Freundii Strains Research Articles

Interactions of FCE 22101 with Class I -lactamases

Hyperproduction of chromosomal Class I beta-lactamase causes resistance to newer cephalosporins in Enterobacter cloacae, Citrobacter freundii, Serratia spp. and indole-positive Proteeae. We examined the interactions of FCE 22101 with these enzymes, measuring (i) antibiotic lability to pure enzyme, (ii) inducing power and (iii) activity against beta-lactamase-inducible strains and their -depressed and -basal mutants. Turnover numbers of FCE 22101 were between 0.7 and 1.5 molecules hydrolysed/min/enzyme molecule, compared with ranges of 0.5-40 for cefotaxime (950 for the Proteus vulgaris cephalosporinase) and 4400-22,000 for cephaloridine. FCE 22101 induced enzyme synthesis strongly below the MIC and antagonized labile weak inducers such as cefotaxime and mezlocillin. Like imipenem, but unlike newer cephalosporins, FCE 22101 had equal activity against inducible strains and derepressed mutants, and did not significantly select derepressed mutants from inducible populations. beta-Lactamase basal mutants of E. cloacae and C. freundii were four- to eight-fold more sensitive than inducible and derepressed organisms whereas beta-lactamase-basal, -inducible and -derepressed Proteeae and Serratia spp. were equally sensitive. This difference reflected the larger amounts of enzyme produced by the E. cloacae and C. freundii strains, rather than kinetic differences in enzyme activity.

Production of Escherichia coli STa-like heat-stable enterotoxin by Citrobacter freundii isolated from humans.

Citrobacter species are often present in the stools of children and are generally considered a normal component of the intestinal microflora. Previous reports suggested that they might act as enteric pathogens. Aiming at defining the role of Citrobacter species in inducing diarrhea, we looked for their presence in the stools of 328 children with diarrhea and in 108 controls. Citrobacter strains were isolated from 46 patients (14%) and 7 controls (6.5%) (P less than 0.05). All isolates were tested for heat-stable (ST) and heat-labile (LT) enterotoxin. No LT-producing organisms were found. Three C. freundii strains, all isolated from children with diarrhea, elaborated an enterotoxin detected by the suckling mouse assay. A crude extract was prepared by acetone precipitation and a sequential ultrafiltration technique. The enterotoxin was heat stable, and its estimated molecular weight was between 2,000 and 10,000. Citrobacter enterotoxin was soluble in methanol and stable at acid and neutral pHs but not above pH 8, and its activity was destroyed by treatment with 2-mercaptoethanol. Citrobacter enterotoxin was inactive in the 18-h rabbit ileal loop test. All these characteristics closely resemble STa produced by Escherichia coli. The time course of Citrobacter enterotoxin-induced intestinal secretion in suckling mice was similar to that of E. coli STa. The enterotoxin produced by C. freundii cross-reacted with monoclonal antibodies raised against E. coli STa. These results suggest that C. freundii is capable of inducing diarrhea through the production of an E. coli-like STa, and its presence in the stools of patients with diarrhea should be considered as that of a possible etiologic agent.

Naturally occurring plasmid coding for heat-labile enterotoxin production and drug resistance from Escherichia coli strain of porcine origin.

A large plasmid of approximately 120 megadaltons (Md) in size isolated from an enterotoxigenic strain of Escherichia coli of porcine origin was found to carry the genes for heat-labile enterotoxin production and drug resistance against tetracycline and kanamycin. This plasmid was non-conjugative itself, but was transmissible to other recipient organisms such as E. coli K-12 and Citrobacter freundii strains simultaneously in the presence of a 60-Md plasmid.

Specific identification of Salmonella serogroup E antigen O3 by immunofluorescence and coagglutination with antiserum elicited by a synthetic trisaccharide-bovine serum albumin glycoconjugate.

Antiserum specific for Salmonella O3 antigen was raised by immunization of rabbits with an artificial glycoconjugate consisting of the synthetic trisaccharide beta-D- Manp (1----4)-alpha-L- Rhap (1----3)-alpha-D-Galp covalently linked to bovine serum albumin (beta- MRG -BSA). Enzyme immunoassays showed that only lipopolysaccharides extracted from Salmonella serogroup E (O3 antigen-containing) bacteria bound the antiserum. The usefulness of the beta- MRG -BSA antiserum for rapid and accurate identification of Salmonella isolates of serogroup E was shown by indirect immunofluorescence tests in which 50 Salmonella strains of serogroup E 1-4 were correctly identified from among 651 intestinal strains investigated. The finding that one strain of Citrobacter freundii was positive in immunofluorescence tests with this antiserum is readily explained by the known cross-reactivity between some C. freundii strains and Salmonella spp. strains of serogroup E. As expected, the specificity of the antiserum for the O3 antigen could further be demonstrated in coagglutination tests with staphylococci sensitized with beta- MRG -BSA antiserum.

Efficacy of BRL 25000 against Serratia marcescens, Enterobacter cloacae, and Citrobacter freundii in urinary tract infections

Synergism between amoxicillin and clavulanic acid was not expected against cephalosporinase-producing bacterial strains because clavulanic acid has little inhibitory action on cephalosporinases. However, in a clinical trial of BRL 25000 (amoxicillin-clavulanic acid), excellent results were obtained in complicated urinary tract infections caused by Serratia marcescens, Enterobacter cloacae, and Citrobacter freundii strains which produced cephalosporinase and were highly resistant to amoxicillin alone. The good clinical efficacy of BRL 25000 in such urinary tract infections was probably due to the fact that the urinary concentration of clavulanic acid was higher than its minimal inhibitory concentrations for these strains.

Study of the efficacy and tolerance of co-trimazine given once or twice daily in urinary tract infections

Forty-one patients with either complicated or uncomplicated urinary tract infections were treated with co-trimazine tablets (410 mg sulphadiazine, 90 mg trimethoprim). Ten were given one tablet once daily, ten were given two tablets once daily and 21 were given one tablet twice daily. 88% were cured. The results were independent of the dosage interval, but depended on the type of infection. Eight patients suffered a reinfection. Two of the sevenE. coli strains isolated were resistant to sulphadiazine and one was resistant to sulphadiazine and trimethoprim. TwoCitrobacter freundii strains were resistant to sulphadiazine and trimethoprim. There was a relapse in five cases in which all isolates were sensitive to sulphadiazine and trimethoprim. There were no subjective side-effects. Eosinophilia was observed in two patients and transitory leucopenia in another two. One case of eosinophilia and one case of transitory leucopenia could possibly be related to the medication.

Antimicrobial susceptibility patterns (antibiograms) as an aid in differentiating Citrobacter species.

The hydrogen sulfide-negative Citrobacter group represents a taxonomic problem. Various investigators have proposed such designations as Padlewskia, Levinea, atypical Enterobacter cloacae, H2S-negative variants of Citrobacter, Citrobacter koseri and Citrobacter diversus. This problem has been investigated with emphasis on antibiograms as a means of discrimination. Clinical isolates fitting the designation Citrobacter were studied and, using the criteria of Ewing and Davis, separated into two groups: C. diversus (40 strains) or C. freundii (25 strains). Susceptibilities to ampicillin, carbenicillin, cefazolin, cephaloridine and cephalothin were determined by the agar-dilution method. C. diversus strains were resistant to 8 mug/ml ampicillin (97.5%) and 32 mug/ml carbenicillin (87.5%), and were susceptible to 8 mug/ml cephalosporins (greater than or equal to 90%). C. freundii strains were moderately susceptible to 8 mug/ml ampicillin (25%) and susceptible to 8 mug/ml carbenicillin (92%), and were resistant to 8 mug/ml cephalosporin (greater than or equal 92%). Using these data one can separate C. diversus from C. freundii with 90% accuracy.

A serological study of strains belonging to Sphaerophorus necrophorus, Sph. varius, and Sph. freundii.

The serological behaviour of 12 strains belonging toSph. necrophorus,Sph. varius, andSph. freundii was studied in cross-agglutination and gel-diffusion experiments using rabbit antisera against whole-cell adjuvant-free antigens. Owing to the spontaneous agglutinability ofSph. necrophorus suspensions, cross-agglutination could only be performed withSph. varius andSph. freundii antigens. In gel-diffusion studies, autoclaved extracts ofSph. necrophorus cultures reacted exclusively with their homologous antisera, whereas intraspecific reciprocal cross-reactions were due to non-heated extracts. Intraspecific cross-reactivity ofSph. varius andSph. freundii strains too was mostly dependant on thermolabile antigens, though the autoclaved extracts of these species gave similarly overlapping reactions. Unilateral interspecific cross-reactions related aSph. necrophorus culture and 3Sph. varius strains to theSph. freundii group. The existence of a close serologic relationship between members of the sameSphaerophorus species supports the biochemical differentiation proposed in a previous communication.

Interaction of various bacteriocins with Klebsiella edwardsii var. edwardsii.

The interaction of four different bacteriocins produced byKlebsiella pneumoniae andCitrobacter freundii strains with cells ofKlebsiella edwardsii var.edwardsii has been studied. All four bacteriocins have different activity spectra. The existence of multi-tolerant and multi-receptor-negative mutants supports the hypothesis that the specific receptor sites for these bacteriocins on sensitive bacteria have some components in common.

Delayed Lactose Fermentation byEnterobacteriaceae

Goodman, R. E. (University of California, Los Angeles), and M. J. Pickett. Delayed lactose fermentation by Enterobacteriaceae. J. Bacteriol. 92:318-327. 1966.-When 171 Citrobacter freundii strains and 14 Paracolobactrum arizonae strains examined, 51 of the C. freundii strains and 13 of the P. arizonae strains were found to be delayed or negative lactose fermenters. Of the slow fermenters, 65% yielded rapidly fermenting mutants in cultures undergoing delayed fermentation. Lactose fermentation could generally be hastened by increasing lactose concentrations. Many organisms which fermented lactose slowly grew readily on a medium containing lactose as the sole carbon source. Regardless of their ability to ferment lactose, all strains of C. freundii and P. arizonae investigated could be shown to possess beta-galactosidase. Delayed fermenters failed to take up lactose from the culture medium, whereas prompt fermenters did so readily. The beta-galactosidases of 12 strains of enteric bacteria were studied in crude cell extracts with respect to specific activity, stability, and activity at varying substrate (o-nitrophenyl-beta-d-galactopyranoside) concentrations, at varying pH, and in the presence of sodium, potassium, and magnesium. The widely varying specific activities and the approximate similarity of the Michaelis constants (about 2 x 10(-4)m) suggested that the strains investigated produced differing amounts of beta-galactosidase. Moreover, qualitative differences in the enzymes provided evidence that these strains synthesized different molecular forms of beta-galactosidase. The results suggested that organisms which ferment lactose only after a prolonged delay do so because they possess multiple defects in their lactose-metabolizing machinery.

Sensitivity to dihydrostreptomycin of Enterobacteriaceae strains on media containing ammonium.

IN the course of investigations of the fertility of some strains of Enterobacteriaceae, it was observed that Citrobacter (E. freundii) strains grew well on minimal medium plates supplemented with galactose 1 per cent and dihydrostreptomycin 100 µgm./ml. On the same medium, E. coli strains showed no growth. The Citrobacter strains were isolated during 1942–43 before the ‘streptomycin era’. On complete medium with the same amount of dihydrostreptomycin, both Citrobacter and E. coli strains were completely inhibited. Even though an explanation of this phenomenon has not been reached as yet, a brief report on the results obtained so far will be given here.