Preliminary Characterization of a Polycaprolactone-SurgihoneyRO Electrospun Mesh for Skin Tissue Engineering

Skin is a hierarchical and multi-cellular organ exposed to the external environment with a key protective and regulatory role. Wounds caused by disease and trauma can lead to a loss of function, which can be debilitating and even cause death. Accelerating the natural skin healing process and minimizing the risk of infection is a clinical challenge. Electrospinning is a key technology in the development of wound dressings and skin substitutes as it enables extracellular matrix-mimicking fibrous structures and delivery of bioactive materials. Honey is a promising biomaterial for use in skin tissue engineering applications and has antimicrobial properties and potential tissue regenerative properties. This preliminary study investigates a solution electrospun composite nanofibrous mesh based on polycaprolactone and a medical grade honey, SurgihoneyRO. The processing conditions were optimized and assessed by scanning electron microscopy to fabricate meshes with uniform fiber diameters and minimal presence of beads. The chemistry of the composite meshes was examined using Fourier transform infrared spectroscopy and X-ray photon spectroscopy showing incorporation of honey into the polymer matrix. Meshes incorporating honey had lower mechanical properties due to lower polymer content but were more hydrophilic, resulting in an increase in swelling and an accelerated degradation profile. The biocompatibility of the meshes was assessed using human dermal fibroblasts and adipose-derived stem cells, which showed comparable or higher cell metabolic activity and viability for SurgihoneyRO-containing meshes compared to polycaprolactone only meshes. The meshes showed no antibacterial properties in a disk diffusion test due to a lack of hydrogen peroxide production and release. The developed polycaprolactone-honey nanofibrous meshes have potential for use in skin applications.

Anti-Staphylococcal Activity of Cinnamomum zeylanicum Essential Oil against Planktonic and Biofilm Cells Isolated from Canine Otological Infections

The aim of this study was to evaluate the phytochemical profile of Cinnamomum zeylanicum essential oil (CZEO) and their antimicrobial and antibiofilm activity against Staphylococcus strains isolated from canine otitis. First, the CZEO chemical composition was determined by gas chromatography-mass spectrometry (CG-MS). External otitis samples collected from dogs were submitted to staphylococcal isolation, followed by MALDI-TOF mass spectrometry identification. The antimicrobial action was tested against the isolates using the disk-diffusion and microdilution methods. The antibiofilm activity was evaluated by CZEO-based concentrations, subMIC for biofilm formation and supraMIC against preformed biofilm, quantified by crystal violet (CV) staining and CFU counting. The chemical analysis revealed that (E)-cinnamaldehyde, eugenol and (E)-cinnamyl acetate were the main compounds in the CZEO, representing 77.42, 8.17 and 4.50%, respectively. Two strains of three different species, S. saprophyticus, S. schleiferi and S. pseudintermedius, were identified. The disk-diffusion test showed an inhibitory zone diameter, ranging from 34.0 to 49.5 mm, while the MIC and MBC values were around 500 and 1000 µg/mL. SubMIC demonstrated an inhibition on biofilm formation against 4 out the 6 strains tested. On mature biofilm, the CZEO-based supraMIC groups had slightly change on biomass, however, the biofilm cell viability decreased the CFU in 3 magnitude orders.

Modulating the Antifungal Activity of Antimycotic Drugs with Farnesol

Introduction. Clinical strains of microorganisms, including opportunistic yeast-like fungi (YLF) of the genus Candida, are resistant to currently used antifungal drugs. In this regard, the search for alternative ways to potentiate the activity of antimicrobial agents in relation to the infectious agent is an important and relevant area of research. The study of combinations of existing antimycotic drugs and a medicinal extract of plant origin – farnesol – is one of the promising approaches in the fight against resistant strains of YLF genus Candida. In our previous studies, farnesol has been shown to exhibit relative activity against YLF Candida albicans biofilms. In this study, we used 6 clinical isolates and one museum strain YLF C. albicans to study the effect of farnesol on the antifungal activity of antimycotic drugs.Aim. To prove that farnesol can increase the antifungal activity of certain antimycotics.Materials and methods. To determine the sensitivity of 7 strains of YLF C. albicans to the antimycotic drugs "Nystatin" (NYS 50 µg), "Ketoconazole" (KET 10 µg), "Clotrimazole" (CTR 10 µg), "Amphotericin B" (AMB 10 µg), "Voriconazole" (VRC 10 µg) disk diffusion test was used. A solution of farnesol in concentrations of 100, 50 and 25 µM in a volume of 25 µl was applied to the disk with the antimycotic drug. Sterile physiological (PhS) solution was used as a control (pH 7.0; V = 25 µl).Results and discussion. In 34.3 % of of experiments we can talk about the modulating effect of farnesol solutions on the antifungal activity of antimycotic drugs. In all these cases, the sensitivity of YLF C. albicans to the antimycotic drug increases.Conclusion. The results of this study provide useful information for understanding the mechanism of QS-molecules action with antifungal activity, as well as they are the basis for the practical application of some QS-molecules in the treatment of infectious diseases caused by YLF of the genus Candida. The study demonstrates that farnesol can be recommended as an active substance that improves the sensitivity of YLF Candida to antimycotic drugs, especially in the case of multi-resistant strains Candida.

Molecular Typing and Antimicrobial Susceptibility Profiles of Streptococcus uberis Isolated from Sheep Milk

Intramammary infections are a major problem for dairy sheep farms, and Streptococcus uberis is one of the main etiological agents of ovine mastitis. Surveys on antimicrobial resistance are still limited in sheep and characterization of isolates is important for acquiring information on resistance and for optimizing therapy. In this study, a sampling of 124 S. uberis isolates collected in Sardinia (Italy) from sheep milk was analyzed by multilocus-sequence typing (MLST) and pulsed field gel electrophoresis (PFGE) for genetic relatedness. All isolates were also subjected to antimicrobial susceptibility analysis by the disk diffusion test using a panel of 14 antimicrobials. Resistance genes were detected by PCR assays. MLST analysis revealed that the isolates were grouped into 86 sequence types (STs), of which 73 were new genotypes, indicating a highly diverse population of S. uberis. The most frequently detected lineage was the clonal complex (CC)143, although representing only 13.7% of all characterized isolates. A high level of heterogeneity was also observed among the SmaI PFGE profiles, with 121 unique patterns. Almost all (96.8%) isolates were resistant to at least one antimicrobial, while all exhibited phenotypic susceptibility to oxacillin, amoxicillin-clavulanic acid and ceftiofur. Of the antimicrobials tested, the highest resistance rate was found against streptomycin (93.5%), kanamycin (79.8%) and gentamicin (64.5%), followed by novobiocin (25%) and tetracycline-TE (19.3%). Seventy-four (59.7%) isolates were simultaneously resistant to all aminoglycosides tested. Seventeen isolates (13.7%) exhibited multidrug resistance. All aminoglycosides-resistant isolates were PCR negative for aad-6 and aphA-3′ genes. Among the TE-resistant isolates, the tetM gene was predominant, indicating that the resistance mechanism is mainly mediated by the protection of ribosomes and not through the efflux pump. Three isolates were resistant to erythromycin, and two of them harbored the ermB gene. This is the first study reporting a detailed characterization of the S. uberis strains circulating in Sardinian sheep. Further investigations will be needed to understand the relationships between S. uberis genotypes, mastitis severity, and intra-mammary infection dynamics in the flock, as well as to monitor the evolution of antimicrobial resistance.

Molecular Typing and Antimicrobial Susceptibility Profiles of Streptococcus uberis Isolated From Sheep Milk

Intrammary infections are a major problem for dairy sheep farms, and Streptococcus uberis is one of the main etiological agents of ovine mastitis. Surveys on antimicrobial resistance are still limited in sheep and characterization of isolates is important for acquiring information on resistance and for optimizing therapy. In this study, a sampling of 124 S. uberis isolates collected in Sardinia (Italy) from sheep milk was analysed by multilocus-sequence typing (MLST) and pulsed field gel electrophoresis (PFGE) for genetic relatedness. All isolates were also subjected to antimicrobial susceptibility analysis by the disk diffusion test using a panel of 14 antimicrobials. Resistance genes were detected by PCR assays. MLST analysis revealed that the isolates were grouped into 86 sequence types (STs), of which 73 were new genotypes, indicating a highly diverse population of S. uberis. The most frequently detected lineage was the clonal complex (CC)143, although representing only 13.7% of all characterized isolates. A high level of heterogeneity was also observed among the SmaI PFGE profiles, with 121 unique patterns. Almost all (96.8%) isolates were resistant to at least one antimicrobial, while all exhibited phenotypic susceptibility to oxacillin, amoxicillin-clavulanic acid and ceftiofur. Of the antimicrobials tested, the highest resistance rate was found against streptomycin (93.5%), kanamycin (79.8%) and gentamicin (64.5%), followed by novobiocin (25%) and tetracycline-TE (19.3%). Seventy-four (59.7%) isolates were simultaneously resistant to all aminoglycosides tested. Seventeen isolates (13.7%) exhibited multidrug resistance. All aminoglycosides-resistant isolates were PCR negative for aad-6 and aphA-3’ genes. Among the TE-resistant isolates, the tetM gene was predominant, indicating that the resistance mechanism is mainly mediated by the protection of ribosomes and not through the efflux pump. Three isolates were resistant to erythromycin, and two of them harboured the ermB gene. This is the first study reporting a detailed characterization of the S. uberis strains circulating in Sardinian sheep. Further investigations will be needed to understand the relationships between S. uberis genotypes, mastitis severity, and intra-mammary infection dynamics in the flock, as well as to monitor the evolution of antimicrobial resistance.

Molecular characterization of enterohemorrhagic Escherichia coli isolated from diarrhea samples from human, livestock, and ground beef in North Jordan

Background and Aim: Enterohemorrhagic Escherichia coli (EHEC) is an important foodborne pathogen with worldwide distribution. Data regarding its presence, distribution, virulence, and antimicrobial susceptibility among various animal species and humans in Jordan are lacking. Therefore, the objectives of this study were to isolate and characterize EHEC from human and animal diarrhea fecal samples and ground beef samples. Materials and Methods: A total of 100 and 270 diarrhea fecal samples from humans and animals, respectively, were collected. In addition, 40 ground beef meat samples were collected from retail markets. EHEC was positively identified by detecting Shiga toxins (stx1 and stx2) genes using multiplex polymerase chain reaction (PCR). Antimicrobial susceptibility patterns were determined using the disk diffusion test. Beta-lactamase production was detected using the double disk diffusion test and the extended-spectrum beta-lactamases (ESBLs) were identified by detection of blaTEM, blaSHV, and OXA-1 genes using multiplex PCR. Pulsed-field gel electrophoresis (PFGE) was used to investigate the relatedness of EHEC isolates from different sources. Results: Out of 410 samples, 194 E. coli isolates were positively identified, of which 57 isolates (29%) were classified as EHEC. Thirty-five (61%) of EHEC isolates were serotyped as O157 (19: O157:H7 and 16: O157:NM). The stx1 gene was detected only among the sheep and goats isolates at a rate of 7.6% and 5.2%, respectively, while the stx2 gene was detected in only one ground beef meat sample. EHEC isolates showed high resistance patterns against amoxicillin, gentamycin, cephalexin, and doxycycline. Twenty-four out of 32 EHEC isolates were determined as ESBL producers, among which 14 isolates expressed the blaSHV gene and 19 isolates expressed the blaTEM while four expressed both genes. PFGE analysis revealed two clusters with high similarity (92%) originated from ground beef meat and cattle fecal samples. No similarities were found between human and animal E. coli isolates. Conclusion: Results of this study indicate widespread ESBL EHEC among humans, animals, and ground beef meat samples. These results represent an important alarm that requires the implementation of appropriate preventative measures by both human and animal health sectors to prevent the transmission of this important foodborne pathogen.

In Vitro Antimicrobial Activity of the Siderophore Cephalosporin Cefiderocol against Acinetobacter baumannii Strains Recovered from Clinical Samples

Background: Cefiderocol is a siderophore cephalosporin that exhibits antimicrobial activity against most multi-drug resistant Gram-negative bacteria, including Enterobacterales, Pseudomonas aeruginosa, Acinetobacter baumannii, and Stenotrophomonas maltophilia. Methods: A total of 20 multidrug-resistant A. baumannii strains were isolated from 2020 to 2021, molecularly characterized and tested to assess the in vitro antibacterial activity of cefiderocol. Thirteen strains were carbapenem-hydrolysing oxacillinase OXA-23-like producers, while seven were non-OXA-23-like producers. Minimum inhibitory concentrations (MICs) were determined by broth microdilution, considered as the gold standard method. Disk diffusion test was also carried out using iron-depleted CAMHB plates for cefiderocol. Results: Cefiderocol MICs ranged from 0.5 to 1 mg/L for OXA-23-like non-producing A. baumannii strains and from 0.25 to >32 mg/L for OXA-23-like producers, using the broth microdilution method. Cefiderocol MIC90 was 8 mg/L. Diameter of inhibition zone of cefiderocol ranged from 18 to 25 mm for OXA-23-like non-producers and from 15 to 36 mm for OXA-23-like producers, using the diffusion disk method. A large variability and a low reproducibility were observed during the determination of diameter inhibition zone. Molecular characterization showed that all isolates presented the ISAba1 genetic element upstream the blaOXA-51. Among OXA-23-like non-producers, four were blaOXA-58 positive and two were negative for all the resistance determinants analyzed. Conclusions: Cefiderocol showed in vitro antimicrobial activity against both carbapenem-susceptible and non-susceptible A. baumannii strains, although some OXA-23-like producers were resistant. Further clinical studies are needed to consolidate the role of cefiderocol as an antibiotic against MDR A. baumannii.

Genome features and antibiotic resistance of Pseudomonas aeruginosa strains isolated in patients with cystic fibrosis in the Russian Federation

Cystic fibrosis (CF) is a common genetic disease, manifested by airway obstruction and chronic respiratory infection. The most prevalent infectious agent in airways of CF patients is Pseudomonas aeruginosa. This study aimed to determine sequence-types, antimicrobial resistance phenotypes and genes defining adaptive antibiotic resistance in P. aeruginosa isolates recovered from CF patients in Russia. In total, 84 P. aeruginosa strains from 64 CF patients were analyzed. Susceptibility to antibiotics was determined by disk diffusion test. Whole-genome sequencing (WGS) was performed on MGISEQ-2000 platform. SPAdes software, Galaxy, ResFinder, PubMLST were used for analysis of WGS data. Examined P. aeruginosa isolates belonged to 53 different sequence-types (STs), including 6 new STs. High-risk epidemic clone ST235 (10%) and clonal CF P. aeruginosa strains ST17, ST242, ST274 (7%) were detected. Non-susceptibility to ticarcillin-clavulanate, cefepime, imipenem was observed in 63%, 12% and 25% of isolates, respectively; to tobramycin - in 24%, to amikacin - in 35%; to ciprofloxacin, levofloxacin - in 35% and 57% of strains, respectively. Multidrug-resistant phenotype was detected in 18% of isolates. In examined strains, genes of beta-lactamases VIM-2 (5 ST235 strains), VEB-1 (two ST2592 strains), GES-1 (1 ST235 strain), PER-1 (1 ST235 strain) were found. Ciprofloxacin-modifying enzyme CrpP gene was detected in 67% of isolates, aminoglycoside-modifying enzymes AAD, ANT, AAC genes - in 7%, 4%, 12% of strains, respectively. P. aeruginosa isolates from CF patients in Russia demonstrate a high clonal diversity, which is similar to other P. aeruginosa infections. The isolates of high-risk clone and clonal CF P. aeruginosa strains are detected.

A Microbiological Study of Acinetobacter calcoaceticus baumannii with Special Reference to Multidrug Resistance

Introduction The outbreak of Acinetobacter calcoaceticus baumannii (ACB) is mainly reported to be a notorious pathogens at health-care settings. It is the major problem on the health-care system with high morbidity and mortality rates because of the broad range of antibiotic resistance and lack of understanding the mechanism of developing new antibiotic resistance rapidly. It emphasizes the importance of local surveillance in describing or understanding and predicting microbial resistance patterns so that there will be limited use of antibiotics by developing strategies to control the extensive use of antimicrobial chemotherapy in clinical environment, which is still considered as one of the factors in the emergence of multidrug resistance microorganisms. Objectives The study aims to detect the occurrence rate of ACB infections from various clinical samples, identify the resistance levels to different groups of antimicrobial agents, and the occurrence rate of multidrug resistant (MDR) ACB clinical isolates from a tertiary hospital in Durgapur, West Bengal, India. Material and Methods The study was performed in the Department of Microbiology of the IQ City Medical College and Hospital, Durgapur, West Bengal, India, for the 24 months duration, that is, from January 1, 2018 to December 31, 2019. Altogether 15,800 clinical samples consisting of endotracheal tube aspirates, sputum, pus, blood, catheter tips, urine, tissue, and other body fluids were studied. ACB from clinical samples were identified by its characteristic colonies (nonlactose fermenting, glistening, small mucoid colonies), Gram-staining pattern (Gram-negative coccobacillus), and standard biochemical reactions. It was further confirmed in the Department of Microbiology of the Healthworld Hospital, Durgapur, West Bengal, India, by Vitek2 compact system (bioMerieux, Inc., Durham, North Carolina, United States). Antibiotic susceptibility testing was performed using automated broth microdilutions by Vitek2 compact system (bioMerieux, Inc.) and Kirby-Bauer disk diffusion test on Mueller-Hinton Agar (HiMedia). Results Nonrepetitive 289 ACB were isolated from various clinical samples. A total of 277 (96%) isolates of ACB were MDR strains. Conclusion ACB was mostly isolated from the intensive care unit department and was found to be the most MDR type in the tertiary care hospital by this study.

Metallo-β Lactamase Producing Non-Fermentative Gram-Negative Bacilli from Various Clinical Isolates in a Tertiary Care Hospital: A Descriptive Cross-sectional Study

Introduction: Non-fermentative gram-negative bacilli are common causes of human infections especially nosocomial infections. These organisms are usually resistant to multiple antimicrobial agents including carbapenems. The study aimed to find out the prevalence of metallo-β-lactamase producing non-fermentative gram-negative bacilli among the samples which yielded growth of bacteria in a tertiary care hospital. Methods: This is a descriptive cross-sectional study conducted in a tertiary care hospital from February 2017 to May 2017. Convenience sampling method was used. Bacterial identification, characterization and antimicrobial susceptibility testing were done by following standard microbiological guidelines. Metallo-β-lactamase production was detected by using combined disk diffusion test and double-disc synergy test. Data were analyzed by using Statistical Package of Social Science software version 16. Point estimate at 95% confidence interval was calculated along with frequency and proportion for binary data. Results: Among 628 samples which yielded growth of bacteria, 118 (18.79%) at 95% Confidence Interval (15.74-21.84) were metallo-β-lactamase producing non-fermentative gram-negative bacilli. Among them, 54 (45.76%) were Pseudomonas aeruginosa and 64 (54.24%) were Acinetobacter baumannii. Conclusions: A high prevalence of metallo-β-lactamase production was observed among the nonfermentative gram-negative bacilli than the study done in similar settings. It is mandatory to perform routine monitoring of metallo-β-lactamase producing isolates in clinical laboratories in order to help the clinicians prescribe proper antibiotics.