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PubMed1995

The nickel resistance determinant cloned from the enterobacterium Klebsiella oxytoca: conjugational transfer, expression, regulation and DNA homologies to various nickel-resistant bacteria.

Klebsiella oxytoca strain CCUG 15788, isolated from a mineral oil emulsion tank in Göteborg, Sweden, was found to be nickel-resistant (tolerating 10 mM NiCl2 in non-complexing mineral-gluconate media; inducible resistance). The nickel resistance determinants were transferred by helper-assisted conjugation to various strains of Escherichia coli and Citrobacter freundii and expressed to between 5 and 10 mM NiCl2. A 4.3 kb HindIII fragment was cloned from the genomic DNA of K. oxytoca. Ligated into the vector pSUP202, the fragment caused constitutive nickel resistance (of up to 3 or 10 mM Ni2+) in various E. coli strains. After cloning into the broad host range vector pVDZ'2 the fragment even expressed low nickel resistance in the transconjugant of Alcaligenes eutrophus AE104. With the 4.3 kb HindIII fragment as a biotinylated DNA probe it was shown by DNA-DNA hybridization that the nickel resistance determinant resides on the chromosome of K. oxytoca and not on its circular plasmid pKO1 (160 kb) or linear plasmid pKO2 (50 kb). Nickel resistance strongly correlated with the presence of the 4.3 kb HindIII fragment in the transconjugants. No homologies were detected when the nickel resistance determinants of other well-known nickel-resistant bacteria, such as A. eutrophus CH34 or A. denitrificans 4a-2, were used as target DNA. Among the 60 strains examined, positive signals only appeared with the 3.1 kb DNA fragment from A. xylosoxydans 31A and the genomic DNA of two enterobacterial strains (5-1 and 5-5) isolated from nickel-rich soil in New Caledonia.

Cloning, Molecular↗
PubMed1991

DNA sequence of a Salmonella-specific DNA fragment and the use of oligonucleotide probes for Salmonella detection.

Hybridization specificity of a 1.8-kb HindIII DNA fragment isolated from Salmonella typhimurium by a molecular cloning technique was confirmed by colony hybridization with 327 Salmonella isolates of various serotypes and 56 non-Salmonella isolates including Enterobacteriaceae closely related to Salmonella, such as Escherichia coli, Klebsiella, Citrobacter and Shigella. It was found that this 1.8-kb DNA fragment was highly specific for all the Salmonella isolates tested. The DNA sequence of this 1.8-kb fragment was then determined by the dideoxynucleotide chain termination method. According to this DNA sequence, six oligonucleotide fragments ranging from 17- to 26-mer were then chemically synthesized and tested for their hybridization specificities. Results show that three of the six oligonucleotide fragments are highly specific for all 327 Salmonella strains tested and can be used as probes for the specific detection of Salmonella in foods or other samples.

Base Sequence↗
PubMed1990

Hemolysin as a marker for Serratia.

All Serratia marcescens strains (total of 33) of different sources were hemolytic including clinical strains previously classified as being nonhemolytic. DNA fragments of the two hemolysin genes hybridized with the chromosomal DNA of S. marcescens, S. liquefaciens, S. kiliensis, S. grimesii, S. proteamaculans, S. plymutica, S. rubridaea which were also hemolytic. The restriction pattern of the hemolysin locus differed in each strain. S. ficaria and S. marinorubra expressed a different hemolysin which was much smaller than the S. marcescens hemolysin since it diffused through dialysis membranes. The DNA of the latter strains did not hybridize with the S. marcescens hemolysin DNA probes. Some S. marcescens strains, S. kiliensis and S. liquefaciens also expressed in addition the small hemolysin. No hybridization was found with DNA of Escherichia coli, Salmonella typhimurium, Proteus mirabilis, Proteus vulgaris, Citrobacter freundii, Enterobacter cloacae, Klebsiella arerogenes, Klebsiella pneumoniae, Shigella dysenteriae, Yersinia enterocolitica, Yersinia pseudotuberculosus, Listeria sp., Aeromonas sp., Legionella sp. and a Meninococcus sp., indicating that the hemolysin DNA probes are specific for Serratia, or that the hemolysin genes occur rarely in genera other than Serratia.

Autoradiography↗
PubMed1995

SlyA, a regulatory protein from Salmonella typhimurium, induces a haemolytic and pore-forming protein in Escherichia coli.

A chromosomal fragment from Salmonella typhimurium, when cloned in Escherichia coli, generates a haemolytic phenotype. This fragment carries two genes, termed slyA and slyB. The expression of slyA is sufficient for the haemolytic phenotype. The haemolytic activity of E. coli carrying multiple copies of slyA is found mainly in the cytoplasm, with some in the periplasm of cells grown to stationary phase, but overexpression of SlyB, a 15 kDa lipoprotein probably located in the outer membrane, may lead to enhanced, albeit unspecific, release of the haemolytic activity into the medium. Polyclonal antibodies raised against a purified SlyA-HlyA fusion protein identified the overexpressed monomeric 17 kDa SlyA protein mainly in the cytoplasm of E. coli grown to stationary phase, although smaller amounts were also found in the periplasm and even in the culture supernatant. However, the anti-SlyA antibodies reacted with the SlyA protein in a periplasmic fraction that did not contain the haemolytic activity. Conversely, the periplasmic fraction exhibiting haemolytic activity did not contain the 17 kDa SlyA protein. Furthermore, S. typhimurium transformed with multiple copies of the slyA gene did not show a haemolytic phenotype when grown in rich culture media, although the SlyA protein was expressed in amounts similar to those in the recombinant E. coli strain. These results indicate that SlyA is not itself a cytolysin but rather induces in E. coli (but not in S. typhimurium) the synthesis of an uncharacterised, haemolytically active protein which forms pores with a diameter of about 2.6 nm in an artificial lipid bilayer. The SlyA protein thus seems to represent a regulation factor in Salmonella, as is also suggested by the similarity of the SlyA protein to some other bacterial regulatory proteins. slyA- and slyB-related genes were also obtained by PCR from E. coli, Shigella sp. and Citrobacter diversus but not from several other gram-negative bacteria tested.

Amino Acid Sequence↗
PubMed1989

Mutagenic DNA repair genes on plasmids from the 'pre-antibiotic era'.

Resistance transfer factors are natural conjugative plasmids encoding antibiotic resistance. Some also encode mutagenic DNA repair genes giving resistance to DNA damage and induced mutagenesis. It has been shown that antibiotic resistance has been acquired by recent transposition events; however, we show here that mutagenic repair genes existed much earlier on these types of plasmids. Conjugative plasmids from eight incompatibility groups from the Murray collection of 'pre-antibiotic era' enterobacteria were tested for complementation of mutagenic repair-deficient Escherichia coli umuC36. Although none of these plasmids carry transposon-encoded drug resistance genes, IncI1 and IncB plasmids were identified which restored ultraviolet resistance and induced mutability to umuC36 mutants. Furthermore they increased the UV resistance and induced mutability of wild-type E. coli, Klebsiella aerogenes and Citrobacter intermedius, thus showing that they could confer a general selective advantage to a variety of hosts. Like known mutagenic repair genes, complementation by these plasmid genes required the SOS response of the host cell. Nucleotide hybridisation showed that these plasmids harboured sequences similar to the impCAB locus, the mutagenic repair operon of modern-day IncI1 plasmids. The evolution of mutagenic repair genes is discussed.

DNA Repair↗
PubMed1989

High efficiency transduction of single strand plasmid DNA into enteric bacteria.

This report demonstrates high efficiency transduction of enteric bacteria using single strand plasmids packaged in M13 phage capsids. Transformation by plasmid DNA is usually a very inefficient process in many enteric bacteria other than Escherichia coli K12. Plasmids carrying an M13 origin of replication can be replicated and packaged when cells carrying such plasmids are infected with M13 or a derivative helper phage. By introducing an F' plasmid into E. coli, Serratia marcescens, Citrobacter freundii, and Enterobacter aerogenes, these species can now be infected at high efficiency with M13 phage and with packaged single strand plasmids, yielding an efficient method to introduce cloned DNA fragments into these bacteria. The titer of colony forming units in a lysate was essentially equivalent in all the bacteria, demonstrating an equal efficiency of transduction of these other enteric bacteria compared to E. coli.

Bacteriophages↗
PubMed1988

A selection cartridge for rapid detection and analysis of spontaneous mutations including insertions of transposable elements in Enterobacteriaceae.

We present a method that allows positive selection and rapid analysis of mutations in Enterobacteriaceae. Mutations are detected in a 2630 bp selection cartridge inserted in two different bacterial multicopy plasmid vectors. Spontaneous mutations in Escherichia coli, Enterobacter cloacae and Citrobacter freundii include insertions, deletions and point mutations. The small size of the target sequence facilitates rapid analysis of DNA rearrangements by cleavage with restriction enzymes and of any type of mutation by DNA sequence analysis. While in E. coli insertions of the mobile elements IS1, IS2 and IS5 were readily found, insertions of putative new transposable elements were detected in Enterobacter cloacae. The selection cartridge can thus serve as a tool for studying the spectrum of insertion mutations in Enterobacteriaceae and probably other Gram-negative bacteria, and the dependency of this spectrum on physiological and environmental factors and the host's genetic background can be investigated.

Base Sequence↗
PubMed1978

Characterization of motile and acetoin-negative Klebsiella pneumoniae strains by DNA: DNA by hybridization.

Hybridization studies were carried out to determine the extent of divergence between different members of the Enterobacteriaceae and group D, previously described by numerical analysis of phenotypic characters, and comprising strains isolated from surface waters. This group, apparently belonging in or related to the genus Citrobacter on the basis of the IMViC tests differs from this taxon by numerical analysis and DNA base composition. Our work reveals a DNA relatedness of 81-89% between the centrotype strain of this group and the genus Klebsiella. Although strains of group D show positive motility and negative Voges-Proskauer reactions, a comparison of their phenotypic characters leads us to consider that the strains with a high level of hybridization within this group belong to K. pneumoniae. These results suggest a complete revision of the traditional classification scheme of the genus Klebsiella.

Acetoin↗
PubMed1978

Studies of relationship among terrestrial Pseudomonas, Alcaligenes, and enterobacteria by an immunological comparison of glutamine synthetase.

Antibody to purified glutamine synthetase from Escherichia coli was prepared and used for an immunological comparison of glutamine synthetases from species of Salmonella, Citrobacter, Enterobacter, Serratia, Proteus, Erwinia, Aeromonas, Pseudomonas, Acinetobacter, Xanthomonas, Alcaligenes, and Paracoccus. The results of Ouchterlony double diffusion experiments and quantitative microcomplement fixation studies indicated that the amino acid sequence of this enzyme was highly conserved in different organisms. The order of relationship to E. coli was found to be similar to that derived from immunological investigations of other enzymes. In addition, congruence was observed between ribosomal RNA homology and the results of the microcomplement fixation experiments. The results also suggested that some species of Alcaligenes were more closely related to species of Pseudomonas than to each other. Immunological comparisons of glutamine synthetases appear to be very useful for the elucidation of relationships among distantly related species and genera.

Alcaligenes↗
PubMed1984

Microbial aspects of the cockroach hindgut.

The cockroach hindgut has a complex, active microbiota, a large portion of which is associated with the chitinous gut wall. It provides a different environment from that of termites and other insects which are dependent on their hindgut microbiota for the digestion of cellulose. The pH of the midgut of Eublaberus posticus was not as high as it is in insects with a primarily cellulolytic nutrition. The hindgut of E. posticus was highly methanogenic, normal adults producing typically 10-25 mumol of methane per hour. The hindgut contained large amounts of the storage products polyphosphate and poly-beta-hydroxybutyrate. Dilution series on nonselective medium yielded 100 times more obligately anaerobic colonies than facultatively anaerobic colonies. The most common facultative isolates were Klebsiella oxytoca, Citrobacter freundii and Enterobacter agglomerans. Treatment of E. posticus with metronidazole caused a dedifferentiation of the different regions of the hindgut. One region of the hindgut is characterized by its visibly black color, a unique microbiota, and electron dense deposits in electron micrographs. Chemical determinations showed high concentrations of ferrous and sulfide ions in the region. X-ray microprobe analysis showed that some of the electron dense deposits consisted of iron, sulfur and lower amounts of copper, aluminium, and chromium associated with ruthenium red staining material. Spectra of other deposits revealed only silicon, which was not associated with ruthenium red.

Anaerobiosis↗
PubMed1986

Salmonellae and other enteropathogenic bacteria in the intestines of wall geckos in Nigeria.

The aerobic bacterial flora of the intestine of 150 wall geckos (Hemidactylus brookei) was investigated. A variety of bacteria was recovered including 35 isolates of Salmonella and several other species of Enterobacteriaceae, viz. Shigella sonnei - 2, Edwardsiella tarda - 4, Enterobacter spp - 8, Citrobacter freudii - 3, Serratia marcescens - 3, Proteus spp - 35, Klebsiella pneumoniae - 13, and Escherichia coli - 17, isolates. Eight Salmonella serotypes were identified, the predominant ones being S. hvittingfoss and S. typhimurium. The significance of these findings for the spread of human enteropathogens is discussed.

Animals↗
PubMed1977

Anaerobic dechlorination and degradation of hexachlorocyclohexane isomers by anaerobic and facultative anaerobic bacteria.

Screening studies with strict and facultative anaerobic bacteria showed that Clostridium app. and several other representatives of Bacillaceae and Enterobacteriaceae actively degraded gamma-hexachlorocyclohexane (gamma-HCH) under anaerobic conditions. Representatives of Lactobacillaceae and Propronibacterium were inactive. With 36Cl-labelled gamma-HCH a nearly complete dechlorination was shown to occur in 4--6 days by Clostridium butyricum, C. pasteurianum and Citrobacter freundii, while other facultative anaerobic species were less active. Aerobically grown facultative anaerobes also dechlorinated actively gamma-HCH during subsequent anaerobic incubation with glucose, pyruvate or formate as substrates. The alpha-, beta- and delta-HCH isomers were also, but more slowly, dechlorinated (gamma larger than alpha larger than beta larger than or equal to delta-HCH). All species active in anaerobic degradation of gamma-HCH formed gamma-tetrachlorocyclohexene (TCH) as the main intermediate metabolite and no gamma-pentachlorocyclohexene (PCH) or other isomers of TCH or PCH have been found. Small amounts of tri- and tetrachlorinated benzenes have been found too. The mechanism of dechlorination is discussed.

Aerobiosis↗
PubMed1982

Comparative in vitro antibacterial activity of seven semi-synthetic penicillins against aerobic gram-negative bacteria and enterococci.

The MICs and MBCs of mecillinam, ticarcillin, mezlocillin, azlocillin and piperacillin were determined by the microdilution method in liquid medium using 700 strains of gram-negative bacilli and enterococci isolated from pathological sources and classified as a function of their sensitivity to ampicillin and carbenicillin. The ampicillin and carbenicillin-sensitive strains were generally sensitive to the other penicillins, although there were differences in activity. The ampicillin and carbenicillin-resistant strains of Escherichia coli that produce a TEM-type penicillinase were sensitive to mecillinam. Mezlocillin, piperacillin and azlocillin had MICs of between 32 and 64 mg/l for 40% of these strains. The Klebsiella strains, whose broad-spectrum penicillinase deactivates ampicillin and carbenicillin, remained sensitive to mecillinam. Mezlocillin, azlocillin and piperacillin had MICs of less than 8 mg/l for 50% of these strains. The carbenicillin-resistant strains of Enterobacter and Citrobacter were also resistant to the other penicillins. Piperacillin and mezlocillin displayed some activity against certain strains of carbenicillin-resistant Serratia, Proteus and Acinetobacter. Azlocillin, piperacillin and, to a lesser degree, mezlocillin were active against the strains of Pseudomonas, for which carbenicillin had an MIC of about 512 mg/l. Ampicillin, mezlocillin and azlocillin showed the best activity against the enterococci, against which mecillinam was inactive. The MBC of these antibiotics is greatly influenced by the density of the bacterial inoculum.

Gram-Negative Aerobic Bacteria↗
PubMed1982

Complementary activity of mezlocillin and the combination of amoxicillin with clavulanic acid on Enterobacteriaceae.

The MICs of amoxicillin, mezlocillin and BRL 25,000, a combination of two parts amoxicillin and one part clavulanic acid (2AM + 1CA), were measured for 331 Enterobacteriaceae strains which produced beta-lactamases as demonstrated by nitrocefin. The MIC values for mezlocillin and the combination 2AM + 1CA were very similar for the total number of the strains investigated. When investigated separately according to the bacterial species, three different sensitivity groups were established for the above-mentioned preparations: 1) species with the same or similar sensitivity to mezlocillin and 2AM + 1CA (Escherichia coli and Shigella spp., amoxicillin-resistant strains); 2) species which were more sensitive to mezlocillin than to the combination 2AM + 1CA (Citrobacter spp., Enterobacter cloacae, Serratia spp. and indole-positive Proteus as well as strains of E. coli and Shigella spp. which produce a cephalosporinase and are sensitive to amoxicillin); 3) species which are more sensitive to 2AM + 1CA than to mezlocillin (amoxicillin-resistant Salmonella spp., Proteus mirabilis and Klebsiella pneumoniae). This complementary activity of mezlocillin and 2AM + 1CA against Enterobacteriaceae depended on the beta-lactamases produced.

Amoxicillin↗
PubMed1982

Antibacterial activity of N-formimidoyl thienamycin in comparison with cefotaxime, lamoxactam, cefoperazone, piperacillin gentamicin.

The in vitro activity of N-formimidoyl thienamycin (MK 0787), a new stable derivative of thienamycin was compared with that of lamoxactam, cefotaxime, cefoperazone, piperacillin and gentamicin against 410 clinical isolates of common bacteria. In comparison with the other agents, MK 0787 was more or equally active against Staphylococcus aureus, Streptococcus faecalis, Escherichia coli, Citrobacter spp., Klebsiella spp., and Serratia spp. It inhibited all isolates at a concentration of 0.5 mg/l, but was less active than cefotaxime and lamoxactam against Proteus mirabilis and less active than cefotaxime, lamoxactam and cefoperazone against Beta-lactamase positive Haemophilus influenzae. Strains of Pseudomonas aeruginosa were more susceptible to N-formimidoyl thienamycin than to other antibiotics.

Anti-Bacterial Agents↗
PubMed1983

In vitro activity of new cephalosporins against multi-resistant gram-negative bacteria.

The in vitro sensitivities of 47 multi-resistant gram-negative bacilli against six third generation cephalosporins were investigated. Overall, moxalactam was the most active compound and cefoperazone the least active. Ceftizoxime and ceftazidime were very active against Enterobacteriaceae. Ceftriaxone and ceftizoxime were active against Escherichia coli and Klebsiella spp., but were inactive against Pseudomonas spp.; Achromobacter sp. was resistant to all drugs, while Citrobacter freundii was sensitive to every antibiotic tested. These drugs may be clinically useful in infections with multi-resistant gram-negative bacteria.

Aeromonas↗
PubMed1978

[Efficacy, tolerance, and pharmacokinetics of sisomicin in urinary tract infections (author's transl)].

Since 1973 30 patients with urinary tract infections (UTI) or pyelonephritis have been treated with sisomicin, a new aminoglycoside, in a daily dose of 2 mg/kg for a period of seven to nine days. From a clinical point of view the result of treatment was good. Complete resolution was achieved in 17 patients, improvement in nine, and there was no effect in four patients. Thirty-five causative organisms (Escherichia coli = 23, Proteus sp. = 7, Klebsiella sp. = 3, Pseudomonas aeruginosa = 1, Citrobacter = 1) were isolated before treatment. Thirty of the organisms were eliminated during treatment, but seven reappeared during the follow-up period; five strains persisted. Side effects observed consisted of reversible increase of serum creatinine in four patients, excretion of granular casts in 14 patients, and a transient rise of alkaline phosphatase, SGOT and/or SGPT in five patients. No signs of ototoxicity or any other adverse reactions were found and local tolerance was good. In 20 patients blood samples for assay were obtained daily one hour after i. m. injection of 1.0 mg/kg. No evidence of drug accumulation in the serum was found: the mean serum concentrations one hour after injection remained between 3.4 and 3,9 microgram/ml during the entire treatment period. Sisomicin is a highly effective antibiotic for the treatment of UTI caused by gramnegative pathogens. On account of its potential toxicity however, it should be used, like other aminoglycosides, only in selected cases.

Anti-Infective Agents, Urinary↗
PubMed1978

In vitro susceptibility of gentamicin and/or tobramycin resistant gram-negative bacilli to seven aminoglycosides.

The in vitro activity of gentamicin, tobramycin, sisomicin, netilmicin, amikacin, kanamycin and streptomycin was tested simultaneously by the agar dilution method against 584 clinical isolates of gram-negative bacilli that were resistant to gentamicin and/or tobramycin. About half of the gentamicin-resistant Pseudomonas were susceptible to tobramycin but cross-resistance was virtually complete between gentamicin and tobramycin for Enterobacteriaceae. Sisomicin was much more active than gentamicin against Klebsiella, Escherichia and Citrobacter species. Only 18.9%, 27.4% and 27.9% of Klebsiella, Enterobacter and Serratia respectively were resistant to netilmicin. Amikacin was the most effective aminoglycoside with an overall resistance of 15.6%. Kanamycin was effective against 40% of Proteus and Providencia species. Surprisingly, more than half of Klebsiella and Enterobacter species and 85.3% of Serratia species were susceptible to streptomycin.

Aminoglycosides↗