmission, and replication of the organism. Beginning 6 days after infection, bacteria were observed to be in close association with the epithelial surface and were also visible scattered throughout the lamina propria and in the submucosa. There was a CD3(+)-cell infiltrate into the colonic lamina propria and epithelium as well as mucosal thickening and crypt hyperplasia. The majority of CD3(+) cells were CD4(+) and were not gammadelta+. Reverse transcription-PCR analysis of cytokines also revealed a highly polarized Th1 response (interleukin-12, gamma interferon, and tumor necrosis factor alpha) in the mucosa and a large increase in the epithelial cell mitogen keratinocyte growth factor. None of the changes were seen in mice inoculated with bacteria lacking intimin (which is necessary for colonization), but they were seen in mice inoculated with C. rodentium complemented with intimin from EPEC. This is the first example of a classically noninvasive bacterial pathogen which elicits a strong mucosal Th1 response and which produces pathology similar to that seen in mouse models of IBD, which is also characterized by a strong Th1 response. These results also suggest that the colonic mucosa responds in a stereotypic way to Th1 responses.

Adhesins, Bacterial↗
PubMed2000

Expression of intimin gamma from enterohemorrhagic Escherichia coli in Citrobacter rodentium.

The carboxy-terminal 280 amino acids (Int280) of the bacterial adhesion molecule intimin include the receptor-binding domain. At least five different types of Int280, designated alpha, beta, gamma, delta, and epsilon, have been described based on sequence variation in this region. Importantly, the intimin types are associated with different evolutionary branches and contribute to distinct tissue tropism of intimin-positive bacterial pathogens. In this study we engineered a strain of Citrobacter rodentium, which normally displays intimin beta, to express intimin gamma from enterohemorrhagic Escherichia coli. We show that intimin gamma binds to the translocated intimin receptor (Tir) from C. rodentium and has the ability to produce attaching and effacing lesions on HEp-2 cells. However, C. rodentium expressing intimin gamma could not colonize orally infected mice or induce mouse colonic hyperplasia. These results suggest that intimin may contribute to host specificity, possibly through its interaction with a receptor on the host cell surface.

ATP-Binding Cassette Transporters↗
PubMed2001

Intimin-specific immune responses prevent bacterial colonization by the attaching-effacing pathogen Citrobacter rodentium.

The formation of attaching and effacing (A/E) lesions on gut enterocytes is central to the pathogenesis of enterohemorrhagic (EHEC) Escherichia coli, enteropathogenic E. coli (EPEC), and the rodent pathogen Citrobacter rodentium. Genes encoding A/E lesion formation map to a chromosomal pathogenicity island termed the locus of enterocyte effacement (LEE). Here we show that the LEE-encoded proteins EspA, EspB, Tir, and intimin are the targets of long-lived humoral immune responses in C. rodentium-infected mice. Mice infected with C. rodentium developed robust acquired immunity and were resistant to reinfection with wild-type C. rodentium or a C. rodentium derivative, DBS255(pCVD438), which expressed intimin derived from EPEC strain E2348/69. The receptor-binding domain of intimin polypeptides is located within the carboxy-terminal 280 amino acids (Int280). Mucosal and systemic vaccination regimens using enterotoxin-based adjuvants were employed to elicit immune responses to recombinant Int280alpha from EPEC strain E2348/69. Mice vaccinated subcutaneously with Int280alpha, in the absence of adjuvant, were significantly more resistant to oral challenge with DBS255(pCVD438) but not with wild-type C. rodentium. This type-specific immunity could not be overcome by employing an exposed, highly conserved domain of intimin (Int388-667) as a vaccine. These results show that anti-intimin immune responses can modulate the outcome of a C. rodentium infection and support the use of intimin as a component of a type-specific EPEC or EHEC vaccine.

Adhesins, Bacterial↗