Agar Spot Test Research Articles

Sol-Gel Synthesis of Silica-Poly (Vinylpyrrolidone) Hybrids with Prooxidant Activity and Antibacterial Properties.

The present work deals with the sol-gel synthesis of silica-poly (vinylpyrrolidone) hybrid materials. The nanohybrids (Si-PVP) have been prepared using an acidic catalyst at ambient temperature. Tetramethyl ortosilane (TMOS) was used as a silica precursor. Poly (vinylpyrrolidone) (PVP) was introduced into the reaction mixture as a solution in ethanol with a concentration of 20%. The XRD established that the as-prepared material is amorphous. The IR and 29Si MAS NMR spectra proved the formation of a polymerized silica network as well as the hydrogen bonding interactions between the silica matrix and OH hydrogens of the silanol groups. The TEM showed spherical particle formation along with increased agglomeration tendency. The efficacy of SiO2/PVP nanoparticles as a potential antimicrobial agent against a wide range of bacteria was evaluated as bacteriostatic, using agar diffusion and spot tests. Combined effects of hybrid nanomaterial and antibiotics could significantly reduce the bactericidal concentrations of both the antibiotic and the particles, and they could also eliminate the antibiotic resistance of the pathogen. The registered prooxidant activity of the newly synthesized material was confirmative and explicatory for the antibacterial properties of the tested substance and its synergetic combination with antibiotics. The effect of new hybrid material on Crustacea Daphnia magna was also estimated as harmless under concentration of 0.1 mg/mL.

Comparative analysis of three methods for screening for colistin resistant <i>Escherichia coli</i>

The usage of colistin regarded as a drug of last resort has increased tremendously in recent years. This increase has been followed by an increase in the development of colistin resistant bacteria. Due the large size of colistin and its ability to adhere to plastic, the broth microdilution method using a special medium is the recommended testing method. Resource limited settings struggle with this method and employ alternate methods. This study therefore set out to determine colistin resistance in a group of Escherichia coli using three different methods comprised of colistin agar spot test (COL-AS), colistin drop test (COL-DT) and a disc diffusion method. A total of 51 Escherichia coli isolated from wound samples were screened for colistin resistance using the COLAS, COL-DT and colistin disc diffusion methods. Results showed a combined resistance rate of 96.1% among test isolates. Actual resistance rates varied between testing methods giving values of ranging from 37.3%, 66.0% and 88.2% for COL- DT, colistin disc diffusion methods and COL-A respectively. An assessment of test performance showed categorical agreement values and very major error values of 57.1%/36.7% for COL-DT and 63.3%/8.2% for COL-A. Results of this study show a high-level occurrence of colistin resistance among clinical Escherichia coli isolates. Furthermore, it demonstrates the superiority of the colistin agar test to the colistin drop test. It also points at a need to use higher concentrations of colistin in the screening tests.

Susceptibility to postbiotics - enterocins of methicillin-resistant Staphylococcus aureus strains isolated from rabbits

There is a major problem with the rising occurrence of highly virulent and multiply-resistant strains, including methicillin-resistant Staphylococcus aureus (MRSA), because of their difficult treatment. This study aimed to evaluate the antibacterial and antibiofilm effect of new enterocins (Ent) against potential pathogenic MRSA strains isolated from rabbits. Staphylococci were identified with PCR and screened for methicillin/oxacillin/cefoxitin resistance (MR) using the disk diffusion method and the PBP2’ Latex Agglutination Test Kit. Enzyme production, hemolysis, DNase activity, slime production, and biofilm formation were tested in MRSA strains. The susceptibility of MRSA to eight partially-purified enterocins (Ent) produced by E. faecium and E. durans strains was checked using agar spot tests. The antibiofilm activity of Ents was tested using a quantitative plate assay. Out of 14 MRSA, PBP testing confirmed MR in 8 strains. The majority of MRSA showed DNase activity and β-hemolysis. Slime production and moderate biofilm formation were observed in all strains. MRSA were susceptible to tested Ents (100–12,800 AU/mL; except Ent4231). The antibiofilm effect of Ents (except Ent4231) was noted in the high range (64.9–97.0%). These results indicate that enterocins offer a promising option for the prevention and treatment of bacterial infections caused by biofilm-forming MRSA.

A Simple In Vitro Test to Select Stools for Fecal Microbiota Transplantation to Limit Intestinal Carriage of Extensively Drug-Resistant Bacteria.

Treatment options for multidrug-resistant bacterial infections are limited and often ineffective. Fecal microbiota transplantation (FMT) has emerged as a promising therapy for intestinal multidrug-resistant bacterial decolonization. However, clinical results are discrepant. The aim of our pilot study was to evaluate the screening performance of a simple diagnostic tool to select fecal samples that will be effective in decolonizing the intestine. Fecal samples from 10 healthy subjects were selected. We developed an agar spot test to evaluate their antagonistic activity toward the growth of VanA Enterococcus faecium and OXA-48-producing Klebsiella pneumoniae, two of the most serious and urgent threats of antibiotic resistance. Most fecal samples were able to limit the growth of both bacteria in vitro but with large inter-individual variation. The samples with the highest and lowest antagonistic activity were used for FMT in a mouse model of intestinal colonization. FMT was not successful in reducing intestinal colonization with VanA Enterococcus faecium, whereas FMT performed with the fecal sample showing the highest activity on the agar spot test was able to significantly reduce the intestinal colonization of mice with Klebsiella pneumoniae OXA-48. The agar spot test could thus serve as a reliable screening tool to select stool samples with the best potential to eradicate/reduce multidrug-resistant bacteria carriage after FMT.

Lugdunin production and activity in Staphylococcus lugdunensis isolates are associated with its genotypes.

Lugdunin produced by Staphylococcus lugdunensis has been shown to have broad inhibitory activity against Gram-positive bacteria; however, lugdunin activity among S. lugdunensis isolates and its association with different agr, SCCmec, and sequence types remain unclear. We used matrix-assisted laser desorption ionization-time-of-flight mass spectrometry to identify S. lugdunensis and collected 202 S. lugdunensis samples for further assays. Agar spot tests were performed to characterize S. lugdunensis lugdunin production and activity. Multilocus sequence typing, SCCmec, and agr genotyping were performed on S. lugdunensis. In all, 91 Staphylococcus aureus strains with varying vancomycin susceptibilities were used to examine lugdunin activity in S. lugdunensis. In total, 48 S. lugdunensis strains (23.8%) were found to be oxacillin-resistant S. lugdunensis (ORSL), whereas 154 (76.2%) were classified as oxacillin-sensitive S. lugdunensis (OSSL). Moreover, 16 (33.3%) ORSL and 35 (22.7%) OSSL strains showed antibacterial activity against S. aureus. Our data showed that most lugdunin-producing ORSL strains (14/48, 29.2%) were of ST3-SCCmec V-agr II genotypes, whereas most lugdunin-producing OSSL strains (15/154, 9.7%) were of ST3-agr II, followed by ST1-agr I (10/154, 6.5%). Our data also revealed that lugdunin exhibited weak inhibitory activity against the VISA ST239 isolate. In addition, we observed that ST239 VSSA was more resistant to lugdunin than ST5, ST59, and ST45 VSSA. Taken together, our data pioneered the epidemiology of lugdunin production in S. lugdunensis isolates and revealed its association with genotypes. However, further molecular and bioinformatics investigations are needed to elucidate the regulatory mechanisms of lugdunin production and activity. IMPORTANCE Lugdunin is active against both methicillin-resistant Staphylococcus aureus and vancomycin-resistant enterococci by dissipating their membrane potential. However, the association of lugdunin activity with the genotypes of Staphylococcus lugdunensis has not been addressed. Here, we show the high prevalence of lugdunin-producing strains among ST1 (83.3%), ST2 (66.7%), and ST3 (53.3%) S. lugdunensis. Moreover, we identified the antibacterial activity of lugdunin-producing strains against VISA and hVISA. These results shed light on the potential application of lugdunin for the treatment of drug-resistant pathogens.

Isolation and Characterization of Pseudomonas aeruginosa Phages with a Broad Host Spectrum from Hospital Sewage Systems and Their Therapeutic Effect in a Mouse Model.

This study aimed to isolate and characterize phages as an alternative treatment of multidrug- or pan-drug-resistant Pseudomonas aeruginosa. Phage titers and bacterial densities correlated, with the phages disappearing after bacteria were eliminated. We isolated phages in filtered sewage water by a double-layered agar spot test. Fifty-eight P. aeruginosa strains were used to screen the host spectrum of the 14 phages isolated. Random amplification of polymorphic DNA-typing polymerase chain reaction was used to analyze the genomic homologies of the 58 host bacteria strains and four phages with a broad host spectrum. Transmission electron microscopy was used to observe the morphology of the four phages with a broad host spectrum. Mice with intraabdominal P. aeruginosa infection were used as an invivo animal model to investigate the therapeutic effect of the selected phage. Four virulent phages with a broad host spectrum specific to P. aeruginosa strains were isolated. They were all double-stranded DNA viruses and belonged to four different genotypes. The test curve showed that phage I had the highest adsorption rate, the shortest latent period, and the largest burst size. The infected mouse model indicated that small doses of phage I could prevent the death of infected mice. Phage titers and bacterial densities correlated, with phages disappearing after bacteria were eliminated. Phage I was the most effective and promising treatment of drug-resistant P. aeruginosa.

Isolation and Characterization of Lytic Bacteriophages from Wastewater with Phage Therapy Potentials Against Gram-Negative Bacteria.

The increase of multidrug resistance in bacteria has increased the efforts in search of alternative methods. The aim of the present study was to isolate and characterize the lytic phages and assess their lytic activity against a number of gram-negative bacteria. The phages and their respective hosts were isolated from wastewater collected from the municipal sewer system of Trabzon, Turkey. The lytic activities of phage were determined using the agar spot test. The identification and antibiotic susceptibility of host bacteria were determined using matrix-assisted laser desorption ionization time-of-flight mass spectrometry and Phoenix 100, respectively. The phages were characterized morphologically using transmission electron microscopy. One of the phages, Enteroc21, which has a broad-host-range, was further characterized by genome restriction endonuclease analysis and burst size. Two phages infected strains of four different species, nine phages were able to infect 2-4 strains belonging to one or two species, and three phages showed lytic activity against only the hosts from which they were isolated. All phages belonged to the Siphoviridae, Myoviridae, and Podoviridae family based on trans- mission electron microscopy morphology. The Enteroc21 had more than 100 kb genome size and a burst size of 180 per infected cell. Most of the host strains were resistant to ampicillin, amoxicillin-clavulanic acid, and in particular, Achromobacter xylosoxidans TRAX 13 was multidrug-resistant showing resistance to cefepime, aztreonam, gentamicin, netilmicin, and ciprofloxacin. This study showed that the isolated phages have the potential to be used in phage therapy against various bacterial infections, including multidrug-resistant bacteria.

Lactic Acid Bacteria from Gamecock and Goat Originating from Phitsanulok, Thailand: Isolation, Identification, Technological Properties and Probiotic Potential.

From independent swab samples of the cloaca of indigenous gamecocks (CIG), anus of healthy baby goats (AHG), and vagina of goats (VG) originating from Phitsanulok, Thailand, a total of 263 isolates of lactic acid bacteria (LAB) were collected. Only three isolates, designated C707, G502, and V202, isolated from CIG, AHG, and VG, respectively, exhibited an excellent inhibitory zone diameter against foodborne pathogenic bacteria when evaluated by agar spot test. Isolates C707 and G502 were identified as Enterococcus faecium, whereas V202 was identified as Pediococcus acidilactici, based on 16S rRNA sequence analysis. When foodborne pathogenic bacteria were co-cultured with chosen LAB in mixed BHI-MRS broth at 39°C, their growth was suppressed. These LAB were found to be capable of surviving in simulated stomach conditions. Only the isolate G502 was able to survive in the conditions of simulated intestinal juice. This research suggests that selected LAB could be used as a food/feed supplement to reduce foodborne pathogenic bacteria and improve the safety of animal-based food or feed.

Effect of enterocins against methicillin-resistant animal-derived staphylococci.

The occurence and spread of animal-derived methicillin resistant staphylococci (MRS) worldwide is acurrent problem, especially due to their increasing incidence in food animals and their products, with possible contamination of food consumers and handlers. Staphylococci isolated from animals (n = 123) were identified with MALDI-TOF mass spectrometry and screened for methicillin/oxacillin/cefoxitin resistance (MR) using the disk diffusion method. Twenty-three phenotypically MRS strains were analysed using PBP2' Latex Agglutination Test Kit to confirm the phenotypic MR and PCR was performed for mecAgene detection; mecA gene positive strains were furtherly confirmed by means of sequencing. The susceptibility of MRS to 11 partially-purified enterocins (Ent) produced by E. faecium, E. durans and E. mundtii strains of animal, feed/food and environmental origin was checked using agar spot tests. Out of 23 MRS, PBP testing confirmed MR in 17 strains. Three Staphylococcus epidermidis and oneS. vitulinus weremecA positive. The majority of MRS, including two mecA gene-positive strains S. epidermidis R44/1 and P3/Tr2a, were susceptible to the tested enterocins, mainly to Ent7420, EntA(P)/EK13, Ent412, Ent55 and Ent9296 (in the range 100 - 12,800 AU/mL). The most susceptible strains appeared to be the mecA gene-positive S. epidermidis SE R44/1 and SE P3/Tr2a strains, inhibited by eight enterocins out of 11 tested (100-200 AU/mL). Only four strains (including mecA gene positive S. epidermidis SE P3/Tr1 and S. vitulinus SV K12PL/1) were resistant to the tested antimicrobial substances. These results indicate that the enterocins used offer a promising option for prevention and treatment of bacterial infection caused by MRS in animals.

278 Role of specific probiotics in skin diseases: new insights from in vitro investigation

The aim of this study is to deeply investigate probiotic strains efficacy in the containment of the opportunistic pathogen Staphylococcus aureus, involved in several diseases, such as non melanoma skin cancer (i.e. actinic keratosis and squamous cell carcinoma) and those inflammatory ones, such as psoriasis and atopic dermatitis. Indeed, during the onset and progression of these skin disorders, the dysbiosis of the microbiota leads to the reduction of protective commensal resident bacteria, favoring pathogenic/opportunistic species proliferation (including S. aureus), and to the variation in the expression of their virulence factors. To this purpose, Lactocaseibacillus rhamnosus GG and Lactobacillus johnsonii, two probiotic strains well described by the literature, were grown independently and as co-culture. Viable probiotics inhibitory efficacy on S. aureus was investigated by agar spot test. S. aureus was also cultured for 24, 48 and 72 hours with probiotics-free supernatants (PFSNs), that were characterized by proteomic analysis. Both pathogen planktonic and biofilm forms were analyzed through the viability alamarBlue test. After that, planktonic form was spotted onto agar plates and single colonies were isolated, while biofilm was visualized by crystal violet staining. Viable probiotic strains demonstrate to inhibit S. aureus growth; interestingly, PFSNs alone (both from single and co-cultured strains) act in reducing pathogen plankton viability and in inducing phenotypic changes. Biofilm form, characteristic virulence feature of the pathogen, was inhibited at all times tested. In conclusion, S. aureus over-colonization, crucial factor in the onset and progression of several skin diseases, could be reduced by both viable Lactobacilli and their PFSNs. The deeper investigation of the cross-talk among microbes will help in clarifying their role in several cutaneous disorders.

Protective effect of Lactobacillus reuteri Lb11 from chicken intestinal tract against Salmonella Enteritidis SE05 in vitro.

Salmonella infections in eggs with increasing morbidity and mortality exhibit worldwide prevalence. The present study intends to evaluate the efficacy of Lactobacillus reuteri Lb11 (L. reuteri Lb11, isolated from chicken intestinal tract) in inhibiting the growth of multi-drug resistant (MDR) Salmonella Enteritidis SE05 (obtained from egg content). The cell-free cell lysates (CFCL) of L. reuteri Lb11 obtained by the agar spot test performed well on inhibition of the MDR (Multi-DrugResistant) Salmonella Enteritidis SE05, The heat-inactivated (HI) fraction of L. reuteri Lb11 showed no inhibition activity. By co-culturing with L. reuteri Lb11 in vitro, the growth of S. Enteritidis SE05 decreased along with time, while, the pH value decreased significantly. Furthermore, In order to evaluate the mechanism of action of CFCL of L.reuteri Lb11, the genes related to the transcription level of AcrAB-TolC efflux pump, outer membrane protein OMPs genes and drug resistance genes have been quantified by real-time PCR, when the S. Enteritidis was SE05 exposed to the CFCL of L. reuteri Lb11 (1 × 1012CFU/mL). Almost all of the AcrAB-TolC efflux pump genes, outer membrane protein genes and antibiotic resistance genes were down-regulated. Especially, the level of ramA, tetA and tetB genes were down-regulated -20.77, -15.85 and -12.42 folds, respectively. L. reuteri Lb11 can effectively prevent the formation of efflux pump to inhibit the production of multidrug-resistant Salmonella Enteritidis in eggs.

Simple Phenotypic Tests To Improve Accuracy in Screening Chromosomal and Plasmid-Mediated Colistin Resistance in Gram-Negative Bacilli.

CLSI and EUCAST recommend that only broth microdilution (BMD) should be used for routine colistin susceptibility testing; however, this technique can be difficult to perform in resource-poor settings. The purpose of this study was to evaluate the accuracy of a colistin agar spot test (COL-AS) and a colistin drop test (COL-DT) compared to BMD. COL-AS and COL-DT were assessed with a collection of 271 Gram-negative bacilli clinical isolates: 195 Enterobacterales (including 63 mcr-1 positive strains), 37 Acinetobacter spp., and 39 Pseudomonas aeruginosa For COL-AS, 3.0 μg/ml (final concentration) of colistin was added to a Mueller-Hinton agar plate and subsequently swabbed with a 0.5 McFarland standard suspension of the tested strain within a 1 cm2 spot. For COL-DT, 10 μl of a 16 μg/ml colistin solution was dripped on the surface of a Mueller-Hinton agar plate, previously inoculated with a lawn of the tested strain (0.5 McFarland standard). Colistin solution was made either by dissolving powder or by disk elution in cation-adjusted Mueller-Hinton broth (CA-MHB). Overall, 141/271 (52%) isolates were categorized as colistin resistant by reference BMD. COL-AS yielded a categorical agreement (CA) of 95.5% compared to BMD, with 0.7% very major errors and 3.8% major errors. COL-DT yielded a CA of 96.2% compared to BMD, with 0.7% and 0% very major errors and 3.1% and 3.8% major errors, for colistin powder and disk elution solutions, respectively. Most major errors occurred for mcr-1 strains with MICs that fluctuated from 2 to 4 μg/ml according to the method used. In conclusion, we developed and validated methods suited to the systematic screening of resistance to colistin in Gram-negative bacilli.

Probiotics and Antimicrobial Effect of Lactiplantibacillus plantarum, Saccharomyces cerevisiae, and Bifidobacterium longum against Common Foodborne Pathogens in Poultry

The probiotic potential and antimicrobial activity of Lactiplantibacillus plantarum, Saccharomyces cerevisiae, and Bifidobacterium longum were investigated against Escherichia coli O157:H7, Salmonella typhimurium and Listeria monocytogenes. Selected strains were subjected to different acid levels (pH 2.5–6.0) and bile concentrations (1.0–3.0%). Strains were also evaluated for their antimicrobial activity by agar spot test. The potential probiotic strains tolerated pH 3.5 and above without statistically significant growth reduction. However, at pH 2.5, a significant (p < 0.05) growth reduction occurred after 1 h for L. plantarum (4.32 log CFU/mL) and B. longum (5.71 log CFU/mL). S. cerevisiae maintained steady cell counts for the entire treatment period without a statistically significant (p > 0.05) reduction (0.39 log CFU/mL). The results indicate at 3% bile concertation, 1.86 log CFU/mL reduction was observed for L. plantarum, while S. cerevisiae, and B. longum growth increased by 0.06 and 0.37 log CFU/mL, respectively. L. plantarum and B. longum demonstrated antimicrobial activity against E. coli O157:H7, S. typhimurium and L. monocytogenes. However, S. cerevisiae did not display any inhibition to any of the pathogens. The results indicate that L. plantarum and B. longum present probiotic potential for controlling E. coli O157:H7, S. and L. monocytogenes in poultry.

Antilisterial Activity by Enterococcus Species Isolated from Traditional Cheeses

In this study, it was aimed to detect the antimicrobial activity of 312 Enterococcus species against Listeria monocytogenes. Antimicrobial activity was detected by agar spot and well diffusion assay. A total of 201 enterococcal strains inhibited the growth of L. monocytogenes strains based on the agar spot test. Only 44 strains showed antimicrobial activity against L. monocytogenes strains using agar well diffusion assay. Of the 44 enterococcal strains screened, 6 E. faecium (2.99%) strains had a high antimicrobial effect against indicator L. monocytogenes strains. The antilisterial activity of 6 E. faecium strains had lost after treatment of proteinase K, trypsin and pepsin. The antimicrobial compounds of these strains could be a protein or peptides nature. E. faecium strains were more active against L. monocytogenes than E. faecalis strains.

Lactococcus lactis Diversity Revealed by Targeted Amplicon Sequencing of purR Gene, Metabolic Comparisons and Antimicrobial Properties in an Undefined Mixed Starter Culture Used for Soft-Cheese Manufacture.

The undefined mixed starter culture (UMSC) is used in the manufacture of cheeses. Deciphering UMSC microbial diversity is important to optimize industrial processes. The UMSC was studied using culture-dependent and culture-independent based methods. MALDI-TOF MS enabled identification of species primarily from the Lactococcus genus. Comparisons of carbohydrate metabolism profiles allowed to discriminate five phenotypes of Lactococcus (n = 26/1616). The 16S sequences analysis (V1–V3, V3–V4 regions) clustered the UMSC microbial diversity into two Lactococcus operational taxonomic units (OTUs). These clustering results were improved with the DADA2 algorithm on the housekeeping purR sequences. Five L. lactis variants were detected among the UMSC. The whole-genome sequencing of six isolates allowed for the identification of the lactis subspecies using Illumina® (n = 5) and Pacbio® (n = 1) technologies. Kegg analysis confirmed the L. lactis species-specific niche adaptations and highlighted a progressive gene pseudogenization. Then, agar spot tests and agar well diffusion assays were used to assess UMSC antimicrobial activities. Of note, isolate supernatants (n = 34/1616) were shown to inhibit the growth of Salmonella ser. Typhimurium CIP 104115, Lactobacillus sakei CIP 104494, Staphylococcus aureus DSMZ 13661, Enterococcus faecalis CIP103015 and Listeria innocua CIP 80.11. Collectively, these results provide insightful information about UMSC L. lactis diversity and revealed a potential application as a bio-protective starter culture.

Resistin-like molecule beta, a colonic epithelial protein, exhibits antimicrobial activity against Staphylococcus aureus including methicillin-resistant strains.

Resistin-like molecule beta (RELMβ) is a small cysteine-rich protein secreted by colonic epithelial cells. RELMβ mRNA and protein expressions are dramatically induced by bacterial exposure in germ-free mice. We hypothesized that RELMβ has antimicrobial activity. The antimicrobial activity of RELMβ was screened by an agar spot test and confirmed by a liquid broth test. The amount of RELMβ in human stools was semi-quantified by Western blot analysis. The induction of RELMβ mRNA and protein expression by bacteria was measured by quantitative RT-PCR using LS174T cells. Electron microscopic immunohistochemistry was performed using polyclonal anti-RELMβ antibody. RELMβ showed antimicrobial activity against S. aureus and all MRSAs examined in a dose- and pH-dependent fashion. Western blot study showed that the amount of RELMβ in healthy human stools was comparable to that exhibiting antimicrobial activity in vitro. Both RELMβ mRNA and protein expression were induced by heat-inactivated S. aureus, but not by E. coli in LS174T cells. Electron microscopic immunohistochemistry showed that RELMβ bound to the cell surface of S. aureus, followed by destruction of the bacterial cytoplasm. RELMβ is a colonic antimicrobial protein and its antibacterial activity is species selective. Because RELMβ is abundant in healthy human stool, RELMβ may modulate gut flora.

Synergistic Effect between Usnic Acid and Polymyxin B against Resistant Clinical Isolates of Pseudomonas aeruginosa.

The present study aimed to characterize the susceptibility profile of Pseudomonas aeruginosa and Acinetobacter spp. clinical isolates to polymyxin B in a public hospital in Recife-PE, Brazil, between the years of 2018 and 2019, as well as to search for the presence of the mcr-1 gene and evaluate the interaction between polymyxin B and usnic acid against these isolates. The strains were identified using the BD Phoenix™ automated system and the agar-spot test was used to determine the susceptibility profile to polymyxin B. The minimum inhibitory concentrations (MICs) of usnic acid and polymyxin B were determined through the broth microdilution method according to the Clinical and Laboratory Standards Institute (CLSI). Subsequently, Polymerase Chain Reaction (PCR) was performed to detect the mcr-1 gene in the isolates. The interaction between usnic acid and polymyxin B was evaluated by the Checkerboard assay. Among 34 isolates of P. aeruginosa, 26.5% (9/34) were positive for the polymyxin B agar-spot test, and 11.8% (4/34) presented an intermediate susceptibility (MIC = 4 μg/mL), while 14.7% (5/34) presented antimicrobial resistance with MIC values ranging from 8 to 32 μg/mL. Among 38 isolates of Acinetobacter spp., 13.2% (5/38) were positive for the polymyxin B agar-spot test and all of them were resistant to polymyxin B with a MIC value > 32 μg/mL. The mcr-1 gene was not detected in the clinical isolates. Regarding usnic acid, it presented a moderate antibacterial activity against two P. aeruginosa isolates (MIC = 250 μg/mL) and no activity was detected against the others. A synergistic effect between usnic acid and polymyxin B was observed against three clinical isolates of P. aeruginosa which were resistant to polymyxin B (FICI ≤ 0.5). Therefore, it was possible to observe that usnic acid is a promising candidate to be used in combination with polymyxin B against infections caused by resistant P. aeruginosa.

In Vitro Antimicrobial Activity and Probiotic Potential of Bifidobacterium and Lactobacillus against Species of Clostridium.

Many Clostridium species are found as commensal members of the intestinal microbiota. However, imbalances of the microbiota may lead to certain infections caused by these microorganisms, mainly Clostridium butyricum, Clostridium difficile, and Clostridium perfringens. In many cases, infection recurrence can occur after antibiotics, indicating the need for novel therapeutic options that act on the pathogens and also restore the microbiota. Herein, the in vitro antimicrobial activity and probiotic potential of clinical and reference strains of Bifidobacterium and Lactobacillus were investigated against Clostridium species. Antimicrobial activity was evaluated by the agar spot test and inhibition of gas production. Then, the probiotic potential of selected strains was assessed by analyzing their coaggregation ability, adhesive properties to host cells and mucin, tolerance to acidic pH and bile salts, and antimicrobial susceptibility profiles. Lactobacillus plantarum ATCC 8014 was the most promising strain based on its inhibitory activity against Clostridium spp. Also, this strain met criteria to be considered a probiotic based on its coaggregation ability, adhesive properties, and tolerance to harsh pH and bile acid salt conditions. The results indicate that among the studied strains, L. plantarum ATCC 8014 presents probiotic potential for controlling infections induced by the studied Clostridium species and should be further evaluated in in vivo animal models.

Relationship between tetracycline antibiotic susceptibility and genotype in oral cavity Lactobacilli clinical isolates

BackgroundAntibiotic resistance, is often conferred by the presence of antibiotic resistance genes. This study aimed to investigate the relationship between tetracycline resistance (Tet-R) and genotype in 31 Lactobacillus isolates from caries-active patients.MethodsThe tetracycline susceptibility of Lactobacillus isolates was determined using the agar spot test and the genetic characteristics associated with tetracycline resistance using whole-genome sequencing (WGS).ResultsThe minimum inhibitory concentration (MIC) values of most isolates were equal to or lower than the breakpoint MIC values. Four strains that were phenotypically more sensitive (L. fermentum B09, S23 and L. rhamonsus B17) or more resistant (L. plantarum B43) than other isolates to tetracycline were subjected to conduct whole-genome sequencing in order to detect the tetracycline resistance genes. The results revealed that the most common Tet-R genes in Lactobacillus strains were tetT, tetW, tetO and tetL. In addition, tetPB, tcr3 and otrA were detected for the first time. There were distinct Tet-R gene mutations in Lactobacillus isolates. Overall, the mean expression values of Tet-R-mutated genes in L. plantarum B43 were elevated, and the relative expression levels of tetT and tetW genes in L. rhamonsus B17 L. fermentum B09 and S23 were decreased relative to reference strains.ConclusionThe results of this study indicate that Lactobacillus isolates from saliva of caries-active patients do not present considerable tetracycline resistance reservoirs. However, genetic compounds associated with tetracycline resistance were identified by whole-genome sequencing, providing meaningful insights into tetracycline resistance mechanisms.

Antibacterial activity and sensory properties of Heracleum persicum essential oil, nisin, and Lactobacillus acidophilus against Listeria monocytogenes in cheese.

Aim:The aim of this study was to evaluate the antibacterial and chemical effect of Heracleum persicum essential oil (EO), nisin, Lactobacillus acidophilus, and their combination against Listeria monocytogenes both in vitro and in Iranian white cheese model.Materials and Methods:Chemical compositions of H. persicum EO were analyzed by gas chromatography–mass spectrometry. After production of Iranian white cheese, minimum inhibitory concentration (MIC) and minimum bactericidal concentration of EO and nisin and agar spot test of L. acidophilus against L. monocytogenes were evaluated.Results:Hexyl butanoate (25.98%), octyl isobutyrate (17.82%), methyl butyrate (14.37%), and pentyl cyclopropane (12.77%) were the main components of the EO. MIC of the EO against L. monocytogenes was 2.5 mg/mL. Combination of nisin (5.3 IU/mL) and H. persicum EO (2500 µg/mL) showed increasing effect against L. monocytogenes (fractional inhibitory concentration = 0.9), while a higher concentration of EO and nisin showed undesirable effect on the cheese flavor. Furthermore, a combination of 1012 CFU/g L. acidophilus with H. persicum EO at the concentration of 2.5 mg/mL (T12) showed acceptable sensorial and also antibacterial results in Iranian white cheese.Conclusion:Combination of H. persicum EO, L. acidophilus, and nisin can be recommended as natural preservatives and flavoring agents in cheese.