Lactobacillus acidophilus VB1 co-aggregates and inhibits biofilm formation of chronic otitis media-associated pathogens.

This study aims to evaluate the antibacterial activity of Lactobacillus acidophilus, alone and in combination with ciprofloxacin, against otitis media-associated bacteria. L. acidophilus cells were isolated from Vitalactic B (VB), a commercially available probiotic product containing two lactobacilli species, L. acidophilus and Lactiplantibacillus (formerly Lactobacillus) plantarum. The pathogenic bacterial samples were provided by Al-Shams Medical Laboratory (Baqubah, Iraq). Bacterial identification and antibiotic susceptibility testing for 16 antibiotics were performed using the VITEK2 system. The minimum inhibitory concentration of ciprofloxacin was also determined. The antimicrobial activity of L. acidophilus VB1 cell-free supernatant (La-CFS) was evaluated alone and in combination with ciprofloxacin using a checkerboard assay. Our data showed significant differences in the synergistic activity when La-CFS was combined with ciprofloxacin, in comparison to the use of each compound alone, against Pseudomonas aeruginosa SM17 and Proteus mirabilis SM42. However, an antagonistic effect was observed for the combination against Staphylococcus aureus SM23 and Klebsiella pneumoniae SM9. L. acidophilus VB1 was shown to significantly co-aggregate with the pathogenic bacteria, and the highest co-aggregation percentage was observed after 24 h of incubation. The anti-biofilm activities of CFS and biosurfactant (BS) of L. acidophilus VB1 were evaluated, and we found that the minimum biofilm inhibitory concentration that inhibits 50% of bacterial biofilm (MBIC50) of La-CFS was significantly lower than MBIC50 of La-BS against the tested pathogenic bacterial species. Lactobacillus acidophilus, isolated from Vitane Vitalactic B capsules, demonstrated promising antibacterial and anti-biofilm activities against otitis media pathogens, highlighting its potential as an effective complementary/alternative therapeutic strategy to control bacterial ear infections.

Beneficial Effects of Spore-Forming Bacillus Probiotic Bacteria Isolated From Poultry Microbiota on Broilers' Health, Growth Performance, and Immune System.

Probiotics are known for their beneficial effects on poultry health and wellbeing. One promising strategy for discovering Bacillus probiotics is selecting strains from the microbiota of healthy chickens and subsequent screening for potential biological activity. In this study, we focused on three probiotic strains isolated from the gastrointestinal tract of chickens bred in different housing types. In addition to the previously reported poultry probiotic Bacillus subtilis KATMIRA1933, three strains with antimutagenic and antioxidant properties Bacillus subtilis KB16, Bacillus subtilis KB41, and Bacillus amyloliquefaciens KB54, were investigated. Their potential effects on broiler health, growth performance, and the immune system were evaluated in vivo. Two hundred newly hatched Cobb500 broiler chickens were randomly divided into five groups (n = 40). Four groups received a standard diet supplemented with the studied bacilli for 42 days, and one group with no supplements was used as a control. Our data showed that all probiotics except Bacillus subtilis KATMIRA1933 colonized the intestines. Treatment with Bacillus subtilis KB54 showed a significant improvement in growth performance compared to other treated groups. When Bacillus subtilis KB41 and Bacillus amyloliquefaciens KB54 were applied, the most significant immune modulation was noticed through the promotion of IL-6 and IL-10. We concluded that Bacillus subtilis KB54 supplementation had the largest positive impact on broilers' health and growth performance.

Antimicrobial and Anti-Biofilm Activity of Polymyxin E Alone and in Combination with Probiotic Strains of Bacillus subtilis KATMIRA1933 and Bacillus amyloliquefaciens B-1895 against Clinical Isolates of Selected Acinetobacter spp.: A Preliminary Study.

Acinetobacter spp., the nosocomial pathogen, forms strong biofilms and is resistant to numerous antibiotics, causing persistent infections. This study investigates the antibacterial and anti-biofilm activity of polymyxin E alone and in combination with the cell-free supernatants (CFS) of the tested probiotic bacilli, Bacillus subtilis KATMIRA1933 and Bacillus amyloliquefaciens B-1895 against the selected Acinetobacter spp. starins. Three isolates of Acinetobacter spp., designated as Acinetobacter spp. isolate 1; Acinetobacter spp. isolate 2, and Acinetobacter spp. isolate 3, were collected from patients with burns, wounds, and blood infections, respectively. Bacterial identification and antibiotic susceptibility testing were conducted using the VITEK2 system. Auto-aggregation and coaggregation of the tested bacilli strains with the selected Acinetobacter spp. isolates were evaluated. A disk diffusion assay was used to identify the microorganism's susceptibility to the selected antibiotics, alone and in combination with the CFS of the bacilli. The MIC and MBIC (minimum inhibitory and minimum biofilm inhibitory concentrations) of polymyxin E combined with bacilli CFS were determined. Acinetobacter spp. isolates were (i) sensitive to polymyxin E, (ii) able to form a strong biofilm, and (iii) resistant to the tested antibiotics and the CFS of tested bacilli. Significant inhibition of biofilm formation was noticed when CFS of the tested bacilli were combined with polymyxin E. The bacilli CFS showed synergy with polymyxin E against planktonic cells and biofilms of the isolated pathogens.

A Review of the Effects and Production of Spore-Forming Probiotics for Poultry.

One of the main problems in the poultry industry is the search for a viable replacement for antibiotic growth promoters. This issue requires a "one health" approach because the uncontrolled use of antibiotics in poultry can lead to the development of antimicrobial resistance, which is a concern not only in animals, but for humans as well. One of the promising ways to overcome this challenge is found in probiotics due to their wide range of features and mechanisms of action for health promotion. Moreover, spore-forming probiotics are suitable for use in the poultry industry because of their unique ability, encapsulation, granting them protection from the harshest conditions and resulting in improved availability for hosts' organisms. This review summarizes the information on gastrointestinal tract microbiota of poultry and their interaction with commensal and probiotic spore-forming bacteria. One of the most important topics of this review is the absence of uniformity in spore-forming probiotic trials in poultry. In our opinion, this problem can be solved by the creation of standards and checklists for these kinds of trials such as those used for pre-clinical and clinical trials in human medicine. Last but not least, this review covers problems and challenges related to spore-forming probiotic manufacturing.

Black cardamom essential oil prevents Escherichia coli O157:H7 and Salmonella Typhimurium JSG 1748 biofilm formation through inhibition of quorum sensing.

This study aimed to investigate the chemical composition, using GC-MS, and anti-biofilm potential of black cardamom essential oil (BCEO) against biofilms of Escherichia coli O157:H7 and Salmonella Typhimurium JSG 1748 through inhibition of bacterial quorum sensing. GC-MS quantification demonstrated that BCEO contains 1,8-cineole (44.24%), α-terpinyl acetate (12.25%), nerolidol (6.03%), and sabinene (5.96%) as the major bioactive compounds. Antioxidant assays for BCEO revealed the total phenolic and flavonoid mean values were 1325.03 ± 7.69 mg GAE 100/g and 168.25 ± 5.26 mg CE/g, respectively. In regards to antimicrobial potential, Candida albicans was the most sensitive species compared to Streptococcus mutans, Staphylococcus aureus, Listeria monocytogenes, Bacillus cereus, and Salmonella Typhimurium with the following zones of inhibition; 14.4 ± 0.52, 13.2 ± 0.42, 11.2 ± 0.28, 11.0 ± 0.52, 8.2 ± 0.24 and 6.6 ± 0.18 mm in diameter, respectively. Biofilm inhibition by BCEO was concentration-dependent, when various concentrations of 0.03, 0.06, 0.12, 0.25 and 0.5% were applied, 33.67, 34.14, 38.66, 46.65 and 50.17% of Salmonella Typhimurium biofilm was inhibited, while 47.31, 54.15, 76.57, 83.36 and 84.63% of Escherichia coli biofilm formation was prevented. Chromobacterium violaceum ATCC 12,472 and its product violacein, was used as a microbial indicator for enhancement or inhibition of quorum sensing. Our data showed that 0.5% of BCEO inhibited violacein production without influencing the growth of Chromobacterium violaceum, while 1% of BCEO, caused 100% inhibtion of violacein production together with 30% inhibition of growth. This study shows that BCEO possesses promising antioxidant and antimicrobial potential, and found anti-biofilm activities linked to the quenching of the quorum sensing system of E. coli and S. Typhimurium.

Anti-biofilm Potential of Elletaria cardamomum Essential Oil Against Escherichia coli O157:H7 and Salmonella Typhimurium JSG 1748.

Foodborne pathogens, microbial recurrent infections, and antibiotic resistance have driven researchers to explore natural compounds as safe alternative antimicrobials. In this study, the chemical profile, antimicrobial, and mutagenic activities of the Elletaria cardamomum essential oil were investigated. GC-MS analysis identified the major bioactive components as α-terpinyl acetate, 1,8-cineole, linalool acetate, and sabinene, at concentrations of 34.95, 25.30, 8.13, and 5.48% respectively, of the essential oil's content. Regarding antimicrobial activity, the minimum inhibitory concentration of green cardamom essential oil was 1% against Escherichia coli O157:H7 and Pseudomonas aeruginosa ATCC 14213. Green cardamom essential oil, when used at concentrations of 0.015, 0.031, 0.062, and 0.125% (v/v) prevented biofilm formation of Escherichia coli O157:H7 by 64.29, 65.98, 70.41, and 85.59%, respectively. Furthermore, these concentrations inhibited 6.13, 45.50, 49.45, and 100%, respectively, of the Salmonella Typhimurium JSG 1748 biofilm. A mutagenicity assay confirmed that green cardamom essential oil has no demonstrable mutagenic activity against the tested strains. The study's findings suggest that green cardamom derived bioactive compounds are safe organic antimicrobials, effective in controlling biofilm formation by Gram-negative pathogens. Moreover, such compounds could possibly be used in the food industry (e.g., bakery, dairy, meat, and other food products) as a safe alternative to chemical preservatives (antimicrobials) to enhance shelf life by improving the antimicrobial status while at the same time imparting a pleasant and appealing aroma for consumers.

Probiotic Bacilli Inhibit Salmonella Biofilm Formation Without Killing Planktonic Cells.

Salmonellosis is a foodborne infection caused by Salmonella. Domestic poultry species are one of the main reservoirs of Salmonella, which causes the foodborne infection salmonellosis, and are responsible for many cases of animal-to-human transmission. Keeping backyard chickens is now a growing trend, increasing the frequency of direct contact with the flock and, by consequence, the incidence of Salmonella infections. Bacillus subtilis KATMIRA1933 and Bacillus amyloliquefaciens B-1895 are probiotic bacilli that produce the bacteriocins subtilosin A and subtilin, respectively. The antimicrobial activity of the two strains was determined against the reference strain Micrococcus luteus ATCC 10420. The cell-free supernatant of B. subtilis KATMIRA1933 inhibited biofilm formation by Salmonella enterica subsp. enterica serovar Hadar, Salmonella enterica subsp. enterica serovar Enteritidis phage type 4, and Salmonella enterica subsp. enterica serovar Thompson by 51.1, 48.3, and 56.9%, respectively. The cell-free supernatant of B. amyloliquefaciens B-1895 inhibited the biofilm formation of these Salmonella strains by 30.4, 28.6, and 35.5%, respectively. These findings suggest that the bacillus strains may have the potential to be used as probiotics and antibiotic alternatives for the control of Salmonella in poultry. The number of planktonic cells was unaffected by treatment with the cell-free supernatant. A co-culture of the Salmonella strains with either bacilli showed no signs of growth inhibition, suggesting that it might have been quorum sensing that is affected by the two Bacillus strains.

Antimicrobial Activity of Bacillus subtilis KATMIRA1933 and Bacillus amyloliquefaciens B-1895 Against Staphylococcus aureus Biofilms Isolated from Wound Infection.

Staphylococcal wound infections range from mild to severe with life-threatening complications. The challenge of controlling such infections is related to bacterial biofilm formation, which is a major factor contributing to antibiotic resistance and infection recurrence. In this study, four clinical isolates of staphylococci species; two isolates of methicillin-resistant Staphylococcus aureus (MRSA) and two methicillin-sensitive Staphylococcus aureus (MSSA) isolates. The identification of bacterial species based on cell morphology, initial biochemical tests, and the VITEK2 system were used to confirm the clinical microbiological diagnosis. Antibiotic sensitivity testing showed that the isolated staphylococci were highly resistant to the following antibiotics, amoxicillin, penicillin G, cefotaxime, and methicillin. Combinations of cefotaxime with the cell-free supernatants (CFS) of Bacillus subtilis KATMIRA1933 and Bacillus amyloliquefaciens B-1895, each one separately showed complementary activity against the tested staphylococci. The co-aggregation capability of the tested bacilli as beneficial bacteria against isolated staphylococci was also evaluated. The data showed a strong co-aggregation with scores (+ 3, + 4) which were reported between the bacilli strains and the isolated staphylococci. Furthermore, the CFS of bacilli strains showed an inhibitory effect against biofilm-associated MRSA and MSSA. These findings confirmed the ability of beneficial bacteria to compete with the pathogens at the site of colonization or for the source of nutrients and, eventually, lead to inhibition of the pathogens' capability of causing a wound infection. Such beneficial bacteria could play an important role in future pharmaceutical and industrial applications.

Sex Hormones and Novel Corona Virus Infectious Disease (COVID-19).

Given the rapid spread of the coronavirus disease 2019 (COVID-19) pandemic and its overwhelming effect on health care systems and the global economy, innovative therapeutic strategies are urgently needed. The proposed primary culprit of COVID-19 is the intense inflammatory response-an augmented immune response and cytokine storm-severely damaging the lung tissue and rendering some patients' conditions severe enough to require assisted ventilation. Sex differences in the response to inflammation have been documented and can be attributed, at least in part, to sex steroid hormones. Moreover, age-associated decreases in sex steroid hormones, namely, estrogen and testosterone, may mediate proinflammatory increases in older adults that could increase their risk of COVID-19 adverse outcomes. Sex hormones can mitigate the inflammation response and might provide promising therapeutic potential for patients with COVID-19. In this article, we explore the possible anti-inflammatory effects of estrogen and testosterone and the anabolic effect of testosterone, with particular attention to the potential therapeutic role of hormone replacement therapy in older men and women with COVID-19.

Potential Probiotics Bacillus subtilis KATMIRA1933 and Bacillus amyloliquefaciens B-1895 Co-Aggregate with Clinical Isolates of Proteus mirabilis and Prevent Biofilm Formation.

A urinary tract infection (UTI) is a multi-factorial disease including cystitis, pyelonephritis, and pyelitis. After Escherichia coli, Proteus mirabilis is the most common UTI-associated opportunistic pathogen. Antibiotic resistance of bacteria and infection recurrence can be connected to biofilm formation by P. mirabilis. In this study, human and sheep isolates of P. mirabilis were investigated for antibiotic sensitivity using an antibiotic disk test. Co-aggregation of the tested potential probiotic bacilli, Bacillus amyloliquefaciens B-1895 and Bacillus subtilis KATMIRA1933, with the isolated pathogen was also evaluated. Then, the anti-biofilm activity of naturally derived metabolites, such as subtilin and subtilosin, in the bacilli-free supernatants was assessed against biofilms of P. mirabilis isolates. The isolated pathogens were sensitive to 30 µg of amikacin and 5 µg of ciprofloxacin but resistant to other tested antibiotics. After 24 h, auto-aggregation of B. amyloliquefaciens B-1895 was at 89.5% and higher than auto-aggregation of B. subtilis KATMIRA1933 (59.5%). B. amyloliquefaciens B-1895 strongly co-aggregated with P. mirabilis isolates from human UTIs. Cell-free supernatants of B. amyloliquefaciens B-1895 and B. subtilis KATMIRA1933 showed higher antimicrobial activity against biofilms of P. mirabilis isolated from humans as compared with biofilms of sheep isolates. According to our knowledge, this is the first report evaluating the anti-biofilm activity of probiotic spore-forming bacilli against clinical and animal UTI isolates of P. mirabilis. Further studies are recommended to investigate the anti-biofilm activity and the mode of action for the antimicrobial substances produced by these bacilli, subtilosin and subtilin.

Benzoyl Peroxide Inhibits Quorum Sensing and Biofilm Formation by Gardnerella vaginalis 14018.

Infection recurrence and antibiotic resistance of bacterial vaginosis-associated pathogenic biofilms underline the need for novel and effective treatment strategies. In this study, we evaluated the antimicrobial, antibiofilm, and quorum sensing inhibitory effects of benzoyl peroxide and salicylic acid against Gardnerella vaginalis ATCC 14018, the predominant pathogen of bacterial vaginosis. While the highest tested concentrations of 250 and 125 µg/mL for both compounds were not sufficient in completely inhibiting the growth of G. vaginalis ATCC 14018, they did prevent biofilm formation by inhibiting the bacterial quorum sensing system in the pathogen. To our knowledge, this report is the first evidence that benzoyl peroxide can have a quorum sensing-mediated biofilm controlling effect, as demonstrated using subinhibitory concentrations of this compound in order to reduce the cost, dosage, and negative side effects associated with current antimicrobial treatments.

Gemini Cationic Amphiphiles Control Biofilm Formation by Bacterial Vaginosis Pathogens.

Antibiotic resistance and recurrence of bacterial vaginosis (BV), a polymicrobial infection, justify the need for novel antimicrobials to counteract microbial resistance to conventional antibiotics. Previously, two series of cationic amphiphiles (CAms) which self-assemble into supramolecular nanostructures with membrane-lytic properties were designed with hydrophilic head groups and nonpolar domains. The combination of CAms and commonly prescribed antibiotics is suggested as a promising strategy for targeting microorganisms that are resistant to conventional antibiotics. Activities of the CAms against Gardnerella vaginalis ATCC 14018, a representative BV pathogen, ranged from 1.1 to 24.4 µM. Interestingly, the tested healthy Lactobacillus species, especially Lactobacillus plantarum ATCC 39268, were significantly more tolerant of CAms than the selected pathogens. In addition, CAms prevented biofilm formation at concentrations which did not influence the normal growth ability of G. vaginalis ATCC 14018. Furthermore, the biofilm minimum bactericidal concentration (MBC-Bs) of CAms against G. vaginalis ATCC 14018 ranged from 58.8 to 425.6 µM, while much higher concentrations (≥850 µM) were required to produce ≥3-log reductions in the number of biofilm-associated lactobacilli. The conventional antibiotic metronidazole synergized strongly with all tested CAms against planktonic cells and biofilms of G. vaginalis ATCC 14018. The synergism between CAms and the tested conventional antibiotic may be considered a new, effective, and beneficial method of controlling biofilm-associated bacterial vaginosis.

Subtilosin Prevents Biofilm Formation by Inhibiting Bacterial Quorum Sensing.

Subtilosin, the cyclic lantibiotic protein produced by Bacillus subtilis KATMIRA1933, targets the surface receptor and electrostatically binds to the bacterial cell membrane. In this study, subtilosin was purified using ammonium sulfate ((NH4)2SO4) precipitation and purified via column chromatography. Subtilosin's antibacterial minimum and sub-minimum inhibitory concentrations (MIC and sub-MIC) and anti-biofilm activity (biofilm prevention) were established. Subtilosin was evaluated as a quorum sensing (QS) inhibitor in Gram-positive bacteria using Fe(III) reduction assay. In Gram-negative bacteria, subtilosin was evaluated as a QS inhibitor utilizing Chromobacterium voilaceum as a microbial reporter. The results showed that Gardnerella vaginalis was more sensitive to subtilosin with MIC of 6.25 µg/mL when compared to Listeria monocytogenes (125 µg/mL). The lowest concentration of subtilosin, at which more than 90% of G. vaginalis biofilm was inhibited without effecting the growth of planktonic cells, was 0.78 µg/mL. About 80% of L. monocytogenes and more than 60% of Escherichia coli biofilm was inhibited when 15.1 µg/mL of subtilosin was applied. Subtilosin with 7.8-125 µg/mL showed a significant reduction in violacein production without any inhibitory effect on the growth of C. violaceum. Subtilosin at 3 and 4 µg/mL reduced the level of Autoinducer-2 (AI-2) production in G. vaginalis. However, subtilosin did not influence AI-2 production by L. monocytogenes at sub-MICs of 0.95-15.1 µg/mL. To our knowledge, this is the first report exploring the relationship between biofilm prevention and quorum sensing inhibition in G. vaginalis using subtilosin as a quorum sensing inhibitor.

Self-assembled cationic amphiphiles as antimicrobial peptides mimics: Role of hydrophobicity, linkage type, and assembly state.

Inspired by high promise using naturally occurring antimicrobial peptides (AMPs) to treat infections caused by antimicrobial-resistant bacteria, cationic amphiphiles (CAms) were strategically designed as synthetic mimics to overcome associated limitations, including high manufacture cost and low metabolic stability. CAms with facially amphiphilic conformation were expected to demonstrate membrane-lytic properties and thus reduce tendency of resistance development. By systematically tuning the hydrophobicity, CAms with optimized compositions exhibited potent broad-spectrum antimicrobial activity (with minimum inhibitory concentrations in low µg/mL range) as well as negligible hemolytic activity. Electron microscope images revealed the morphological and ultrastructure changes of bacterial membranes induced by CAm treatment and validated their membrane-disrupting mechanism. Additionally, an all-atom molecular dynamics simulation was employed to understand the CAm-membrane interaction on molecular level. This study shows that these CAms can serve as viable scaffolds for designing next generation of AMP mimics as antimicrobial alternatives to combat drug-resistant pathogens.

Control of Biofilm Formation: Antibiotics and Beyond.

Biofilm-associated bacteria are less sensitive to antibiotics than free-living (planktonic) cells. Furthermore, with variations in the concentration of antibiotics throughout a biofilm, microbial cells are often exposed to levels below inhibitory concentrations and may develop resistance. This, as well as the irresponsible use of antibiotics, leads to the selection of pathogens that are difficult to eradicate. The Centers for Disease Control and Prevention use the terms "antibiotic" and "antimicrobial agent" interchangeably. However, a clear distinction between these two terms is required for the purpose of this assessment. Therefore, we define "antibiotics" as pharmaceutically formulated and medically administered substances and "antimicrobials" as a broad category of substances which are not regulated as drugs. This comprehensive minireview evaluates the effect of natural antimicrobials on pathogens in biofilms when used instead of, or in combination with, commonly prescribed antibiotics.

Natural antimicrobials subtilosin and lauramide arginine ethyl ester synergize with conventional antibiotics clindamycin and metronidazole against biofilms of Gardnerella vaginalis but not against biofilms of healthy vaginal lactobacilli.

The purpose of this study was to evaluate the ability of clindamycin and metronidazole to synergize with natural antimicrobials against biofilms of bacterial vaginosis (BV)-associated Gardnerella vaginalis. Minimum bactericidal concentrations for biofilm cells (MBCs-B) were determined for each antimicrobial. The MBCs-B of lauramide arginine ethyl ester (LAE), subtilosin, clindamycin and metronidazole were 50, 69.5, 20 and 500 µg mL(-1), respectively. A checkerboard assay and isobologram were used to analyze the type of interactions between these antimicrobials. The combination of metronidazole with natural antimicrobials did not inhibit planktonic lactobacilli. Clindamycin with either LAE or with subtilosin was inhibitory for planktonic but not for biofilm-associated lactobacilli. All tested antimicrobial combinations were inhibitory for BV-associated Mobiluncus curtisii and Peptostreptococcus anaerobius. LAE and subtilosin synergized with clindamycin and metronidazole against biofilms of G. vaginalis but not biofilm-associated vaginal lactobacilli. The biofilms of BV-associated pathogens can be controlled by synergistically acting combinations of conventional antibiotics and natural antimicrobials which will help better management of current antibiotics, especially considering robust bacterial resistance. Our findings create a foundation for a new strategy in the effective control of vaginal infections.

Susceptibility of Gardnerella vaginalis biofilms to natural antimicrobials subtilosin, ε-poly-L-lysine, and lauramide arginine ethyl ester.

Bacterial vaginosis is a common vaginal infection associated with numerous gynecological and obstetric complications. This condition is characterized by the presence of thick adherent vaginal biofilms, composed mainly of Gardnerella vaginalis. This organism is thought to be the primary aetiological cause of the infection paving the way for various opportunists to colonize the niche. Previously, we reported that the natural antimicrobials subtilosin, ε-poly-L-lysine, and lauramide arginine ethyl ester selectively inhibit the growth of this pathogen. In this study, we used plate counts to evaluate the efficacy of these antimicrobials against established biofilms of G. vaginalis. Additionally, we validated and compared two rapid methods (ATP viability and resazurin assays) for the assessment of cell viability in the antimicrobial-treated G. vaginalis biofilms. Out of the tested antimicrobials, lauramide arginine ethyl ester had the strongest bactericidal effect, followed by subtilosin, with clindamycin and polylysine showing the weakest effect. In comparison to plate counts, ATP viability and resazurin assays considerably underestimated the bactericidal effect of some antimicrobials. Our results indicate that these assays should be validated for every new application.