Potentiation of the humoral immune response elicited by a commercial vaccine against bovine respiratory disease by Enterococcus faecalis CECT7121.

Vaccination against pathogens involved in bovine respiratory disease (BRD) is a useful tool to reduce the risk of this disease however, it has been observed that the commercially available vaccines only partially prevent the infections caused by Pasteurella multocida and Mannheimia haemolytica. Therefore, it is recommended to search for new adjuvant strategies to minimise the economic impact of this respiratory syndrome. A possibility to improve the conventional vaccine response is to modulate the immune system with probiotics, since there is accumulating evidence that certain immunomodulatory strains administered around the time of vaccination can potentiate the immune response. Considering veterinary vaccines are frequently tested in murine models, we have developed an immunisation schedule in BALB/c mice that allows us to study the immune response elicited by BRD vaccine. In order to evaluate a potential strategy to enhance vaccine efficacy, the adjuvant effect of Enterococcus faecalis CECT7121 on the murine specific humoral immune response elicited by a commercial vaccine against BRD was studied. Results indicate that the intragastric administration of E. faecalis CECT7121 was able to induce an increase in the specific antibody titres against the bacterial components of the BRD vaccines (P. multocida and M. haemolytica). The quality of the humoral immune response, in terms of antibody avidity, was also improved. Regarding the cellular immune response, although the BRD vaccination induced a low specific secretion of cytokines in the spleen cell culture supernatants, E. faecalis CECT7121-treated mice showed higher interferon-γ production than immunised control mice. Our results allowed us to conclude that the administration of E. faecalis CECT7121 could be employed as an adjuvant strategy to potentiate humoral immune responses.

Probiotic activity of Enterococcus faecalis CECT7121: effects on mucosal immunity and intestinal epithelial cells.

AIMS: To analyse the effect of Enterococcus faecalis CECT7121 on intestinal epithelial cells (IECs) and its effects on the mucosal immune response. METHODS AND RESULTS: Enterococcus faecalis CECT7121 showed a high adhesion capacity to completely and heterogeneously differentiated human intestinal epithelial cell line (Caco-2 cells). In addition, the contact of this bacterium with Caco-2 cells did not induce inflammatory chemokines (IL-8 and CCL-20). The presence of IgA(+) and IL-6(+) cells in the small intestine, as well as the production of inflammatory cytokines (TNFα, IL-6 and IL-12) in the gut, was determined after intragastric inoculation of Ent. faecalis CECT7121 in BALB/c mice. The administration of Ent. faecalis CECT7121 increased the number of IgA(+) cells in the intestinal lamina propria without modifying the percentage of IL-6(+) cells. No differences were observed in the cytokines measured in the intestinal extracts between probiotic-treated and control mice. CONCLUSIONS: Enterococcus faecalis CECT7121 stimulates local mucosal immunity and adheres to IECs without inducing inflammatory signals. SIGNIFICANCE AND IMPACT OF THE STUDY: Our results indicate that, apart from its already reported systemic immune activity, Ent. faecalis CECT7121 has a modulatory effect at a local level.

Bio-preservation of ground beef meat by Enterococcus faecalis CECT7121.

Meat and particularly ground beef is frequently associated with Food Poisoning episodes and breeches in Food Safety. The main goal of this research was to evaluate the bactericide effect of the probiotic Enterococcus faecalis CECT7121, against different pathogens as: Escherichia coli O157:H7, Staphylococcus aureus, Clostridium perfringens and Listeria monocytogenes, inoculated in ground beef meat. Three studies were performed to evaluate the inhibition of E. faecalis CECT7121 on ground beef meat samples inoculated with pathogens: Study I: Samples (100 g meat) were inoculated with pathogens (10(3) CFU/g)) and E. faecalis CECT7121 (10(4) CFU/g) simultaneously. Study II: Samples were inoculated with E. faecalis CECT7121 24 h before the pathogens. Study III: E. faecalis CECT7121were inoculated 24 h after pathogens. The viable counts were performed at 0, 24, 48 and 72 h post-inoculation. The simultaneous inoculation of E. faecalis CECT7121 with E. coli O157:H7 strains resulted in the absence of viable counts of bacteria at 72 h post-treatment. However, when the probiotic was added 24 h before and 24 h after the pathogen E. coli O157:H7, viable cells were not detected at 24 h and 48 h post-treatment, respectively. Consistently, neither S. aureus nor Cl. perfringens viable bacteria were detected at 48 h in whole assays when inoculated with E. faecalis CECT7121. The same trend than described before was obtained after applying the 3 models assayed for L. monocytogenes. The current assays demonstrated the bactericide activity of E. faecalis CECT7121 strain on bacterial pathogens in ground beef meat.

Bio-preservation of ground beef meat by Enterococcus faecalis CECT7121

Meat and particularly ground beef is frequently associated with Food Poisoning episodes and breeches in Food Safety. The main goal of this research was to evaluate the bactericide effect of the probiotic Enterococcus faecalis CECT7121, against different pathogens as: Escherichia coli O157:H7, Staphylococcus aureus, Clostridium perfringens and Listeria monocytogenes, inoculated in ground beef meat. Three studies were performed to evaluate the inhibition of E. faecalis CECT7121 on ground beef meat samples inoculated with pathogens: Study I: Samples (100 g meat) were inoculated with pathogens (10³ CFU/g)) and E. faecalis CECT7121 (10(4) CFU/g) simultaneously. Study II: Samples were inoculated with E. faecalis CECT7121 24 h before the pathogens. Study III: E. faecalis CECT7121were inoculated 24 h after pathogens. The viable counts were performed at 0, 24, 48 and 72 h post-inoculation. The simultaneous inoculation of E. faecalis CECT7121 with E. coli O157:H7 strains resulted in the absence of viable counts of bacteria at 72 h post-treatment. However, when the probiotic was added 24 h before and 24 h after the pathogen E. coli O157:H7, viable cells were not detected at 24 h and 48 h post-treatment, respectively. Consistently, neither S. aureus nor Cl. perfringens viable bacteria were detected at 48 h in whole assays when inoculated with E. faecalis CECT7121. The same trend than described before was obtained after applying the 3 models assayed for L. monocytogenes. The current assays demonstrated the bactericide activity of E. faecalis CECT7121 strain on bacterial pathogens in ground beef meat.

High-level resistance to gentamicin: genetic transfer between Enterococcus faecalis isolated from food of animal origin and human microbiota.

AIMS: To investigate the in vivo gene transfer of high-level gentamicin resistance (HLRG) from Enterococcus faecalis isolated from the food of animal origin to a human isolate, using a mouse model of intestinally colonized human microbiota. METHODS AND RESULTS: In vitro study: The presence of plasmids involved in HLRG coding was investigated. After the conjugation experiment, the recipient strain, Ent. faecalis JH2-SS, acquired a plasmid responsible for HLRG [minimal inhibitory concentration (MIC) >800 µg ml(-1) ], in a similar position to the donor cells. In vivo study: Seven BALB/c mice were dosed with ceftriaxone (400 mg kg(-1) ) and then inoculated with a dilution of 1/100 of human faeces (HFc). After 72 h, Ent. faecalis JH2-SS (recipient) was inoculated and then, after a further 72 h, the animals were given Ent. faecalis CS19, isolated from the food of animal origin, involved in HLRG (donor). The presence of transconjugant strains in HFc was subsequently recorded on a daily basis until the end of the experiment. The clonal relationship between Ent. faecalis and Escherichia coli in faeces was assessed by RAPD-PCR. Both the in vitro and in vivo studies showed that the receptor strain acquired a plasmid responsible for HLRG (MICs >800 µg ml(-1) ), which migrated with a similar relative mobility value. Transconjugant strains were detected from 24 h after the donor strain inoculation and persisted until the end of the experiment. CONCLUSIONS: The in vivo gene transfer of HLRG from Ent. faecalis strains, isolated from the food of animal origin, to human microbiota has been demonstrated in a mouse model. SIGNIFICANCE AND IMPACT OF THE STUDY: The complexity found on the therapeutic responses of invasive infectious diseases caused by Ent. faecalis facilitates the assessment of food of animal origin as a resistant pathogen reservoir. In addition, this study may contribute to the understanding of antimicrobials' resistance gene transfer between Ent. faecalis strains from food and human GI tract.

Enrofloxacin-based therapeutic strategy for the prevention of endometritis in susceptible mares.

Enrofloxacin (EFX) is often used empirically to prevent uterine infections in mares in order to improve efficiency on Commercial Embryo Transfer Farms. This study investigated the uterine distribution of EFX and its metabolite ciprofloxacin (CFX) in mares and assessed the minimal inhibitory concentrations (MIC) of EFX against various common pathogens as a basis for establishing a rational dosing schedule. Plasma and uterine pharmacokinetic (PK) studies were performed in two groups (n = 5) of healthy mares following intravenous (i.v.) administration of EFX at either 2.5 and at 5 mg/kg bodyweight. Plasma and endometrial tissue samples, taken before for up to 48 h after treatment were analysed by Reverse Phase HPLC. MIC values for wild strains of Gram-negative (Escherichia coli, Pseudomonas aeruginosa) and Gram-positive bacteria (beta-haemolytic streptococci) ranged from 0.25-2 and 1.5-3.0 microg/mL respectively. In terms of tissue distribution, the sum of the endometrial concentrations of the parent drug (EFX) and its active metabolite (CFX) (in terms of AUC), exceeded those in plasma by 249% and 941% following administration of EFX at 2.5 and 5 mg/kg respectively. After i.v. treatment with EFX at 5 mg/kg, endometrial concentrations of EFX and CFX above the MIC value were detected for 36-48 and 22-43 h posttreatment for Gram-negative and -positive isolates respectively. Concentrations above MIC were maintained for much shorter periods at the lower (2.5 mg/kg) treatment dose. Based on these results, a conventional dose (5 mg/kg) of EFX given prebreeding followed by two further doses at 36-48 h postbreeding are proposed as a rational strategy for using of EFX as a preventative therapy against a variety of common bacterial strains associated with equine endometritis.

[Molecular characterization of Streptococcus pyogenes from invasive disease and streptococcal toxic shock syndrome episodes]. / Caracterización molecular de Streptococcus pyogenes causantes de enfermedad invasora y síndrome de shock tóxico estreptocócico.

Streptococcus pyogenes causes a variety of common human diseases, including pharyngitis, scarlet fever and impetigo. Nevertheless, the past decades have witnessed a worldwide resurgence in invasive disease and streptococcal toxic shock syndrome (STSS). The objective of the present study is to evaluate the genetic diversity, virulence gene distribution (spe, sme and ssa genes) and susceptibility pattern of 10 S. pyogenes isolates causing invasive disease and STSS. The isolates were recovered from blood cultures of hospitalized patients at Hospital Santamarina and Nueva Clínica Chacabuco, Tandil, Buenos Aires, Argentina between 12/2000-04/2005. Two pulse field gel electrophoretic patterns predominated. The most frequent one included 5 characteristic isolates of emm1-T1 type, toxin gene profile speA, speB, speF, speG and smeZ. The second pattern included 2 characteristic isolates of emm3-TNT type (speB, speF, speG). The other 3 isolates corresponded to types emm49-TNT (speB, speC, speF, speG), emm75-T25 (speB, speF, speG) and emm83-TNT (speB, speF, speG, ssa, smeZ). All isolates were susceptible to penicillin, cefotaxime, erythromycin, clindamycin, chloramphenicol, tetracycline and rifampicin. The data from the present study demonstrated genetic diversity among the strains. Types emm1 and emm3 were prevalent in invasive disease. The empirical treatment with the combination of penicillin and clindamicin is still valid.

Assessment of the in vitro efficacy of the novel antimicrobial peptide CECT7121 against human Gram-positive bacteria from serious infections refractory to treatment.

BACKGROUND: Resistant Gram-positive bacteria are causing increasing concern in clinical practice. This work investigated the efficacy of AP-CECT7121 (an antimicrobial peptide isolated from an environmental strain of Enterococcus faecalis CECT7121) against various pathogenic Gram-positive bacteria. METHODS: Strains were isolated from intensive care unit patients unresponsive to standard antibiotic treatments. Inhibitory activity of AP-CECT7121 was assessed using the agar-well diffusion method. The most resistant isolates from each species screened (Enterococcus faecium, Enterococcus faecalis,Staphylococcus aureus, Streptococcus pneumoniae, Streptococcus pyogenes, Clostridium perfringens and Clostridium difficile) were further examined in time-killing curve studies. RESULTS: These bactericidal kinetic experiments demonstrated a rapid killing effect with no viable bacteria being detected within 30 and 90 min for enterococcal and streptococcal strains and 180 min for community-acquired methicillin-resistant S. aureus and C. perfringens: viable counts for C. difficile were threefold decreased after 90 min. CONCLUSIONS: AP-CECT7121 may provide a novel strategy for treating potentially fatal clinical infections in hospitalized patients.

In vitro efficacy of the novel peptide CECT7121 against bacteria isolated from mastitic dairy cattle.

AIMS: To evaluate the in vitro bactericidal activity of the novel antimicrobial peptide (AP) CECT7121 against Gram-positive bacteria from mastitic dairy cattle. METHODS AND RESULTS: A total of 15 Staphylococcus aureus, 10 Streptococcus dysgalactiae, 7 Strep. uberis, 1 Strep. agalactiae strains were isolated from 33 different mastitic dairy cattle, sourced from two dairies in Tandil-Argentina. Isolates from each of the bacterial species screened which developed the lowest inhibition zones in response to the peptide, were further evaluated in a series of time-killing curve studies. No survivors were detected in whole strains (from the three Streptococcal species isolated) within 120 min of incubation in presence of the peptide. The Staph. aureus isolates were less sensitive but, nevertheless, a drop in viable counts to below the detection limit was achieved for all the test strains by the final postincubation sampling point at 180 min. CONCLUSIONS: The study demonstrated the in vitro efficacy of the AP-CECT7121 against a variety strains of Gram positives isolated from mastitic dairy cattle. SIGNIFICANCE AND IMPACT OF THE STUDY: There is urgent global interest in the development of natural alternatives for the control and prevention of mastitis. Confirmation of the in vitro activity of the novel AP-CECT7121 against Gram-positive isolates encourages further research.

Characteristics of an environmental strain, Enterococcus faecalis CECT7121, and its effects as additive on craft dry-fermented sausages.

Lactic acid bacteria are the most adequate microorganisms for natural preservation of food. In the present work, the strain of Enterococcus faecalis CECT7121 was employed in the manufacture of craft dry-fermented sausages and its performance as a biopreservative was analysed. This strain is devoid of the genes for haemolysin and gelatinase and does not produce biogenic amines. It is sensitive to almost all the antibiotics tested and opsonophagocytic assays showed that it is devoid of a capsule. This strain had a high LD50 (10(11)CFU ml(-1)) in mice. No statistical differences were found between control and sausages inoculated with E. faecalis CECT7121 regarding the production of lactic acid, pH variation over time, reaching a minimum pH value of 5.1, and sensory analysis in both series. Sausages inoculated with E. faecalis CECT7121 had lower viable counts of Enterobacteriaceae, Staphylococcus aureus and other Gram-positive cocci at the end of fermentation and 7 days and no viable enterobacteria and S. aureus were recovered at the end of drying. E. faecalis CECT7121 did not affect the growth of Lactobacillus spp. but it displaced the autochthonous populations of enterococci. E. faecalis CECT7121 was recovered in each time point as assessed by its inhibitory activity on Listeria monocytogenes and S. aureus. These results would indicate that the addition of E. faecalis CECT7121 during the manufacture of craft dry-fermented sausages offers an interesting alternative for biopreservation.

Partial characterization of enterocin MR99 from a corn silage isolate of Enterococcus faecalis.

AIMS: To assess the inhibitory activity on Gram-positive and Gram-negative bacteria of several species of enterococci recovered from a natural corn silage. METHODS AND RESULTS: The inhibitory activity of strains of Enterococcus faecalis (58), Enterococcus faecium (35), Enterococcus gallinarum (3) and Enterococcus casseliflavus (4) were studied employing indicator strains from various sources (clinical, food and ATCC). Enterococcus faecalis MR99, the only strain with inhibitory activity, inhibited other enterococci, Listeria spp., Staphylococcus aureus, Clostridium spp., Bacillus spp., Escherichia coli, Shigella sonnei and Shigella flexneri. The bacterium contained only one conjugative pheromone-responsive plasmid. The partially chromatography-purified MR99 enterocin (PPE) had a molecular weight of approx. 5000 Da and a pI of 6.2, was sensitive to proteolytic enzymes and could be extracted in benzene and butanol. It appeared stable to adjustment of pH 4.0, 5.0, 6.0, 7.0 and 8.0 and was resistant to heat. Inactivation was at 15 min at 121 degrees C. Enterocin MR99 was bactericidal on strains of Listeria monocytogenes, Staph. aureus, and bovine mastitis agents, it was bacteriostatic on E. coli. Although enterocins MR99 and AS48 have inhibitory activity on Gram-negative bacilli, PCR studies demonstrated a lack of relationship between them. CONCLUSIONS: The active component had a protein nature, was resistant to heat and presented a wide inhibitory spectrum. SIGNIFICANCE AND IMPACT OF THE STUDY: The biological properties of Ent. faecalis MR99 suggest that this strain merits further investigations so it can be applied in human and veterinary health programmes.

[Evaluation of the bacterial flora in natural corn silage]. / Evaluación de la flora bacteriana en un ensilado natural de maíz.

Silage is the best method for forage conservation, keeping it in a fresh condition and with high nutrient content. In order to study the natural evolution of maize silage without additives during 50 days after sealed, pH, temperature changes, number and type of the indigenous bacteria were studied every 5 days. The initial pH decreased from 6.40 to 4.10 and temperature stabilized at 26 degrees C. The mesophilic aerobic bacteria were quickly reduced across time. Enterococci were descended gradually. Clostridia remained in a low quantity. The microorganisms most frequently isolated were Escherichia coli, Clostridium butyricum, Enterococcus faecalis and Lactobacillus plantarum; this acid lactic bacteria was the principal cause of the abundant production of anaerobic fermentation of sugars in the forage reaching the pH which maintained the silage in adequate conditions.

Evaluación de la flora bacteriana en un ensilado natural de maíz / Evaluation of the bacterial flora in natural corn silage

Silage is the best method for forage conservation, keeping it in a fresh condition and with high nutrient content. In order to study the natural evolution of maize silage without additives during 50 days after sealed, pH, temperature changes, number and type of the indigenous bacteria were studied every 5 days. The initial pH decreased from 6.40 to 4.10 and temperature stabilized at 26 degrees C. The mesophilic aerobic bacteria were quickly reduced across time. Enterococci were descended gradually. Clostridia remained in a low quantity. The microorganisms most frequently isolated were Escherichia coli, Clostridium butyricum, Enterococcus faecalis and Lactobacillus plantarum; this acid lactic bacteria was the principal cause of the abundant production of anaerobic fermentation of sugars in the forage reaching the pH which maintained the silage in adequate conditions.(AU)

Evaluación de la flora bacteriana en un ensilado natural de maíz / Evaluation of the bacterial flora in natural corn silage

Silage is the best method for forage conservation, keeping it in a fresh condition and with high nutrient content. In order to study the natural evolution of maize silage without additives during 50 days after sealed, pH, temperature changes, number and type of the indigenous bacteria were studied every 5 days. The initial pH decreased from 6.40 to 4.10 and temperature stabilized at 26 degrees C. The mesophilic aerobic bacteria were quickly reduced across time. Enterococci were descended gradually. Clostridia remained in a low quantity. The microorganisms most frequently isolated were Escherichia coli, Clostridium butyricum, Enterococcus faecalis and Lactobacillus plantarum; this acid lactic bacteria was the principal cause of the abundant production of anaerobic fermentation of sugars in the forage reaching the pH which maintained the silage in adequate conditions.

An unusual transmission of Neisseria meningitidis: neonatal conjunctivitis acquired at delivery from the mother's endocervical infection.

BACKGROUND: Primary meningococcal conjunctivitis is assumed to be due to the direct inoculation of Neisseria meningitidis into the conjunctival sac from an exogenous source. According to a literature review, no case of neonatal conjunctivitis infection acquired at delivery from maternal endocervicitis has been published. GOAL: To report a case of meningococcal neonatal conjunctivitis acquired at delivery because of the mother's endocervical infection and cross-transmission of the strain with her partner. STUDY DESIGN: Strains were characterized by bacteriologic and serologic methods including grouping (agglutination), typing, and subtyping (enzyme-linked immunoabsorbent assay). Molecular analysis was done by pulsed-field gel electrophoresis. RESULTS: The three strains (newborn infant, mother, partner) were of the same antigenic formula (C:NT:P1.NT) and exhibited similar NheI and SpeI pulsed-field gel electrophoresis patterns. CONCLUSION: The identical phenotypic and genomic analysis of strains is the evidence for N meningitidis transmission at delivery from a maternal endocervical infection to the newborn infant and cross transmission between sexual partners.

Evaluación de la flora bacteriana en un ensilado natural de maíz. / [Evaluation of the bacterial flora in natural corn silage]

Silage is the best method for forage conservation, keeping it in a fresh condition and with high nutrient content. In order to study the natural evolution of maize silage without additives during 50 days after sealed, pH, temperature changes, number and type of the indigenous bacteria were studied every 5 days. The initial pH decreased from 6.40 to 4.10 and temperature stabilized at 26 degrees C. The mesophilic aerobic bacteria were quickly reduced across time. Enterococci were descended gradually. Clostridia remained in a low quantity. The microorganisms most frequently isolated were Escherichia coli, Clostridium butyricum, Enterococcus faecalis and Lactobacillus plantarum; this acid lactic bacteria was the principal cause of the abundant production of anaerobic fermentation of sugars in the forage reaching the pH which maintained the silage in adequate conditions.

Characterization of an outbreak of tetM-containing Neisseria gonorrhoeae in Argentina.

Phenotypic and molecular characterization of an outbreak of 9 Neisseria gonorrhoeae (NG) isolates exhibiting high-level plasmid mediated resistance to penicillin and tetracycline (PP-TRNG) that took place in Tandil, Argentina between February and April 1995. Comparison with the patterns of the 3 PP-TRNG strains previously isolated were made. We determined the following markers for each strain: antimicrobial susceptibility, serogroup, auxotype, plasmid profile, presence of tetM determinant and restriction pattern of the tetM-containing plasmid. Antimicrobial tests values were: tetracycline disk diameter 12-14 mm, minimum inhibitory concentration (MIC) 32 micrograms/ml; penicillin disk diameter 6 mm, MIC 32 micrograms/ml and sensitive by both methods to spectinomycin, cefuroxime, ceftriaxone and ciprofloxacin. All isolates were of the same serogroup (WI). Ten of the strains, including the 9 from Tandil outbreak, were arginine-requiring, while the other 2 were methionine and arginine-requiring. All of them demonstrate the same plasmid profile (2.6, 3.2, 25.2 MDa). They were positive for the tetM determinant and the restriction analysis identified it is a Dutch-type plasmid. In spite of the temporal and geographical dispersion, PP-TRNG strains in Argentina seem to be highly homogeneous in terms of antimicrobial susceptibility, serogroup, plasmid profiles and even auxotype.

PIP: This study determines the phenotypic and molecular characterization of an outbreak of 9 Neisseria gonorrhea isolates exhibiting high-level plasmid-mediated resistance to penicillin and tetracycline (PP-TRNG) that took place in Tandil, Argentina. Subjects included patients attending Tandil's Sanatamarina Hospital. Individuals infected with sexually transmitted disease were examined using a standardized questionnaire. Results show that all isolates produced B-lactamase, were penicillin resistant (disk zone diameter 6 mm, MIC 32 mcg/ml) and high-level tetracycline resistant (disk zone diameter 12-14 mm, MIC 32 mcg/ml). Moreover, all strains were fully sensitive to cefuroxime, ceftriaxone, ciprofloxacin, and spectinomycin; while autotype and serogroup results showed that all the strains were arginine-requiring and of serogroup WI. In spite of the temporal and geographical dispersion, PP-TRNG strains in Argentina seem to be highly homogeneous in terms of antimicrobial susceptibility, serogroup, plasmid profiles, and even auxotype.

Argentinian collaborative study on prevalence of erythromycin and penicillin susceptibility in Streptococcus pyogenes. The Argentinian Streptococcus Study Group.

Two monthly studies on the prevalence of penicillin and erythromycin susceptibility of Streptococcus pyogenes were performed in May and October of 1994 in Argentina. A total of 58 centers from 27 cities participated in these studies. A total of 1072 isolates were tested by a diffusion method, although 595 isolates were tested both by the diffusion and an agar dilution method (n = 1767 isolates). No penicillin-resistant streptococci were found in our study (MIC100 = 0.03 microgram/ml). Only four isolates were confirmed as erythromycin resistant S. pyogenes (prevalence 0.14 and 0.28% in May and October 1994, respectively). Resistance in three isolates was due to an inducible mechanism, although in one strain a different phenotype was observed.