A highly specific Serratia-infecting T7-like phage inhibits biofilm formation in two different genera of the Enterobacteriaceae family

Due to the emergence of multidrug-resistant bacteria, bacteriophages have become a viable alternative in controlling bacterial growth or biofilm formation. Biofilm is formed by extracellular polymeric substances (EPS) and is one of the factors responsible for increasing bacterial resistance. Bacteriophages have been studied as a bacterial control agent by use of phage enzymes or due to their bactericidal activities. A specific phage against Serratia marcescens was isolated in this work and was evaluated its biological and genomic aspects. The object of this study was UFV01, a bacteriophage belonging to the Podoviridae family, genus Teseptimavirus (group of lytic viruses), specific to the species S. marcescens, which may be related to several amino acid substitutions in the virus tail fibers. Despite this high specificity, the phage reduced the biofilm formation of several Escherichia coli strains without infecting them. UFV01 presents a relationship with phages of the genus Teseptimavirus, although it does not infect any of the E. coli strains evaluated, as these others do. All the characteristics make the phage an interesting alternative in biofilm control in hospital environments since small breaks in the biofilm matrix can lead to a complete collapse.     

Stationary biofilm growth normalizes mutation frequencies and mutant prevention concentrations in Pseudomonas aeruginosa from cystic fibrosis patients

Bacterial biofilms play an important role in the persistent colonization of the respiratory tract in cystic fibrosis (CF) patients. The trade‐offs among planktonic or sessile modes of growth, mutation frequency, antibiotic susceptibility and mutant prevention concentrations (MPCs) were studied in a well‐defined collection of 42 CF Pseudomonas aeruginosa isolates. MICs of ciprofloxacin, tobramycin, imipenem and ceftazidime increased in the biofilm mode of growth, but not the MPCs of the same drugs. The mutation frequency median was significantly higher in planktonic conditions (1.1 × 10^?8) than in biofilm (9.9 × 10^?9) (p 0.015). Isolates categorized as hypomutable increased their mutation frequency from 3.6 × 10^?9 in the planktonic mode to 6 × 10^?8 in biofilm, whereas normomutators (from 9.4 × 10^?8 to 5.3 × 10^?8) and hypermutators (from 1.6 × 10^?6 to 7.7 × 10^?7) decreased their mutation frequencies in biofilm. High and low mutation frequencies in planktonic growth converge into the normomutable category in the biofilm mode of growth of CF P. aeruginosa, leading to stabilization of MPCs. This result suggests that once the biofilm mode of growth has been established, the propensity of CF P. aeruginosa populations to evolve towards resistance is not necessarily increased.     

Secondary multidrug efflux pump mutants alter Escherichia coli biofilm growth in the presence of cationic antimicrobial compounds

Escherichia coli possesses many secondary active multidrug resistance transporters (MDTs) that confer overlapping substrate resistance to a broad range of antimicrobials via proton and/or sodium motive force. It is uncertain whether redundant MDTs uniquely alter cell survival when cultures grow planktonically or as biofilms. In this study, the planktonic and biofilm growth and antimicrobial resistance of 13 E. coli K-12 single MDT gene deletion strains in minimal and rich media were determined. Antimicrobial tolerance to tetracycline, tobramycin and benzalkonium were also compared for each ΔMDT strain. Four E. coli MDT families were represented in this study: resistance nodulation and cell division members acrA, acrB, acrD, acrE, acrF and tolC; multidrug and toxin extruder mdtK; major facilitator superfamily emrA and emrB; and small multidrug resistance members emrE, sugE, mdtI and mdtJ. Deletions of multipartite efflux system genes acrB, acrE and tolC resulted in significant reductions in both planktonic and biofilm growth phenotypes and enhanced antimicrobial susceptibilities. The loss of remaining MDT genes produced similar or enhanced (acrD, acrE, emrA, emrB, mdtK, emrE and mdtJ) biofilm growth and antimicrobial resistance. ΔMDT strains with enhanced antimicrobial tolerance also enhanced biofilm biomass. These findings suggest that many redundant MDTs regulate biofilm formation and drug tolerance.     

Propagation method for persistent high yield of diverse Listeria phages on permissive hosts at refrigeration temperatures

The efficient production of a high concentration of bacteriophage in large volumes has been a limiting factor in the exploration of the true potential of these organisms for biotechnology, agriculture and medicine. Traditional methods focus on generating small volumes of highly concentrated samples as the end product of extensive mechanical and osmotic processing. To function at an industrial scale mandates extensive investment in infrastructure and input materials not feasible for many smaller facilities. To address this, we developed a novel, scalable, generic method for producing significantly higher titer psychrophilic phage (P < 2.0 × 10⁻⁶), 2- to 4-fold faster than traditional methods. We generate renewable high yields from single source cultures by propagating phage under refrigeration conditions in which Listeria, Yersinia and their phages grow in equilibrium. Diverse Yersinia and Listeria phages tested yielded averages of 3.49 × 10⁸ to 3.36 × 10¹² PFU/ml/day compared to averages of 1.28 × 10⁵ to 1.30 × 10¹⁰ PFU/ml/day by traditional methods. Host growth and death kinetics made this method ineffective for extended propagation of mesophilic phages.     

Escherichia coli enhances the virulence factors of Candida albicans,the cause of vulvovaginal candidiasis,in a dual bacterial/fungal biofilm

Co-infection with other microorganisms can promote the Candida albicans to be invasive. In this study, Escherichia coli and C. albicans were co-isolated from the women with candidiasis symptoms. The in vitro effects of E. coli on C. albicans hypha development, biofilm formation, antibiotic susceptibility, dispersion from the biofilm, expression of Als3, Hwp1, and Tup1 genes, and pathogenesis in Galleria mellonella were investigated. Electron microscopic images revealed that hypha induction was markedly increased in the bacteria-fungi co-culture. Biofilm formation was increased 2.2 fold in the presence of E. coli. The minimum inhibitory concentration of nystatin against Candida was increased from (μg mL^?1) 25 to 50 in the dual biofilm. Candida dissemination was increased up to 2.7 fold from the mixed fungi/bacteria biofilm. The expression of ALS3 and HWP1 genes was increased (5.9 and 2.0 fold, respectively) while the TUP1 gene expression was decreased (0.4 fold) when C. albicans was incubated with E. coli. The simultaneous injection of C. albicans and E. coli to the insect larvae increased Galleria mortality up to 40%. This study demonstrated the effects of E. coli to promote fungi virulence factors, which suggest polymicrobial interaction should be considered during treatment of fungal infections.     

Antimicrobial activities against biofilm formed by Proteus mirabilis isolates from wound and urinary tract infections

Background: Bacterial species are capable of living as biofilm and/or planktonic forms. There is increasing evidence for the role of bacterial biofilm in various wound and urinary tract infections (UTIs). The aim of the present study was to evaluate the ability of the bacteria, isolated from urinary tract infections (UTIs) and wound infections, to form biofilm and correlate the role of biofilm with their antimicrobial resistance. Materials and Methods: All the isolated bacteria were screened for their ability to form biofilm using the microtitre plate method. Results: Wound isolates of Staphylococcus aureus and Enterobacter sp. had more biofilm forming capacity than the UTI isolates. Proteus mirabilis isolates were among the strongest biofilm forming bacteria and were chosen for antimicrobial study. In sub-MIC concentrations of antimicrobial agents used, ciprofloxacin was found to be the most effective in decreasing biofilm formation. On the other hand, ceftriaxone and ciprofloxacin were effective in partial removal of preformed biofilm biomass. Conclusion: Ciprofloxacin was more effective in killing bacterial cells especially at high antimicrobial concentrations that could be reached in urine levels and can be used in impregenating catheters.     

Nosocomial pathogen biofilms on biomaterials: Different growth medium conditions and components of biofilms produced in vitro

Background/Purpose (s)Nosocomial pathogens can develop biofilms on hospital surfaces and medical devices; however, few studies have focused on the evaluation of mono-and dual-species biofilms developed by nosocomial pathogens under different growth conditions.MethodsThis study investigated biofilm development by nosocomial pathogens (Acinetobacter baumannii, Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa) on biomaterials in different culture media and their components of the extracellular matrix biofilm.ResultsThe mono-species biofilms showed cell densities from 7.50 to 9.27 Log₁₀ CFU/cm² on natural rubber latex type I (NLTI) and from 7.58 to 8.79 Log₁₀ CFU/cm² on stainless steel (SS). Dual-species biofilms consisted of S. aureus + P. aeruginosa (7.87–8.27 Log₁₀ CFU/cm² in TSBP and TSBME onto SS; p < 0.05), E. coli + P. aeruginosa (8.32–8.86 Log₁₀ CFU/cm² in TSBME onto SS and TSBP onto NLTI; p < 0.05), and S. aureus + E. coli (7.82 Log₁₀ CFU/cm² in TSBME onto SS; p < 0.05). Furthermore, biofilm detachment after proteinase K treatment was 5.54–32.81% compared to 7.95–24.15% after DNase I treatment in the mono-dual species biofilm matrix. Epifluorescence microscopy and scanning electron microscopy (SEM) enabled visualizing the bacteria and extracellular polymeric substances of biofilms on SS and NLTI.ConclusionNosocomial pathogens can develop biofilms on biomaterials. Mono-species biofilms of Gram-negative bacteria showed lower densities than dual-species biofilms in TSBME and TSBP. Additionally, dual-species biofilms showed a higher concentration of proteins and eDNA in the extracellular matrix.     

Evaluation of Combined Ciprofloxacin and azithromycin free and nano formulations to control biofilm producing Pseudomonas aeruginosa isolated from burn wounds

BackgroundNanoparticles are becoming increasingly important against resistant superbugs including Pseudomonas aeruginosa infections.AimsExploration of Azithromycin as an adjunctive therapy to Ciprofloxacin for treatment of P. aeruginosa infections. Also, preparation of Ciprofloxacin-Azithromycin nanoparticles on chitosan nanocarrier (Cipro-AZM-CS) and assessment of its antimicrobial effect in vitro and in vivo.MethodsDetection of biofilm production and biofilm-specific antibiotic resistance ndvB and tssC1 genes was attempted. Minimal inhibitory concentration (MIC) and Minimum biofilm eradication concentration (MBEC) were done in vitro for assessment of P. aeruginosa planktonic and biofilm forms eradication, respectively. In In vivo study, Cipro-AZM-CS and free form were used to evaluate survival rate, wound contraction and bacterial load in mice after third degree burn.ResultsAll isolates were positive for biofilm production and ndvB and tssC1 genes. Majority of isolates (37, 74%) were extensively drug resistant. In the planktonic state, MIC values of Cipro-AZM free and CS forms were significantly lower than free Cipro MIC (P = 0.015 and P < 0.001 respectively). Also, Cipro-AZM free and CS MBEC values were significantly lower than that of free Cipro (P < 0.010 and P < 0.001 respectively). Furthermore, The MIC and MBEC values of free Cipro-AZM decreased significantly when challenged with Cipro-AZM-CS (P = 0.009 and P < 0.001 respectively). In vivo study combined free and Cipro-AZM-CS treated subgroups showed 100% mice survival with early resolution of infection and wound contraction (75%, 77.5% respectively) VS 45% for Cipro CS (P < 0.001).ConclusionCombined free and Cipro-AZM-CS showed promising results in vitro and in vivo overcoming high resistance of biofilm producing P. aeruginosa.     

Protective and therapeutic application of the depolymerase derived from a novel KN1 genotype of Klebsiella pneumoniae bacteriophage in mice

Klebsiella pneumoniae is one of the major Gram-negative bacterial pathogens causing hospital-acquired multidrug-resistant infections, and the antimicrobial treatment options are scarce. The lack of available antimicrobials has prompted the development of alternative strategies for the treatment of these infections. In this study, a K. pneumoniae bacteriophage (vB_KpnP_IME321) targeting a KN1 capsular type strain, Kp409, was isolated, characterized and sequenced. This bacteriophage has a latent period of 20 min and a burst size of approximately 410 pfu/cell. It contained 49 predicted open reading frames, of which ORF42 was identified as encoding the putative capsule depolymerase. The enzyme expressed and purified in the Escherichia coli BL21 system, namely Dp42, could depolymerize the capsular polysaccharide of Kp409 and form translucent halos on the plates. The phage-encoded depolymerase could increase the inhibitory effect of serum on the growth of bacteria in vitro. Pre-treated with Dp42 rescued 100% of mice following lethal Kp409 challenge, and administration of this enzyme after infection significantly increased survival rates of infected mice in the animal experiment. In conclusion, the phage-encoded depolymerase Dp42 represents a potential alternative strategy for controlling infections mediated by K. pneumoniae expressing the KN1 capsular polysaccharide.     

Detection of somatic coliphages through a bioluminescence assay measuring phage mediated release of adenylate kinase and adenosine 5′-triphosphate

The feasibility of detecting somatic coliphages by phage infection of Escherichia coli WG5 and measurement of phage propagation by the lysis mediated release of the bacterial host adenylate kinase (AK) and adenosine 5′-triphosphate (ATP) detected by a bioluminescent signal was evaluated. After 2 h of incubation, all cultures infected with reference bacteriophage X174 showed a significant increase in the bioluminescent signal, even with number of phages as low as less of 10 plaque forming units (PFU). Naturally occurring somatic coliphages ensured a significant bioluminescent signal after 3 h of infection when >10 PFU were inoculated. These results indicate that an easy and reliable method to detect low numbers of coliphages in less than 3 h is feasible.     

Hospital Trichosporon asahii isolates with simple architecture biofilms and high resistance to antifungals routinely used in clinical practice

Infections by Trichosporon spp. are increasing worldwide and its treatment remains a challenge. Colonization of medical devices has been considered as a predisposing factor for trichosporonosis, which is related to fungal biofilm production. Thus, this study aimed to evaluate the ability of six hospital T. asahii isolates to form biofilm on abiotic surface, as well as to investigate the impact of three classic antifungals on both planktonic and biofilm forms. The fungal identification was based on macro and micromorphological characteristics, biochemical tests and confirmation by mass spectrometry assisted by the flight time desorption/ionization matrix (MALDI-TOF MS). Antifungal susceptibility assay of planktonic cells showed inhibitory and fungicidal concentrations ranging from 2.5 to 10 µg/mL for voriconazole, 2 to 8 µg/mL for fluconazole, and 1 to 4 µg/mL for amphotericin B. All T. asahii strains were able to form biofilms on the polystyrene microplates surface within 24 h, showing a simple architecture when compared with Candida spp. biofilm. On the other hand, the same antifungals did not show action in neither the inhibition of biofilm formation nor on the formed biofilm. Concluding, the present study reinforced the relevance of the MALDI-TOF MS methodology for a safe identification of T. asahii. Classic antifungals were active on the planktonic form, but not on the biofilms. All isolates formed biofilms on the polystyrene microplates and showed a simple architecture.     

Block copolymer mixtures as antimicrobial hydrogels for biofilm eradication

Current antimicrobial strategies have mostly been developed to manage infections due to planktonic cells. However, microbes in their nature state will tend to exist by attaching to and growing on living and inanimate surfaces that result in the formation of biofilms. Conventional therapies for treating biofilm-related infections are likely to be insufficient due to the lower susceptibility of microbes that are embedded in the biofilm matrix. In this study, we report the development of biodegradable hydrogels from vitamin E-functionalized polycarbonates for antimicrobial applications. These hydrogels were formed by incorporating positively-charged polycarbonates containing propyl and benzyl side chains with vitamin E moiety into physically cross-linked networks of “ABA”-type polycarbonate and poly(ethylene glycol) triblock copolymers. Investigations of the mechanical properties of the hydrogels showed that the G′ values ranged from 1400 to 1600 Pa and the presence of cationic polycarbonate did not affect the stiffness of the hydrogels. Shear-thinning behavior was observed as the hydrogels displayed high viscosity at low shear rates that dramatically decreased as the shear rate increased. In vitro antimicrobial studies revealed that the more hydrophobic VE/BnCl(1:30)-loaded hydrogels generally exhibited better antimicrobial/antifungal effects compared to the VE/PrBr(1:30) counterpart as lower minimum biocidal concentrations (MBC) were observed in Staphylococcus aureus (Gram-positive), Escherichia coli (Gram-negative) and Candida albicans (fungus) (156.2, 312.5, 312.5 mg/L for VE/BnCl(1:30) and 312.5, 2500 and 625 mg/L for VE/PrBr(1:30) respectively). Similar trends were observed for the treatment of biofilms where VE/BnCl(1:30)-loaded hydrogels displayed better efficiency with regards to eradication of biomass and reduction of microbe viability of the biofilms. Furthermore, a high degree of synergistic antimicrobial effects was also observed through the co-delivery of antimicrobial polycarbonates with a conventionally-used antifungal agent, fluconazole. These hydrogels also displayed excellent compatibility with human dermal fibroblasts with cell viability >80% after treatment with hydrogels loaded with cationic polymers and/or fluconazole at minimum biocidal concentrations (MBC).     

Mycobacterium avium ssp. hominissuis biofilm is composed of distinct phenotypes and influenced by the presence of antimicrobials

Mycobacterium avium ssp. hominissuis, hereafter referred to as M. avium, forms biofilm, a property that, in mice, is associated with lung infection via aerosol. As M. avium might co‐inhabit the respiratory tract with other pathogens, treatment of the co‐pathogen‐associated infections, such as in bronchiectasis, would expose M. avium to therapeutic compounds that may have their origin in other organisms sharing the natural environments. Incubation of M. avium with two compounds produced by environmental organisms, streptomycin and tetracycline, in vitro at subinhibitory concentrations increased biofilm formation in a number of M. avium strains, although exposure to ampicillin, moxifloxacin, rifampin and trimethoprim–sulphamethoxazole had no effect on biofilm formation. No selection of genotypically resistant clones was observed. Although incubation of bacteria in the presence of streptomycin upregulates the expression of biofilm‐associated genes, the response to the antibiotics had no association with the expression of a regulator (LysR) linked to the formation of biofilm in M. avium. Biofilms are composed of planktonic and sessile bacteria. Whereas planktonic M. avium is susceptible to clarithromycin and ethambutol (clinically used antimicrobials), sessile bacteria are at least three‐fold to four‐fold more resistant to antibiotics. The sessile phenotype, however, is reversible, and no selection of resistant clones was observed. Mice infected through the airway with both phenotypes were infected with a similar number of bacteria, demonstrating no phenotype advantage. M. avium biofilm formation is enhanced by commonly used compounds and, in the sessile bacterial phenotype, is resistant to clarithromycin and ethambutol, in a reversible manner.     

Characterization of a bacteriophage,isolated from a cow with mastitis,that is lytic against <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> strains

Methicillin-resistant strains of Staphylococcus aureus (MRSA) are now the most commonly reported antibiotic-resistant bacterium in clinical settings. Therefore, there is an urgent need to develop novel antibacterial agents to control this pathogen. Bacteriophage therapy is a potential alternative treatment for MRSA infections. The objective of this study was characterization of a novel virulent bacteriophage (MSA6) isolated from a cow with mastitis. Electron microscopy showed its resemblance to members of the family Myoviridae, with an isometric head (66 nm) and a long contractile tail (173 nm). The genome of phage MSA6 was tested by pulsed-field gel electrophoresis and estimated to be about 143 kb. It exhibited rapid adsorption (>82% in 5 min), a short latent period (15 min) and a relatively small burst size (23 PFU/cell). Isolated phage was capable of infecting a wide spectrum of staphylococcal strains of both human and bovine origin. The results of this investigation indicate that MSA6 is similar to other bacteriophages belonging to the family Myoviridae (Twort, K, G1, 812) that have been successfully used in bacteriophage therapy.     

Some characteristics of infectious RNA from potato virus Y.

High molecular weight (greater than 10⁸–10⁹) host DNA present in Escherichia coli cells infected with nuclear disruption-deficient and host DNA degradation-negative T4 bacteriophage multiple mutants is not packed into T4 progeny particles.     

Novel lytic bacteriophage AB7-IBB1 of Acinetobacter baumannii: isolation, characterization and its effect on biofilm

Acinetobacter baumannii infections are difficult to treat due to biofilm formation. The literature shows paucity of data on A. baumannii bacteriophages and their application in biofilm control. In this report, we have isolated a new lytic bacteriophage, AB7-IBB1, infecting A. baumannii. Transmission electron microscopy revealed its resemblance to members of the family Siphoviridae, with a tail size of 240 × 10 nm and an icosahedral head 50 nm in diameter. Plaques were 3-5 mm in diameter after 24 h, increasing to 7-9 mm in three days. The phage genome size was determined to be ~75 kb. AB7-IBB1 could lyse 23 of 39 (59 %) clinical isolates of A. baumannii. It exhibited rapid adsorption (>99 % adsorbed in 5 min), a latency period of 30 min and a burst size of 125 PFU/infected cell. The phage affected A. baumannii biofilm formation on an abiotic surface (polystyrene) and a biotic surface (human embryonic kidney 293 cell line). It also showed biofilm control ability on an abiotic surface (polystyrene). FESEM visualization studies confirmed the detrimental effect of phage AB7-IBB1 on host biofilm. In conclusion, this study reports a novel lytic bacteriophage, AB7-IBB1, belonging to family Siphoviridae, with promising anti-biofilm properties.     

Spermagglutinating Escherichia coli and its role in infertility: In vivo study

PurposeAssociation of Escherichia coli with its detrimental action on spermatozoa is well established in vitro. Therefore, an attempt was made to clarify the effect of presence of E. coli in Balb/c mouse vagina on fertility outcome.Materials and methodsAll the mice in experimental groups received intravaginal administration of either spermagglutinating E. coli, PBS or standard E. coli strain (MTCC 1687; non-spermagglutinating/spermimmobilizing). Different doses and durations of administration were 10⁴, 10⁶, or 10⁸ cfu for 10 consecutive days; 10⁴ or 10⁶ for 3 consecutive days. Subgroups were created to evaluate cytokine level in reproductive organ and histopathological changes in both reproductive and non-reproductive organs.ResultsAll the animals receiving either 10⁴, 10⁶ or 10⁸ cfu of spermagglutinating E. coli for 3 or 10 consecutive days were rendered infertile in contrast to groups receiving PBS or standard strain (MTCC 1687) of E. coli. Another group of mice receiving spermagglutinating E. coli when mated after the clearance of organism from mouse vagina under natural circumstances or with use of antibiotic remained fertile. No other clinical manifestation could be seen apparently or histologically, except minor rise in IL-10 level and mild leukocyte infiltration in vagina of animals inoculated with spermagglutinating E. coli.ConclusionsOur data suggests that presence of spermagglutinating strain of E. coli in vagina/vaginal tract might be playing significant role in fertility outcome.     

Determination of the duration of a primary immune response and the ID50 of ALA rabbit rotavirus in rabbits

Summary.  The rabbit model of rotavirus infection has been used to examine the immune response to rotavirus infection and to evaluate strategies for rotavirus vaccine development. To determine the 50% infectious does (ID₅₀) of tissue culture adapted ALA virus, rabbits were orally inoculated with 10¹–10³ PFU of ALA rotavirus. The ID₅₀ of ALA virus was determined to be 1.7×10² PFU. The immune response induced in rabbits infected at low virus doses (10²–10³ PFU) was of similar magnitude to the immune responses induced with a high dose (10⁶ PFU) inoculum, indicating that the immune response to ALA rotavirus in rabbits is not dose dependent. To determine if a single rotavirus inoculation would induce a long lasting immune response, four rabbits were inoculated once with ALA virus (3.5 × 10⁵ PFU) and their serologic and mucosal antirotavirus titers were monitored at intervals for 1.5–2 years. The infected rabbits maintained serologic and mucosal rotavirus antibody titers until the final time point more than 700 days post inoculation. These data are important because they indicate that the antigenic load achieved following a single oral inoculation is sufficient to achieve long lasting immunity, the goal of any potential vaccine. Received October 10, 1996 Accepted June 11, 1997     

Comparative antimicrobial susceptibility of biofilm versus planktonic forms of Salmonella enterica strains isolated from children with gastroenteritis

In the present study, 194 Salmonella enterica strains, isolated from infected children and belonging to various serotypes, were investigated for their ability to form biofilms and the biofilm forms of the isolated strains were compared to their corresponding planktonic forms with respect to the antimicrobial susceptibility. For the biofilm-forming strains, the minimum inhibitory concentration for bacterial regrowth (MICBR) from the biofilm of nine clinically applicable antimicrobial agents was determined, and the results were compared to the respective MIC values of the planktonic forms. One hundred and nine S. enterica strains out of 194 (56%) belonging to 13 serotypes were biofilm-forming. The biofilm forms showed increased antimicrobial resistance compared to the planktonic bacteria. The highest resistance rates of the biofilm bacteria were observed with respect to gentamicin (89.9%) and ampicillin (84.4%), and the lowest rates with respect to ciprofloxacin and moxifloxacin (2.8% for both). A remarkable shift of the MICBR₅₀ and MICBR₉₀ toward resistance was observed in the biofilm forms as compared to the respective planktonic forms. The development of new consensus methods for the determination of the antimicrobial susceptibility of biofilm forms seems to be a major research challenge. Further studies are required in order to elucidate the biofilm antimicrobial resistance mechanisms of the bacterial biofilms and their contribution to therapeutic failure in infections with in vitro susceptible bacteria.