Plant lectin-like bacteriocin from a rhizosphere-colonizing Pseudomonas isolate

Rhizosphere isolate Pseudomonas sp. strain BW11M1, which belongs to the Pseudomonas putida cluster, secretes a heat- and protease-sensitive bacteriocin which kills P. putida GR12-2R3. The production of this bacteriocin is enhanced by DNA-damaging treatment of producer cells. We isolated a TnMod mutant of strain BW11M1 that had lost the capacity to inhibit the growth of strain GR12-2R3. A wild-type genomic fragment encompassing the transposon insertion site was shown to confer the bacteriocin phenotype when it was introduced into Escherichia coli cells. The bacteriocin structural gene was identified by defining the minimal region required for expression in E. coli. This gene was designated llpA (lectin-like putidacin) on the basis of significant homology of its 276-amino-acid product with mannose-binding lectins from monocotyledonous plants. LlpA is composed of two monocot mannose-binding lectin (MMBL) domains. Several uncharacterized bacterial genes encoding diverse proteins containing one or two MMBL domains were identified. A phylogenetic analysis of the MMBL domains present in eukaryotic and prokaryotic proteins assigned the putidacin domains to a new bacterial clade within the MMBL-containing protein family. Heterologous expression of the llpA gene also conveyed bacteriocin production to several Pseudomonas fluorescens strains. In addition, we demonstrated that strain BW11M1 and heterologous hosts secrete LlpA into the growth medium without requiring a cleavable signal sequence. Most likely, the mode of action of this lectin-like bacteriocin is different from the modes of action of previously described Pseudomonas bacteriocins.     

Diagnostic and epidemiological significance of F-donor activity of Enterobacteria strains

The study of the circulating strains of enteropathogenic Escherichia coli (EPEC), shigellae and salmonellae for the presence of F-factor (the conjunction factor) was carried out. The study revealed that 85% of Shigella flexneri strains 2a had conjugative (F)-factor. The proportion of such strains was 63% among EPEC of different serovars and 15% among Salmonella enteritidis. As 63% EPEC strains had hemolytic activity and were found to carry F-conjugative plasmid it should be taken into consideration in the identification of microorganisms. The presence of conjugation plasmids is an important epidemiological marker.     

Stress-related Pseudomonas genes involved in production of bacteriocin LlpA

Pseudomonas sp. BW11M1 produces a novel type of bacteriocin that inhibits the growth of Pseudomonas putida GR12-2R3 and some phytopathogenic fluorescent Pseudomonas. A collection of mutants was screened for altered bacteriocin production phenotypes. Strongly reduced bacteriocin production was found to be caused by inactivation of the recA gene or the spoT gene. Conversely, in a recJ mutant, the bacteriocin was constitutively overproduced. The same phenotype was observed for a mutant hit in a gene of unknown function. The predicted gene product belongs to a distinct subgroup of prokaryotic helicase-like proteins within the SWI/SNF family of regulatory proteins. One mutant that also exhibited a bacteriocin overproducer phenotype was deficient in the production of the peptidoglycan-associated lipoprotein OprL. This study shows that various environmental stress response pathways are involved in controlling expression of the Pseudomonas sp. BW11M1 bacteriocin.     

Antagonistic action of Pseudomonas aeruginosa against Staphylococci, coliform bacilli and various types of Proteus

Antagonistic activity of 2 fresh isolates and 3 collection strains of Pseudomonas aeruginosa against 177 microbial strains was determined with the method of late antagonism. Among the microbial strains there were 56 staphylococcal strains isolated from patents and carriers. 38 nontypable colon bacilli isolated from healthy persons, 59 enteropathogenic colon bacilli of various serogroups, 12 strains of Proteus and 12 colon bacilli, carriers of multiple drug resistance factors (R factors). All the cultures were sensitive to the antagonistic action of 5 or at least 3 strains of Pseudomonas used in the study. The most active antagonists were the fresh isolates of Pseudomonas as compared to the collection strains. Among the staphylococci S. aureus proved to be the most resistant to the antagonistic action of Pseudomonas as compared to S. epidermidis, the same as the strains isolated from carriers as compared to the strains isolated from patients. As for the enteric bacilli the most resistant were the strains of Proteus. Acquiring of transmissive R factors by the colon bacilli markedly increased their sensitivity to the antagonistic action of Pseudomonas.     

A study of enteropathogenic organisms isolated in the public health laboratory, Lusaka.

A total of 328 specimens of stools were examined in the Public Health Laboratory during January and February 1973. Enteropathogens were isolated from 117 of these specimens. Besides these, 12 strains of Salmonellae were isolated from blood and 8 from urine. An occasional Salmonella was isolated from the pleural fluid (S. paratyphi A) pus from the knee (S. enteritidis) and from the C.S.F. of an infant (S. paratyphi C.). Salmonella typhi and Salmonella paratyphi A are the predominant Salmonella species. No Salmonella paratyphi B has been isolated. Shigella, was isolated with slightly less frequency than Salmonella, and Shigella flexneriis was the predominant species. E. coli 0112/K66 is the most common enteropathogenic E. coli. The majority of the Shigella and Salmonella species are sensitive to the common antibiotics used. The E. coli organisms show multiple resistance to a number of antibiotics.     

Bacterial antagonists of Shigellae

Bacterial antagonism between a microorganisms and Shigella sonnei strains was studied in model experiments simulating conditions of the natural aquatic environment. In these studies surface waste samples from the river Vltava served as the experimental environment. To ensure bacteriologically defined conditions all water samples were heat-sterilized prior to antagonism testing. Consistently with the literature data and author's own observations the following bacterial species and genera were chosen as test organisms to be tested for antagonism against Shigella sonnei strains in water; E. coli, Citrobacter, Enterobacter, Klebsiella pneumoniae, Proteus, Pseudomonas aeruginosa and the fecal streptococci S. fecalis and S. faecium. Presence or absence of microbial antagonism against shigellae was determined in the experimental water medium contaminated with shigella-test organism mixtures of density ratios within the range 1 : 1 through 1 : 10(4). The highest degree of antagonism was observed with Pseudomonas aeruginosa that at density ratio 1 : 1 inhibited the Shigella sonnei growth in water within 42 hours of incubation. A similar degree of antagonism was also observed with Klebsiella pneumoniae at the density ratio 1 : 10(1) and with Enterobacter aerogenes at 1 : 10(2). At lower density ratios the antagonism exhibited by these two species was also present, but occurred much later, i.e. after 72 hours up to 5 days. The remaining test organisms used showed no antagonistic action Shigella sonnei strain in the model aquatic environment.     

Type III secretion: a bacterial device for close combat with cells of their eukaryotic host

Salmonella, Shigella, Yersinia, Pseudomonas aeruginosa, enteropathogenic Escherichia coli and several plant-pathogenic Gram-negative bacteria use a new type of systems called 'type III secretion' to attack their host. These systems are activated by contact with a eukaryotic cell membrane and they allow bacteria to inject bacterial proteins across the two bacterial membranes and the eukaryotic cell membrane to reach a given compartment and destroy or subvert the target cell. These systems consist of a secretion apparatus made up of about 25 individual proteins and a set of proteins released by this apparatus. Some of these released proteins are 'effectors' that are delivered by extracellular bacteria into the cytosol of the target cell while the others are 'translocators' that help the 'effectors' to cross the membrane of the eukaryotic cell. Most of the 'effectors' act on the cytoskeleton or on intracellular signalling cascades. One of the proteins injected by the enteropathogenic E. coli serves as a membrane receptor for the docking of the bacterium itself at the surface of the cell.     

Molecular characterization of antimicrobial compound produced by Lactobacillus acidophilus AA11

Approximately 63 strains of Lactobacillus acidophilus were isolated from Egyptian home-made cheese and examined for production of antagonism. Only eight strains demonstrated inhibitory activity against spoilage microorganisms (i.e. Staphylococcus aureus and Bacillus cereus) and pathogens (i.e. E. coli, Salmonella sp. and Shigella sp.). Lactobacillus acidophilus AA11 produced higher antimicrobial activity with a wide range of inhibition. The agent AA11 was sensitive to proteolytic enzymes and retained full activity after 30 min at 100 degrees C. Activity against sensitive cells was bactericidal but not bacteriolytic. The compound was produced during growth phase and could be extracted from the culture supernatant fluids with n-butanol. 12% SDS-PAGE analysis of 40% ammonium sulphate precipitated agent showed two peptides with molecular weights of approximately 36 kDa and approximately 29 kDa. No plasmid was identified in Lactobacillus acidophilus AA11 indicating that the genes encoding the inhibitory agent were located on the chromosome. These characteristics identify the inhibitory substance as a bacteriocin, designated acidocin AA11 and confer the agent an application potential as a biopreservative.     

Antibacterial and bactericidal activities of Japanese green tea

We found that extracts of Japanese green tea leaves inhibited the growth of various bacteria causing diarrheal diseases. All tea samples tested showed antibacterial activity against Staphylococcus aureus, S. epidermidis, Vibrio cholerae O1, V. cholerae non O1. V. parahaemolyticus, V. mimicus, Campylobacter jejuni and Plesiomonas shigelloides. None of the tea samples had any effect on the growth of V. fluvialis, Aeromonas sobria, A. hydrophila, Pseudomonas aeruginosa, Salmonella enteritidis, enteroinvasive Escherichia coli, enterohemorrhagic E. coli, enteropathogenic E. coli, enterotoxigenic E. coli, Enterobacter cloacae or Yersinia enterocolitica. Salmonella and Shigella showed susceptibilities different depending on the kind of Japanese green tea. Japanese green tea showed also bactericidal activity over S. aureus, V. parahaemolyticus and even enteropathogenic E. coli which was not sensitive when tested by cup method. The bactericidal activity was shown even at the drinking concentration in daily life.     

Epidemiological study on Jordanian patients suffering from diarrhoea

Stool specimens from 1400 Diarrhoeal patients from the Jordanian population were examined for bacterial pathogens and Rotavirus during a four- year period (1997-2000). Pathogenic bacteria were identified in 343 patients (24.5%), most often from children. Salmonella spp. was the most frequent isolated organism in 10.7% of the patient's cultures, followed by enteropathogenic Escherichia coli (EPEC) in 3.9%, Shigella spp. in 0.8% and Campylobacter spp. in 0.9%. Vibrio spp. was not identified in the stools tested. Resistance to ampicillin was observed in 42.2% of the Salmonella, 77.0% of the Shigella, and 31.0% of the EPEC isolates. Cotrimoxazole resistance was observed in 34.0% of the Shigella and 13.0% of the EPEC isolates and 77.0% of Campylobacter isolates. Rotavirus was identified in 373 samples (26.6%) of the patients     

Distribution of insertion sequence IS1 in multiple-antibiotic resistant clinical Enterobacteriaceae strains

The presence of insertion sequence IS1 in 70 multiple-antibiotic resistant clinical strains was determined. This 70-strain collection comprised 46 Escherichia coli, 18 Salmonella and 6 Shigella strains. The presence of IS1 was detected in the chromosome and plasmids of 73% and 63% of the strains, respectively, and 51% of the strains carried IS1 in both. The frequency of IS1 was higher in Salmonella than in E. coli and Shigella strains. A total of 31 strains carried large plasmids with IS1; 10 of these strains (32.3%) were able to transfer all or some of the antibiotic resistance markers to E. coli K12 or S. typhimurium recipient strains. Resistance markers of all clinical strains were maintained stably after several generations of growth. The presence of IS1 in a relatively high percentage of plasmids of multiple-antibiotic resistant clinical isolates, suggests a role for this sequence in the dissemination of genes which code for antibiotic resistance.     

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.     

Evolutionary genetics of a new pathogenic Escherichia species: Escherichia albertii and related Shigella boydii strains

A bacterium originally described as Hafnia alvei induces diarrhea in rabbits and causes epithelial damage similar to the attachment and effacement associated with enteropathogenic Escherichia coli. Subsequent studies identified similar H. alvei-like strains that are positive for an intimin gene (eae) probe and, based on DNA relatedness, are classified as a distinct Escherichia species, Escherichia albertii. We determined sequences for multiple housekeeping genes in five E. albertii strains and compared these sequences to those of strains representing the major groups of pathogenic E. coli and Shigella. A comparison of 2,484 codon positions in 14 genes revealed that E. albertii strains differ, on average, at approximately 7.4% of the nucleotide sites from pathogenic E. coli strains and at 15.7% from Salmonella enterica serotype Typhimurium. Interestingly, E. albertii strains were found to be closely related to strains of Shigella boydii serotype 13 (Shigella B13), a distant relative of E. coli representing a divergent lineage in the genus Escherichia. Analysis of homologues of intimin (eae) revealed that the central conserved domains are similar in E. albertii and Shigella B13 and distinct from those of eae variants found in pathogenic E. coli. Sequence analysis of the cytolethal distending toxin gene cluster (cdt) also disclosed three allelic groups corresponding to E. albertii, Shigella B13, and a nontypeable isolate serologically related to S. boydii serotype 7. Based on the synonymous substitution rate, the E. albertii-Shigella B13 lineage is estimated to have split from an E. coli-like ancestor approximately 28 million years ago and formed a distinct evolutionary branch of enteric pathogens that has radiated into groups with distinct virulence properties.     

Antimicrobial activity of 2,5-dihydroxy-3-methyl-1,4-benzoquinone from Embelia schimperi

Chromatographic separation of an ethyl acetate extract from Embelia schimperi led to the isolation of a new compound identified as 2,5-dihydroxy-3-methyl-1,4-benzoquinone (1) on the basis of spectroscopic and physical data. The plant's crude extract and pure compound 1 were assayed for in vitro antimicrobial activity against clinical strains of Salmonella spp., Proteus spp., Pseudomonas aeruginosa, Klebsiella pneumoniae, Escherichia coli, Cryptococcus neoformans, Shigella dysentriae and Staphylococcus aureus. Disc diffusion method was used and zones of inhibition, after respective incubation periods, were used to quantify antimicrobial activity. Standard antibiotics namely: augmentin, cotrimoxazole, gentamycin, tetracycline and lyncomycin were used as controls. The crude extract was inactive while the pure compound 1 showed significant activities against Salmonella spp., Proteus spp., Pseudomonas aeruginosa, Klebsiella pneumoniae, Escherichia coli, Cryptococcus neoformans, Shigella dysentriae and Staphylococcus aureus with zones of inhibition ranging from 10-20 mm. The most sensitive microorganism was P aeruginosa while C. neoformans was insensitive to both the crude extract and compound 1.     

金樱子茎提取物体外抑菌活性研究

采用纸片扩散法和试管稀释法,研究金樱子茎不同溶剂粗提取物对临床常见病原细菌的抑菌活性。结果表明,金樱子茎水提取物对绿脓杆菌、痢疾杆菌、大肠杆菌、变形杆菌无抑菌活性;对伤寒杆菌的最低抑菌浓度（MIC）为20 mg（生药）/mL;对金黄色葡萄球菌的MIC为10 mg（生药）/mL。金樱子茎75%乙醇提取物对绿脓杆菌、伤寒杆菌、痢疾杆菌、大肠杆菌、变形杆菌无抑菌活性;对金黄色葡萄球菌MIC为10 mg（生药）/mL。金樱子茎乙酸乙酯、氯仿、石油醚提取物对绿脓杆菌、伤寒杆菌、痢疾杆菌、大肠杆菌、金黄色葡萄球菌、变形杆菌无抑菌活性。     

Inactivation of gram-negative bacteria by lysozyme, denatured lysozyme, and lysozyme-derived peptides under high hydrostatic pressure

We have studied the inactivation of six gram-negative bacteria (Escherichia coli, Pseudomonas fluorescens, Salmonella enterica serovar Typhimurium, Salmonella enteritidis, Shigella sonnei, and Shigella flexneri) by high hydrostatic pressure treatment in the presence of hen egg-white lysozyme, partially or completely denatured lysozyme, or a synthetic cationic peptide derived from either hen egg white or coliphage T4 lysozyme. None of these compounds had a bactericidal or bacteriostatic effect on any of the tested bacteria at atmospheric pressure. Under high pressure, all bacteria except both Salmonella species showed higher inactivation in the presence of 100 microg of lysozyme/ml than without this additive, indicating that pressure sensitized the bacteria to lysozyme. This extra inactivation by lysozyme was accompanied by the formation of spheroplasts. Complete knockout of the muramidase enzymatic activity of lysozyme by heat treatment fully eliminated its bactericidal effect under pressure, but partially denatured lysozyme was still active against some bacteria. Contrary to some recent reports, these results indicate that enzymatic activity is indispensable for the antimicrobial activity of lysozyme. However, partial heat denaturation extended the activity spectrum of lysozyme under pressure to serovar Typhimurium, suggesting enhanced uptake of partially denatured lysozyme through the serovar Typhimurium outer membrane. All test bacteria were sensitized by high pressure to a peptide corresponding to amino acid residues 96 to 116 of hen egg white, and all except E. coli and P. fluorescens were sensitized by high pressure to a peptide corresponding to amino acid residues 143 to 155 of T4 lysozyme. Since they are not enzymatically active, these peptides probably have a different mechanism of action than all lysozyme polypeptides.     

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.     

Demonstration of cell division by septation in a variety of gram-negative rods.

Through use of an initial fixative employing a combination of crotonaldehyde and glutaraldehyde, septa were preserved in thin sections of dividing cells of strains of Pseudomonas aeruginosa, Salmonella typhimurium, Shigella sonnei, and Escherichia coli when grown at 30 C in a dilute basal medium. The same procedures, however, revealed only a constrictive division process in Proteus vulgaris and Erwinia sp. This adds to the evidence that septation, although difficult to demonstrate, is the process of cell division in the enteric gram-negative rods and the pseudomonads and that constriction is a fixation artifact in these organisms.     

Lactobacilli in human dental caries and saliva

Samples (98 plaque and 72 saliva) from 93 patients with dental caries were investigated for Lactobacillus species which comprised 65 (62.5%) of 104 isolates. Yeasts (20.1%), Streptococcus spp. (8.7%), Staphylococcus spp. (2.9%) and a few unidentified species (5.8%), were also found. The Lactobacillus isolates were L. brevis (24.6%) L. fermentum (18.5%) L. casei (16.9%), L. delbrueckii (15.4%), L. plantarum (9.23%), L. acidophilus (7.69%), L. jensenii (4.62%), L. salivarius (1.54%) and L. gasseri (1.54%). The most common species was L. brevis (24.6%). The strains tested for beta-lactamase production showed 75.4% positive. All the Lactobacillus strains were tested for bacteriocin production against Escherichia coli, Salmonella spp., Shigella dysenteriae, S. sonnei, Klebsiella spp. and Campylobacter sp. All the lactobacilli except L. jensenii produced bacteriocin against at least one of the indicator organisms. The involvement of Lactobacillus in dental caries was established, although its role and mechanism is not well understood. The ability of Lactobacillus spp. to protect their host against certain diseases by inhibiting the growth of potential pathogens was evident.     

In-vitro antibacterial, antifungal and cytotoxic properties of metal-based furanyl derived sulfonamides

A new series of antibacterial and antifungal furanyl-derived sulfonamides and their cobalt (II), copper (II), nickel (II) and zinc (II) metal complexes have been synthesized, characterized and screened for their in-vitro antibacterial activity against four Gram-negative (Escherichia coli, Shigella flexneri, Pseudomonas aeruginosa and Salmonella typhi) and two Gram-positive (Bacillus subtilis and Staphylococcus aureus) bacterial strains and, for in-vitro antifungal activity against Trichophyton longifusus, Candida albicans, Aspergillus flavus, Microsporum canis, Fusarium solani and Candida glaberata. The results of these studies revealed that all compounds showed significant to moderate antibacterial activity. However, the zinc (II) complexes were found to be comparatively much more active as compared to the others. For antifungal activity generally, compounds (22) and (24) showed significant activity against Escherichia coli (a), (6) against Shigella flexeneri (b), (16) and (22) against Pseudomonas aeruginosa (c), (14) and (16) against Salmonella typhi (d), (9) against Staphylococcus aureus (e) and, (14) and (16) against Bacillus subtilis (f) fungal strains. The brine shrimp (Artemia salina) bioassay was also carried out to study their in-vitro cytotoxic properties. Only three compounds, (6), (10) and (23) displayed potent cytotoxic activity with LD50 = 1.8535 x 10(-4), 1.8173 x 10(-4) and 1.9291 x 10(-4) respectively.