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PubMed1994

Formation of B-vitamins by bacteria during the soaking process of soybeans for tempe fermentation.

The formation of B-vitamins (nicotinic acid and nicotinamide, thiamine, vitamin B6 and vitamin B12) during the soaking of soybeans by bacteria, isolated from tempe, was investigated. Among the isolates examined no vitamin B6 producer was found. After inoculation of the soaking soybeans with Citrobacter freundii, Klebsiella pneumoniae, Pseudomonas fluorescens and Streptococcus spp. the concentrations of vitamin B12 increased significantly. For the first time it was found that soakings inoculated with C. freundii showed an increased vitamin B12 content. Nicotinic acid and nicotinamide were produced by Lactobacillus spp. and C. freundii. C. freundii synthesized thiamine as well.

Citrobacter freundii↗
PubMed1995

Polymicrobial gram-negative bacteremia associated with saline solution flush used with a needleless intravenous system.

BACKGROUND: During a 2-week period, seven cases of nosocomial polymicrobial gram-negative rod bacteremia occurred on a 39-bed medical and cardiac step-down unit. Combinations of Enterobacter cloacae (seven isolates), Klebsiella pneumoniae (five isolates), and Citrobacter freundii (two isolates) were isolated from blood cultures. METHODS: Concurrent and retrospective chart reviews were used to look for further cases and common exposures. Epidemiologic methods were used to refine determination of common exposure. Restriction enzyme DNA analysis was performed on the isolates. RESULTS: Concurrent and retrospective chart reviews revealed four additional possible cases during the same period. All case patients were exposed, through peripheral saline solution locks or central venous catheters, to saline solution "flush" from a central 0.9% saline solution bag and a needleless dispensing pin. Epidemiologic methods implicated probable extrinsic contamination of a single bag and pin used during a 24-48-hour period (Fisher's Exact Test, p < 0.002). There were no other common exposures. Restriction enzyme DNA analysis of the isolates further supported a common source for the outbreak. CONCLUSIONS: The introduction of needleless intravascular systems has been embraced for employee protection. Our report is the first to raise the question of patient safety with such systems. This outbreak highlights the inherent risks in rapid introduction of new technologies and points out the delicate balance among patient health, employee safety, and cost containment.

Bacteremia↗
PubMed1994

O-acetylation affects the binding properties of the carboxyl groups on the Vi bacterial polysaccharide.

The capsular polysaccharide of Salmonella typhi and Citrobacter freundii (Vi) is a linear homopolymer of (1-->4)-linked 2-acetamido-2-deoxy-alpha-D-galactopyranosyluronic acid partially O-acetylated at the C-3 position. The physico-chemical properties of the carboxyl groups of the Vi polysaccharide, as a function of different degrees of O-acetylation, were studied by potentiometric titration, circular dichroism, and their reaction with the bulky nucleophile 2-nitro-phenylhydrazine (NPH). Potentiometric titrations with K+ and Ca2+ hydroxides showed that the difference in the free energy of binding between the two cations (delta GKCa) was inversely proportional to the degree of O-acetylation. Similar cationic effects were found when measuring circular dichroism. Moreover there was also an inverse relation between the degree of O-acetylation and the extent of binding of NPH to the carboxyl groups. These data all indicate that O-acetyl groups hinder the association of carboxyls with cations and nucleophilic reagents. This provides a possible explanation for the importance of the O-acetyl and the relative unimportance of the carboxyl groups in contributing to the immunologic properties of the Vi.

Acetylation↗
PubMed1991

Molecular cloning of the ViaB region of Salmonella typhi.

The ViaB region required for Vi antigen production in Salmonella typhi was cloned. The plasmid pGBM124 containing a 14-kb S. typhi chromosomal DNA fragment conferred the ability to produce Vi antigen on Escherichia coli HB101 and ViaB-deleted S. typhi GIFU10007-3. Tn5 insertion analysis showed that the 14-kb DNA was split into three regions. Region 1 and region 2 are involved in the biosynthesis of Vi polysaccharide. Region 3 is involved in translocation of the Vi polysaccharide to the cell surface. Southern blot hybridization showed that regions 2 and 3 but not region 1, were considerably homologous to the DNA of Vi-positive Citrobacter freundii.

Antigens, Bacterial↗
PubMed1994

Cloning and analysis of translational control for genes encoding the Cfr9I restriction-modification system.

The complete type-II Cfr9I restriction-modification (R-M) system of Citrobacter freundii strain RFL9, recognizing the DNA sequence CCCGGG, has been cloned and expressed, and functionally active enzymes have been produced in Escherichia coli. Both the methyltransferase (MTase; M.Cfr9I) and restriction endonuclease (ENase; R.Cfr9I) were found to be encoded on a 2.3-kb cloned fragment in the same transcriptional orientation, but differing in translational phases. The last codon (underlined) (ATGA) of the MTase-encoding gene (Cfr9IM) overlaps with the start codon for the ENase-encoding gene (overlined) (cfr9IR). A nucleotide sequence complementary to a predicted Shine-Dalgarno sequence preceding cfr9IR is within this gene. Predicted free energy (delta G) for formation of the mRNA secondary structure involving these complementary sequences was found to be -16.1 kcal/mol. Amino-acid sequence homology of 80% was found between R.Cfr9I and R.XcyI.

Amino Acid Sequence↗
PubMed1988

Bacteriological studies with ampicillin/sulbactam on selected strains of gram-negative bacteria.

Antibacterial activity of sulbactam or ampicillin alone and in combination on ampicillin-resistant, beta-lactamase-producing Gram-negative bacteria (Citrobacter freundii and Escherichia coli) was studied. Inhibition of beta-lactamase activity by sulbactam was investigated using intact and disrupted cells. Minimal inhibitory concentrations of ampicillin were high but decreased significantly in the presence of sulbactam. Similar enzyme inhibition was observed with intact and disrupted bacterial cells, thus indicating efficient penetration by sulbactam into the periplasmic space. Bacterial killing was achieved in approximately 4 hrs with ampicillin/sulbactam at concentrations that neither killed nor inhibited the same strains when the drugs were used alone. Sulbactam was more effective against plasmid-cured strain of E. coli than the same plasmid-containing organism.

Ampicillin↗
PubMed2004

Digestion of cellulose and xylan by symbiotic bacteria in the intestine of the Indian flying fox (Pteropus giganteus).

Bats (Order Chiroptera) are a widely distributed group of mammals. Pteropus giganteus belongs to the Suborder Megachiroptera. This bat consumes fruits and leaves as their major food. Cellulose and xylan are the major composition of leaves. As they consume leaves in their diet, their digestive tract must contain cellulolytic and xylanolytic bacteria which help in the digestion of cellulose and xylan. The cellulolytic and xylanolytic bacteria were isolated and screened on Berg's agar containing cellulose and xylan. The bacteria isolated were characterized biochemically and found to be Proteus vulgaris, Proteus mirabilis, Citrobacter freundii, Serratia liquefaciens and Klebsiella oxytoca. These bacteria help in digestion of cellulose and xylan in the diet of the bat, P. giganteus. Here we show that leaves are also used as a carbohydrate source by these bats. An insectivorous bat, Hipposideros fulvus, was used as a control and does not possess cellulolytic and xylanolytic bacteria.

Animals↗
PubMed2004

Shiga toxin-encoding bacteriophages--genomes in motion.

Shiga toxins (Stx) represent a group of bacterial toxins that are involved in human and animal disease. Stx are mainly produced by Escherichia coli isolated from human and non-human sources, Shigella dysenteriae type 1, and sporadically, by Citrobacter freundii, Enterobacter cloacae and Shigella flexneri. The genes encoding Stx are encoded in the genome of heterogeneous lambdoid prophages (Stx-converting bacteriophages; Stx-phages). They are located in a similar position in the late region of the prophage genome and stx is under control of phage genes. Therefore, induction of Stx-converting prophages triggers increased production of Stx. Following induction, Stx-phages can infect other bacteria in vivo and in vitro. Stx-phages may be considered to represent highly mobile genetic elements that play an important role in the expression of Stx, in horizontal gene transfer, and hence in genome diversification.

Animals↗
PubMed2004

Nonpseudomonal ecthyma gangrenosum.

Ecthyma gangrenosum is a cutaneous infection associated most commonly with pseudomonal sepsis in the patient who is immunocompromised. We describe an 8-month-old girl with acute myelocytic leukemia who developed perineal ecthyma gangrenosum caused by Citrobacter freundii, a gram-negative pathogen that has been rarely associated with cutaneous disease. We also review the literature to categorize the range of pseudomonal and nonpseudomonal pathogens associated with ecthyma gangrenosum.

Biopsy↗
PubMed2004

Molecular biology of extended-spectrum beta-lactamase-producing Enterobacteriaceae responsible for digestive tract colonization.

Twenty-nine extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae strains (14 Klebsiella pneumoniae, 10 Escherichia coli and five Citrobacter diversus) isolated from April to July 1996 from faecal carriers in a surgical intensive care unit at the university hospital of Casablanca (Morocco) were studied. Plasmid content and DNA macrorestriction polymorphism determined by pulsed-field gel electrophoresis (PFGE) were used to compare the strains. Restriction profiles of total genomic DNAs cleaved by XbaI and compared by PFGE revealed nine, four and two clones in K. pneumoniae, E. coli and C. diversus, respectively. Plasmid profile analysis of ESBL-producing strains of K. pneumoniae showed that only seven of 14 isolates had a plasmid; four different plasmid profiles were observed. Three different plasmid profiles were observed in E. coli and two in C. diversus. Plasmids responsible for ESBL production could be transferred by conjugation to E. coli K(12) J53-2 from all E. coli isolates and from four of seven K. pneumoniae. No plasmid transfer could be obtained from C. diversus strains. Restriction enzyme digests of plasmids from transconjugants (four transconjugants of K. pneumoniae and five transconjugants of E. coli) showed different patterns. In the surgical intensive care unit where the survey was conducted, the dissemination of ESBLs was due to a mix of strain spread and strain diversity rather than to plasmid dissemination.

Carrier State↗
PubMed2006

Crystal structure of the nucleotide-binding subunit DhaL of the Escherichia coli dihydroxyacetone kinase.

Dihydroxyacetone (Dha) kinases are a family of sequence-related enzymes that utilize either ATP or phosphoenolpyruvate (PEP) as source of high energy phosphate. The PEP-dependent Dha kinase of Escherichia coli consists of three subunits. DhaK and DhaL are homologous to the Dha and nucleotide-binding domains of the ATP-dependent kinase of Citrobacter freundii. The DhaM subunit is a multiphosphorylprotein of the PEP:sugar phosphotransferase system (PTS). DhaL contains a tightly bound ADP as coenzyme that gets transiently phosphorylated in the double displacement of phosphate between DhaM and Dha. Here we report the 2.6A crystal structure of the E.coli DhaL subunit. DhaL folds into an eight-helix barrel of regular up-down topology with a hydrophobic core made up of eight interlocked aromatic residues and a molecule of ADP bound at the narrower end of the barrel. The alpha and beta phosphates of ADP are complexed by two Mg2+ and by a hydrogen bond to the imidazole ring of an invariant histidine. The Mg ions in turn are coordinated by three gamma-carboxyl groups of invariant aspartate residues. Water molecules complete the octahedral coordination sphere. The nucleotide is capped by an alpha-helical segment connecting helices 7 and 8 of the barrel. DhaL and the nucleotide-binding domain of the C.freundii kinase assume the same fold but display strongly different surface potentials. The latter observation and biochemical data indicate that the domains of the C.freundii Dha kinase constitute one cooperative unit and are not randomly interacting and independent like the subunits of the E.coli enzyme.

Adenosine Diphosphate↗
PubMed2004

The application of peptide nucleic acid probes for rapid detection and enumeration of eubacteria, Staphylococcus aureus and Pseudomonas aeruginosa in recreational beaches of S. Florida.

A novel chemiluminescent in situ hybridization technique using peptide nucleic acids (PNA) was adapted for the detection of bacteria in beach sand and recreational waters in South Florida. The simultaneous detection and enumeration of eubacteria and the novel indicators, Staphylococcus aureus and Pseudomonas aeruginosa, was achieved within 6-8 h of processing. Following 5 h of incubation on TSA, soybean peroxidase-labeled peptide nucleic acid probes (Boston Probes, Boston, MA) targeting species-specific 16S rRNA sequences of P. aeruginosa and S. aureus were used to hybridize microcolonies of the target species in-situ. In addition, a universal probe for 16S rRNA sequences was used to target the eubacteria. Probes were detected after a light generating reaction with a chemiluminescent substrate and their presence recorded on Polaroid film. The probes showed limited cross-reactivity with mixed indigenous bacteria extracted from seawater and sand by shaking with phosphate-buffered saline (PBS). Specificity and cross-reactivity was tested on the reference bacterial genera Pseudomonas, Staphylococcus, Vibrio, Shigella, Salmonella, Acinetobacter, Enterobacter, Escherichia and Citrobacter. These tests confirmed that the probes were specific for the microorganisms of interest and were unaffected by high salt levels. The results of the PNA chemiluminescent in situ hybridization were compared with traditional plate count methods (PCM) for total 'freshwater' eubacteria, S. aureus and P. aeruginosa. Counts of eubacteria and S. aureus were comparable with numbers obtained from traditional plate counts but levels of P. aeruginosa were higher with PNA than with PCM. It is possible that PNA is more sensitive than PCM because it can detect microcolonies on the agar surface that never fully develop with the plate count method. We conclude that the in situ hybridization technique used here represents an important potential tool for the rapid monitoring of novel indicator organisms in beaches and recreational waters.

Acinetobacter↗
PubMed2006

Functional analysis of replication and stability regions of broad-host-range conjugative plasmid CTX-M3 from the IncL/M incompatibility group.

Plasmid CTX-M3 (89 kb) isolated from Citrobacter freundii from a Warsaw hospital is a mosaic plasmid with replication functions 100% identical with those of pMU407.1 of the IncL/M group, conjugative operons with up to 60% homology to ColIb-P9 (IncI) and stability functions originating either from NR1(R100) (IncFII) or ColIb-P9 /R1/NR1 plasmids. We established the broad-host-range for pCTX-M3 and defined its minireplicon in Escherichia coli. We analyzed the role of stability cassettes and showed that the par operon consists of three orfs parA (stbA), parB (stbB) and nuc with a centromere-like region located upstream of the operon. Deletion of the par operon strongly destabilized pCTX-M3 despite the presence of the pemIK toxin-antidote system identical to that on NR1(R100) plasmids. Deletion of the pemIK operon had no effect on plasmid stability.

Amino Acid Sequence↗
PubMed1997

Distribution of a sub-class of bacterial ABC polar amino acid transporter and identification of an N-terminal region involved in solute specificity.

A new sub-class of binding protein-dependent transporter with specificity for a broad range of polar amino acids has been identified by sequence comparison, in Rhizobium leguminosarum, Rhodobacter capsulatus, Escherichia coli and Pseudomonas fluorescens. Southern blotting and PCR analysis has shown that transporters from this new sub-class are widely distributed in Gram-negative bacteria, including, in addition to the above, Citrobacter freundii, Erwinia carotovorum and Rhizobium meliloti. ABC transporters of polar amino acids can be divided into two groups: those with narrow solute specificity and the newly identified sub-class with broad solute specificity. The binding and inner membrane proteins from transporters with a broad solute specificity are larger by approximately 30% than those with a narrow solute specificity. Multiple alignment of the inner membrane proteins from all sequenced polar amino acid transporters indicates there is an N-terminal conserved region that may be involved in solute specificity. A conserved arginine or lysine at residue 30 of this region is changed to glutamate in arginine transporters. Residue 53 also has a strong correlation with the charge on the transported solute, with basic amino acid transporters replacing an aliphatic amino acid at this position with a negatively charged amino acid. The general amino acid permease from R. leguminosarum, which will transport aliphatic as well as basic and acidic amino acids, juxtaposes two prolines at residues 52 and 53 of the N-terminal conserved region.

ATP-Binding Cassette Transporters↗
PubMed1976

Toxigenic bacterial diarrhea: nursery outbreak involving multiple bacterial strains.

An outbreak of watery diarrhea occurred in 14 of 15 infants in a special care unit over a four-day period. Using the CHO cell assay for enterotoxin, we found that 11 of these patients had toxigenic bacteria in their stools. These bacteria comprised nine different serotypes of three species of organisms: Escherichia coli, Klebsiella, and Citrobacter. None of the three serotypes of E. coli were classic enteropathogenic serotypes. Rectal swab specimens from all 15 infants were examined for the presence of viruses by electron microscopic and cell culture techniques as well as by studies in suckling mice. None had parvovirus- or reovirus-like agents and two had adenoviruses. No other viral agents were detected. Of 38 bacterial strains isolated from ten control infants without diarrhea, three Klebsiella strains from two individuals were found to be toxigenic. Analysis of a total of 136 enteric isolates showed that toxigenicity as measured by the CHO assay was strongly associated with strains isolated during acute diarrheal illness when compared with strains isolated in convalescence (p less than 10(-5)) or with strains from control infants without diarrhea (p less than 10(-5)). This study raises the possibility of an outbreak of disease caused by a transmissible plasmid responsible for a cholera-like enterotoxin production in several enteric bacterial strains.

Adenoviruses, Human↗
PubMed1978

Bacterial colonization of neonates admitted to an intensive care environment.

In order to elucidate some of the factors responsible for the high rate of nosocomial infection associated with neonatal intensive care, we studied bacterial colonization in 63 infants admitted to a neonatal intensive care unit. In a six-month period, cultures of nose, throat, umbilicus, and stool were obtained on admission and every three days from all infants staying in the NICU greater than or equal to 3 days. Study infants did not develop "normal" aerobic flora. Forty-eight percent of infants grew Escherichia coli from stool, but 52% had stool colonization with Klebsiella, Enterobacter, or Citrobacter, the only other Enterobacteriaceae encountered. KEC were also isolated from throat, nose, and umbilicus in 22%, 22%, and 24% of patients, respectively. The risk of stool colonization with KEC increased with duration of hospitalization: 2% of infants were colonized on admission, 60% after 15 days, and 91% after 30 days. Stool colonization with E. coli seemed to protect infants from colonization with other gram-negative bacilli. Thirteen of 20 infants, however, developed pharyngeal GNB colonization in spite of pre-existing abundant growth of alpha streptococci. Antibiotic therapy for greater than 3 days was associated with the isolation of KEC in stool and GNB in the throat, but birth weight less than 2,500 gm and lack of breast milk feedings were not.

Boston↗
PubMedDate not supplied

Royal Society of Tropical Medicine and Hygiene meeting at Manson House, London, 14 December 1995. Enteropathogenic Escherichia coli--mucosal infection models.

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-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 and C. rodentium. In this study we found similar ultrastructural damage in small intestinal biopsies from an EPEC-infected child and large bowel specimens from C. rodentium-infected mice. The C. rodentium-infected large bowel biopsies revealed massive hyperplastic reactions and the infected human small intestinal biopsies showed an increase in total crypt cell number and mitotic index. EPEC-infected small intestinal organ cultures revealed bacteria adhering in a localized pattern and evidence of A/E lesions. Covaspheres coated with a biologically active cell-binding domain of intimin also adhered to cells in a localized fashion but did not induce the characteristic A/E lesions.

Adhesins, Bacterial↗
PubMed2003

Evaluation of increased incubation temperature and cefixime-tellurite treatment for the isolation of Escherichia coli O157:H7 from minced beef.

To improve enrichment and isolation of Escherichia coli O157:H7, this study evaluated increased incubation temperature and cefixime-tellurite (CT) on five strains of each of the following bacteria, E. coli, Hafnia alvei, Enterobacter spp., Citrobacter freundii and E. coli O157:H7, and two strains of E. coli O157:nH7. These were grown in pure culture in LST broth with varying cefixime-tellurite concentrations. A range of incubation temperatures from 37 to 46 degrees C was investigated for the inhibition of cohabitant microorganisms. Minced beef, spiked with E. coli O157:H7 and cohabitant microorganisms was investigated. Increased incubation temperature (42 degrees C) and treatment with half of the prescribed amount of cefixime-tellurite by BAM for SMAC agar in enrichment step were the most effective in selectively growing E. coli O157:H7. The results show that E. coli O157:H7 is more resistant to these two conditions than the other cohabitant bacteria.

Animals↗