Gallinarum Strains Research Articles

Biological decolourization of C.I. Direct Black 38 by E. gallinarum

In the present study, an Enterococcus gallinarum strain was isolated from effluent treatment plant of a textile industry based on its ability to decolourize C.I. Direct Black 38 (DB38), a benzidine-based azo dye. Effects of dye concentration and medium composition on dye decolourization were studied. The strain was found to decolourize DB38 even under aerobic conditions. Kinetics of DB38 decolourization was also examined, and V max and K s of decolourization were found to be higher in Luria broth (12.8 mg l −1 h −1 and 490.6 mg l −1) than in minimal medium (4.09 mg l −1 h −1 and 161.84 mg l −1). However, decolourization rate/biomass was found to be higher in minimal medium than in Luria broth, indicating greater decolourization efficiency of biomass in the former. The study also revealed biodegradation of DB38 to benzidine and its further deamination to 4-aminobiphenyl (4-ABP) by the culture. Ammonia released during this process was used as nitrogen source for growth of the culture.

Gram-Positive Bloodstream Infections in Liver Transplant Recipients: Incidence, Risk Factors, and Impact on Survival

The objective of the study was to assess the incidence, risk factors, and survival of gram-positive bloodstream infections (GP-BSI s) among liver transplant recipients during the first year after transplantation. Between October 2000 and September 2006, 42 episodes of GP-BSI s occurred in 205 patients with an overall incidence of 0.20 episodes/patient. Coagulase-negative staphylococci were detected in 45.2% of cases, Enterococcus species in 42.9% ( E faecalis, eight; E faecium, seven; E avium, two; E gallinarum, one) and Staphylococcus aureus in 11.9%. Retransplantation was the only independent risk factor for GP-BSI (odds ratio [OR], 0.253; 95% confidence interval (CI), 0.089 to 0.715; P = .009). Thirty-day mortality rate was 28.5% and S aureus infections were related to a poorer outcome. It is noteworthy that all the isolates of S aureus were methicillin-resistant. Ampicillin was inactive against all the strains of E faecium and 50% of E avium isolates, but active against all E faecalis and E gallinarum strains. All the isolates were glycopeptide-susceptible. No significant differences in mortality rate were observed in relation to sex, etiologies of end-stage liver disease, cytomegalovirus infection/reinfection, type of donor, rejection, or retransplantation. GP-BSI, the only independent risk factor for death (OR, 0.262; 95% CI, 0.106 to 0.643; P = .003), reduced the survival rate by 26% in the first year posttransplant. In conclusion, GP-BSI s impact significantly on morbidity and mortality posttransplant, particularly among retransplantations. Control measures are required to reduce the incidence of GP-BSI s in liver transplant recipients. These findings must be considered when empirical antimicrobial therapy is indicated while awaiting blood-culture results.

Specificity of L,D-Transpeptidases from Gram-positive Bacteria Producing Different Peptidoglycan Chemotypes

We report here the first direct assessment of the specificity of a class of peptidoglycan cross-linking enzymes, the L,D-transpeptidases, for the highly diverse structure of peptidoglycan precursors of Gram-positive bacteria. The lone functionally characterized member of this new family of active site cysteine peptidases, Ldt(fm) from Enterococcus faecium, was previously shown to bypass the D,D-transpeptidase activity of the classical penicillin-binding proteins leading to high level cross-resistance to glycopeptide and beta-lactam antibiotics. Ldt(fm) homologues from Bacillus subtilis (Ldt(Bs)) and E. faecalis (Ldt(fs)) were found here to cross-link their cognate disaccharide-peptide subunits containing meso-diaminopimelic acid (mesoDAP(3)) and L-Lys(3)-L-Ala-L-Ala at the third position of the stem peptide, respectively, instead of L-Lys(3)-d-iAsn in E. faecium. Ldt(fs) differed from Ldt(fm) and Ldt(Bs) by its capacity to hydrolyze the L-Lys(3)-D-Ala(4) bond of tetrapeptide (L,D-carboxypeptidase activity) and pentapeptide (L,D-endopeptidase activity) stems, in addition to the common cross-linking activity. The three enzymes were specific for their cognate acyl acceptors in the cross-linking reaction. In contrast to Ldt(fs), which was also specific for its cognate acyl donor, Ldt(fm) tolerated substitution of L-Lys(3)-D-iAsn by L-Lys(3)-L-Ala-L-Ala. Likewise, Ldt(Bs) tolerated substitution of mesoDAP(3) by L-Lys(3)-D-iAsn and L-Lys(3)-L-Ala-L-Ala in the acyl donor. Thus, diversification of the structure of peptidoglycan precursors associated with speciation has led to a parallel evolution of the substrate specificity of the L,D-transpeptidases affecting mainly the recognition of the acyl acceptor. Blocking the assembly of the side chain could therefore be used to combat antibiotic resistance involving L,D-transpeptidases.

Inhibitory Effects of Enterococcus Strains Obtained from a Probiotic Product on In Vitro Growth of Salmonella enterica Serovar Enteritidis Strain IFO3313

Enterococcus faecium and Enterococcus gallinarum strains were isolated from a commercial probiotic product and the effects of these strains on the growth of Salmonella enterica serovar Enteritidis strain IFO3313 were investigated. Viable cell counts of Salmonella Enteritidis in mixed cultures with the probiotic product isolate of E. faecium were significantly (P < 0.05) lower than those in pure cultures after 6, 8, and 24 h when the cultures were incubated in heart infusion broth at 37 and 41°C. Significant differences in viable cell counts of Salmonella Enteritidis in mixed cultures with the probiotic product isolate of E. gallinarum and those in pure cultures were also observed after 8 and 24 h at 37 and 41°C. Similar observations were shown in mixed cultures of Salmonella Enteritidis with the reference strains of E. faecium GIFU8355 and E. gallinarum ATCC 49573. Significant differences in viable cell counts of these enterococcal strains were not shown among pure and mixed cultures with Salmonella Enteritidis. The pH values in pure and mixed cultures were 7.0 or 7.5 throughout the experiments. E. faecium strains were found to harbor the genes encoding enterocins A and B and showed inhibitory zones with a diameter of 4 to 6 mm against growth of Salmonella Enteritidis in the enterocin production assays. However, the E. gallinarum strains possessed neither of the enterocin genes tested and exhibited no inhibition zone in the enterocin production assays. These results indicated that enterococcal strains exhibit inhibitory effects on the growth of Salmonella Enteritidis and these effects were due to both enterocin and nonenterocin factors.

Virulence of Broad- and Narrow-Host-RangeSalmonella entericaSerovars in the Streptomycin-PretreatedMouse Model

Salmonella enterica subspecies I serovars are common bacterial pathogens causing diseases ranging from enterocolitis to systemic infections. Some serovars are adapted to specific hosts, whereas others have a broad host range. The molecular mechanisms defining the virulence characteristics and the host range of a given S. enterica serovar are unknown. Streptomycin pretreated mice provide a surrogate host model for studying molecular aspects of the intestinal inflammation (colitis) caused by serovar Typhimurium (S. Hapfelmeier and W. D. Hardt, Trends Microbiol. 13:497-503, 2005). Here, we studied whether this animal model is also useful for studying other S. enterica subspecies I serovars. All three tested strains of the broad-host-range serovar Enteritidis (125109, 5496/98, and 832/99) caused pronounced colitis and systemic infection in streptomycin pretreated mice. Different levels of virulence were observed among three tested strains of the host-adapted serovar Dublin (SARB13, SD2229, and SD3246). Several strains of host restricted serovars were also studied. Two serovar Pullorum strains (X3543 and 449/87) caused intermediate levels of colitis. No intestinal inflammation was observed upon infection with three different serovar Paratyphi A strains (SARB42, 2804/96, and 5314/98) and one serovar Gallinarum strain (X3796). A second serovar Gallinarum strain (287/91) was highly virulent and caused severe colitis. This strain awaits future analysis. In conclusion, the streptomycin pretreated mouse model can provide an additional tool to study virulence factors (i.e., those involved in enteropathogenesis) of various S. enterica subspecies I serovars. Five of these strains (125109, 2229, 287/91, 449/87, and SARB42) are subject of Salmonella genome sequencing projects. The streptomycin pretreated mouse model may be useful for testing hypotheses derived from this genomic data.

Glycogen production by different Salmonella enterica serotypes: contribution of functional glgC to virulence, intestinal colonization and environmental survival

In enteric bacteria, the contribution of endogenous energy sources to survival both inside and outside the host is poorly understood. The contribution of glycogen production to the virulence, colonization and environmental survival of different Salmonella enterica serotypes was assessed. Of 19 serotypes (339 strains) tested for glycogen production, 17 (256 strains) were positive. The avian-specific serovars S. Gallinarum (62 strains) and S. Pullorum (21 strains) did not produce glycogen. The sequence of glgC in three S. Gallinarum strains tested revealed an identical deletion of 11 consecutive bases, which was not present in S. Pullorum, and a CCC insertion after position 597. Transduction of S. Gallinarum and S. Pullorum to a glycogen-positive phenotype did not change the ability to colonize the intestine or affect virulence in the chicken. Mortality rates in chickens following oral infection with a S. Typhimurium glycogen mutant (glgC : : km) were not significantly reduced, although colonization of the intestine was reduced over the first 4 weeks of the trial. Growth and yield of the glgC : : km mutant were comparable to the parent. The glgC mutant survived less well in faeces and in water at 4 degrees C when the strain was grown in LB broth containing 0.5 % glucose, and in saline it died off more rapidly after 7 days. The data suggest that glycogen has a complex but comparatively minor role in virulence and colonization, but a more significant role in survival.

The Genome of Salmonella enterica Serovar Gallinarum: Distinct Insertions/Deletions and Rare Rearrangements

Salmonella enterica serovar Gallinarum is a fowl-adapted pathogen, causing typhoid fever in chickens. It has the same antigenic formula (1,9,12:--:--) as S. enterica serovar Pullorum, which is also adapted to fowl but causes pullorum disease (diarrhea). The close relatedness but distinct pathogeneses make this pair of fowl pathogens good models for studies of bacterial genomic evolution and the way these organisms acquired pathogenicity. To locate and characterize the genomic differences between serovar Gallinarum and other salmonellae, we constructed a physical map of serovar Gallinarum strain SARB21 by using I-CeuI, XbaI, and AvrII with pulsed-field gel electrophoresis techniques. In the 4,740-kb genome, we located two insertions and six deletions relative to the genome of S. enterica serovar Typhimurium LT2, which we used as a reference Salmonella genome. Four of the genomic regions with reduced lengths corresponded to the four prophages in the genome of serovar Typhimurium LT2, and the others contained several smaller deletions relative to serovar Typhimurium LT2, including regions containing srfJ, std, and stj and gene clusters encoding a type I restriction system in serovar Typhimurium LT2. The map also revealed some rare rearrangements, including two inversions and several translocations. Further characterization of these insertions, deletions, and rearrangements will provide new insights into the molecular basis for the specific host-pathogen interactions and mechanisms of genomic evolution to create a new pathogen.

Differentiation of Salmonella Gallinarum biovar Gallinarum from Salmonella Gallinarum biovar Pullorum by PCR‐RFLP of the fimH gene

In our studies on FimH adhesins expressed by different Salmonella serovars, we cloned and sequenced the fimH genes from Salmonella enterica ssp. Enterica ser. Gallinarum biovar Gallinarum and S. enterica ssp. Enterica ser. Gallinarum biovar Pullorum. Comparison of the nucleotide sequences revealed the presence of a single-nucleotide polymorphism (SNP) at position 544 bp from the A of the start codon of the fimH open reading frame (ORF). Further analysis of the restriction enzyme sites in fimH gene showed that the SNP at this position is responsible for a sequence specifically recognized by SacI in S. Gallinarum biovar Gallinarum only, making it possible to differentiate both biovars with the use of polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Digestion of PCR amplicons of the fimH gene from S. Gallinarum biovar Gallinarum strains with SacI gave two DNA fragments of 554 and 472 bp and only one fragment of 1026 bp for S. Gallinarum biovar Pullorum. This allows a clear differentiation between these two biovars.

Development of a pcr system for the characterisation of Salmonella flagellin genes

Analysis of flagellin genes was carried out on strains of Salmonella Typhimurium, Salmonella Hadar, Salmonella Abortusequi, Salmonella Enteritidis and Salmonella Gallinarum serovars, using a PCR system designed in this study. The purpose of these studies was to explore the flagellin genes of biphasic and monophasic Salmonellae for future targeted genetic interventions. The PCR primers were designed for two different structural genes of flagellin (fliC, fljB), for the repressor of fliC (fljA), for the operator region of fliC, and for the invertase system responsible for phase variation in Salmonella (hin, hixL, hixR). PCR analysis revealed that all of the examined genes (fliC, fliC-operator, fljB, fljA, hin, hixL, hixR) were present in all S. Typhimurium (n = 10) and S. Hadar (n = 10) strains tested. The results obtained on S. Typhimurium and S. Hadar strains confirmed their biphasic character at DNA level. However, the S. Enteritidis (n = 46) and S. Gallinarum (n = 5) strains lacked the invertase system (hin, hixL, hixR) as well as the fljA and fljB genes, while fliC and its operator were detectable. Consequently, the S. Enteritidis strains could only express fliC gene resulting in phase H1 flagellin. The examined S. Gallinarum strains were also demonstrated to have a cryptic flagellin gene (fliC). On the other hand, PCR results on S. Abortusequi (n = 2) indicated that both flagellin genes (fliC, fljB) and the whole phase variation system were present in both strains tested but only the H2 phase gene (fljB) was expressed. The phenotype of these strains could be clarified by motility test and/or by classical flagellar serology. The findings are also substantiated by the results of serovar-specific PCR for S. Typhimurium and S. Enteritidis. In conclusion, the PCR system developed in this study proved to be suitable for characterisation of Salmonella flagellin genes and confirmed serological results regarding all S. Typhimurium, S. Hadar and S. Enteritidis strains. This system could also identify cryptic flagellar genes of S. Abortusequi and S. Gallinarum.

Transcriptional analysis of the vanC cluster from Enterococcus gallinarum strains with constitutive and inducible vancomycin resistance.

The vanC glycopeptide resistance gene cluster encodes enzymes required for synthesis of peptidoglycan precursors ending in D-Ala-D-Ser. Enterococcus gallinarum BM4174 and SC1 are constitutively and inducibly resistant to vancomycin, respectively. Analysis of peptidoglycan precursors in both strains indicated that UDP-MurNAc-tetrapeptide and UDP-MurNAc-pentapeptide[D-Ser] were synthesized in E. gallinarum SC1 only in the presence of vancomycin (4 microg/ml), whereas the "resistance" precursors accumulated in the cytoplasm of BM4174 cells under both inducing and noninducing conditions. Northern hybridization and reverse transcription-PCR experiments revealed that all the genes from the cluster, vanC-1, vanXY(C), vanT, vanR(C), and vanS(C), were transcribed from a single promoter. In the inducible SC1 isolate, transcriptional regulation appeared to be responsible for inducible expression of resistance. Promoter mapping in E. gallinarum BM4174 revealed that the transcriptional start site was located 30 nucleotides upstream from vanC-1 and that the -10 promoter consensus sequence had high identity with that of the vanA cluster. Comparison of the deduced sequence of the vanS(C) genes from isolates with constitutive and inducible resistance revealed several amino acid substitutions located in the X box (R200L) and in the region between the F and G2 boxes (D312N, D312A, and G320S) of the putative sensor kinase proteins from isolates with constitutive resistance.

Molecular characterization of multidrug-resistant Enterococcus spp. from poultry and dairy farms: detection of virulence and vancomycin resistance gene markers by PCR

Thirty multidrug-resistant Enterococcus spp. strains, including two from the milk of cows with mastitis, nine from chicken litter and 19 from turkey litter, were isolated. Twenty-five were identified by biochemical methods as E. gallinarum and five as E. faecalis. Most of the isolates were resistant to vancomycin, gentamicin, streptomycin, tetracycline, erythromycin, bacitracin, kanamycin and nalidixic acid but sensitive to ciprofloxacin, sulfamethoxazole, chloramphenicol, ampicillin and ofloxacin. Attempts were made by partial amplification of the gene sequences to detect the vancomycin resistance markers vanA (734-bp), vanB (420-bp), vanC1 (531-bp), and vanC2-C3 (673-bp); virulence markers cylA (427-bp) and cylB (225-bp) for enterococcal cytolysin and a biofilm-forming surface protein (Esp). Individual and multiplex-PCR assays for vancomycin resistance markers revealed the vanC1 gene in 22 E. gallinarum strains. None of the remaining isolates including five E. faecalis strains (MIC=2 microg ml(-1)) and three E. gallinarum strains (MIC=8 microg ml(-1)) had any of the van genes tested. Analysis by pulsed-field gel electrophoresis (PFGE) and a comparison of smaI banding profiles showed 11 different patterns. Probing with a DIG-labeled vanC1 PCR product indicated a common 38.0 kb SmaI DNA fragment in all the E. gallinarum strains harboring the vanC1 gene. The genes cylA and cylB were detected only in one clinical E. gallinarum isolate and two quality control clinical strains of E. faecalis (ATCC 51299 and 29212). None of the virulence factors were found in milk or poultry isolates. Intermediate level resistance to vancomycin in enterococci from the US animal farms was predominantly due to the presence of vanC1 gene.

Molecular study of vancomycin-resistant enterococci isolated from humans and from food in a cattle-rearing area of France.

Study possible links between vancomycin-resistant enterococci strains isolated from human stool samples and from pork or poultry food products. One hundred and eleven vancomycin-resistant enterococci strains (15 VanA and 96 VanC) were isolated from human stool samples and from pork or poultry food products. Characterization of the Tn1546-like element of the 15 VanA strains was realized by restriction analysis of PCR products and polymorphism study. The 96 strains of VanC phenotype (75 Enterococcus gallinarum and 21 Enterococcus casseliflavus) were analysed by pulsed-field gel electrophoresis (PFGE). In the study described here, polymorphism of the Tn1546-like element enabled the establishment of five groups. Groups III, IV and V were found only in human strains. Groups I and II were found to occur in strains isolated from humans and from food, suggesting a possible transfer of the Tn1546-like element. The isolates from Group I harboured the whole Tn1546 element. In Group II, the five strains possessed a novel Tn1546-like element, characterized by a single-nucleotide difference in the vanX gene and a deletion upstream from the nucleotide 164. Analysis by PFGE of the 75 E. gallinarum strains revealed 20 different patterns. One pattern was shared by isolates from pork food and human samples. None of the 21 E. casseliflavus strains tested was found to share similar PFGE patterns. Results tend to support the possible transfer of the Tn1546-like element between strains of VanA phenotype. Concerning VanC phenotype strains, the transfer was not demonstrated.

Mechanism of metronidazole-resistance by isolates of nitroreductase-producing Enterococcus gallinarum and Enterococcus casseliflavus from the human intestinal tract

Enterococcus casseliflavus and Enterococcus gallinarum strains resistant to metronidazole, nitrofurantoin and nitrofurazone were isolated from fecal samples of a patient with recurrent ulcerative colitis treated with metronidazole. Unlike other metronidazole-resistant bacteria, these strains produced nitroreductase but metabolized metronidazole to compounds that could not be detected by liquid chromatography with UV or mass spectral analysis. Metronidazole-susceptible Clostridium perfringens grew equally well in spent cultures of Enterococcus spp. incubated with or without metronidazole. These data indicate that the nitroreductases produced by these Enterococcus strains did not activate metronidazole to bactericidal metabolites and these bacteria may reduce the effectiveness of metronidazole. We have indirect evidence for an alternative pathway that results in metronidazole resistance. These strains of enterococcus had nitroreductase so resistance should not have occurred.

Biochemical Characteristics and Antimicrobials Susceptibility of Salmonella gallinarum Isolated in Korea

Fowl typhoid (FT) reported since 1992 in Korea is a septicemic disease of domestic birds caused by Salmonella gallinarum (S. gallinarum). The purpose of this study was to investigate the biochemical characteristics and antimicrobials susceptibility of field isolates of S. gallinarum isolated by year in Korea. A total of 258 isolates of S. gallinarum from 1995 to 2001 showed the same pattern in the majority of biochemical test such as IMViC (indole, methyl red, Voges-Proskauer and citrate utilization), carbohydrate fermentation and amino acid decarboxylation, and these results were almost in accordance with the traditional biochemical characteristics of S. gallinarum strain. When the antimicrobial susceptibility test against 258 isolates of S. gallinarum was performed by the disk diffusion method using 12 antimicrobial agents, all isolates from 1995 appeared to be susceptible to all of the antimicrobial agents tested except for tetracycline and oxytetracycline, whereas the vast majority of isolates from 2001 showed the reduced susceptibility to ampicillin (13.0%), gentamicin (43.4%), kanamycin (69.6%), enrofloxacin (6.5%), ciprofloxacin (10.9%), norfloxacin (52.5%) and ofloxacin (82. 6%). The prevalence of the prevalence of completely resistant isolates resistant isolates to one or more drugs rapidly increased from 0% in 1995 to 93.5% in 2001. The minimal concentrations range of the majority of antimicrobial agents to inhibit 50% (MIC50s) against S. gallinarum isolates increased from 0.06 approximately 8 microg/ml in 1997 to 2 approximately 256 microg/ml in 2001. Especially, MIC50s for gentamicin and fluoroquinolones of isolates from 2001 increased over 10-fold than those of isolates from 1997. Therefore, our results indicate that sorbitol fermentation and arginine decarboxylation showed the diversity by isolates and the vast majority of isolates from 2001 showed the reduced susceptibility to antimicrobials tested.

Virulence analysis of a Salmonella gallinarum strain by oral inoculation of 20-day-old chickens.

In order to know the effect of in vitro passages on the pathogenicity of the Salmonella gallinarum strain INTA 91, a lyophilized culture was compared with the same strain recently isolated from a sick bird. The mean lethal dose (LD50) of the orally administered lyophilized culture was determined as 2.04 x 10(8) colony-forming units (CFU)/chicken. There was no correlation between the LD50 dose and the degree of disease produced; doses 10 or 100 times higher than the calculated LD50 did not produce a more severe disease. In trial 1, chickens were challenged with 1.02 x 10(9) CFU per chicken (5LD50) of the lyophilized strain and reached 52.2% mortality at the end of the assay. In trial 2, three different groups of chickens were infected with a recent isolate of the same strain: 2.04 x 10(8) CFU/chicken, 4.1 x 10(8) CFU/chicken, and 2.1 x 10(9) CFU/chicken (i.e., 1LD50, 2LD50, and 10LD50 of the dose calculated for the lyophilized strain, respectively). These chicken groups presented higher mortality rates (90%, 100%, and 95%, respectively) than previous trials, showing that the S. gallinarum strain used here increased its virulence by in vivo infected chicken passage. In all assays, the disease started after an incubation period of around 5-6 days. To obtain reliable and reproducible results in future challenge experiments, a fixed limited number of in vitro passages of the S. gallinarum strain must be determined.

Protection against experimental fowl typhoid by parenteral administration of live SL5828, an aroA-serC (aromatic dependent) mutant of a wild-type Salmonella Gallinarum strain made lysogenic for P22 sie

A wild-type strain of Salmonella enterica serotype Gallinarum, lysogenized with P22 sie (superinfection-exclusion defective) was greatly attenuated for newly hatched or 21-day-old chickens. An aroA transductant of the lysogenic strain and an aroA-serC tetracycline-sensitive deletion or deletioninversion mutant of the latter were equally attenuated. Intramuscular administration of the aroA-serC strain to 21-day-old chickens protected them against oral challenge with 106 colony forming units of a highly virulent Gallinarum strain (no deaths in the 30 vaccinated chickens versus 14 of 30 in the control group). There was evidence of protection in the contents, mucosa and lymphoid tissue of the alimentary tract, in addition to that which occurred in the liver and spleen.A weak serological response was detected by enzyme-linked immunosorbent assay, indicating that use of such a strain is compatible with serological monitoring and would be a useful adjunct to control schemes for fowl typhoid.

Enterocin 012, a bacteriocin produced by Enterococcus gallinarum isolated from the intestinal tract of ostrich.

Enterococcus gallinarum strain 012, isolated from the duodenum of ostrich, produced enterocin 012 which is active against Ent. faecalis, Lactobacillus acidophilus, Lact. sake, Listeria innocua, Propionibacterium acidipropionici, Propionibacterium sp., Clostridium perfringens, Pseudomonas aeruginosa and Salmonella typhimurium. One of the four pathogenic strains of Escherichia coli isolated from the intestinal tract of ostrich was inhibited by enterocin 012. No antimicrobial activity was recorded against Bacillus cereus, Cl. sporogenes, Cl. tyrobutyricum, Leuconostoc cremoris, Pediococcus pentosaceus, Staphylococcus carnosus and Streptococcus thermophilus. Enterocin 012 was resistant to treatment with lysozyme, catalase, lipase and papain, but sensitive to Proteinase K, alpha-chymotrypsin, trypsin and pepsin. Treatment of enterocin 012 with gastric juice from the duodenum resulted in a 50% loss of antibacterial activity. Half of the activity was lost when incubated at 80 degrees C for 30 min, or when kept overnight at a pH of 1.0-5.0 and pH 11.0 and 12.0, respectively. Enterocin 012 production started in mid-logarithmic growth and reached a maximum of 800 AU ml-1, but increased further to 1600 AU ml-1 in the stationary growth phase. The peptide is approximately 3.4 kDa in size, as determined after partial purification with Amberlite XAD-1180 and ammonium sulphate precipitation, followed by tricine-sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The mechanism of antimicrobial activity against Lact. sake LMG 13558 is bactericidal and caused cell lysis of active growing cells.

The presence of genes homologous to the K88 genes faeH and faeI on the virulence plasmid of Salmonella gallinarum

A Tn 3 insertion mutation was produced in the virulence plasmid of a strain of Salmonella gallinarum which conferred avirulence by parenteral and oral routes but which was also less invasive following oral inoculation. The transposon was found to have inserted near an open reading frame (ORF) with no homologies in the data banks. This ORF was adjacent to two additional ORF's with a high degree of homology to Escherichia coli genes encoding the minor structural subunits (FaeH and FaeI) of the K88 fimbria. A similar region of homology was found by DNA-DNA hybridization on the virulence plasmids of S. pullorum, S. dublin and other S. gallinarum strains but not in the plasmids of S. typhimurium, S. enteritidis or S. cholerae-suis.

The presence of genes homologous to the K88 genes faeH and faeI on the virulence plasmid of Salmonella gallinarum.

A Tn3 insertion mutation was produced in the virulence plasmid of a strain of Salmonella gallinarum which conferred avirulence by parenteral and oral routes but which was also less invasive following oral inoculation. The transposon was found to have inserted near an open reading frame (ORF) with no homologies in the data banks. This ORF was adjacent to two additional ORF's with a high degree of homology of Escherichia coli genes encoding the minor structural subunits (FaeH and FaeI) of the K88 fimbria. A similar region of homology was found by DNA-DNA hybridization on the virulence plasmids of S. pullorum, S. dublin and other S. gallinarum strains but not in the plasmids of S. typhimurium, S. enteritidis or S. choleraesuis.

Identification of VanB-Type Vancomycin Resistance in Enterococcus gallinarum from Japan

In late January 1996, pus obtained from a periproctal abscess on an acute myeloid leukemia patient in Toho University Omori Hospital yielded Enterococcus gallinarum strain TUH327. Antimicrobial susceptibility testing by the agar dilution method showed that this strain was resistant to low levels of vancomycin but remained susceptible to teicoplanin, indicating resistance of the VanC type that is, the intrisic glycopeptide resistance of motile enterococci such as Enterococcus. casseliflavus and E. gallinarum. Subsequently, to confirm the existence of the VanC in E. gallinarum TUH 327, we carried out PCR using primer sets specific to vanA, vanB and vanC. Unexpectedly, results showed the coexistence of vanB and vanC in E. gallinarum TUH327. This appears to be the first reported isolation anywhere in the world of motile enterococci harboring vanB, and also the first report of vanB in any enterococci from Japan.