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PubMed2005

Ciprofloxacin, salicylate, and 2,4-dinitrophenol decrease production of AmpC-type beta-lactamase in two Citrobacter freundii clinical isolates.

The effect of ciprofloxacin and two mar RAB inducers on the susceptibility to beta-lactam antibiotics in two AmpC beta-lactamase semi-constitutive producer Citrobacter freundii clinical isolates (the DM 1 and DM 2 strains) was studied. Possible changes in outer membrane protein expression, permeability to cephaloridine, active efflux, and hydrolytic activity of beta-lactamase-crude extracts were evaluated under the influence of ciprofloxacin, sodium salicylate, and 2,4-dinitrophenol. Results were compared with those of the effect of the same three chemicals on a normally beta-lactamase-inducible wild-type C. freundii strain. The three assayed compounds decreased beta-lactamase hydrolysis on cephaloridine in both the two clinical isolates as well as in the wild-type strain. However, only the DM 1 and DM 2 strains showed increased susceptibility to beta-lactams. Sodium salicylate and 2,4-dinitrophenol, but not ciprofloxacin, reduced the expression of a 45-kDa outer membrane protein in the three studied strains, which was accompanied by a 4- to 20-fold diminution in permeability to cephaloridine. In conclusion, two mar RAB inducers and ciprofloxacin induced the Mar phenotype and repressed AmpC beta-lactamase synthesis in the DM 1 and DM 2 clinical isolates.

2,4-Dinitrophenol↗
PubMed1978

Hydrogen sulfide-producing Citrobacter diversus. A re-emphasis of the potential ability of all Enterobacteriaceae to manifest this quality.

Among 68 strains of Citrobacter diversus isolated in a community hospital in a year and a half, six were characterized by late H2S production in iron-agar slants, while otherwise typical. Although not recorded in standard tabulations, the potential for Enterobacteriaciae to develop this quality by plasmid transfer appears to be virtually unlimited, and dictates caution in the use of failure to produce H2S as a criterion for exclusion. In the present group, the use of the adonitol reaction as a means of distinguishing these organisms from more typical H2S producers is emphasized.

Citrobacter↗
PubMedDate not supplied

Amino acid sequence, active-site residue, and effect of suicide inhibitors on cephalosporinase of Citrobacter freundii GN346.

The structural gene for a cephalosporinase of Citrobacter freundii GN346 was sequenced. From the nucleotide sequence, the entire amino acid sequence of the mature enzyme with 361 amino acids and a molecular weight of 39,867 was determined. The active-site serine was directly confirmed to be serine 64 by studies in which the enzyme was labeled with dansylpenicillin. In investigations comparing the inhibitory effect of sulbactam (penicillanic acid sulfone) and cloxacillin sulfone on the cephalosporinase and on TEM-2-type penicillinase, sulbactam was found to be an effective progressive inhibitor but a poor competitive inhibitor for the cephalosporinase. The cephalosporinase and the inhibitor formed a long-lived complex with a half-life of 550 minutes. Cloxacillin sulfone could not inactivate the cephalosporinase progressively but irreversibly inactivated the penicillinase.

Amino Acid Sequence↗
PubMed1993

Recurring ventriculitis due to Citrobacter diversus: clinical and bacteriologic analysis.

Neonatal meningitis due to Citrobacter diversus is usually accompanied by the development of brain abscesses and tends to have high rates of morbidity and mortality. We report a case of a newborn with C. diversus meningitis and brain abscesses who relapsed after initial antibiotic therapy and from whom C. diversus was recovered from cerebrospinal fluid 4 years later during a neurosurgical procedure. Genetic differences in the early and late isolates were discovered, and explanations for this phenomenon are suggested. Possible mechanisms for prolonged persistence in the central nervous system are explored. This unusual case and the analysis of the organisms illustrate the unique features of C. diversus as a pathogen and underscore the need for developing optimal therapeutic strategies.

Cerebrospinal Fluid↗
PubMed1981

Epidemic nosocomial meningitis due to Citrobacter diversus in neonates.

Five infants born at one hospital over a two-year period developed meningitis due to a serotype O2 strain of Citrobacter diversus; four infants developed brain abscesses due to this organism. The initial prevalence of stool colonization in infants was 79%; eventually 140 infants (10%) and six nurses (6%) were found to be colonized. One colonized infant remained in the hospital for the entire two-year period. The strains were of two biotypes marked by the presence (biotype d) or absence (biotype a) of fermentation of sucrose and dulcitol. The biotype d strain was found in the five infants with meningitis, 110 asymptomatic infants, and five nurses. The biotype a strain, which was isolated from 30 infants and one nurse, did not cause disease. Colonized infants were distinguished by intensive care therapy (P = 10-31), gavage feeding (P = 0.036), and prenatal intrauterine monitoring (P = 0.037). These findings suggest a fecal reservoir and person-to-person transmission of C. diversus. Measures to control the outbreak cost about $110,000.

Birth Weight↗
PubMed1984

Nosocomial meningitis due to Citrobacter diversus in neonates: new aspects of the epidemiology.

Two term neonates born within four days of each other at a small hospital developed sepsis and meningitis caused by a unique strain of Citrobacter diversus not previously reported to cause meningitis. Eleven (27.5%) of 40 other infants admitted to the nursery during the epidemic period developed rectal or umbilical colonization by C. diversus. Contact soon after birth with either of two nurses was more common among infected or colonized infants than among infants who were not infected or colonized. Hand cultures of both nurses and a rectal culture of one of the nurses yielded the epidemic strain. C. diversus may have been introduced into the nursery by the rectal carrier and spread person to person. Six weeks later continued surveillance identified a second cluster (of four colonized infants) associated with a mother who was a carrier of C. diversus and whose newborn infant became colonized at birth. The outbreak ended after strict control measures were used.

Carrier State↗
PubMed1986

Molecular epidemiology of neonatal meningitis due to Citrobacter diversus: a study of isolates from hospitals in Maryland.

Six cases of neonatal meningitis due to Citrobacter diversus were diagnosed in three Baltimore (Maryland) hospitals between 1983 and 1985. Using plasmid profiles, biotypes, serotypes, and chromosomal restriction endonuclease digests as epidemiological markers, we studied 63 isolates of C. diversus (including four isolates from cerebrospinal fluid) from these and seven other hospitals in Maryland. Within two of the three hospitals with meningitis cases, the same strain of C. diversus was isolated from case infant(s), healthy neonates, and nursery personnel. In all three hospitals, C. diversus strains different from those implicated as a cause of meningitis were also isolated. Other than the meningitis-associated strains, 15 different strains of C. diversus were isolated from infants in the hospitals studied, with several distinct clusters of asymptomatic, colonized infants identified.

Citrobacter↗
PubMed1988

Characterization of Citrobacter diversus strains causing neonatal meningitis.

We studied 17 strains of Citrobacter diversus isolated from the cerebrospinal fluid of infants with meningitis and compared these strains with 21 strains isolated from other body sites. The two groups of strains were similar with respect to biotype, piliation, hemolysin production, and resistance to the killing effects of serum. By using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, we found that 14 (82%) of 17 strains from cerebrospinal fluid, but only two (10%) of 21 strains isolated from other body sites, possessed a minor outer membrane protein with a molecular weight of 32,000 (P less than .0001). This protein may serve as a marker for strains of C. diversus that are likely to cause meningitis or brain abscess in human neonates.

Bacterial Outer Membrane Proteins↗
PubMed1988

Pathogenesis of brain abscess formation in an infant rat model of Citrobacter diversus bacteremia and meningitis.

The pathogenesis of Citrobacter diversus meningitis and brain abscess was studied in infant rats. Two-day-old rats were inoculated intraperitoneally and intranasally with C. diversus. C. diversus strain 4277, lacking the 32,000-molecular-weight outer membrane protein that appears to be a marker for strains causing meningitis in human neonates, was more likely to produce bacteremia, meningitis, and death in rats than was strain 4036, which possesses this outer membrane protein. Strain 4036 was, however, more likely than strain 4277 to produce ventriculitis and brain abscess. In the infant rat, central nervous system involvement by C. diversus begins with bacteremia and leptomeningitis, followed by ventriculitis and direct extension of infection into periventricular brain parenchyma. Large numbers of bacteria persist inside inflammatory cells, an observation suggesting resistance to intraphagocytic killing. Bacterial strain differences, possibly related to the presence of a 32,000-molecular-weight outer membrane protein, may account for histopathologic differences in the brains of infant rats with C. diversus meningitis.

Animals↗
PubMed1992

Interaction of Citrobacter diversus strains with HEp-2 epithelial and human umbilical vein endothelial cells.

More than 75% of neonates with Citrobacter diversus meningitis develop brain abscesses. Interaction of C. diversus strains with HEp-2 and human umbilical vein endothelial cells (HUVEC) was studied to examine mechanisms related to brain abscess formation. Two of 9 strains invaded HEp-2 cells and 0 of 6 invaded HUVEC better than the others. C. diversus survived at least 20 h within HEp-2 cells (in decreasing numbers). Adhesion to HEp-2 cells was increased in 3 of 4 strains expressing type 1 fimbriae, but this did not correlate with increased invasion. Inhibition of RNA or protein synthesis blocked invasion but not adhesion. Thus, invasion requires ongoing protein synthesis, and adhesion to and invasion of HEp-2 cells by type-1-fimbriated strains are independent steps. Invasion was inhibited by cytochalasin D. A 32-kDa protein found in cerebrospinal fluid isolates of C. diversus was not related to invasion of either cell line. Ability to invade HEp-2 cells was not increased among strains isolated from central nervous system sources.

Bacterial Adhesion↗
PubMed1987

Citrobacter diversus ULA27 produces two forms of a chromosomal beta-lactamase.

A multi-resistant clinical isolate of Citrobacter diversus, particularly resistant to penicillins and cephalosporins, has been studied. The strain produced constitutively high levels of beta-lactamase which efficiently hydrolysed cephaloridine but not ampicillin. The apparent Km value for cephaloridine (125 +/- 25 microM) was within the range usually observed for beta-lactamases in similar bacteria. The strain possessed two forms of the enzyme as indicated by isoelectric focusing patterns, HPLC analysis and two pH optima. SDS-PAGE analysis indicated a single protein responsible for both forms. The strain possessed a single plasmid, as revealed by agarose gel electrophoresis, but beta-lactamase production was not plasmid encoded. The available data indicate the beta-lactamase(s) of C. diversus ULA27 to be chromosomally encoded.

Anti-Bacterial Agents↗
PubMed1990

Broad spectrum beta-lactamases of Citrobacter diversus.

Citrobacter diversus NF85 produced a chromosomal beta-lactamase that was induced by a variety of beta-lactam antibiotics. Two major forms of the enzyme, with isoelectric points (pI's) of 5.7 and 6.2, were found in crude cell extracts. Derepressed mutants of NF85, generated by nitrosoguanidine treatment, displayed different levels of beta-lactamase expression to the parent strain and had different patterns of resistance to a range of beta-lactam antibiotics. Those mutants of NF85 that were totally derepressed, expressing high, constitutive levels of enzyme, were found to have an additional beta-lactamase activity with a pI of 6.8.

Anti-Bacterial Agents↗
PubMed1995

The ability of beta-lactam antibiotics to select mutants with derepressed beta-lactamase synthesis from Citrobacter freundii.

The ability of beta-lactam compounds to induce synthesis of the class C beta-lactamase of Citrobacter freundii was assessed both directly and indirectly, the latter by measurement of the different susceptibilities of strains with inducible and depressed beta-lactamase synthesis and those of a beta-lactamase-negative strain. Most beta-lactam compounds were poor inducers but ampicillin, amoxycillin and cephalexin were moderately good inducers and cefoxitin, imipenem and meropenem were strong inducers. The ability of the compounds to select mutants in which the synthesis of the beta-lactamase was derepressed was also assessed. Imipenem and temocillin, which had a high degree of stability to the beta-lactamase failed to select such mutants or were very poor selectors. In contrast, most other compounds showed considerable lability to the beta-lactamase and readily selected derepressed mutants, whether they were strong inducers of beta-lactamase synthesis (for example cefoxitin) or poor inducers (cefotaxime, ceftazidime and many other compounds). Thus it appears that lability to the beta-lactamase is a more important factor in determining whether or not a compound will select derepressed mutants than the power of the compound as an inducer of beta-lactamase synthesis, since induction results in the production of less beta-lactamase than derepression as a result of mutation.

Anti-Bacterial Agents↗
PubMed1998

Transferable class C beta-lactamases in Escherichia coli strains isolated in Greek hospitals and characterization of two enzyme variants (LAT-3 and LAT-4) closely related to Citrobacter freundii AmpC beta-lactamase.

Among 2133 isolates of Escherichia coli obtained during 1996 from 10 Greek hospitals, 63 (3%) were resistant to cefoxitin. Typing by ERIC2-PCR indicated that the cefoxitin-resistant (FOXr) isolates were distinct. beta-Lactamase studies and hybridization experiments showed that most strains produced beta-lactamases related to the AmpC chromosomal cephalosporinase of Citrobacter freundii. The enzymes were encoded by similar non-self-transmissible plasmids. The bla genes encoding two beta-lactamases (LAT-3 and LAT-4) with isoelectric points 8.9 and 9.4, respectively, were cloned and sequenced. The deduced amino acid sequences displayed a high degree of homology (>95%) with the AmpC beta-lactamase of C. freundii. The patterns of resistance to beta-lactams of the FOXr E. coli depended on the quantity of class C enzymes and the simultaneous expression of other beta-lactamases. In a few isolates a 36 kDa outer-membrane protein, presumably a porin, was not expressed at detectable quantities. These isolates were resistant to cefoxitin, and their susceptibility to the other beta-lactams tested was not significantly decreased.

Amino Acid Sequence↗
PubMed2000

In vitro selected fluoroquinolone-resistant mutants of Citrobacter freundii: analysis of the quinolone resistance acquisition.

Ten quinolone-resistant mutants of Citrobacter freundii, which were selected in vitro with fluoroquinolones from two clinical isolates, were studied. The parent isolates were susceptible to quinolones in spite of showing a single substitution in the GyrB (His-417 --> Leu). No change was observed in the outer membrane proteins or in the lipopolysaccharide in any of the ten mutants studied with respect to their parent isolates. The development of quinolone resistance in selected mutants was associated with the appearance of a substitution in the GyrA (Thr-83 --> Ile) in nine of the ten mutants plus enhanced active efflux in all of them.

Anti-Infective Agents↗
PubMed2002

Metallo-beta-lactamase-producing Enterobacteriaceae isolates in a university hospital in Taiwan: prevalence of IMP-8 in Enterobacter cloacae and first identification of VIM-2 in Citrobacter freundii.

A total of 9082 clinical isolates of Enterobacteriaceae other than Klebsiella spp. collected in 1999 and 2000 at a university hospital in Taiwan were investigated for the production of metallo- beta-lactamases (MBLs). Thirty-six (2.9%) of the 1261 Enterobacter cloacae isolates and one (0.3%) of the 340 Citrobacter freundii isolates were found to carry bla(IMP-8) and bla(VIM-2), respectively, by colony hybridization, PCR and sequence analysis. The IMP-8 producers were recovered from 20 patients and four of them had recently transferred from other hospitals, implying spread of IMP-8-producing E. cloacae among different healthcare settings. Of the 20 non-repetitive IMP-8 producers, 17 (85%) isolates also harboured bla(SHV-12), which was on the same transferable plasmids with bla(IMP-8). The bla(VIM-2)-positive isolate and all non-repetitive bla(IMP-8)-positive isolates appeared susceptible to imipenem (MICs < 8 mg/L) and meropenem (MICs < 4 mg/L), indicating the difficulty in detection of MBLs in Enterobacteriaceae by routine susceptibility testing. Ribotyping of the IMP-8-producing E. cloacae isolates indicated that the dissemination of bla(IMP-8) was due largely to the spread of an epidemic clone, but horizontal transfer among unrelated strains also occurred.

Citrobacter freundii↗
PubMed2006

Intracellular accumulation of linezolid in Escherichia coli, Citrobacter freundii and Enterobacter aerogenes: role of enhanced efflux pump activity and inactivation.

OBJECTIVES: The oxazolidinone class of antibiotics such as linezolid have a narrow spectrum of activity that targets Gram-positive bacteria. We hypothesized that the poor activity of linezolid in Gram-negative bacteria is in part caused by relatively low intracellular concentration due to efflux. METHODS: Using whole cell accumulation assays we estimated the intracellular concentration of linezolid in Escherichia coli and other Enterobacteriaceae. We included test strains with enhanced RND-type multidrug efflux pump activity and with genetic inactivation of the pump or functional inhibition by carbonyl cyanide m-chlorophenylhydrazone as inhibitor of the proton motive force or 1-(1-naphthylmethyl)-piperazine (NMP), an efflux pump inhibitor. RESULTS: Consistent with susceptibility studies, enhanced pump activity caused decreased accumulation, and pump inactivation and inhibition caused increased accumulation, of linezolid. The accumulation levels in test strains of E. coli, Citrobacter freundii and Enterobacter aerogenes with functional pumps were lower than in control strains of Staphylococcus aureus and Enterococcus faecium, but were higher after pump inactivation and correlated with ethidium bromide and pyronin Y accumulation. CONCLUSIONS: The intracellular concentration of linezolid is comparatively low owing to efficient efflux of the drug and could be increased substantially by inhibition of RND-type efflux pumps.

ATP Binding Cassette Transporter, Subfamily B↗
PubMed2001

A cryptic melibiose transporter gene possessing a frameshift from Citrobacter freundii.

Wild-type Citrobacter freundii cannot grow on melibiose as a sole source of carbon. The melibiose transporter gene melB was cloned from a C. freundii mutant M4 that could utilize melibiose as a sole carbon source. Although the cloned melB gene is closely similar to the melB genes of other bacteria, it is cryptic because of a frameshift mutation. Site-directed mutagenesis was used to construct a functional melB gene by deleting one nucleotide, resulting in the production of an active melibiose transporter. The active MelB transporter could utilize Na(+) and H(+) as coupling cations to melibiose transport. The amino acid sequence of the C. freundii MelB was found to be most similar to those of Salmonella typhimurium and Escherichia coli MelB. These facts are consistent with the phylogenetic relationship of bacteria and the cation coupling properties of the melibiose transporters.

Amino Acid Sequence↗