Microcin-mediated interactions between Klebsiella pneumoniae and Escherichia coli strains

Amensal indirect interactions between a Klebsiella pneumoniae microcin-producing strain and several Escherichia coli strains, all of intestinal origin, were studied. Mixed batch cultures of both microcin-producing and microcin-sensitive strains showed that microcin production and excretion into the medium allowed the producer strain to prevail over sensitive strains, even when initial competition conditions were highly unfavourable for the producer. Mixed cultures also showed the production of a microcin-antagonist by the same microcin-producing strain when the nutrients in the medium had been depleted. The antagonist apparently promoted the viability of sensitive cells already damaged by microcin. These results have likely ecological implications.     

Antibacterial activity evaluation of microcin J25 against diarrheagenic Escherichia coli

The inhibitory activities of known microcins were evaluated against some diarrheagenic Escherichia coli strains. Some antibacterial properties of microcin J25, the most active one, were studied. A rapid two-step purification was performed. The MIC and the minimum bactericidal concentration of J25 against E. coli O157:H7 were 1 and 100 microg ml(-1), respectively. A 10(4)-CFU ml(-1) contamination by this strain was destroyed in milk and meat extract by 6.25 microg of J25 ml(-1) and in half-diluted egg yolk by 50 microg of J25 ml(-1).     

A Fusobacterium mortiferum strain produces a bacteriocin-like substance(s) inhibiting Salmonella enteritidis

Seven strictly anaerobic strains showing anti-Salmonella enteritidis activity were isolated from poultry caecal contents. Among them, the most inhibitory one, a strain of Fusobacterium mortiferum, called FM1025, was selected. Biochemical tests, showing the proteinaceous structure of the antagonist(s) produced, indicated that the strain Fus. mortiferum FM1025 synthesized (a) bacteriocin-like compound(s) active against Salm. enteritidis. Among the other strains tested, both Gram-negative and Gram-positive bacteria were inhibited. These preliminary results suggested the important role of the Fusobacterium strains against pathogenic bacteria among the intestinal flora.     

In vitro inhibition activity of different bacteriocin-producing Escherichia coli against Salmonella strains isolated from clinical cases

Aims:  To compare in vitro the inhibitory activity of four bacteriocin-producing Escherichia coli to a well-characterized panel of Salmonella strains, recently isolated from clinical cases in Switzerland.
Methods and Results:  A panel of 68 nontyphoidal Salmonella strains was characterized by pulsed-field gel electrophoresis analysis and susceptibility to antibiotics. The majority of tested strains were genetically different, with 40% resistant to at least one antibiotic. E. coli Mcc24 showed highest in vitro activity against Salmonella (100%, microcin 24), followed by E. coli L1000 (94%, microcin B17), E. coli 53 (49%, colicin H) and E. coli 52 (21%, colicin G) as revealed using a cross-streak activity assay.
Conclusions:  Escherichia coli Mcc24, a genetically modified organism producing microcin 24, and E. coli L1000, a natural strain isolated from human faeces carrying the mcb -operon for microcin B17-production, were the most effective strains in inhibiting in vitro both antibiotic resistant and sensitive Salmonella isolates.
Significance and Impact of the Study:  Due to an increasing prevalence of antibiotic resistant Salmonella strains, alternative strategies to fight these foodborne pathogens are needed. E. coli L1000 appears to be a promising candidate in view of developing biotechnological alternatives to antibiotics against Salmonella infections.     

The activity of microcin E492 from Klebsiella pneumoniae is regulated by a microcin antagonist

Abstract Microcin E492 is a polypeptide antibiotic that is produced and excreted by Klebsiella pneumoniae . Different growth conditions of the producer strain affect microcin activity. The production of a microcin antagonist is responsible for the changes in microcin activity. The microcin antagonist is induced when cells are iron-deprived, resulting in a low microcin activity. The microcin antagonist was purified using a procedure developed for the isolation of a catechol-type siderophore, and its activity was titrated using purified microcin. The inhibitory effect of the microcin antagonist is not observed when this compound is forming a complex with iron. The same inhibitory effect on microcin activity was obtained using purified enterochelin from Escherichia coli . The microcin antagonist was identified as enterochelin through thin-layer chromatography.     

Inhibition of Salmonella enterica serovars by microcin J25

Escherichia coli microcin J25 (MccJ25) is a 2107-Da peptide antibiotic whose uptake into E. coli is mediated by the outer-membrane receptor FhuA and the inner membrane proteins TonB, ExbB, ExbD, and SbmA. A survey of the sensitivity of several Salmonella enterica serovars showed that the antibiotic was highly active against some serovars, while S. Typhimurium, S. Derby, and some S. Enteritidis strains were completely resistant. Resistant strains became hypersensitive to MccJ25 when given the fhuA gene of E. coli, indicating that insensitivity is due to the inability of the FhuA protein to mediate penetration of MccJ25. Whereas in E. coli MccJ25 targets RNA polymerase, in S. Typhimurium it inhibits not only RNA synthesis but also cell respiration. Fluorescence viability staining showed that S. Typhimurium cells exposed to MccJ25 remain viable but are unable to form colonies.     

Antagonistic effects of intestinal Lactobacillus isolates on pathogens of chicken

L.Z. JIN, Y.W. HO, N. ABDULLAH, M.A. ALI AND S. JALALUDIN. 1996. Twelve Lactobacillus strains isolated from chicken intestine, which demonstrated a strong and moderate capacity to adhere to the ileal epithelial cells in vitro , were used to investigate their inhibitory ability against five strains of salmonella, i.e. Salmonella enteritidis 935/79, Salm. pullorum, Salm. typhimurium, Salm. blockley and Salm. enteritidis 94/448, and three serotypes of Escherichia coli , viz. E. coli O1 : K1, O2 : K1 and O78 : K80. The results showed that all the 12 Lactobacillus isolates were able to inhibit the growth of the five strains of salmonella, and the three strains of E. coli in varying degrees. Generally, they were more effective in inhibiting the growth of salmonella than E. coli . Inhibition of the pathogenic bacteria was probably due to the production of organic acids by the Lactobacillus isolates.     

The cyclic structure of microcin J25, a 21-residue peptide antibiotic from Escherichia coli.

Microcin J25 (MccJ25) is the single representative of the immunity group J of the microcin group of peptide antibiotics produced by Enterobacteriaceae. It induces bacterial filamentation in susceptible cells in a non-SOS-dependent pathway [R. A. Salomon and R. Farias (1992) J. Bacteriol. 174, 7428-7435]. MccJ25 was purified to homogeneity from the growth medium of a microcin-overproducing Escherichia coli strain by reverse-phase HPLC. Based on amino acid composition and absolute configuration determination, liquid secondary ion and electrospray mass spectrometry, extensive two-dimensional NMR, enzymatic and chemical degradations studies, the structure of MccJ25 was elucidated as a 21-residue peptide, cyclo(-Val1-Gly-Ile-Gly-Thr- Pro-Ile-Ser-Phe-Tyr-Gly-Gly-Gly-Ala-Gly-His-Val-Pro-Glu-Tyr-Phe21- ). Although MccJ25 showed high resistance to most of endoproteases, linearization by thermolysin occurred from cleavage at the Phe21-Val1 bond and led to a single peptide, MccJ25-L. While MccJ25 exhibited remarkable antibiotic activity towards Salmonella newport and several E. coli strains (minimal inhibitory concentrations ranging between 0.01 and 0.2 microgram.mL-1), the thermolysin-linearized microcin showed a dramatic decrease of the activity, indicating that the cyclic structure is essential for the MccJ25 biological properties. As MccJ25 is ribosomally synthesized as a larger peptide precursor endowed with an N-terminal extremity, the present study shows that removal of this extension and head-tail cyclization of the resulting propeptide are the only post-translational modifications involved in the maturation of MccJ25, that appears as the first cyclic microcin.     

Identification and characterization of microcin S, a new antibacterial peptide produced by probiotic Escherichia coli G3/10

Escherichia coli G3/10 is a component of the probiotic drug Symbioflor 2. In an in vitro assay with human intestinal epithelial cells, E. coli G3/10 is capable of suppressing adherence of enteropathogenic E. coli E2348/69. In this study, we demonstrate that a completely novel class II microcin, produced by probiotic E. coli G3/10, is responsible for this behavior. We named this antibacterial peptide microcin S (MccS). Microcin S is coded on a 50.6 kb megaplasmid of E. coli G3/10, which we have completely sequenced and annotated. The microcin S operon is about 4.7 kb in size and is comprised of four genes. Subcloning of the genes and gene fragments followed by gene expression experiments enabled us to functionally characterize all members of this operon, and to clearly identify the nucleotide sequences encoding the microcin itself (mcsS), its transport apparatus and the gene mcsI conferring self immunity against microcin S. Overexpression of cloned mcsI antagonizes MccS activity, thus protecting indicator strain E. coli E2348/69 in the in vitro adherence assay. Moreover, growth of E. coli transformed with a plasmid containing mcsS under control of an araC PBAD activator-promoter is inhibited upon mcsS induction. Our data provide further mechanistic insight into the probiotic behavior of E. coli G3/10.     

Exoproducts of the Escherichia coli strain H22 inhibiting some enteric pathogens both in vitro and in vivo

AIMS: The antagonistic activity of the Escherichia coli strain H22 against enteric bacteria was studied both in vitro and in vivo. METHODS AND RESULTS: In vitro, bacterial strains belonging to seven of nine genera of the family Enterobacteriaceae (Enterobacter, Escherichia, Klebsiella, Morganella, Salmonella, Shigella and Yersinia) were inhibited by the strain H22. Six days after simultaneous oral inoculation in germ-free mice, E. coli strain H22 reduced the faecal population of Shigella flexneri 4 to undetectable levels (P < 0.05). In ex vivo assay, inhibitory zones against Sh. flexneri 4 were observed around faecal samples from mice inoculated with E. coli strain H22. The in vitro inhibition of Sh. flexneri 4 was shown to be mediated by microcin C7. In addition to microcin C7, strain H22 was shown to produce aerobactin, new variants of colicins E1 and Ib, and bacteriophage particles with morphology similar to the phages of the family Myoviridae. CONCLUSIONS: Altogether, the properties of E. coli H22, observed both under in vitro and in vivo conditions, suggest its potential use as a probiotic strain for livestock and humans. SIGNIFICANCE AND IMPACT OF THE STUDY: The strain H22 was shown to produce several antimicrobial compounds with inhibitory capabilities against pathogenic or potentially pathogenic enterobacteria.     

Antibacterial Activity Evaluation of Microcin J25 against Diarrheagenic Escherichia coli

The inhibitory activities of known microcins were evaluated against some diarrheagenic Escherichia coli strains. Some antibacterial properties of microcin J25, the most active one, were studied. A rapid two-step purification was performed. The MIC and the minimum bactericidal concentration of J25 against E. coli O157:H7 were 1 and 100 μg ml⁻¹, respectively. A 10⁴-CFU ml⁻¹ contamination by this strain was destroyed in milk and meat extract by 6.25 μg of J25 ml⁻¹ and in half-diluted egg yolk by 50 μg of J25 ml⁻¹.     

The action of a preparation of Escherichia coli M-17 growth autostimulants (Actoflor) on the growth of pure and mixed bacterial cultures

The effect of the preparation of E. coli M-17 low-molecular exometabolites (Actoflor), containing growth autostimulators, on the growth of pure cultures of E. coli M-17 E. coli K-12, Salmonella enteritidis, Serratia marcescens and Bifidobacterium adolescentis MC-42 was studied. This preparation was shown to stimulate the growth of all above-mentioned bacteria. The addition of Actoflor also led to the acceleration of growth in the cultivation of mixed cultures of E. coli M-17 with E. coli K-12 (or S. enteritidis), the producer strain (E. coli M-17) showing the highest degree of acceleration. Moreover, the action of Actoflor led to the elimination of competitor strains and to the increase of the antagonistic activity of E. coli M-17. Actoflor may be supposedly used as a therapeutic or prophylactic remedy.     

YojI of Escherichia coli functions as a microcin J25 efflux pump

In the present study, we showed that yojI, an Escherichia coli open reading frame with an unknown function, mediates resistance to the peptide antibiotic microcin J25 when it is expressed from a multicopy vector. Disruption of the single chromosomal copy of yojI increased sensitivity of cells to microcin J25. The YojI protein was previously assumed to be an ATP-binding-cassette-type exporter on the basis of sequence similarities. We demonstrate that YojI is capable of pumping out microcin molecules. Thus, one obvious explanation for the protective effect against microcin J25 is that YojI action keeps the intracellular concentration of the peptide below a toxic level. The outer membrane protein TolC in addition to YojI is required for export of microcin J25 out of the cell. Microcin J25 is thus the first known substrate for YojI.     

The microcin J25 beta-hairpin region is important for antibiotic uptake but not for RNA polymerase and respiration inhibition

The antibiotic microcin J25 (MccJ25) was cleaved by hydrolysis with thermolysin giving a two-chain peptide (MccJ25-Th19) of 10 and 9 amino acid residues. MccJ25-Th19 with deep modifications in beta-hairpin region had no effect on Escherichia coli growth, but still inhibited RNA polymerase in vitro and oxygen consumption in Salmonella strains. MccJ25-Th19 showed antibiotic activity on E. coli transformed with plasmids containing either fhuA or sbmA genes, which code for proteins involved in MccJ25 transport. These results suggest that an intact beta-hairpin region is crucial for MccJ25 import but not for inhibition of E. coli RNA polymerase or oxygen consumption in Salmonella strains.     

Microcin J25 has dual and independent mechanisms of action in Escherichia coli: RNA polymerase inhibition and increased superoxide production

Microcin J25 (MccJ25) uptake by Escherichia coli requires the outer membrane receptor FhuA and the inner membrane proteins TonB, ExbD, ExbB, and SbmA. MccJ25 appears to have two intracellular targets: (i) RNA polymerase (RNAP), which has been described in E. coli and Salmonella enterica serovars, and (ii) the respiratory chain, reported only in S. enterica serovars. In the current study, it is shown that the observed difference between the actions of microcin on the respiratory chain in E. coli and S. enterica is due to the relatively low microcin uptake via the chromosomally encoded FhuA. Higher expression by a plasmid-encoded FhuA allowed greater uptake of MccJ25 by E. coli strains and the consequent inhibition of oxygen consumption. The two mechanisms, inhibition of RNAP and oxygen consumption, are independent of each other. Further analysis revealed for the first time that MccJ25 stimulates the production of reactive oxygen species (O(2)(*-)) in bacterial cells, which could be the main reason for the damage produced on the membrane respiratory chain.     

Chemical modification of microcin J25 with diethylpyrocarbonate and carbodiimide: evidence for essential histidyl and carboxyl residues

In this paper we compared the antibacterial activity of native microcin J25, a peptide antibiotic, with the activities of two analogues obtained by chemical modifications. In the first analogue, the negative charge of glutamic carboxyl group was specifically blocked with an L-glycine methyl ester and in the second the histidine imidazole ring was carbethoxylated. Both analogues decreased notably its antibiotic activity against Escherichia coli and Salmonella newport, strains sensible to the native microcin J25. The biological activity of the carbethoxylated analogue was completely recovered after treatment with hydroxylamine. The extreme importance of both polar residues could be interpreted as specific structural features indispensable for the peptide transportation into the cell, extrusion outside the cell or alternatively to inhibit the RNA-polymerase.     

Cross-protection against Salmonella enteritidis infection in mice

Mice were vaccinated with six strains of Salmonella and two strains of Escherichia coli, as well as with Pseudomonas aeruginosa, Proteus vulgaris, and Serratia marcescens. The amount of in vivo growth of each organism was followed by viable counting techniques on organ homogenates. The vaccinated mice, along with unvaccinated controls, were challenged intravenously with 1,000 ld(50) of a streptomycin-resistant strain of Salmonella enteritidis. The ability of the vaccine to protect the mice against virulent challenge correlated with the ability of the strain to establish a persisting population in the liver and spleen. Enumeration of the liver and spleen populations in the challenged mice revealed that extensive growth of S. enteritidis occurred in animals which showed "protection," as assessed by progressive mortality data. No evidence was obtained for a major role of humoral factors in the cross-protection against intravenous S. enteritidis challenge.     

Distribution of pathogen inhibition in the Lactobacillus isolates of a commercial probiotic consortium

Pure strains of Lactobacillus ssp. isolated from a commercial probiotic consortium were checked in a double layer solid medium for their inhibition activities against selected pathogenic bacteria including serotypes of Listeria monocytogenes, Escherichia coli and Salmonella. The antagonistic properties of the Lactobacillus strains may be related to the production of bacteriocin-like compounds. All the pathogens tested were inhibited by one or a few strains of Lactobacillus, the best inhibition was observed against L. monocytogenes but the inhibition was also satisfactory against E. coli, Salm. typhimurium and Salm. enteritidis.