High Biofilm Production Research Articles

Biofilm Eradication of Stenotrophomonas maltophilia by Levofloxacin and Trimethoprim-Sulfamethoxazole.

Stenotrophomonas maltophilia is a nonfermenting Gram-negative drug-resistant pathogen that causes healthcare-associated infections. Clinical isolates from Mexico were assessed for biofilm formation using crystal violet staining. Antimicrobial susceptibility was evaluated in planktonic and biofilm cells using the broth microdilution method. The effects of antibiotics on biofilms were visualized using fluorescence microscopy. Fifty isolates were included in this study, of which 14 (28%) were biofilm producers (9 [64%] from blood and 5 [36%] from respiratory samples). In planktonic cells 4/50 (8%) of isolates were resistant to levofloxacin (8.0%) and 22/50 (44%) were resistant to trimethoprim-sulfamethoxazole. All isolates were resistant to levofloxacin and trimethoprim-sulfamethoxazole in biofilm cells. Bacterial biofilms treated with different concentrations of both antibiotics were completely disrupted. In conclusion, S. maltophilia isolated from blood had higher biofilm production than those isolated from respiratory samples. Biofilm production was associated with increased antibiotic resistance. Antibiotic monotherapy might not be the best course of action for the treatment of S. maltophilia infections in Mexico, because it might cause biofilm production and antimicrobial resistance.

Genetic, virulence, and antimicrobial resistance characteristics associated with distinct morphotypes in ST11 carbapenem-resistant Klebsiella pneumoniae

ABSTRACT ST11 is the most common lineage among carbapenem-resistant Klebsiella pneumoniae (CRKP) infections in Asia. Diverse morphotypes resulting from genetic mutations are associated with significant differences in microbial characteristics among K. pneumoniae isolates. Here, we investigated the genetic determinants and critical characteristics associated with distinct morphotypes of ST11 CRKP. An ST11-KL47 CRKP isolate carrying a pLVPK-like virulence plasmid was isolated from a patient with a bloodstream infection; the isolate had the “mcsw” morphotype. Two distinct morphotypes (“ntrd” and “msdw”) were derived from this strain during in vitro passage. Whole genome sequencing was used to identify mutations that cause the distinct morphotypes of ST11 CRKP. Transmission electron microscopy, antimicrobial susceptibility tests, growth assays, biofilm formation, virulence assays, membrane permeability assays, and RNA-seq analysis were used to investigate the specific characteristics associated with different morphotypes of ST11 CRKP. Compared with the parental mcsw morphotype, the ntrd morphotype resulted from mutation of genes involved in capsular polysaccharide biosynthesis (wza, wzc, and wbaP), a result validated by gene knockout experiments. This morphotype showed capsule deficiency and lower virulence potential, but higher biofilm production. By contrast, the msdw morphotype displayed competition deficiency and increased susceptibility to chlorhexidine and polymyxin B. Further analyses indicated that these characteristics were caused by interruption of the sigma factor gene rpoN by insertion mutations and deletion of the rpoN gene, which attenuated membrane integrity presumably by downregulating the phage shock protein operon. These data expand current understanding of genetic, virulence, and antimicrobial resistance characteristics associated with distinct morphotypes in ST11 CRKP.

Evaluation of Biofilm Production and Antifungal Susceptibility to Fluconazole in Clinical Isolates of Candida spp. in Both Planktonic and Biofilm Form.

Candida spp. are an important opportunistic pathogen that can represent a possible cause of severe infections, especially in immunocompromised individuals. The clinical impact of Candida spp. depends, in part, on the ability to form biofilms, communities of nestled cells into the extracellular matrix. In this study, we compared the biofilm formation ability of 83 strains of Candida spp. isolated from blood cultures and other materials, such as respiratory samples, urine, and exudate, and their sensitivity to fluconazole (FLZ). Strains were divided into tertiles to establish cut-offs to classify isolates as low, moderate, or high biofilm producers (<0.26, 0.266-0.839, >0.839) and biofilms with low, moderate, or high metabolic activity (<0.053, 0.053-0.183, >0.183). A non-linear relationship between biofilm production and metabolic activity was found in C. glabrata and C. tropicalis. In addition, the increase in minimum biofilm eradication concentrations (MBEC50) compared to the Minor Inhibitory Concentration (PMIC) of the planktonic form in Candida spp. confirms the role of biofilm in the induction of resistance to FLZ.

Investigating Lemna minor and microorganisms for the phytoremediation of nanosilver and microplastics

Due to improper disposal of materials, the amount of pollution that enters waterways has greatly increased. Pollutants that can escape into waterways include microplastics and nanosilver - which are used in protective equipment and disinfectants, respectively. Phytoremediation, a method of using plants to remove pollutants from the environment, has shown great potential for remediating such contaminated aquatic environments. In addition, bacteria that promote plant growth have been shown to alleviate stress from plants and make biofilms that can capture small particles. Here, we employed Lemna minor, an aquatic floating plant, in combination with three plant growth-promoting bacteria to see which combination could effectively remove more pollutants from a simulated environment. We measured the pollutant-removal efficiency of L. minor, chlorophyll content, biofilm efficiency, and water toxicity. The ability of L. minor to accumulate nanosilver and microplastics increased significantly with the addition of bacteria. The bacteria with the highest biofilm production, measured through crystal violet staining, was associated with an increase in the amount of pollutants removed. When L. minor was exposed to just the pollutants, chlorophyll content significantly decreased from 4.17mg/mL to as low as 2.86 mg/mL; however, with the addition of the bacteria, chlorophyll increased to a point where there was not significant damage. When exposed to the initial and post-remediation concentrations of nanosilver particles, the negative impacts on zebrafish embryos were reduced. These forms of bacteria-assisted phytoremediation provide insight into how treatment plants can effectively remove pollutants from wastewater with the addition of bacteria.

The effect of NaCl, pH, and phosphate on biofilm formation and exopolysaccharide production by high biofilm producers of Bacillus strains.

Biofilm formation is an effective survival strategy of plant-associated microorganisms in hostile environments, so the application of biofilm-forming and exopolysaccharide (EPS)-producing beneficial microbes to plants has received more attention in recent years. This study examined the ability of biofilm and EPS production of Bacillus subtilis and Bacillus thuringiensis strains under different NaCl concentrations (0, 50, 100, 200, and 400mmol/L), pH values (5.5, 6.5, 7.5, and 8.5), and phosphate levels (0, 25, 50, and 100mmol/L at 0 and 400mmol/L NaCl). B. subtilis BS2 and B. thuringiensis BS6/BS7 strains significantly increased biofilm formation in a similar pattern to EPS production under salt stress. B. subtilis BS2/BS3 enhanced biofilm production at slightly acidic pH with a lower EPS production but the other strains formed considerably more amount of biofilm and EPS at alkaline pH. Interestingly, higher levels of phosphate substantially decreased biofilm and EPS production at 0mmol/L NaCl but increased biofilm formation at 400mmol/L salt concentration. Overall, contrary to phosphate, salt and pH differently influenced biofilm and EPS production by Bacillus strains. EPS production contributed to biofilm formation to some extent under all the conditions tested. Some Bacillus strains produced more abundant biofilm under salt and pH stress, indicating their potential to form in vivo biofilms in rhizosphere and on plants, particularly under unfavorable conditions.

Potential of some microbial isolates on diesel hydrocarbons removal, bio surfactant production and biofilm formation.

Potential of Arthrobacter citreus B27Pet, Bacillus thuringiensis B48Pet and Candida catnulata to produce biosurfactant using four different carbon sources (naphthalene, hexadecane, diesel and petroleum crude oil) was investigated. Removal of petroleum crude oil from aqueous culture and degradation of diesel were also determined using single and mixed culture of strains. The biofilm existence in single and mixed culture of strains was considered using naphthalene, hexadecane and diesel in culture medium. Cell surface hydrophobicity of A. citreus was higher than other isolates which also showed maximum surface tension reduction and emulsification index. As a whole, remarkable biosurfactant production occurred using petroleum crude oil as a carbon source in medium. A. citreus was found to be more robust than other tested strains in removal efficiency of crude oil due to its biosurfactant production capability. Statistically significant positive correlation was observed between biofilm existence and surface tension using diesel and hexadecane as carbon source. Overall diesel biodegradation efficiency by the mix culture of three applied strains was about 75% within a short period of time (10 days) which was accompanied with high biofilm production.

Capacity of biofilm production by filamentous fungi isolated from the uterus of susceptible mares

Fungi are opportunistic microorganisms that adhere to surfaces. Their intrauterine growth is influenced by impaired immunity, indiscriminate use of antibiotics, and poor vulvar conformation. Many publications use yeast research models, but only some report biofilm production by filamentous fungi. This work aimed to biofilm capacity from isolates of filamentous fungi found in uterine samples from susceptible mares. Uterine saline lavage samples from 41 mares with a history of subfertility were collected and sent to LABRAPE-UFRPE, Brazil. Samples were cultured on Sabouraud Dextrose Agar plates with 10% chloramphenicol and incubated at 25°C for up to 15 days, inspecting every 24 hours. After development of the colonies, identification used the macro and micromorphological aspects of the somatic and reproductive structures, comparing them with the literature. To induce biofilm production, isolates were re-cultured on Sabouraud dextrose agar at 37°C for 72 h. Conidia were collected by washing the surface of the cultures using 5mL PBS, pH 7.2, supplemented with 0.025% (v/v) Tween 20. Subsequently, conidia were counted in a Neubauer chamber, and the inoculum was adjusted to the concentration of 1 × 105 cells in RPMI 1640and buffered to pH 7.0. Biofilms were produced in 96-well microtiter plates by adding 200µL of standardized cell suspension in MOPS-RPMI 1640 buffer in each well, at each determined period (24, 48, 72, and 96h) then incubated at 37°C. At each time interval, biofilms were washed three times with PBS pH 7.2 then quantified using crystal violet technique (absorbance 570nm). Of the samples, 12.2% were positive for fungal endometritis, with Cladosporium spp. isolated in 60% of the positive samples, Trichosporon spp. and Penicillium spp. in 20% of the positive samples each. After induction of biofilm production, fungi of the genus Cladosporium spp. and Penicillium spp. showed a high biofilm production capacity, with a growth peak at 48 hours after incubation and a gradual decrease in this production in the subsequent hours. Cladosporium spp. showed low biofilm production until 96h of incubation, and Penicillium spp zeroed its formation simultaneously. In conclusion filamentous fungi isolated from the uterus of mares have a greater biofilm production capacity after 48 hours incubation, which tends to decrease over time but can be maintained for up to 96 hours, depending on the gender involved in the infection, as well as the quantity and quality of the substrate available.

Determination of the ability of matrix-assisted laser desorption ionization time-of-flight mass spectrometry to identify high-biofilm-producing strains.

The traditional method for assessing the capacity of a microorganism to produce biofilm is generally a static in vitro model in a multi-well plate using the crystal violet (CV) binding assay, which takes 96 h. Furthermore, while the method is simple to perform, its reproducibility is poor. We evaluated whether matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) could make it possible to differentiate between high-and low-biofilm-producing microorganisms on 24-h cultures of Staphylococcus aureus and Candida albicans. We included 157 strains of S. aureus and 91 strains of C. albicans obtained from the blood cultures of patients with bacteremia/candidemia. We tested biofilm production using the CV binding assay as the gold standard to classify strains as low or high biofilm producers. We then applied MALDI-TOF MS to create a machine learning-based predictive model using 40 strains of S. aureus and C. albicans, each with extreme absorbance values, and validated this approach with the remaining 117 and 51 strains using the random forest algorithm and the support vector machine algorithm, respectively. Overall, 81.2% of the S. aureus strains (95/117) and 74.5% of the C. albicans strains (38/51) used for validation were correctly categorized, respectively, as low and high-biofilm-producing. Classification based on MALDI-TOF MS protein spectra enables us to predict acceptable information about the capacity of 24-h cultures of S. aureus and C. albicans to form biofilm.

Acinetobacter baumannii in suspected bacterial infections: Association between multidrug resistance, virulence genes, & biofilm production.

Acinetobacter baumannii has emerged as a nosocomial pathogen with a tendency of high antibiotic resistance and biofilm production. This study aimed to determine the occurrence of A. baumannii from different clinical specimens of suspected bacterial infections and furthermore to see the association of biofilm production with multidrug resistance and expression of virulence factor genes in A. baumannii. A. baumannii was confirmed in clinical specimens by the detection of the blaOXA-51-like gene. Biofilm production was tested by microtitre plate assay and virulence genes were detected by real-time PCR. A. baumannii was isolated from a total of 307 clinical specimens. The isolate which showed the highest number of A. baumannii was an endotracheal tube specimen (44.95%), then sputum (19.54%), followed by pus (17.26%), urine (7.49%) and blood (5.86%), and <2 per cent from body fluids, catheter-tips and urogenital specimens. A resistance rate of 70-81.43 per cent against all antibiotics tested, except colistin and tigecycline, was noted, and 242 (78.82%) isolates were multidrug-resistant (MDR). Biofilm was detected in 205 (66.78%) with a distribution of 54.1 per cent weak, 10.42 per cent medium and 2.28 per cent strong biofilms. 71.07 per cent of MDR isolates produce biofilm (P<0.05). Amongst virulence factor genes, 281 (91.53%) outer membrane protein A (OmpA) and 98 (31.92%) biofilm-associated protein (Bap) were detected. Amongst 100 carbapenem-resistant A. baumannii, the blaOXA-23-like gene was predominant (96%), the blaOXA-58-like gene (6%) and none harboured the blaOXA-24-like gene. The metallo-β-lactamase genes blaIMP-1 (4%) and blaVIM-1(8%) were detected, and 76 per cent showed the insertion sequence ISAba1. The majority of isolates studied were from lower respiratory tract specimens. The high MDR rate and its positive association with biofilm formation indicate the nosocomial distribution of A. baumannii. The biofilm formation and the presence of Bap were not interrelated, indicating that biofilm formation was not regulated by a single factor. The MDR rate and the presence of OmpA and Bap showed a positive association (P<0.05). The isolates co-harbouring different carbapenem resistance genes were the predominant biofilm producers, which will seriously limit the therapeutic options suggesting the need for strict antimicrobial stewardship and molecular surveillance in hospitals.

Exploring the Diversity of Biofilm Formation by the Food Spoiler Brochothrix thermosphacta.

Brochothrix thermosphacta is considered as a major spoiler of meat and seafood products. This study explores the biofilm formation ability and the biofilm structural diversity of 30 multi-origin B. thermosphacta strains using a set of complementary biofilm assays (biofilm ring test, crystal violet staining, and confocal laser scanning microscopy). Two major groups corresponding to low and high biofilm producers were identified. High biofilm producers presented flat architectures characterized by high surface coverage, high cell biovolume, and high surface area.

Smoothie Drinks: Possible Source of Resistant and Biofilm-Forming Microorganisms.

Smoothie drinks are currently very popular drinks sold especially in fast food establishments. However, smoothies are a significant source of microorganisms. The aim of this study was to evaluate the microbiological quality of smoothies purchased in Eastern Bohemia. A higher prevalence of mesophilic aerobic bacteria (5.4-7.2 log CFU/mL), yeast (4.4-5.9 log CFU/mL) and coliform bacteria (3.1-6.0 log CFU/mL) was observed in vegetable smoothies, in which even the occurrence of enterococci (1.6-3.3 log CFU/mL) was observed. However, the occurrence of S. aureus, Salmonella spp. and Listeria spp. was not observed in any samples. Nevertheless, antimicrobial resistance was observed in 71.8% of the isolated strains. The highest level of resistance was found in isolates from smoothie drinks with predominantly vegetable contents (green smoothie drinks). Considerable resistance was observed in Gram-negative rods, especially to amoxicillin (82.2%) and amoxicillin with clavulanic acid (55.6%). Among enterococci, only one vancomycin-resistant strain was detected. The vast majority of isolated strains were able to form biofilms at a significant level, which increases the clinical importance of these microorganisms. The highest biofilm production was found in Pseudomonas aeruginosa, Kocuria kristinae and Klebsiella pneumoniae. Overall, significant biofilm production was also noted among isolates of Candida spp.

Production of biofilm by Staphylococcus aureus: Association with infective endocarditis?

ObjectivesStaphylococcus aureus is a well-known biofilm-producing pathogen that is capable of causing chronic infections owing to its ability to resist antibiotic treatment and obstruct the immune response. However, the possible association between high biofilm production and infective endocarditis (IE) has not been assessed. Our objective was to compare production of biofilm by S. aureus strains isolated from patients with bacteremia and IE, catheter-related bloodstream infection (C-RBSI), or non-device associated bacteremia. MethodsWe isolated 260 S. aureus strains from the blood of patients with bacteremia who were diagnosed during hospital admission between 2012 and 2015. Patients were divided into 3 groups according to whether they had IE, C-RBSI, or non-device associated bacteremia. Biofilm production was measured in terms of biomass and metabolic activity using the crystal violet and XTT assays, respectively. High biomass and metabolic activity rates (based on tertile ranks classification) were compared between the 3 groups. ResultsThe high biomass and metabolic activity rates of each group were 41.9% and 37.2% for IE, 32.5% and 35.0%, for C-RBSI, and 29.0% and 33.3% for non-device associated bacteremia (p=0.325 and p=0.885, respectively). ConclusionsHigh biomass and metabolic activity levels for S. aureus isolates from IE were similar to those of S. aureus isolates from C-RBSI or non-device associated bacteremia.

Exhaustive diagnosis of breast implants with capsular contracture: The microbiology laboratory as a major support

The most frequent complications of post-mastectomy reconstructions are breast implant (BI) infection and capsular contracture (CC). The diagnosis of BI colonization is based on cultures from the sonicated BI and from the capsule tissue. Therefore, we first aimed to assess the yield of conventional culture and molecular techniques in periprosthetic fluid, in addition to BI and capsular tissue. Moreover, we compare colonization and biofilm production between patients with and without CC. During 19 months, we prospectively included patients whose BIs had been removed and divided them into two groups: A (CC, Baker III-IV) and B (no CC). Samples were obtained for conventional culture, 16s rRNA PCR, and MALDI-TOF. Biofilm production was also evaluated. We included 81 BIs from 69 patients with CC (22) and without CC (53). Forty-three (53.1%) of the 81 BIs had ≥1 positive culture. The culture was positive in 57.1% and 50.9% in groups A and B, respectively (p=0.645). The highest 16s rRNA PCR positivity rate was detected in capsular tissue (40.5%). MALDI-TOF was unable to detect colonization in any of the samples. High biofilm production was the following: high biomass: A, 29.8%; B, 39.7% (p=0.293); high metabolic activity: A, 36.2%; B, 34.5% (p=0.857). We confirm that cultures from different sites are mandatory to ensure a proper diagnosis of BI colonization. Our study is the first to demonstrate that CC was not associated with BI colonization or high biofilm production. The application of molecular techniques in BI samples was not substantially useful for predicting colonization.

Significant in-Vitro and in-Vivo Antimicrobial and Antibiofilm Activity of Colloidal Silver Nanoparticles (cAgNPs) in Chronic Diabetic Foot Ulcers.

Infection is a foremost challenge in the cases of wound care, especially in cases of chronic wounds. The present study was conducted to determine the antimicrobial and antibiofilm activity of the colloidal silver nanoparticles (cAgNPs) on Gram positive organisms and to evaluate the in-vivo response of cAgNPs on patients of chronic diabetic foot ulcers (DFUs). cAgNPs were tested against selected Gram-positive organisms like methicillin-sensitive and resistant Staphylococcus aureus (MSSA, MRSA), Enterococcus faecalis and vancomycin resistant enterococci (VRE) using microbroth dilution assay to estimate minimum inhibitory/bactericidal concentration (MIC/MBC). Biofilm inhibition capacity and time kill assay was performed. Further, the in-vivo response of topical application of cAgNPs was evaluated on patients of DFUs. The susceptibility testing demonstrated the MIC and MBC values of the cAgNPs ranging from 0.5μg/ml to 1.0 μg/ml and 1.0 μg/ml to 8 μg/ml against the tested organisms respectively. The cAgNPs showed inhibition of biofilm formation in the low, medium and high biofilm producers by 91%, 83% and 75% respectively at the highest concentration (52ppm). The time kill kinetics showed significant reduction in the number of viable cells (p < 0.0001). Significant reduction in microbial load (p = 0.0062) and in the number of moderate to strong biofilm producing organisms (p = 0.0069) after treatment with cAgNPs was seen. cAgNPs exhibited significant in-vitro bactericidal and bacteriostatic activity against MRSA, MSSA and VRE respectively along with anti-biofilm activity. Additionally, cAgNPs showed significant reduction in microbial load of the chronic DFUs.

The classification of Staphylococcus aureus strains by biofilm production differs depending on the method used

IntroductionStrains can be classified in terms of biofilm production from quantitative absorbance values collectively by dividing strains into tertile ranks or individually by calculating the optical density for the negative control. However, these methods have not been compared in a large sample of Staphylococcus aureus strains. Therefore, our objective was to analyze the agreement between both methods in terms of biomass production and metabolic activity of their biofilm. MethodsWe classified 233 S. aureus strains by biomass production and metabolic activity using the crystal violet and XTT assays, respectively. Strains were classified as low, moderate, or high biofilm producers according to tertile or optical density. ResultsWe found no agreement between both methods (p<0.001 and p=0.028, respectively). ConclusionsWe consider strains’ biofilm classification by optical density to be a more reliable method, as it depends on the individual absorbance of each strain.

Drought-Tolerant Landrace Rice Norungan Harbors Novel Putative Competitive Endophytes

The landraces and wild species wisely choose their microbiome and explored them as a second genome for nutrient acquisition from the limited resources of the soil ecosystem and can withstand drought and diseases. Hence, the present study aimed to explore the drought-tolerant rice landrace of Tamil Nadu for the putative competitive endophytes to develop a plant-growth-promoting endophytic bacterial inoculant. Twenty two putative competitive endophytes were authenticated from plant tissues and seeds of Norungan. The 16S rRNA gene sequencing revealed that Norungan rice was colonized predominantly by Firmicutes, including six species of Bacillus, four species of Priestia, and one species each of Micrococcus, Geobacillus, and Lysinibacillus, along with two Proteobacteria, Enterobacter cloacae and Kosakonia oryzae. These endophytes were screened for nutrient transformation, growth hormone production, drought mitigation, and antagonism against plant pathogens. Bacillus cereus NE07, B. paralicheniformis NE18, B. subtilis NE20, and Priestia flexa NE09 showed potential nutrient-transforming capability, while Bacillus cereus NE07 and Priestia flexa NE09 showed high plant growth hormones (indole-3-acetic acid and gibberellic acid production). Similarly, Bacillus cereus NE07 and Priestia endophytica (NE14) showed high ACC deaminase, proline accumulation, biofilm production, and potential candidates for drought mitigation. Bacillus hayneshii NE04 and Priestia aryabhattai NE10, and Bacillus cereus NE07 showed antagonism against fungal pathogen Rhizoctonia solani and bacterial leaf blight causing Xanthomonas oryzae. The results confirmed that no single strain with all plant-growth-promoting and stress-mitigating traits found in Norungan, and these strains were performed division of labor basis. Hence, developing a putative competitive endophytic bacterial consortium with compatible strains representing cumulative beneficial traits as inoculants would be a novel approach for improving rice productivity

Activity of Moxifloxacin Against Biofilms Formed by Clinical Isolates of Staphylococcus aureus Differing by Their Resistant or Persister Character to Fluoroquinolones.

Staphylococcus aureus biofilms are poorly responsive to antibiotics. Underlying reasons include a matrix effect preventing drug access to embedded bacteria, or the presence of dormant bacteria with reduced growth rate. Using 18 clinical isolates previously characterized for their moxifloxacin-resistant and moxifloxacin-persister character in stationary-phase culture, we studied their biofilm production and matrix composition and the anti-biofilm activity of moxifloxacin. Biofilms were grown in microtiter plates and their abundance quantified by crystal violet staining and colony counting; their content in polysaccharides, extracellular DNA and proteins was measured. Moxifloxacin activity was assessed after 24 h of incubation with a broad range of concentrations to establish full concentration-response curves. All clinical isolates produced more biofilm biomass than the reference strain ATCC 25923, the difference being more important for those with high relative persister fractions to moxifloxacin, most of which being also resistant. High biofilm producers expressed icaA to higher levels, enriching the matrix in polysaccharides. Moxifloxacin was less potent against biofilms from clinical isolates than from ATCC 25923, especially against moxifloxacin-resistant isolates with high persister fractions, which was ascribed to a lower concentration of moxifloxacin in these biofilms. Time-kill curves in biofilms revealed the presence of a moxifloxacin-tolerant subpopulation, with low multiplication capacity, whatever the persister character of the isolate. Thus, moxifloxacin activity depends on its local concentration in biofilm, which is reduced in most isolates with high-relative persister fractions due to matrix effects, and insufficient to kill resistant isolates due to their high MIC.

Occurrence of Pseudomonas spp. in Raw Vegetables: Molecular and Phenotypical Analysis of Their Antimicrobial Resistance and Virulence-Related Traits.

Pseudomonas is characterized by its great capacity to colonize different ecological niches, but also by its antimicrobial resistance and pathogenicity, causing human, animal, or plant diseases. Raw and undercooked food is a potential carrier of foodborne disease. The aim of this study was to determine the occurrence of Pseudomonas spp. among raw vegetables, analysing their antimicrobial resistance, virulence, and molecular typing. A total of 163 Pseudomonas spp. isolates (12 different species) were recovered from 77 of the 145 analysed samples (53.1%) and were classified into 139 different pulsed-field gel electrophoresis patterns. Low antimicrobial resistance levels, but one multidrug-resistant isolate, were found. Among the 37 recovered P. aeruginosa strains, 28 sequence-types and nine serotypes were detected. Eleven OprD patterns and an insertion sequence (ISPa1635) truncating the oprD gene of one imipenem-resistant strain were found. Ten virulotypes were observed, including four exoU-positive and thirty-one exoS-positive strains. The lasR gene was absent in three ST155 strains and was truncated by different insertion sequences (ISPre2, IS1411, and ISPst7) in other three strains. High biofilm, motility, pigment, elastase, and rhamnolipid production were detected. Our study demonstrated a low occurrence of P. aeruginosa (18%) and low antimicrobial resistance, but a high number of virulence-related traits in these P. aeruginosa strains, highlighting their pathological importance.

Discovery of Polyvalent Myovirus (vB_STM-2) Phage as a Natural Antimicrobial System to Lysis and Biofilm Removal of Salmonella Typhimurium Isolates from Various Food Sources

New and natural antimicrobials as an alternative control system are now an urgent need to overcome stubborn bacterial infections. Salmonella Typhimurium has become the most frequent serovar responsible for salmonellosis in humans around the world. The high antimicrobial resistance and biofilm production make this pathogen more dangerous. We aimed to isolate a broad lytic phage to prevent Salmonella infection and reduce its biofilms. Using Salmonella Typhimurium (ST-4) as a host, seven phages were isolated, and only three phages showed clear lytic plaques, two members of the Siphoviridae family (vB_STS-1 and vB_STS-3) and one of the Myoviridae family (vB_STM-2). The vB_STM-2 phage was the most potent broad lytic phage, infecting 100% of tested Salmonella Typhimurium serovars and non-Salmonella strains. Additionally, the vB_STM-2 phage was thermostable at −20 to 55 °C up to 24 h, while at 65 and 75 °C, a significant (p &lt; 0.05) titer reduction was observed after 7 days. Moreover, the phage seemed to be stable at different pHs (4–11) after one to twelve hours (hrs), while increasing the time made the phage more sensitive to the alkaline medium rather than the acidic medium. Interestingly, the vB_STM-2 phage had the capacity to diminish or eradicate the biofilms of tested Salmonella Typhimurium, e.g., ST-4, ST-19, ST-30, ST-37, ST-45 and ST-49 by 81.2%, 76.4%, 43.6%, 41%, 39.8% and 93.4%, respectively, at a titer concentration of 106 PFU/mL. Eventually, the vB_STM-2 phage showed significant (p &lt; 0.05) efficacy in the elimination of Salmonella Typhimurium (ST-4) from contaminated chicken breasts at both storage periods with high titer stability. The Salmonella count showed a severe decline from 7.00 ± 0.63 log10 CFU/cm2 to 0.88 ± 0.17 log10 CFU/cm2 on the seventh day of the short-term storage, and from 5.13 ± 0.44 log10 CFU/cm2 to 1.10 ± 0.12 log10 CFU/cm2 on day 27 of the long-term assay. In both periods, the phage titers remained stable, with insignificant (p &lt; 0.05) loss. Therefore, this phage is considered a prime candidate to combat multi-drug-resistant Salmonella Typhimurium and its biofilms.

Growth conditions affect biofilms of Staphylococcus aureus producing mastitis: Contribution of MALDI-TOF-MS to strain characterization.

Bovine mastitis is a disease of dairy cattle prevalent throughout the world that causes alterations in the quality and composition of milk, compromising technological performance. Staphylococcus aureus is one of the most important pathogens that produce clinical, subclinical, and chronic mastitis. Biofilms are considered a virulence factor necessary for the survival of S. aureus in the mammary gland. Its zoonotic potential is important not only for the dairy industry sector but also for public health. This study aimed to evaluate the effect of different growing culture conditions on the biofilm formation of S. aureus isolated from mastitis and to test the MALDI-TOF-MS's ability to discriminate among different biofilm formation levels. Fluids commonly found in the dairy environment were incorporated to approach the pathogen's behavior in natural surroundings. PIA production was also evaluated. All strains were able to form high biofilms in TSB, TSBg, and milk. Milk changed the behavior of some strains which formed more biofilms in this medium than in TSBg. The free iron medium CTSBg and milk whey inhibited the biofilm formation of the most strains. MALDI-TOF-MS performance was an excellent tool to discriminate between high, moderate, and low biofilm producers strains of S. aureus in each media, confirming the results of crystal violet assay. PIA production was variable among the strains and showed a media-dependent behavior. Our data highlights the importance of considering the growing conditions that mimic the natural ones to the study of biofilm formation in vitro.