Rolf Reissbrodt

Evaluation of new broth media for microdilution antibiotic susceptibility testing of lactobacilli, pediococci, lactococci, and bifidobacteria

ABSTRACT Nine pure or mixed broth media were evaluated for their suitabilities to determine MICs in a microdilution test of 19 antibacterial agents for lactic acid bacteria (LAB) of the genera Lactobacillus, Pediococcus, Lactococcus, and Bifidobacterium. A mixed formulation of Iso-Sensitest broth (90%) and deMan-Rogosa-Sharpe broth (10%) with or without supplementation with l-cysteine, referred to as the LAB susceptibility test medium, provided the most optimal medium basis in terms of growth support of nonenterococcal LAB and correct indication of MICs of international control strains.

Role of Receptor Proteins for Enterobactin and 2,3-Dihydroxybenzoylserine in Virulence of Salmonella enterica

ABSTRACT Single, double, and triple mutants of an enterobactin-deficient mutant strain of Salmonella enterica serovar Typhimurium were constructed that were defective in the expression of the iron-regulated outer membrane proteins (IROMPs) FepA, IroN, and Cir, which are proposed to function as catecholate receptors. Uptake of naturally occurring and chemically synthesized catecholate molecules by these mutants was assessed in standard growth promotion assays. Unique patterns of uptake were identified for each IROMP; specifically, FepA and IroN were confirmed to be required for transport of enterobactin, and all three proteins were shown to function as receptors for the enterobactin breakdown product 2,3-dihydroxybenzoylserine. The fepA, iroN, and cir alleles were transduced to enterobactin-proficient strains of S. enterica serovar Typhimurium and S. enterica serovar Enteritidis, and the resulting phenotypes were confirmed by analysis of outer membrane protein profiles, by sensitivity to KP-736, a catecholate-cephalosporin conjugate, and by growth promotion tests on egg white agar. Intragastric infections of mice with the S. enterica serovar Typhimurium strains indicated that the parental strain and the fepA iroN double mutant were similarly virulent but that the fepA iroN cir triple mutant was significantly attenuated. Moreover, in mixed infections, the fepA iroN mutant showed similar cecal colonization and invasion of the liver to the parental strain, while the triple mutant showed significantly reduced cecal colonization and no measurable spread to the liver. Infections of 4-day-old chicks with S. enterica serovar Enteritidis strains also indicated that mutation of the fepA iroN genes did not significantly reduce cecal colonization and systemic spread compared with those of the parental strain. The results indicate that, while enterobactin uptake is not essential for the virulence of S. enterica serovars in mouse and chicken infection models, the ability to take up 2,3-dihydroxybenzoylserine via any of the three catecholate siderophore receptors appears to play an important role, since the S. enterica serovar Typhimurium triple mutant was significantly attenuated in the mouse model. Salmochelins appear not to be involved in the virulence of S. enterica.

Further differentiation of enterobacteriaceae by means of siderophore-pattern analysis

By means of a combination of 5 siderophore bioassays using several indicator strains, genera, species and subspecies of Enterobacteriaceae can be further differentiated. Enterobactin, aerobactin and other siderophores produced can be detected. Each strain shows specific pattern which we called siderophore-pattern. It is easy to separate Morganella, Proteus, Providencia, Yersinia strains from the genera Salmonella, Shigella, Escherichia coli, Enterobacter, Citrobacter, Klebsiella, Serratia. Enterobacter agglomerans strains differ from other Enterobacter species with respect to their siderophore pattern. In Salmonella strains there are differences between the subspecies I and IV. Additionally the most strains of Salmonella subspecies I from nosocomial infections produced aerobactin, in the most cases determined by plasmids. Among Shigella strains different siderophore pattern exist according to other epidemiological markers. S. flexneri strains of serovar 6 produced contrary to the strains of other serovars enterobactin. By means of the siderophore-pattern analysis E. coli strains of serovars 01; 02; 018 can be further differentiated. E. coli 01:K1 strains containing the fimbrial antigen F11 produced aerobactin whereas the F9 strains did not. All Hafnia alvei strains produced a uniform siderophore pattern, different from all other members of the enterobacteriaceae family. With this aim Hafnia alvei strains can be easily separated under practical conditions.

High Prevalence of Multidrug Resistance and Random Distribution of Mobile Genetic Elements Among Uropathogenic Escherichia coli (UPEC) of the Four Major Phylogenetic Groups

One hundred and ten UTI Escherichia coli strains, from Ljubljana, Slovenia, were analyzed for antibiotic resistances, mobile DNA elements, serotype, and phylogenetic origin. A high prevalence of drug resistance and multidrug resistance was found. Twenty-six percent of the isolates harbored a class 1 integron, while a majority of the strains (56%) harbored rep sequences characteristic of F-like plasmids. int as well as rep sequences were found to be distributed in a random manner among strains of the four major phylogenetic groups indicating that all groups have a similar tendency to acquire and maintain mobile genetic elements frequently associated with resistance determinants.

Inhibition of growth of Shiga toxin‐producing Escherichia coli by nonpathogenic Escherichia coli

During routine quality control testing of diagnostic methods for Shiga toxin-producing Escherichia coli (STEC) using stool samples spiked with STEC, it was observed that the Shiga toxin could not be detected in 32 out of 82 samples tested. Strains of E. coli isolated from such stool samples were shown to be responsible for this inhibition. One particular isolate, named E. coli 1307, was intensively studied because of its highly effective inhibitory effect; this strain significantly reduced growth and Shiga toxin levels in coculture of several STEC strains regardless of serovar or Shiga toxin type. The probiotic E. coli Nissle 1917 inhibited growth and reduced Shiga toxin levels in STEC cultures to an extent similar to E. coli 1307, but commensal E. coli strains and several other known probiotic bacteria (enterococci, Bacillus sp., Lactobacillus acidophilus) showed no, or only small, inhibitory effects. Escherichia coli 1307 lacks obvious fitness factors, such as aerobactin, yersiniabactin, microcins and a polysaccharide capsule, that are considered to promote the growth of pathogenic bacteria. We therefore propose strain E. coli 1307 as a candidate probiotic for use in the prevention and treatment of infections caused by STEC.

Population structure of Francisella tularensis.

We have sequenced fragments of five metabolic housekeeping genes and two genes encoding outer membrane proteins from 81 isolates of Francisella tularensis, representing all four subspecies. Phylogenetic clustering of gene sequences from F. tularensis subsp. tularensis and F. tularensis subsp. holarctica aligned well with subspecies affiliations. In contrast, F. tularensis subsp. novicida and F. tularensis subsp. mediasiatica were indicated to be phylogenetically incoherent taxa. Incongruent gene trees and mosaic structures of housekeeping genes provided evidence for genetic recombination in F. tularensis.

Isolation and identification of ferrioxamine G and E inHafnia alvei

SummaryUnder conditions of iron-deprivationHafnia alvei (Enterobacteriaceae) produces ferrioxamine G as the principal siderophore. Maximum hydroxamate siderophore production occurred at medium iron limitation. The ferrioxamines were extracted, purified by gel filtration and chromatography on silica gel yielding a major and a minor siderophore fraction. The minor siderophore fraction contained three siderophores, among which ferrioxamine E could be identified by HPLC and FAB mass spectrometry. Reductive hydrolysis of the ferrioxamine G fraction yielded succinic acid and a mixture of diaminopentane and diaminobutane, as determined by gas-liquid chromatography and GLC/MS. HPLC and FAB mass spectrometry confirmed that the ferrioxamine G fraction consisted of two different species, G1 and G2, possessing molecular masses of 671 Da and 658 Da respectively.

SELECTIVE GROWTH PROMOTION AND GROWTH INHIBITION OF GRAM-NEGATIVE AND GRAM-POSITIVE BACTERIA BY SYNTHETIC SIDEROPHORE-BETA -LACTAM CONJUGATES

Conjugates of a carbacephalosporin with hydroxamate, spermexatol, N,N-bis(2,3-dihydroxybenzoyl)-L-lysine, mixed catecholate/hydroxamate and cyanuric acid-based siderophores were investigated for their potential to promote growth of siderophore indicator strains of Gram-negative and Gram-positive bacteria under iron depleted conditions, for their antibacterial activity and for their ability to use iron transport path-ways to penetrate the Gram-negative bacterial outer membrane. The selective growth promotion of enter-obacterial and pseudomonas strains by hydroxamate, spermexatol and mixed catecholate-hydroxamate siderophore-based conjugates bearing a L- or D-amino acid spacer was correlated with TonB dependent uptake routes. The preferred outer membrane siderophore receptor used in Escherichia coli was found to be Fiu, followed by Cir. Antagonistic effects of siderophores administered with the conjugates to determine antibacterial activity confirmed the active transport of conjugates via siderophore receptors. All of the conjugates were still able to diffuse through the porin proteins OmpC and OmpF. Nevertheless, strong inhibition of E. coli and Pseudomones aeruginosa outer membrane mutants DC2 and K799/61 compared to the parent strains indicated inefficient penetrability of all types of conjugates tested. Mycobacterium smegmatis SG 987 was able to use all of the siderophore-cephalosporin conjugates as growth promotors. Consequently there was no growth inhibition of this strain.

Iron uptake and intracellular metal transfer in mycobacteria mediated by xenosiderophores

Growth promotion was tested using M. smegmatis wild type strain, an exochelin-deficient mutant, and M. fortuitum employing a broad variety of xenosiderophores including hydroxamates, catecholates and a-hydroxy carboxylic acids. The experiments revealed that utilization of siderophore-bound iron is substrate specific suggesting high-affinity siderophore receptor and transport systems. Concentration-dependent uptake of a selected xenosiderophore (fericrocin) in M. smegmatis showed saturation kinetics and uptake was inhibited by respiratory poisons. In situ Mössbauer spectroscopy of ferricrocin uptake in M. smegmatis indicated rapid intracellular reductive removal of the metal excluding intracellular ferricrocin accumulation. The ultimate intracellular iron pool is represented by a compound (δ = 0.43 mm s, DE = 1.03 mm s) which has also been found in many other microorganisms and does not represent a bacterioferritin, cytochrome or iron-sulfur cluster. By contrast, iron uptake via citrate - a compound exhibiting a very low complex stability constant - involves ligand exchange with mycobactin. Mycobactin has merely a transient role. The ultimate storage compound is an E.coli-type bacterioferritin, in which over 90% of cellular iron is located.

Ferrioxamine E-supplemented pre-enrichment and enrichment media improve various isolation methods for Salmonella

Supplementation of pre-enrichment broth and enrichment broth media with ferrioxamine E (1 microgram/ml) significantly improved the recovery of Salmonella from artificially or naturally contaminated foods. Based on the selectivity of ferrioxamine E, Salmonella enteritidis and S. typhimurium could be isolated also from various mixed cultures (one Salmonella cell in 10(3)-10(4)-fold concentration of cells of competitors) by shaking for 6 h in supplemented buffered peptone water followed by cultivation on XLD- or XLT-4 agars. Isolation of Salmonella from these pre-enrichment cultures by use of Dynabeads-Anti-Salmonella was highly effective. 27 S. typhimurium strains were isolated from 762 naturally infected chicken giblets by use of unsupplemented Tetrathionate broth. However, 33 S. typhimurium isolates were obtained with ferrioxamine E-supplemented Tetrathionate broth from the same samples. Three Salmonella isolates out of 50 evenly divided meat meal samples were obtained by use of ferrioxamine E-supplemented buffered peptone water followed by direct streaking onto XLD- and Rambach agars, no Salmonella isolates could be detected by the conventional method.