Products of defective lysogeny in Serratia marcescens SMG 38 and their activity against Escherichia coli and other Enterobacteria

From a series of Serratia marcescens clinical isolates analysed with respect to bacteriocin production, one strain (SMG 38) was exceptional in that it produced two distinct phage-tail-like bacteriocins differing in morphology, sedimentation, heat sensitivity, and host range. The more active component (bc25) was effective against Serratia, while the other component (McG) inhibited growth of Escherichia coli, Salmonella typhimurium and Shigella sonnei, but not Serratia. Plaque formation on tested strains was negative except in the single case of the lysate of a subclone of SMG 38 which caused the production of a virulent phage, phi epsilon, in E. coli K12 RH 5108. This seems to be a rare event. Like the bacteriocin McG, phage phi epsilon used the same receptor protein, coded at about 30 min (locus fig) on the E. coli chromosome, as does the temperate and serologically unrelated phage phi gamma. Both McG and UV-irradiated phi epsilon killed sensitive bacteria. The survival rate depended on the input multiplicity and also on the indicator strain, and was increased by the presence of prophage phi 80 in the cell. When survivors were allowed to resume their growth under normal conditions, they showed cell elongation whatever their RecA phenotype. No difference was observed between the two agents with respect to these observations, except that McG, unlike irradiated phi epsilon, was inactive against Klebsiella pneumoniae UNF 5023, which possessed the Fig receptor.     

A simple method for semi-preparative-scale production and recovery of enterocin AS-48 derived from Enterococcus faecalis subsp. liquefaciens A-48-32

Production of enterocin AS-48 by Enterococcus faecalis A-48-32 was compared between standard and high-cell density batch fermentations. In high-cell density cultures, bacteriocin production was 2.47-fold higher, provided that the pH was controlled during the fermentation. A two-step procedure for recovery of milligram quantities of purified bacteriocin was developed, based on adsorption of the bacteriocin on Carboxymethyl Sephadex CM-25 followed by reversed-phase chromatography on a semi-preparative column. The purified bacteriocin was active on all the Gram-positive bacteria tested (for example, species of Bacillus, Paenibacillus, Staphylococcus, and Listeria). Strains E. coli U-9, E. coli CECT 102, E. coli CECT 104, E. coli CECT 432, E. coli CECT 543, E. coli CECT 877 and Shigella sonnei CECT 542 were sensitive, while seven other E. coli strains as well as Salmonella choleraesuis CECT 722, S. choleraesuis CECT 916, Enterobacter cloacae CECT 194 and Aeromonas hydrophila CECT 398 were resistant.     

Plasmid-mediated bacteriocin production by Shigella flexneri isolated from dysenteric diarrhoea and their transformation into Escherichia coli

AIMS: To determine the production of bacteriocin by Shigella flexneri strains, to relate their production to the presence of dysenteric diarrhoea and to asses the genetic determination of the bacteriocin. METHODS AND RESULTS: One hundred and sixteen strains of Sh. flexneri were isolated from patients with diarrhoea and 49 of them produced bacteriocin active against several Escherichia coli and abacteriocinogenic Sh. flexneri strains. The extrachromosomal DNA isolated from bacteriocinogenic Sh. flexneri strains were used as a substrate to transform E. coli HB-101 cells by means of electroporation. CONCLUSIONS: Only the Sh. flexneri strains isolated from dysenteric diarrhoea produced bacteriocin. It was demonstrated that a plasmid of approx. 3 kb was responsible for the genetic determination of these anti-bacterial substances. Significance and IMPACT OF THE STUDY: A 3-kb plasmid that harboured information for the production of bacteriocin by Sh. flexneri strains was described. The production of this bacteriocin may be related to dysenteric diarrhoea produced by these bacterial strains.     

Sequence analysis of the plasmid pColG from the Escherichia coli strain CA46

The complete 4715 nucleotide sequence of the plasmid pColG from the Escherichia coli strain CA46, which was originally assumed to code for colicin G activity, has been determined. Based on the nucleotide sequence homology of the 1828bp replication region, with an average G+C content of 48%, pColG was classified as a ColE1-like plasmid. Computer assisted analysis of the remaining 2887bp nucleotide sequence with an average G+C content of 34% revealed three putative OFRs. To find out whether one or all of the three ORFs code for a possible bacteriocin, a DNA fragment encompassing these ORFs has been cloned and the recombinant colonies tested for bacteriocin production. None of the colonies had an inhibitory activity against E. coli strains DH5, HB101 and MC4100. The assumption that the plasmid pColG from the E. coli strain CA46 codes for a bacteriocin thus could not be confirmed.     

Isolation and identification of a bacteriocin with antibacterial and antibiofilm activity from Citrobacter freundii

Multi- and pan-antibiotic-resistant bacteria area major health challenge in hospital settings. Furthermore,when susceptible bacteria establish surface-attached biofilm populations, they become recalcitrant to antimicrobial therapy. Therefore, there is a need for novel antimicrobials that are effective against multi-drug-resistant and surface-attached bacteria. A screen to identify prokaryote-derived antimicrobials from a panel of over 100 bacterial strains was performed. One compound isolated from Citrobacter freundii exhibited antimicrobial activity against a wide range of Gram-negative bacteria and was effective against biofilms. Random transposon mutagenesis was performed to find mutants unable to produce the antimicrobial compound.Transposons mapped to a bacteriocin gene located on a small plasmid capable of replication in Escherichia coli. The plasmid was sequenced and found to be highly similar to a previously described colicinogenic plasmid.Expression of the predicted bacteriocin immunity gene conferred bacteriocin immunity to E. coli. The predicted bacteriocin gene, colA-43864, expressed in E. coli was sufficient to generate anti-microbial activity, and purified recombinant ColA-43864 was highly effective in killing E. coli, Citrobacter species, and Klebsiella pneumoniae cells in a planktonic and biofilm state. This study suggests that bacteriocins can be an effective way to control surface-attached pathogenic bacteria.     

Pathogenic significance of Acinetobacter calcoaceticus: analysis of experimental infection in mice

The phenomenon that mixed infection with certain species of bacteria and Acinetobacter calcoaceticus is more virulent than single infection was analyzed experimentally. In mixed infections with A. calcoaceticus paired with either Escherichia coli, Serratia marcescens, or Pseudomonas aeruginosa, the virulence of the latter three organisms was markedly increased over that of single infections only by slime-producing strains of A. calcoaceticus. Of the 100 strains of A. calcoaceticus tested, 14 had slime-producing ability. There was scarcely any difference in the chemical components of the slimes of the two strains tested, but the components of the slime of P. aeruginosa were different from those of these strains. The slime of these two strains exhibited lethal activity in mice, but no correlation was found between the amount of slime produced and the virulence. The slime enhanced the virulence of E. coli, S. marcescens, and P. aeruginosa when it was inoculated along with their viable cells. Furthermore, the slime exhibited potent cell-impairing activity against mouse neutrophils both in vitro and in vivo. This activity was considered to be mainly responsible for the enhancement of virulence in mixed infections.     

Cloning and characterization of two Serratia marcescens genes involved in core lipopolysaccharide biosynthesis.

Bacteriocin 28b from Serratia marcescens binds to Escherichia coli outer membrane proteins OmpA and OmpF and to lipopolysaccharide (LPS) core (J. Enfedaque, S. Ferrer, J. F. Guasch, J. Tomás, and M. Requé, Can. J. Microbiol. 42:19-26, 1996). A cosmid-based genomic library of S. marcescens was introduced into E. coli NM554, and clones were screened for bacteriocin 28b resistance phenotype. One clone conferring resistance to bacteriocin 28b and showing an altered LPS core mobility in polyacrylamide gel electrophoresis was found. Southern blot experiments using DNA fragments containing E. coli rfa genes as probes suggested that the recombinant cosmid contained S. marcescens genes involved in LPS core biosynthesis. Subcloning, isolation of subclones and Tn5tac1 insertion mutants, and sequencing allowed identification of two apparently cotranscribed genes. The deduced amino acid sequence from the upstream gene showed 80% amino acid identity to the KdtA protein from E. coli, suggesting that this gene codes for the 3-deoxy-manno-octulosonic acid transferase of S. marcescens. The downstream gene (kdtX) codes for a protein showing 20% amino acid identity to the Haemophilus influenzae kdtB gene product. The S. marcescens KdtX protein is unrelated to the KdtB protein of E. coli K-12. Expression of the kdtX gene from S. marcescens in E. coli confers resistance to bacteriocin 28b.     

Efficient transformation of Serratia marcescens with pBR322 plasmid DNA

Eight Serratia marcescens strains tested could be transformed with the plasmid pBR322. Transformants were selected on the basis of resistance to high levels of ampicillin (400 to 500 micrograms/ml). For six of the strains, the CaCl2- mediated transformation procedure developed for Escherichia coli was successful. For the other two strains, no transformants were obtained with the CaCl2-mediated transformation procedure unless the cells first received a heat treatment. Transformation frequency was dependent on DNA concentration, and no transformation was detected with linear pBR322 DNA. The stability and copy number of pBR322 were similar in S. marcescens and E. coli. As in E. coli, the pBR322 DNA was amplified in S. marcescens after inhibition of proteins synthesis. Based on these results, cloning in S. marcescens should be possible and pBR322 should be a useful cloning vehicle.     

Escherichia coli bacteriocins: antimicrobial efficacy and prevalence among isolates from patients with bacteraemia

Bacteriocins are antimicrobial peptides generally active against bacteria closely related to the producer. Escherichia coli produces two types of bacteriocins, colicins and microcins. The in vitro efficacy of isolated colicins E1, E6, E7, K and M, was assessed against Escherichia coli strains from patients with bacteraemia of urinary tract origin. Colicin E7 was most effective, as only 13% of the tested strains were resistant. On the other hand, 32%, 33%, 43% and 53% of the tested strains exhibited resistance to colicins E6, K, M and E1. Moreover, the inhibitory activity of individual colicins E1, E6, E7, K and M and combinations of colicins K, M, E7 and E1, E6, E7, K, M were followed in liquid broth for 24 hours. Resistance against individual colicins developed after 9 hours of treatment. On the contrary, resistance development against the combined action of 5 colicins was not observed. One hundred and five E. coli strains from patients with bacteraemia were screened by PCR for the presence of 5 colicins and 7 microcins. Sixty-six percent of the strains encoded at least one bacteriocin, 43% one or more colicins, and 54% one or more microcins. Microcins were found to co-occur with toxins, siderophores, adhesins and with the Toll/Interleukin-1 receptor domain-containing protein involved in suppression of innate immunity, and were significantly more prevalent among strains from non-immunocompromised patients. In addition, microcins were highly prevalent among non-multidrug-resistant strains compared to multidrug-resistant strains. Our results indicate that microcins contribute to virulence of E. coli instigating bacteraemia of urinary tract origin.     

Experimental Administration of the Probiotic Escherichia coli Strain Nissle 1917 Results in Decreased Diversity of E. coli Strains in Pigs

The strain Escherichia coli Nissle 1917 (EcN) is widely used as an efficient probiotic in therapy and prevention of human infectious diseases, especially of the intestinal system. Concurrently, small adult pigs are being used as experimental omnivore models to study human gastrointestinal functions. EcN bacteria were applied to 6 adult healthy female pigs in a 2-week trial. 6 Control animals remained untreated. Altogether, 164 and 149 bacterial strains were isolated from smear samples taken from gastrointestinal mucosa in the experimental and control group, respectively. Each individual E. coli strain was then tested for the presence of 29 bacteriocin-encoding determinants as well as for DNA markers of A, B1, B2 and D phylogenetic groups. A profound reduction of E. coli genetic variance (from 32 variants to 13 ones, P?=?0.0006) was found in the experimental group, accompanied by a lower incidence of bacteriocin producers in the experimental group when compared to control (21.3 and 34.9%, respectively; P?=?0.007) and by changes in the incidence of individual bacteriocin types. The experimental administration of EcN strain was not sufficient for stable colonization of porcine gut, but induced significant changes in the enterobacterial microbiota.     

Epidemiological Differentiation of Serratia marcescens: Typing by Bacteriocin Sensitivity

Strains of Serratia marcescens were compared and differentiated by a new method. Bacteriocin lysates were prepared from mitomycin-induced S. marcescens and added to lawns of test strains. From 100 bacteriocin producers, 12 were chosen with the aid of computer analysis as the most useful in differentiation. Uniform drops of the 12 standard bacteriocins were added simultaneously with a bacteriocin-bacteriophage dropper to each strain to be typed. All 93 strains of S. marcescens tested were typable and were differentiated into 79 different sensitivity patterns. One pattern had three strains, 12 patterns had two strains each, and 66 patterns had only one strain. The bacteriocins also inhibited Shigella, Klebsiella, and Enterobacter, but no other Enterobacteriaceae. Bacteriocin sensitivity was less stable as an epidemiological marker than bacteriocin production. Several colonial mutants had sensitivity patterns different from the wild types, but most mutants were identical. In three different instances when cross-infection had been shown by other methods, bacteriocin sensitivity also gave the correct epidemiological results. Until the significance and frequency of genetic variations are known, a more stable epidemiological technique should be used in conjunction with bacteriocin sensitivity.     

Genetics of Sensitivity of Salmonella Species to Colicin M and Bacteriophages T5, T1, and ES18

Nearly all of 62 strains of Salmonella paratyphi B were sensitive to colicin M and phage T5 but resistant to phages T1 and ES18 and to colicin B. All tested S. typhimurium strains were resistant to colicin M and phage T5, and many were sensitive to phage ES18. A rough S. typhimurium LT2 strain given the tonA region of Escherichia coli or S. paratyphi B became sensitive to colicin M and phage T5. We infer that the tonA allele of S. paratyphi B, like that of E. coli, determines an outer membrane protein that adsorbs T5 and colicin M but not phage ES18, whereas the S. typhimurium allele determines a protein able to adsorb only ES18. The partial T1 sensitivity of a rough LT2 strain with a tonA allele from E. coli or S. paratyphi B and also the tonB(+) phentotype of an E. coli B trp-tonB Delta mutant carrying an F' trp of LT2 origin showed that S. typhimurium LT2 has a tonB allele like that of E. coli with respect to determination of sensitivity to colicins and phage T1. Rough S. paratyphi B, although T5 sensitive, remained resistant to T1 even when given F' tonB(+) of E. coli origin. Classes of Salmonella mutants selected as resistant to colicin M included: T5-resistant mutants, probably tonA(-); mutants unchanged except for M resistance, perhaps tolerant; and Exb(+) mutants, producing a colicin inhibitor (presumably enterochelin). Some Exb(+) mutants were resistant to a bacteriocin inactive on E. coli but active on all tested S. paratyphi B and S. typhimurium strains (and on nearly all other tested Salmonella). A survey showed sensitivity to colicin M in several other species of Salmonella.     

Use of genetic and molecular characteristics of R plasmids as an epidemiological marker in an outbreak of hospital infection

Antibiotic sensitivity of 38 strains of enteric bacteria, such as Serratia marcescens Klebsiella pneumoniae and others and Ps. aeruginosa isolated during an outbreak of meningitis in a premature infant resuscitation department was studied. It was shown that all the isolates were multiple resistant, most frequently to 7 antibiotics. All the resistance markers were transferred on conjugation, segregation of some markers being observed. Investigation of the plasmid composition of the clinical strains and transconjugants of E. coli revaled the presence of 2 plasmids with the molecular weights of 40 and 60 Md or one of them. The restriction analysis demonstrated that the plasmids with the same molecular weights isolated from different strains were identical. It was suggested that such plasmids originated from the same source and were distributed by conjugation. The possible part of R plasmids in epidemiological analysis of hospital infections is discussed: the possible part as an additional marker in determination of the infection source and the possible part through its ability to change the host cell phenotype, including the phage and bacteriocin types.     

Bacteriocinogenicity of brucellae isolated in foci in the Caucasus and their evaluation from taxonomic viewpoints

The aim of the study was to elucidate the possibility of using bacteriocinogenicity of Brucella as taxonomic feature, to determine their phylogenetic relation to other microorganisms by their bacteriocinogenic properties and to investigate the physicochemical properties of brucellacin and conditions for its stable detection. The Brucella cultures were isolated in the Caucasus. Investigation of their capacity for production of bacteriocin according to the procedure described by M.A. Konstantinova and A.D. Garmazova (1979) revealed that 62.1 per cent of the 216 cultures tested produced brucellacin. Isolation of bacteriocin with the methods developed was shown possible in all of the tested strains of B. melitensis, B. abortus, B. suis and in most of the strains of B. ovis. The methods also provided an increase in the synthesis and activity of brucellacin. The analysis of the characteristic features of bacteriocinogenicity and the properties of bacteriocin allowed recommending the use of additional taxonomic features for identification and differentiation of Brucella. Sensitivity of the indicator strains of Brucella to bacteriocins of other species (F. tularensis, Campylobacter fetus intestinalis B-8833, Y. enterocolitica, Vibrio cholerae and E. coli Fredericq) was noted which was additional evidence of the phylogenic relation between the above organisms. Investigation of the physicochemical properties of brucellacin confirmed the suggestion of the protein nature of the active principle of brucellacin and its similarity in different Brucella species.     

Physical properties of L-asparaginase from Serratia marcescens.

Purified L-asparaginase from Serratia marcescens had an apparent-weight average molecular weight of 171,000 to 180,000 as determined by electrophoresis on polyacrylamide gels and by sedimentation equilibrium at low speed in an analytical ultracentrifuge. A subunit molecular weight of 31,500 +/- 1,500 was estimated for the enzyme after treatment with sodium dodecyl sulfate and urea and electrophoresis on polyacrylamide gels; a similar value was obtained by high-speed sedimentation equilibrium in the presence of guanidine hydrochloride. Our data indicate that the Serratia enzyme could have five or six subunits of 32,000 daltons, compared to four subunits of 32,000 daltons in the Escherichia coli enzyme. The Serratia L-asparaginase also appears to be a larger molecule than the enzyme from Erwinia carotovora, Proteus vulgaris, Acinetobacter glutaminasificans, and Alcaligenes eutrophus. The Serratia enzyme, like that from E. caratovora, was more resistant than the E. coli enzyme to dissociation by sodium dodecyl sulfate. This resistance could be due to the finding that the Serratia enzyme had a relatively high hydrophobicity, similar to the enzyme from E. caratovora, when compared with the hydrophobicity of the E. coli enzyme. The isoelectric point of the Serratia enzyme was approximately 5.2. The influence of certain physical characteristics of the enzyme on the biological properties is discussed.     

Genetic analysis of bacteriocin 43 of vancomycin-resistant Enterococcus faecium

A total of 636 vancomycin-resistant Enterococcus faecium (VRE) isolates obtained between 1994 and 1999 from the Medical School Hospital of the University of Michigan were tested for bacteriocin production. Of the 277 (44%) bacteriocinogenic strains, 21 were active against E. faecalis, E. faecium, E. hirae, E. durans, and Listeria monocytogenes. Of those 21 strains, a representative bacteriocin of strain VRE82, designated bacteriocin 43, was found to be encoded on mobilizable plasmid pDT1 (6.2 kbp). Nine open reading frames (ORFs), ORF1 to ORF9, were presented on pDT1 and were oriented in the same direction. The bacteriocin 43 locus (bac43) consists of the bacteriocin gene bacA (ORF1) and the immunity gene bacB (ORF2). The deduced bacA product is 74 amino acids in length with a putative signal peptide of 30 amino acids at the N terminus. The bacB gene encodes a deduced 95-amino-acid protein without a signal sequence. The predicted mature BacA protein (44 amino acids) showed sequence homology with the membrane-active class IIa bacteriocins of lactic acid bacteria and showed 86% homology with bacteriocin 31 from E. faecalis YI717 and 98% homology with bacteriocin RC714. Southern analysis with a bac43 probe of each plasmid DNA from the 21 strains showed hybridization to a specific fragment corresponding to the 6.2-kbp EcoRI fragment, suggesting that the strains harbored the pDT1-like plasmid (6.2 kb) which encoded the bacteriocin 43-type bacteriocin. The bac43 determinant was not identified among non-VRE clinical isolates.     

Differentiation of Serratia from Enterobacter on the Basis of Nucleoside Phosphotransferase Production

Precoated cellulose thin-layer chromatograms were used to detect the production of guanosine 5'-phosphate from guanosine and p-nitrophenylphosphate by whole-cell preparations. One-hundred per cent of Serratia (163 strains) and 84% of E. liquefaciens (15 of 18 strains) produced the nucleotide. All other Enterobacter (23 strains), Klebsiella (10 strains), and E. coli (10 strains) were negative for the production of this nucleotide. The entire test procedure could be carried out in 4 hr. It is proposed that E. liquefaciens is more closely related to Serratia than Enterobacter and that reclassification of these organisms should be investigated.     

Genome sequence of E. coli O104:H4 leads to rapid development of a targeted antimicrobial agent against this emerging pathogen

A recent widespread outbreak of Escherichia coli O104:H4 in Germany demonstrates the dynamic nature of emerging and re-emerging food-borne pathogens, particularly STECs and related pathogenic E. coli. Rapid genome sequencing and public availability of these data from the German outbreak strain allowed us to identify an O-antigen-specific bacteriophage tail spike protein encoded in the genome. We synthesized this gene and fused it to the tail fiber gene of an R-type pyocin, a phage tail-like bacteriocin, and expressed the novel bacteriocin such that the tail fiber fusion was incorporated into the bacteriocin structure. The resulting particles have bactericidal activity specifically against E. coli strains that produce the O104 lipopolysaccharide antigen, including the outbreak strain. This O-antigen tailspike-R-type pyocin strategy provides a platform to respond rapidly to emerging pathogens upon the availability of the pathogen's genome sequence.