Lactoferricin B causes depolarization of the cytoplasmic membrane of Escherichia coli ATCC 25922 and fusion of negatively charged liposomes.

Antimicrobial peptides have been extensively studied in order to elucidate their mode of action. Most of these peptides have been shown to exert a bactericidal effect on the cytoplasmic membrane of bacteria. Lactoferricin is an antimicrobial peptide with a net positive charge and an amphipatic structure. In this study we examine the effect of bovine lactoferricin (lactoferricin B; Lfcin B) on bacterial membranes. We show that Lfcin B neither lyses bacteria, nor causes a major leakage from liposomes. Lfcin B depolarizes the membrane of susceptible bacteria, and induces fusion of negatively charged liposomes. Hence, Lfcin B may have additional targets responsible for the antibacterial effect.

Long PCRs of transposons in the structural analysis of genes encoding acquired glycopeptide resistance in enterococci.

Glycopeptide-resistant enterococci (GRE) associated with multiple antibiotic resistance present a major challenge to clinical practice and infection control due to limited or nonexistent antimicrobial treatment options. The genes encoding VanA- and VanB-type glycopeptide resistance have been shown to reside on transposons Tn1546 and Tn1547, respectively. These transferable genetic elements may carry the resistance determinants between and within different ecological niches. Molecular epidemiological studies of nosocomial outbreaks of VanA- and VanB-type GRE indicate horizontal transfer of glycopeptide resistance genes as an important mechanism for the spread of GRE. To target infection control and better understand the epidemiology of GRE, outbreak investigations and molecular epidemiological studies should therefore apply at least two different approaches, i.e., molecular-typing methods to analyze bacterial genomic heterogeneity and structural analysis of mobile resistance determinants. Here we describe the development and use of long PCRs in the structural analysis of vanA and vanB gene clusters in GRE.

Antimicrobial resistance of enteropathogenic Escherichia coli strains from a nosocomial outbreak in Kenya.

The majority of the 78 enteropathogenic (EPEC) and the 151 non-EPEC Escherichia coli strains isolated from preterm neonates during an outbreak of gastroenteritis in a hospital in Nairobi, Kenya, were resistant to trimethoprim-sulfamethoxaxole, chloramphenicol, oxytetracycline and ampicillin, but only a few strains were resistant to cefazolin, cefamandole, cefotaxime, amikacin and nalidixic acid. Fourteen different antimicrobial resistance patterns were observed in the 229 strains of E. coli analysed. Eighty-two percent of the EPEC strains belonged to two resistance pattern compared with 79% of non-EPEC strains which exhibited three resistance patterns. There was no consistent relationship between plasmid profile group and antimicrobial resistance pattern, although one resistance pattern was more frequently observed in EAF-positive strains belonging to the dominant plasmid profile group. Nine percent of the EPEC strains were resistant to gentamicin compared to 37% in the non-EPEC group. No correlation was observed between administration of gentamicin and percentage of resistant strains isolated. None of the nine neonates receiving gentamicin died during the outbreak. Gentamicin resistance was observed in E. coli strains from six out of these nine neonates. Five out of fourteen neonates who received other antimicrobials, or no antibiotic treatment at all, died.

A unique plasmid profile characterizing Salmonella enteritidis isolates from patients and employees in a hospital.

Plasmid profiling was used as an epidemiological tool during a period of frequent Salmonella enteritidis infection in a hospital. S. enteritidis was isolated from 22 patients and employees. Isolates from 18 persons harbored one 29 and one 36 megadalton (MDa) plasmid. The 29 MDa plasmid has not been previously described in this species and was not found in 54 control strains of S. enteritidis from other sources. The respective restriction endonuclease digest fragments of the 36 and the 29 MDa plasmids were always identical. This plasmid pattern thus served as a marker for the isolates from the outbreak.

Comparison of thymidine kinase and A-type inclusion protein gene sequences from Norwegian and Swedish cowpox virus isolates.

During the last decades, cowpox virus, a member of the genus Orthopoxvirus within the Poxviridae family, has appeared as a pathogen in domestic cats, zoo animal species, and humans. At the same time, vaccinia virus, another orthopoxvirus, has been used as a recombinant vaccine vector with foreign genes inserted in the thymidine kinase (TK) gene. By PCR and cycle sequencing, we have determined the nucleotide sequences of the TK gene and the A-type inclusion protein (ATIP) gene of virus isolates from two human cowpox cases in Sweden, as well as a human and a feline case from Norway. We also obtained the corresponding sequences from ectromelia virus (strain Moscow), cowpox virus (strain Brighton) and vaccinia virus (strain Western Reserve). The new virus isolates differed from ectromelia virus and vaccinia virus, and were confirmed to be cowpox virus strains. Isolates originating from the same country had nearly identical TK sequences and fully identical ATIP sequences. They probably represent local geographical strains of cowpox virus.

Tetracycline resistance genes in Kenyan hospital isolates of Salmonella typhimurium.

All 97 strains of Salmonella typhimurium isolated from patients at a hospital in Nairobi, Kenya, during 1988-90 were resistant to tetracycline. The minimum inhibitory concentration (MIC) showed a large distribution range from 1 microgram/ml to 128 micrograms/ml. The strains were heterogeneous with respect to plasmid content, but initially all strains possessed, in addition to other plasmids, a large 60-, 63- or 65-MDa plasmid. The tetracycline resistance genes were characterized using oligonucleotide probes, and 20% of the resistant strains possessed tetracycline type A (tetr A), 6% tetr B, and 4% tetrC genes. Three strains possessed both type A and B tetracycline resistance determinants, which were shown to be located on the large 65-MDa plasmid. There was no correlation between strains isolated from stools, blood, cerebrospinal or epidural fluids, pus, or urine, with respect to the tetracycline genotypes, MIC values or plasmid content.

Characteristics of four cowpox virus isolates from Norway and Sweden.

We report the first isolation of cowpox virus from a domestic cat in Norway, and the first confirmed isolation of cowpox virus from a human case in Norway. These two Norwegian cowpox virus isolates, as well as two Swedish human isolates, were partially characterized and compared with each other and with cowpox virus Brighton and vaccinia virus strain Western Reserve. Restriction enzyme analysis of the genomes revealed differences between all six viruses examined, but suggested that the two Norwegian isolates are closely related, as are the two Swedish isolates. Restriction endonuclease digestion of genomic DNA demonstrated that one of the Swedish isolates and the two Norwegian isolates have larger genomes than vaccinia virus strain Western Reserve, but smaller than cowpox Brighton. All four Scandinavian isolates lacked a 72 base-pair region within the A-type inclusion body protein gene which is present in the prototype cowpox virus Brighton.

A transferable multiple drug resistance plasmid from Vibrio cholerae O1.

Ten multiple antimicrobial-resistant isolates of Vibrio cholerae O1 isolated from patients in Uganda were characterized, and the transferability of resistance to bacteria of diverse origins was investigated. The isolates were toxigenic and belonged to biotype E1 Tor, serotype Ogawa, and ribotype 8, and possessed a 130-MDa plasmid of incompatibility group 6-C. This plasmid, designated pRVC1, was shown to confer resistance to trimethoprim (mediated by a dhfrI gene), sulfonamides (a suII gene), tetracycline [a tet(C) gene], chloramphenicol (a catI gene), ampicillin (a beta-lactamase gene other than blaTEM or blaSHV), and streptomycin. pRVC1 proved to be transmissible at frequencies between 1 x 10(-1) and 5 x 10(-6) transconjugants per recipient to a variety of bacterial pathogens, including those of humans, animals, and fish. Most efficient transfer was observed from V. cholerae to strains of Shigella flexneri, Escherichia coli, Vibrio parahaemolyticus, and three Aeromonas species. The present in vitro study suggests that pRVC1 may spread from V. cholerae to other bacteria pathogenic to man, animals, and fish in natural environments.

Transmission of VanA-type vancomycin-resistant enterococci and vanA resistance elements between chicken and humans at avoparcin-exposed farms.

The genetical relatedness between epidemiologically linked fecal VRE strains from poultry farmers (n = 5) and their broilers (n = 7) at five avoparcin-exposed Norwegian farms was examined. Pulsed-field gel electrophoresis (PFGE) of bacterial chromosomal digests and structural analysis of vanA resistance elements was performed. Animal and human Enterococcus faecium strains at one farm were genetically closely related with indistinguishable vanA elements and a single band position difference in PFGE analysis. Examination of the vanA elements in genetically unrelated strains by restriction enzyme digestion of Tn1546 long-PCR amplicons and ORF2-vanR intergenic sequencing revealed a pool of at least two distinct vanA gene cluster groups in the two reservoirs. The results indicate that transmission of VanA glycopeptide resistance in enterococci between human and animal at avoparcin-exposed farms can occur by direct transfer of VRE strains as well as horizontal spread of resistance genes between strains.

Typeability of Tn1546-like elements in vancomycin-resistant enterococci using long-range PCRs and specific analysis of polymorphic regions.

Molecular subtyping of the VanA-type resistance element Tn1546 in an international collection of 81 genomically diverse vancomycin-resistant enterococci (VRE) from human, animal, and environmental reservoirs was evaluated by restriction analysis of long-range PCR amplicons (PCR-RFLP), single gene PCRs, Southern blot analysis of genomic digests, and partial DNA sequencing. A dominant Tn1546-RFLP in accordance with Enterococcus faecium BM4147 was detected in 43 of the 49 long-range PCR positive strains from ecologically diverse sources in several European countries and the US. Tn1546-like elements from the 32 (40%) long-range PCR negative strains were typed into 17 different groups by single-gene PCRs and Southern blot analysis of the ORF1, ORF2, vanS-vanH, vanX-vanY, and vanZ regions. All these isolates showed deletions in the ORF1 and/or vanZ primer binding regions explaining the failure of long-range PCR amplification. Enlarged vanS-vanH or vanX-vanY fragments were detected in 7 (22%) and 16 (50%) of the long-range PCR negative strains, respectively. The enlarged vanS-vanH regions of five clinical isolates from the US (n = 2), Ireland (n = 2), and Norway (n = 1) contained identical IS1251-like insertions indicating intercontinental spread of the vanA gene cluster. Intergenic vanS-vanH IS1251 insertions have so far not been reported in European studies. Structural rearrangements of Tn1546-like elements may represent single recombination events that can serve as fingerprints in the molecular examination of vanA gene cluster evolution and transmission. The optimal strategy for such analysis has yet to be determined. Two alternative long-range PCRs with subsequent RFLP analysis were successfully used to type the majority of vanA gene clusters in an ecologically and geographically heterogeneous VRE strain collection, but failed to detect and type a group of variant Tn1546-like elements truncated in the left-end ORF1/ORF2 region. Further subtyping of such variants should specifically target the polymorphic vanS-vanH and vanX-vanY regions.

Chlorpromazine inhibition of enterotoxin-induced fluid secretion and cAMP production in rat ileum.

A heat-labile enterotoxin prepared from E. coli (EcLT) increased fluid secretion and cAMP production by segments of rat ileum in vivo and in vitro. The effect of this toxin was compared to that of cholera toxin (VcLT). The increase of cAMP occurred more rapidly after EcLT than after VcLT indicating a difference in the kinetics of uptake or action of the two toxins. Chlorpromazine (CPZ) 5 mg/kg given by intramuscular injection 1 h before application of the toxins inhibited the increase in cAMP levels and the increase in fluid secretion in vivo. CPZ 10(-4) M given together with the toxins to intestinal loops in vitro inhibited the increase in cAMP levels and fluid secretion by this preparation. Scanning electron microscopy revealed that CPZ caused extensive shedding of the fluid-producing mucosal cells.

Immunomagnetic separation of Salmonella from foods.

Salmonella could be separated from different inoculated foods using antibody-coated immunomagnetic beads. When applied on suitable foods, the immunomagnetic separation technique showed a sensitivity of 10-20 Salmonella cells/g of the original sample. The technology appeared less useful for some food items.

Identification of thermotolerant Campylobacter species from poultry using an enzyme-labelled oligonucleotide DNA probe.

A commercially available enzyme-labelled DNA probe for human Campylobacter strains has been tested and also found to hybridize with DNA from C. jejuni and C. coli isolates from poultry. DNA from 11 enteric, non-campylobacter organisms, included in the test as negative controls, failed to hybridize with the probe indicating that the probe might be used for identification of campylobacter from poultry too.

Pathogenic Escherichia coli found in food.

The bacteria constituting the species Escherichia coli are commonly found in the intestinal flora of man and animals, and were until late 1950s recognized as non-pathogenic normal cohabitants. However, certain strains might induce disease, and E. coli should therefore be regarded as a potential pathogenic organism. The pathogenic strains can cause distinct disease syndrome as different diarrheal diseases, wound infections, meningitis, septicemia, artherosclerosis, hemolytic uremic syndrome and immunological diseases such as reactive and rheumatoid arthritis. Several different groups of diarrhea-inducing strains are known. The enterotoxigenic E. coli (ETEC) strains produce one or more of toxins from the heat-labile and the heat-stable enterotoxin families. These strains possess specific adhesion fimbria for intestinal attachment and colonization. Some enteropathogenic E. coli strains (EPEC) produce one or more of the cytotoxins, but adhere also to intestinal cells interfering with the electrolyte transport system. The group of strains possessing invasive properties are designated enteroinvasive E. coli (EIEC). Recently, the enterohemorrhagic E. coli (EHEC) strains have been identified and shown to produce one or more of the cytotoxins (vero-cytotoxins, shiga-like toxins). Food originating from warm-blooded animals may be contaminated with E. coli, but contamination from human sources are more common for food involved in outbreak of disease. In general, strains causing disease in animals do possess other colonization factors than those found on human pathogenic strains. EIEC strains are, like Shigella, only known to induce disease in man. However, both healthy and sick cattle are suspected to be a major reservoir for EHEC strains, and several outbreaks have been associated with consumption of meat or meat products. Cheeses have been the source of outbreaks of both ETEC and EIEC in Europe and the USA, while water seems to be a major source for the different diarrheic E. coli strains affecting children and tourists in the 3rd world. Strains causing non-enteric disease are less known as being transmitted to humans with food as a vector, but the importance of some of these diseases, should implicate further research on what role food plays in spreading these organisms. The recipient of the potential pathogenic E. coli through food, the humans, are also of different risk of contracting diseases.(ABSTRACT TRUNCATED AT 400 WORDS)

Detection of Clostridium perfringens type A enterotoxin in faecal and food samples using immunomagnetic separation (IMS)-ELISA.

A simple, rapid and sensitive immunoassay, based on immunomagnetic particles (Dynabeads M-280) was developed for detection and quantitation of Clostridium perfringens type A enterotoxin from faecal and food extracts. The assay had a detection limit of 2.5 ng/ml enterotoxin in homogenates of faeces and inoculated meat extracts. The specificity was confirmed by both crossed immunoelectrophoresis and Western immunoblotting techniques, using a purified enterotoxin as standard.

Probes and polymerase chain reaction for detection of food-borne bacterial pathogens.

DNA-hybridization and the polymerase chain reaction (PCR) are techniques commonly used to detect pathogenic bacteria. In this paper, the use of these techniques for detection of Salmonella, E. coli, V. cholerae, non-O1 Vibrio, Yersinia enterocolitica, Campylobacter, Listeria monocytogenes, Staphylococcus aureus, Bacillus cereus, Clostridium perfringens, and C. botulinum is reviewed with emphasis on application in food microbiology. In food control, DNA-techniques have most often been used in a 'culture confirmation' fashion, i.e. bacteria are enriched and sometimes even purified by traditional culture procedures and thereafter identified by the use of DNA-based methods. The most desirable approach is, however, to detect organisms directly in the food, but major problems remain to be solved before this can be routinely performed.

Characterization of Escherichia coli strains isolated from pigs with edema disease.

Escherichia coli strains belonging to serogroups O 138 and O 139 isolated from pigs with edema disease, were characterized with respect to the presence of genes encoding Shiga-like toxin I, Shiga-like toxin II and Shiga-like toxin IIv (SLT I, SLT II and SLT IIv). Genes coding for the heat-stable and heat-labile enterotoxins (ST I and LT I) were also detected. Plasmid profiling, restriction enzyme digestion of total DNA, and ribotyping were performed for further characterization of the strains. The oligonucleotide probes applied in this study appeared to be useful tools for detecting genes coding cytotoxins and enterotoxins. DNA from 12 of 16 strains hybridized with two SLT II probes, and DNA from two SLT IIv encoding strains also hybridized with the ST I probe. DNA from one SLT IIv negative strain hybridized with the LT I probe. The results from plasmid profiling, restriction enzyme digestion, and ribotyping were compared with serogrouping in attempts to distinguish between the different E. coli edema disease isolates. Fourteen different plasmid profiles were identified, and as restriction patterns barely did, and ribotyping patterns did not, reveal any information useful for differentiation of the strains beyond serogroup level, plasmid profiling seemed to be the most suitable method for discrimination between the edema disease strains investigated here.

Detection of infectious pancreatic necrosis virus (IPNV) RNA by hybridization with an oligonucleotide DNA probe.

Marine farming of Atlantic salmon (Salmo salar) is growing rapidly in Norway, and fish diseases have now become one of the biggest obstacles for this new industry. Infectious pancreatic necrosis virus (IPNV) is commonly found in fish from Norwegian sea farms. Diagnosis of IPNV infection is, at present, mainly based on virus isolation in cell cultures and identification by neutralization tests (NT). A DNA-RNA hybridization assay was developed using a 24 base DNA oligonucleotide probe. This is homologous to a part of the nucleotide sequence of the IPNV genome coding for a protease. RNA extracted from IPNV and harvest from infected cell cultures were fixed to nylon filters and hybridized with the 32P end-labelled probe. The results showed that the probe specifically identified IPNV from these two materials, both for the three different virus strains (Ab, Sp and VR-299) used, and for several different field isolates. It did not hybridize with reoviruses or non-infected cell cultures used as controls. These results indicate that the probe is not serotype specific, and furthermore that RNA extraction is not required before hybridization. This method may be a useful alternative to NT for routine identification of IPNV, particularly when non-radioactive labelling of the probe is introduced.

Serosurvey for orthopoxviruses in rodents and shrews from Norway.

Two hundred and twenty one blood samples representing eight different rodent species and the common shrew (Sorex araneus), collected in Norway between 1993 and 1995, were examined for anti-orthopoxvirus antibodies by a competition enzyme linked imunnosorbent assay (ELISA) and, when possible, an indirect immunofluorescence assay. The serological results indicated that the bank vole (Clethrionomys glareolus), woodmouse (Apodemus sylvaticus) and Norway lemming (Lemmus lemmus) may be reservoir species for orthopoxviruses in Norway, with antibody prevalences of 17 (12/69), 30 (24/81) and 56% (19/34), respectively. Orthopoxvirus infection in lemmings has not been reported previously. On some other small rodent species such as field voles (Microtus agrestis), common rats (Rattus norvegicus), and common shrews, seropositive individuals were detected. However, the total number of tested animals was low, and the role of these species in the epidemiology of orthopoxvirus infections remains unclear. Attempts to isolate orthopoxviruses from these small mammals failed, although orthopoxvirus specific DNA sequences were detected previously in the same animals by the polymerase chain reaction (PCR). The serological results were compared with and discussed in the context of the occurrence of orthopoxvirus-specific DNA sequences, and it is concluded that orthopoxviruses are widely distributed among wildlife in Norway.

Antibodies against orthopoxviruses in wild carnivores from Fennoscandia.

Two hundred and three sera obtained in 1993-96 from red foxes (Vulpes vulpes), lynx (Lynx lynx), brown bears (Ursus arctos) and wolverines (Gulo gulo) in Fennoscandia (Norway, Sweden, and Finland) were examined for the presence of anti-orthopoxvirus antibodies by a competition enzyme linked immunosorbent assay (ELISA). High prevalences were found for the red foxes in Norway (7/62, 11%) and Finland (7/14, 50%). While only one of 73 (1%) lynx from Finland had anti-orthopoxvirus antibodies, a high prevalence was found in sera from the Sarek National Park in Sweden (5/17, 29%). In addition, anti-orthopoxvirus antibodies were found in one brown bear from the same area (1/45, 2%), whereas none of the 14 wolverines were seropositive. This is the first report of anti-orthopoxvirus antibodies in the brown bear and the lynx, and the first screening for such antibodies in Sweden and Finland. These results indicate that orthopoxviruses are distributed in Sweden and Finland as well as in Norway, and that the red fox and the European lynx may serve as indicator species for the presence of orthopoxviruses in the local populations of small mammals.