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

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

Antimicrobial Susceptibility Profile of Several Bacteria Species Identified in the Peritoneal Exudate of Cows Affected by Parietal Fibrinous Peritonitis after Caesarean Section

The aim of this study was to identify the species and antimicrobial susceptibility of bacteria involved in parietal fibrinous peritonitis (PFP). We studied 156 peritoneal fluid samples from cows presenting PFP after caesarean section. Bacteria were cultured in selective media and their antimicrobial susceptibility was tested by disk diffusion assay. Bacteria were isolated in the majority (129/156; 83%) of samples. The majority (82/129; 63%) of positive samples contained one dominant species, while two or more species were cultured in 47/129 (36%) samples. Trueperella pyogenes (T. Pyogenes) (107 strains) was the most identified species, followed by Escherichia coli (E. coli) (38 strains), Proteus mirabilis (P. mirabilis) (6 strains), and Clostridium perfringens (C. perfringens) (6 strains). Several other species were sporadically identified. Antimicrobial susceptibility was tested in 59/185 strains, predominantly E. coli (38 strains) and P. mirabilis (6 strains). Antibiotic resistance, including resistance to molecules of critical importance, was commonly observed; strains were classified as weakly drug resistant (22/59; 37%), multidrug resistant (24/59; 41%), extensively drug resistant (12/59; 20%), or pan-drug resistant (1/59; 2%). In conclusion, extensive antibiotic resistance in the isolated germs might contribute to treatment failure. Ideally, antimicrobial therapy of PFP should be based upon bacterial culture and susceptibility testing.

Comparative analysis of Bacillus cereus group isolates' resistance using disk diffusion and broth microdilution and the correlation between resistance phenotypes and genotypes

A collection of 85 Bacillus cereus group isolates were screened for phenotypic resistance to nine antibiotics using disk diffusion and broth microdilution. The broth microdilution antimicrobial results were interpreted using the CLSI M45 breakpoints for Bacillus spp. Due to the lack of Bacillus spp. disk diffusion breakpoints, the results obtained with the disk diffusion assay were interpreted using the CLSI M100 breakpoints for Staphylococcus spp. We identified significant (p < 0.05) discrepancies in resistance interpretation between the two methods for ampicillin, gentamicin, rifampicin, tetracycline, and trimethoprim/sulfamethoxazole. Antimicrobial resistance genes were detected using unassembled and assembled whole-genome sequences with Ariba and Abricate, respectively, to assess the sensitivity and specificity for predicting phenotypic resistance based on the presence of antimicrobial resistance genes. We found antimicrobial resistance gene presence to be a poor indicator for phenotypic resistance, calling for further investigation of mechanisms underlying antimicrobial resistance in the B. cereusgroup. Genes with poor sensitivity and/or specificity, as determined based on broth microdilution results included rph(rifampicin, 0%, 95%), mphgenes (erythromycin, 0%, 96%), and all vangenes (vancomycin, 100%, 35%). However, Bc(ampicillin, 64%, 100%) andtet genes (tetracycline, 67%, 100%) were highly specific, albeit moderately sensitive indicators of phenotypic resistance based on broth microdilution results. Only beta-lactam resistance genes (Bc, BcII, and blaTEM) were highly sensitive (94%) and specific (100%) markers of resistance to ceftriaxone based on the disk diffusion results, providing further evidence of these beta-lactams' role in nonsusceptibility of Bacillus cereus group isolates to ceftriaxone.

Relationship between type III secretion toxins, biofilm formation, and antibiotic resistance in clinical Pseudomonas aeruginosa isolates

Background and aim. Pseudomonas aeruginosa is considered as a notorious pathogen due to its multidrug resistance and life threatening infections. We investigated the relationship between type III secretion toxins, biofilm formation, and antibiotic resistance among clinical P. aeruginosa isolates. Methods. A total of 70 genetically distinct clinical P. aeruginosa isolates were characterized for antibiotic resistance by disk diffusion assay. Biofilm formation was evaluated by microtiter plate method and presence of four exo genes (exoS, exoU, exoT and exoY) was investigated by PCR. A p-value < 0.05 was regarded statistically significant. Results. The most effective antibiotics were Meropenem and Piperacillin. Multidrug resistance was more prevalent in the ciprofloxacin-resistant isolates than in the susceptible isolates. The most frequently identified exo was exoS (37.1%). Genotype exoS/exoT was found in 4 isolates, while genotype exoU/exoT was not found. Prevalence of exoS was generally higher in the susceptible isolates than in the resistant isolates. A significant association was found between the formation of strong biofilm and resistance to antibiotics (p < 0.05). Prevalence of exoY and exoU was higher in the non-strong biofilm producers compared to the strong biofilm producers. Conclusion. Our study revealed formation of strong biofilm along with antibiotic resistance and the presence of exo genes in P. aeruginosa isolates. Knowledge of virulence gene profiles and biofilm formation may be useful in deciding appropriate treatment.

Detection of Salmonella spp. in exotic pigeons of North Kerala and its antibiogram

Pigeon breeding has transformed from being a mere hobby to becoming established as an industry. The increased trade of pigeons inadvertently invites the risk of dissemination of infections including zoonoses like salmonellosis. Pigeons once infected remain carriers for life. This coupled with the ability of the organism to acquire antimicrobial resistance makes salmonellosis, particularly from pigeons an important, public health risk for pigeon handlers. Cloacal swabs from a total of 200 exotic pigeons belonging to 24 lofts from Northern districts of Kerala were collected and attempted to isolate Salmonella and understand its antimicrobial resistance profile. Five isolates of salmonella could be obtained from four of the lofts studied. A prevalence of 2.5 per cent was identified for salmonellosis with 16.67 per cent of the lofts affected. Antimicrobial sensitivity based on disk diffusion assay revealed that all the five isolates were sensitive to amoxicillin-clavulanate and all were resistant to tetracycline and streptomycin. Sixty per cent of the isolates were sensitive to co-trimoxazole, chloramphenicol, ampicillin, cefoperazone, amikacin and gentamicin.

Niche Preference of Escherichia coli in a Peri-Urban Pond Ecosystem

Escherichia coli comprises diverse strains with a large accessory genome, indicating functional diversity and the ability to adapt to a range of niches. Specific strains would display greatest fitness in niches matching their combination of phenotypic traits. Given this hypothesis, we sought to determine whether E. coli in a peri-urban pond and associated cattle pasture display niche preference. Samples were collected from water, sediment, aquatic plants, water snails associated with the pond, as well as bovine feces from cattle in an adjacent pasture. Isolates (120) were obtained after plating on Membrane Lactose Glucuronide Agar (MLGA). We used the uidA and mutS sequences for all isolates to determine phylogeny by maximum likelihood, and population structure through gene flow analysis. PCR was used to allocate isolates to phylogroups and to determine the presence of pathogenicity/virulence genes (stxI, stxII, eaeA, hlyA, ST, and LT). Antimicrobial resistance was determined using a disk diffusion assay for Tetracycline, Gentamicin, Ciprofloxacin, Meropenem, Ceftriaxone, and Azithromycin. Our results showed that isolates from water, sediment, and water plants were similar by phylogroup distribution, virulence gene distribution, and antibiotic resistance while both snail and feces populations were significantly different. Few of the feces isolates were significantly similar to aquatic ones, and most of the snail isolates were also different. Population structure analysis indicated three genetic backgrounds associated with bovine, snail, and aquatic environments. Collectively these data support niche preference of E. coli isolates occurring in this ecosystem.

High-Level Carbapenem Resistance among OXA-48-Producing Klebsiella pneumoniae with Functional OmpK36 Alterations: Maintenance of Ceftazidime/Avibactam Susceptibility

The aim of this work was to analyze outer membrane porin-encoding genes (ompK35 and ompK36) in a collection of OXA-48 producing Klebsiella pneumoniae, to assess the effect of porin alterations on the susceptibility to ceftazidime/avibactam, and to describe a screening methodology for phenotypic detection of OXA-48-producing K. pneumoniae with disrupted porins. Antimicrobial susceptibility was tested by Microscan and Etest. The genomes of 81 OXA-48-producing K. pneumoniae were sequenced. MLST, detection of antimicrobial resistance genes, and analysis of ompK35 and ompK36 were performed in silico. Tridimensional structures of the OmpK36 variants were assessed. Receiver operating characteristics curves were built to visualize the performance ability of a disk diffusion assay using carbapenems and cefoxitin to detect OmpK36 functional alterations. A wide variety of OmpK36 alterations were detected in 17 OXA-48-producing K. pneumoniae isolates. All displayed a high-level meropenem resistance (MIC ≥ 8 mg/L), and some belonged to high-risk clones, such as ST15 and ST147. Alterations in ompK35 were also observed, but they did not correlate with high-level meropenem resistance. All isolates were susceptible to ceftazidime/avibactam and porin alterations did not affect the MICs of the latter combination. Cefoxitin together with ertapenem/meropenem low inhibition zone diameters (equal or lower than 16 mm) could strongly suggest alterations affecting OmpK36 in OXA-48-producing K. pneumoniae. OXA-48-producing K. pneumoniae with porin disruptions are a cause of concern; ceftazidime/avibactam showed good in vitro activity against them, so this combination could be positioned as the choice therapy to combat the infections caused by this difficult-to-treat isolates.

Antimicrobial Activity and Mode of Action of Aspergillus terreus Strain (MTCC9618) Mediated Biosynthesized Silver Nanoparticles-AgNPs Against Staphylococcus aureus and Escherichia coli

Fungal approaches bio-synthesis of silver nanoparticles (AgNPs) have been given attention because of their cost effectiveness and environment friendliness. Therefore, fungal Aspergillus terreus (MTCC 9618) intermediated biosynthesis of AgNPs was conducted, and the SEM, EDS and XRD reports confirmed the synthesis of well-organized, structured and stabilized AgNPs. Antimicrobial efficacy of disk diffusion assay of AgNPs (10, 20 and 30[Formula: see text][Formula: see text]g/ml) dosage was confirmed against Staphylococcus aureus and Escherichia coli and found that has bactericidal effects against both strains. Broth assay was also confirmed through LB broth media and colony forming units (CFU) which defined that biosynthesized AgNPs had much potential effect against gram negative ([Formula: see text]ve) than gram positive ([Formula: see text]ve) due to its peptidoglycan thickness. AgNPs had adverse mode of action on both bacterial strains and resulting was found damaged site on cell wall, necrosis, shrinkage, influx out and ruptures of the cells according to SEM scanning profiles. This study promised to green and economical way of AgNPs biosynthesis along with targeted antimicrobial effects in food preservative, biomedical coating tools, fabrics and pharmaceutical industries.

A study of antifungal susceptibility pattern of dermatophytes at tertiary care center

Dermatophytes are fungi that can cause infections of the skin, hair & nails due to their ability to invade keratin. Dermatophytosis is the most common superficial fungal infection worldwide; it is common in tropics and subtropical regions. It may present in epidemic proportions in areas of high humidity: The present study aimed to identify various species causing dermatophytosis & to determine the invitro susceptibility pattern against commonly used systemic antifungal agents in our tertiary care center. A total of 149 samples were collected of infected skin, hair and nails in a period of 1 year from January 2020 to December 2020. Samples were collected under aseptic condition by skin scrapping, nail and hair clipping by using scalpel or forceps. Identification of the causative pathogen was done by performing slide culture, lacto-phenol cotton blue mount, hair perforation tests and urease tests. We adopted a newly developed agar based disk diffusion assay to test susceptibility of clinically isolated dermatophytes for antifungal susceptibility testing. Microbiological investigations revealed the presence of dermatophytic fungi in 71.8% of the samples. Trichophyton rubrum was the predominant pathogen isolated. The study showed Itraconazole to be most effective antifungal drugs against dermatophytes followed by terbinafine and fluconazole.Further intensive epidemiological and invitro antifungal susceptibility studies of dermatophytes are required which will have more public health significance.

In Vitro Antimicrobial Activity Evaluation of a Novel Fitostimoline® Plus Spray Formulation

Wound contaminants are the main cause of healing delay and infection in both chronic and acute wounds; for this reason, the microbial infection management in wound healing is one of the most important components for an effective standard of care. The wound contaminants are most likely to originate from the environment and from the surrounding skin lesion, and to date, the most frequent bacteria isolated are Staphylococcus aureus, Pseudomonas aeruginosa, and Klebsiella pneumoniae. In order to counteract and control these contaminants, the standard care includes topical antiseptic agents. The most commonly used include iodine-releasing agents (e.g., povidone-iodine), hydrogen peroxide, and polyhexanide. This study aims to investigate the in vitro antibacterial activity of a novel topical spray (Fitostimoline® Plus spray) based on 0.1% polyhexanide and Rigenase® against S. aureus, P. aeruginosa, K. pneumoniae, and the combination of S. aureus and K. pneumoniae. The in vitro antimicrobial activity of Fitostimoline® Plus spray was evaluated by the agar disk diffusion assay, quantitative suspension test, and quantitative carrier test, demonstrating that Fitostimoline® Plus spray is able to kill 99.9% bacteria. These results support the microbiological characterization of Fitostimoline® Plus spray confirming the antibacterial activity of polyhexanide (PHMB).