Ocular Isolates Research Articles

Synthetic lincosamides iboxamycin and cresomycin are active against ocular multidrug-resistant methicillin-resistant Staphylococcus aureus carrying erm genes

ObjectiveAntimicrobial resistance is a global pandemic that poses a major threat to vision health as ocular bacteria, especially methicillin-resistant Staphylococcus aureus (MRSA), are becoming increasingly resistant to first-line therapies. Here we evaluated the antimicrobial activity of new synthetic lincosamides in comparison to currently used antibiotics against clinical ocular MRSA isolates. MethodsAntimicrobial susceptibility testing was performed by broth microdilution for two novel synthetic lincosamides (iboxamycin and cresomycin) and eight comparator antibiotics against a collection of 50 genomically characterised ocular MRSA isolates, including isolates harbouring erm genes (n = 25). ResultsBoth drugs were active against widespread MRSA clonal complexes CC8 and CC5. The MIC50 and MIC90 of iboxamycin were 0.06 and 2 mg/L, respectively. Cresomycin (MIC50 = 0.06 mg/L) also displayed good activity with an in vitro potency four-fold higher (MIC90 = 0.5 mg/L) than iboxamycin. In isolates harbouring erm genes, MIC90 were >16, 2, and 0.5 mg/L for clindamycin, iboxamycin, and cresomycin, respectively. The in vitro potencies of iboxamycin and cresomycin were similar or higher than that of comparator agents and were not impacted by multidrug-resistance phenotypes or by the presence of erm genes when compared with clindamycin. ConclusionsOur results demonstrate that iboxamycin and cresomycin display potent in vitro activity against ocular MRSA isolates, including multidrug-resistant isolates harbouring erm genes.

334 Metagenomic- and culture-based characterization of the ocular microbiome in cattle with infectious bovine keratoconjunctivitis

Abstract Pinkeye, clinically known as infectious bovine keratoconjunctivitis (IBK), is one of the most highly contagious diseases impacting cattle at all ages. Despite extensive use of antibiotics and vaccines targeting Moraxella bovis, the primary IBK pathogen, the incidence of pinkeye has been increasing in North American beef cow-calf operations. This highlights the incomplete understanding of the pathogenesis of this disease and the need for novel and alternative approaches to control IBK. The objectives of this study were to: 1) characterize the ocular microbiome of beef cattle with and without IBK using shotgun metagenomic sequencing; 2) investigate the genetic diversity of M. bovis, Moraxella bovoculi, and commensal ocular bacterial isolates; 3) evaluate whether selected ocular probiotic candidate strains inhibit Moraxella pathogen growth in the presence of the ocular microbiome in culture using qPCR. A total of 143 ocular swabs were collected from cattle with (n = 102) or without IBK (n = 42) and before antibiotic treatment from beef herds across North Dakota, USA. Fifty samples from IBK (n = 38) and healthy cattle (n = 12) were also subjected to shotgun metagenomic sequencing. Seven bacterial species were relatively more abundant (P < 0.05) in the healthy cattle ocular microbiome including Bifidobacterium globosum, Bacillus licheniformis, Pasteurella multocida, Ruminococcus sp900316555, and Ruminococcus sp900100595. Arthrobacter luteus was the only bacterial species significantly associated with IBK-affected cattle. From the metagenomes, 37 high-quality dereplicated metagenome-assembled genomes (MAGs) were recovered, nearly a third of which were identified as Mesomycoplasma bovoculi (n = 10). M. bovis (n = 5) and M. bovoculi (n = 13) were also isolated from the ocular swabs of both healthy and IBK-affected cattle. These isolates represented 9 M. bovoculi and 5 M. bovis strains (average nucleotide identity < 99.99%). We also isolated and sequenced several potentially beneficial bacterial species from healthy cattle including Bacillus pumilus, Lactiplantibacillus plantarum, Lentilactobacillus buchneri, Levilactobacillus brevis, and Weizmannia coagulans. Four of these probiotic candidates were also tested for their inhibitory effects against M. bovoculi in co-culture with the ocular microbiome after 24 h incubation using qPCR. The B. pumilus, L. buchneri, L. plantarum, and W. coagulans strains, as well as a mixture of all four isolates, significantly reduced the growth of Moraxella. In summary, our results provide important information on the culturable and non-culturable fraction of the bovine ocular microbiome potentially associated with IBK and suggest that certain bacterial species in the eye may inhibit the growth of Moraxella spp.

Comparison of susceptibility of Pseudomonas aeruginosa isolated from keratitis to antibiotics and multipurpose disinfecting solutions

PurposeThis study aimed to assess the susceptibility of ocular isolates of Pseudomonas aeruginosa to two multi-purpose disinfectant solutions (MPDS) and to determine if there was a relationship with resistance to antibiotics. MethodsTwenty-three strains of P. aeruginosa isolated from microbial keratitis cases in Australia were utilized in this study. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of two commercially available MPDSs, Biotrue and cleadew MPS were determined. Additionally, the MIC of the strains to five antibiotics, ciprofloxacin, levofloxacin, gentamicin, ceftazidime, and imipenem, were analyzed. ResultsAll strains were susceptible to 100 % of each of the MPDS. However, when MPDSs were diluted, cleadew MPS had significantly lower median MIC (median = 12.5 vs 25; p = 0.00008) and MBC (median = 25 vs 50; p = 0.0027) compared to Biotrue. All tested strains were susceptible to levofloxacin and gentamicin. Susceptibility rates to ciprofloxacin, imipenem, and ceftazidime were 52.2 %, 30.4 %, and 91.3 %, respectively. There were no significant relations between MIC or MBC to either MPDS and resistance to the antibiotics (p≧0.23). ConclusionBoth MPDSs were active against P. aeruginosa isolated from microbial keratitis in Australia. However, after dilution, cleadew MPS remained active against P. aeruginosa at lower concentrations. Certain strains of P. aeruginosa were resistant to imipenem, ceftazidime or ciprofloxacin. The lack of association of susceptibility to MPDS and antibiotics suggest that resistance to one did not predispose to resistance to the other.

Mycolyltransferase is important for biofilm formation and pathogenesis of Tsukamurella keratitis

ABSTRACT Tsukamurella, a group of multi-drug resistant, Gram-positive, aerobic, and partially acid-fast bacteria, are emerging causes of bacterial conjunctivitis and keratitis. However, the pathogenesis of Tsukamurella keratitis is largely unknown. To address this, we used New Zealand White rabbits to develop the first eye infection model and conducted in vitro tests to study the pathogenesis mechanisms of Tsukamurella. There is increasing evidence that biofilms play a significant role in ocular infections, leading us to hypothesize that biofilm formation is crucial for effective Tsukamurella infection. In order to look for potential candidate genes which are important in biofilm formation and Tsukamurella keratitis. We performed genome sequencing of two ocular isolates, T. pulmonis-PW1004 and T. tyrosinosolvens-PW899, to identify potential virulence factors. Through in vitro and in vivo studies, we characterized their biological roles in mediating Tsukamurella keratitis. Our findings confirmed that Tsukamurella is an ocular pathogen by fulfilling Koch's postulates, and using genome sequence data, we identified tmytC, encoding a mycolyltransferase, as a crucial gene in biofilm formation and causing Tsukamurella keratitis in the rabbit model. This is the first report demonstrating the novel role of mycolyltransferase in causing ocular infections. Overall, our findings contribute to a better understanding of Tsukamurella pathogenesis and provide a potential target for treatment. Specific inhibitors targeting TmytC could serve as an effective treatment option for Tsukamurella infections.

Unveiling the ocular battlefield: Insights into Pseudomonas aeruginosa virulence factors and their implications for multidrug resistance

The research investigates the virulence factors of Pseudomonas aeruginosa (P. aeruginosa), a pathogen known for its ability to cause human infections by releasing various exoenzymes and virulence factors. Particularly relevant in ocular infections, where tissue degeneration can occur, even after bacterial growth has ceased due to the potential role of secreted proteins/enzymes. Clinical isolates of P. aeruginosa, both ocular (146) and non-ocular (54), were examined to determine the frequency and mechanism of virulence factors. Phenotypic characterization revealed the production of alginate, biofilm, phospholipase C, and alkaline protease, while genotypic testing using internal uniplex PCR identified the presence of Exo U, S, T, Y, and LasB genes. Results showed a significant prevalence of Exo U and Y genes in ocular isolates, a finding unique to Indian studies. Additionally, the study noted that ocular isolates often contained all four secretomes, suggesting a potential link between these factors and ocular infections. These findings contribute to understanding the pathogenesis of P. aeruginosa infections, particularly in ocular contexts, and highlights the importance of comprehensive virulence factor analysis in clinical settings.

Virulence-related genotypic differences among Bacillus cereus ocular and gastrointestinal isolates and the relationship to endophthalmitis pathogenesis.

Bacillus cereus (Bc) can cause self-limiting gastrointestinal infections, but when infecting the eye, can cause rapid and irreversible blindness. This study investigated whether clinical ocular and gastrointestinal Bc isolates differed in terms of virulence-related genotypes and endophthalmitis virulence. Twenty-eight Bc ocular, gastrointestinal, and laboratory reference isolates were evaluated. Hemolysis assays were performed to assess potential differences in hemolytic activity. The presence of twenty Bc virulence-related genes was assessed by PCR. A subset of ocular and gastrointestinal isolates differing in PCR positivity for 5 virulence genes was compared to strain ATCC14579 in an experimental murine model of endophthalmitis. At 8 hours post infection, retinal function was evaluated by electroretinography, and intraocular bacterial concentrations were determined by plate counts. Gastrointestinal Bc isolates were more hemolytic than the Bc ocular isolates and ATCC14579 (p < 0.0001). Bc ocular isolates were more frequently PCR-positive for capK, cytK, hblA, hblC, and plcR compared to the gastrointestinal isolates (p ≤ 0.0002). In the endophthalmitis model, mean A-wave retention did not differ significantly between eyes infected with ATCC14579 and eyes infected with the selected ocular or gastrointestinal isolates (p ≥ 0.3528). Similar results were observed for mean B-wave retention (p ≥ 0.0640). Only one diarrheal isolate showed significantly greater B-wave retention when compared to ATCC14579 (p = 0.0303). No significant differences in mean A-wave (p ≥ 0.1535) or B-wave (p ≥ 0.0727) retention between the selected ocular and gastrointestinal isolates were observed. Intraocular concentrations of ATCC14579 were significantly higher than the selected ocular isolate and 3 of the gastrointestinal isolates (p ≤ 0.0303). Intraocular concentrations of the selected ocular isolate were not significantly different from the gastrointestinal isolates (p ≥ 0.1923). Among the subset of virulence-related genes assessed, 5 were significantly enriched among the ocular isolates compared to gastrointestinal isolates. While hemolytic activity was higher among gastrointestinal isolates, retinal function retention and intraocular growth was not significantly different between the selected ocular and gastrointestinal isolates. These results suggest that Bc strains causing gastrointestinal infections, while differing from ocular isolates in hemolytic activity and virulence-related gene profile, are similarly virulent in endophthalmitis.

168 Characterization of the Culturable Fraction of Bovine Ocular Microbiota and Its in Vitro Antimicrobial Activities Against Pinkeye Pathogens

Abstract Pinkeye, clinically known as infectious bovine keratoconjunctivitis (IBK), is considered one of the most important ocular diseases in cattle, causing an estimated $226 million in annual losses to U.S. cow-calf producers. While Moraxella bovis is considered the principal IBK pathogen, Moraxella bovoculi has also been associated with the pathogenesis of IBK. Thus, these Moraxella species are often the target for antibiotic and/or vaccine-based treatment and prevention strategies. Despite extensive use of antibiotics, IBK infections are challenging to treat, highlighting the increased need for an antibiotic-alternative approach to control IBK. Recent evidence suggests that the microbial community associated with the bovine oculus is important for eye health, and that the ocular microbiota can be a target for enhancing disease resistance against IBK in cattle. The objectives of this study were to characterize the ocular microbiota of healthy and IBK infected cattle using sequencing and culturing techniques, and to isolate and screen commensal ocular isolates for their antimicrobial activity against M. bovis and M. bovoculi. Ocular swabs (n = 64) were collected using eSwabs from cattle diagnosed with IBK or without IBK (healthy control) before antibiotic treatment from beef herds across North Dakota. Swabs were plated onto four different agar plates (blood, Columbia blood, MRS and multi-sliced agar plates), and cultured aerobically and anaerobically. A total of 658 bacterial isolates were obtained via culturing and cryopreserved. Taxonomic identification was performed on a subset of banked isolates (n = 277) by near full-length sequencing of the 16S rRNA gene. These isolates taxonomically belong to 6 different bacterial phyla: Bacillus (42%), Firmicutes (27%), Actinomycetota (13%), Pseudomonadota (11%), Actinobacteria (5%), and Proteobacteria (2%). A total of 55 different genera were identified with Bacillus (25%), Streptococcus (13%), Staphylococcus (10%), Moraxella (6%), Macrococcus (5%), Paenibacillus (3%), Caldibacillus (3%), and Rummeliibacillus (3%) being the most dominant genera. Next, a subset of these commensal isolates (n 53) were screened for their growth inhibition against M. bovis and M. bovoculi using an agar slab method. Among the screened isolates, 6 isolates showed inhibition against M. bovis and M. bovoculi with zones of inhibition ranging from 12 mm to 28 mm. Ocular isolates identified as Weizmannia coagulans (43Y-MRSC), Lentillactobacillus buchneri (23D-MRSA) and Paenibacillus polymyxa (785-WCF) exhibited relatively strong inhibition against both Moraxella isolates. Scanning electron microscopy revealed noticeable structural and morphological damages in M. bovis and M. bovoculi cells due to Weizmannia coagulans culture-supernatant treatment. Overall, our results indicate that ocular mucosa of beef cattle is inhabited by relatively rich and diverse culturable bacterial communities, and that some ocular commensals can inhibit IBK pathogens. This suggests the potential for the development of bacterial therapeutics based on commensal ocular bacteria to mitigate IBK in cattle.

Molecular and Phenotypic Characterization of Ocular Methicillin-Resistant Staphylococcus epidermidis Isolates in Taiwan.

Staphylococcus epidermidis, a commensal, has emerged as an important opportunistic pathogen, particularly methicillin-resistant S. epidermidis (MRSE). The mechanism behind this transformation remains unclear. This study aimed to investigate the molecular and phenotypic characteristics of MRSE isolated from healthy conjunctiva and ocular infections. We collected MRSE isolates from two groups: healthy conjunctiva from patients undergoing cataract surgeries and ocular infections at our hospital. Genotypic analysis included pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), staphylococcal cassette chromosome mec (SCCmec), and biofilm-related genes (icaA, aap, and bhp). Additionally, phenotypic data on biofilm production and antibiotic susceptibility were recorded. A total of 86 isolates, including 42 from healthy conjunctiva and 44 from ocular infections, were analyzed. MLST identified 21 sequence types (STs), with ST59 being the most frequent (n = 33, 39.5%), followed by ST130 (n = 10, 11.6%), ST57 (n = 6, 7.0%), and ST2 (n = 6, 7.0%). All isolates were categorized in 23 PFGE types, and SCCmec IV was the most prevalent SCCmec type (n = 52, 60.5%). The two sources of isolates exhibited overlapping molecular types and phenotypic traits, although the ocular infection isolates exhibited significantly higher multidrug resistance compared to healthy conjunctiva isolates (P = 0.032). When contrasting ST59 with non-ST59, ST59 displayed a significantly higher presence of aap (100%) and bhp (69.7%) while lacking icaA (0%). ST59 also showed lower susceptibility to fluoroquinolones compared to non-ST59 (42.4%-54.5% vs. 75.5%-83.0%; P < 0.01). MRSE isolates from healthy conjunctiva and ocular infections demonstrated a degree of resemblance. Specific strains, notably ST59, exhibited distinctive characterizations.

Phylogenetic and Genomic Characterization of Whole Genome Sequences of Ocular Herpes Simplex Virus Type1 Isolates Identifies Possible Virulence Determinants in Humans.

There are limited data on the prevalence and genetic diversity of herpes simplex virus type 1 (HSV-1) virulence genes in ocular isolates. Here, we sequenced 36 HSV-1 ocular isolates, collected by the Bascom Palmer Eye Institute, a university-based eye hospital, from three different ocular anatomical sites (conjunctiva, cornea, and eyelid) and carried out a genomic and phylogenetic analyses. The PacBio Sequel II long read platform was used for genome sequencing. Phylogenetic analysis and genomic analysis were performed to help better understand genetic variability among common virulence genes in ocular herpetic disease. A phylogenetic network generated using the genome sequences of the 36 Bascom Palmer ocular isolates, plus 174 additional strains showed that ocular isolates do not group together phylogenetically. Analysis of the thymidine kinase and DNA polymerase protein sequences from the Bascom Palmer isolates showed multiple novel single nucleotide polymorphisms, but only one, BP-K14 encoded a known thymidine kinase acyclovir resistance mutation. An analysis of the multiple sequence alignment comprising the 51 total ocular isolates versus 159 nonocular strains detected several possible single nucleotide polymorphisms in HSV-1 genes that were found significantly more often in the ocular isolates. These genes included UL6, gM, VP19c, VHS, gC, VP11/12, and gG. There does not seem to be a specific genetic feature of viruses causing ocular infection. The identification of novel and common recurrent polymorphisms may help to understand the drivers of herpetic pathogenicity and specific factors that may influence the virulence of ocular disease.

Ocular Bacterial Infections: A Ten-Year Survey and Review of Causative Organisms Based on the Oklahoma Experience.

Ocular infections can be medical emergencies that result in permanent visual impairment or blindness and loss of quality of life. Bacteria are a major cause of ocular infections. Effective treatment of ocular infections requires knowledge of which bacteria are the likely cause of the infection. This survey of ocular bacterial isolates and review of ocular pathogens is based on a survey of a collection of isolates banked over a ten-year span at the Dean McGee Eye Institute in Oklahoma. These findings illustrate the diversity of bacteria isolated from the eye, ranging from common species to rare and unique species. At all sampled sites, staphylococci were the predominant bacteria isolated. Pseudomonads were the most common Gram-negative bacterial isolate, except in vitreous, where Serratia was the most common Gram-negative bacterial isolate. Here, we discuss the range of ocular infections that these species have been documented to cause and treatment options for these infections. Although a highly diverse spectrum of species has been isolated from the eye, the majority of infections are caused by Gram-positive species, and in most infections, empiric treatments are effective.

Microbiological spectrum and antibiotic susceptibility patterns of non-viral conjunctivitis over 16years at a tertiary eye care center in Southern India.

To determine the trends in the microbial spectrum and antibacterial susceptibility patterns of non-viral conjunctivitis over 16years. Microbiology data were reviewed from 2006-2021 for all the patients with clinically and culture-proven infectious conjunctivitis. Conjunctival swabs and/or conjunctival scrapings were collected for microbiological workup, and the demographic and antibiotic susceptibility data were extracted from the EMR (Electronic Medical Record). For statistical analysis, χ2-test was performed. Of the 1711 patients, 814 (47.57%) were culture positive and 897 (52.43%) were culture negative. Of the total culture-proven conjunctivitis cases, 775/814 (95.20%) were bacteria, and 39/814 (4.80%) were fungi. Among these bacterial isolates, 75.74% were gram-positive bacteria, while 24.26% were gram negative. The predominant gram-positive pathogens isolated were S. epidermidis (16.7%), S. aureus (17.9%) (p < 0.05), and S. pneumoniae (18.2%), while Haemophilus spp. (36.2%) (p < 0.05) was the most often isolated gram-negative bacteria (36.2%), and Aspergillus spp. was the most commonly isolated fungus (50%). The susceptibility of gram-positive bacteria to cefazoline increased from 90.46 to 98% (p = 0.01), whereas the susceptibility for gatifloxacin decreased in both gram-positive (81-41%; p < 0.0001) and gram-negative bacteria (73-58%; p = 0.02). Increasing resistance of ocular isolates to mainstay antibiotics is a concern, and this data can assist healthcare practitioners in making informed choices regarding the treatment of ocular infections with ophthalmic antibiotics.

Ciprofloxacin resistance and tolerance of Pseudomonas aeruginosa ocular isolates

PurposeTolerance to antibiotics may occur due to changes in bacterial growth patterns and can be a precursor to development of resistance. However, there is a lack of information on the ability of ocular bacteria isolates to develop tolerance. This paper explores the tolerance to 8 different antibiotics of 61 microbial keratitis isolates of Pseudomonas aeruginosa from Australia and India using the MBC/MIC ratio, with tolerance defined by a ratio ≥ 32, and tolerance to ciprofloxacin by an agar diffusion assay. MethodsAntibiotics used were ciprofloxacin, levofloxacin, gentamicin, tobramycin, piperacillin, imipenem, ceftazidime and polymyxin B. Isolates were sourced from microbial keratitis infections in Australia and India. Minimum bactericidal and minimum inhibitory concentration (MBC and MIC) were obtained using broth microdilution and compared to breakpoints from the Clinical Laboratory Standards Institute (CLSI) and European Committee on Antimicrobial Susceptibility Testing (EUCAST) to determine bacterial susceptibility. Tolerance was assessed as MBC/MIC ≥ 32. An alternative method for tolerance detection (TD) was assessed with 13P. aeruginosa sensitive isolates by agar disk diffusion assay of ciprofloxacin followed by application of glucose to the agar and observation of re-growth of colonies. ResultsThirty-three isolates were resistant to imipenem, 20 to ciprofloxacin, 14 to tobramycin and piperacillin, 12 to levofloxacin and ceftazidime, 8 to gentamicin, and 5 to polymyxin B. The percentage of strains resistant to levofloxacin (7 vs 30 %; p = 0.023), gentamicin (0 vs 24 %; p = 0.005) and tobramycin (4 vs 33 %; p = 0.004) was significantly greater in isolates from India.On average, strains from India exhibited notably greater MIC and MBC values compared to strains obtained from Australia. Out of 61 isolates, none displayed an MBC/MIC ratio ≥ 32. However, three sensitive isolates had low tolerance, nine had medium tolerance and one had high tolerance to ciprofloxacin with the TDtest. ConclusionsThis study used two methods to determine whether P. aeruginosa strains could show tolerance to antibiotics. Using the MBC/MIC criteria no strain was considered tolerant to any of the eight antibiotics used. When 13 strains were tested for tolerance against ciprofloxacin, the most commonly used monotherapy for keratitis, one had high tolerance and nine had medium tolerance. This demonstrates the capacity of P. aeruginosa to develop tolerance which may result in therapeutic failures if inappropriate dosing regimens are used to treat keratitis.

Activity of Colloidal Silver Solution against Microorganisms Implicated in Ocular Infections

Endophthalmitis most likely originates from both planktonic bacteria suspended in the tear film and bacteria adherent to the conjunctiva and the eyelid. This study aimed to expand the research on the effectiveness of a colloidal silver solution (Silverix®) against ocular microorganisms. The activity of Silverix® was evaluated against methicillin-resistant Staphylococcus aureus, S. epidermidis, ofloxacin-resistant Pseudomonas aeruginosa, and Candida albicans strains, previously characterized for their antibiotic resistance and biofilm-forming capabilities. The microbial killing was estimated at various times in the presence and absence of colloidal silver solution against planktonic and biofilm-embedded cells. The results documented the efficacy of Silverix® on planktonic cells of S. aureus and S. epidermidis (2.49–2.87 Log CFU/mL reduction) and P. aeruginosa strains (3–4.35 Log CFU/mL reduction). On the contrary, C. albicans showed mild susceptibility. Regarding early biofilm, the ocular isolates were harder to kill (2–2.6 Log CFU/mL reduction) than the reference strains, whereas a similar decrease (3.1 Log CFU/mL reduction) was estimated for P. aeruginosa strains. The light microscope images of biofilms treated with colloidal solution confirmed the ability of Silverix® to destroy the biofilm.

Comparative In Vitro Activity of New Lipoglycopeptides and Vancomycin Against Ocular Staphylococci and Their Toxicity on the Human Corneal Epithelium.

The purpose of this study was to assess the potential of new lipoglycopeptides as novel topical therapies for improved treatment of recalcitrant ocular infections. We evaluated the in vitro antimicrobial activity of oritavancin, dalbavancin, and telavancin compared with vancomycin (VAN) against a large collection of ocular staphylococcal isolates and their cytotoxicity on human corneal epithelial cells (HCECs). Antimicrobial susceptibility testing was performed by broth microdilution against 223 Staphylococcus spp. clinical isolates. Time-kill kinetics were determined for methicillin-resistant strains of Staphylococcus aureus (MRSA) (n = 2) and Staphylococcus epidermidis (MRSE) (n = 1). In vitro cytotoxicity assays were performed with AlamarBlue and live/dead staining on HCECs. All new lipoglycopeptides showed strong in vitro potency against ocular staphylococci, including multidrug-resistant MRSA strains, with dalbavancin showing a slightly higher potency overall [minimum inhibitory concentration (MIC) 90 0.06 μg/mL] compared with telavancin and oritavancin (MIC 90 0.12 μg/mL), whereas VAN had the lowest potency (MIC 90 2 μg/mL). Oritavancin exerted rapid bactericidal activity within 1 h for MRSA and 2 h for MRSE. All other drugs were bactericidal within 24 h. At a concentration commonly used for topical preparations (25 mg/mL), cytotoxicity was observed for VAN after 5 min of incubation, whereas reduction in HCEC viability was not seen for telavancin and was less affected by oritavancin and dalbavancin. Cytotoxicity at 25 mg/mL was seen for all drugs at 30 and 60 min but was significantly reduced or undetected for lower concentrations. Our study demonstrates that new lipoglycopeptides have substantially better in vitro antimicrobial activity against ocular staphylococcal isolates compared with VAN, with a similar or improved toxicity profile on HCECs.

The Relationship between Ciprofloxacin Resistance and Genotypic Changes in S. aureus Ocular Isolates.

Staphylococcus aureus (S. aureus) is a frequent cause of eye infections with some isolates exhibiting increased antimicrobial resistance to commonly prescribed antibiotics. The increasing resistance of ocular S. aureus to ciprofloxacin is a serious concern as it is a commonly used as a first line antibiotic to treat S. aureus keratitis. This study aimed to analyse genetic mutations in the genomes of 25 S. aureus isolates from infections or non-infectious ocular conditions from the USA and Australia and their relationship to ciprofloxacin resistance. Overall, 14/25 isolates were phenotypically resistant to ciprofloxacin. All isolates were analyzed for mutations in their quinolone resistance-determining regions (QRDRs) and efflux pump genes. Of the fourteen resistant isolates, 9/14 had ciprofloxacin resistance mutations within their QRDRs, at codons 80 or 84 within the parC subunit and codon 84 within the gyrA subunit of DNA gyrase. The highest resistance (MIC = 2560 μg/mL) was associated with two SNPs in both gyrA and parC. Other resistant isolates (3/14) had mutations within norB. Mutations in genes of other efflux pumps and their regulator (norA, norC, mepA, mdeA, sepA, sdrM, mepR, arlR, and arlS) or the DNA mismatch repair (MMR) system (mutL and mutS) were not associated with increased resistance to ciprofloxacin. The functional mutations associated with ciprofloxacin resistance in QRDRs (gyrA and parC) and norB suggests that these are the most common reasons for ciprofloxacin resistance in ocular isolates. Novel SNPs of gyrA Glu-88-Leu, Asn-860-Thr and Thr-845-Ala and IIe-855-Met, identified in this study, need further gene knock out/in studies to better understand their effect on ciprofloxacin resistance.

Assessment of genotypes, endosymbionts and clinical characteristics of Acanthamoeba recovered from ocular infection

IntroductionAcanthamoeba is an emerging pathogen, infamous for its resilience against antiprotozoal compounds, disinfectants and harsh environments. It is known to cause keratitis, a sight-threatening, painful and difficult to treat corneal infection which is often reported among contact lens wearers and patients with ocular trauma. Acanthamoeba comprises over 24 species and currently 23 genotypes (T1-T23) have been identified.AimsThis retrospective study was designed to examine the Acanthamoeba species and genotypes recovered from patients with Acanthamoeba keratitis (AK), determine the presence of endosymbionts in ocular isolates of Acanthamoeba and review the clinical presentations.MethodologyThirteen culture-confirmed AK patients treated in a tertiary eye care facility in Hyderabad, India from February to October 2020 were included in this study. The clinical manifestations, medications and visual outcomes of all patients were obtained from medical records. The Acanthamoeba isolates were identified by sequencing the ribosomal nuclear subunit (rns) gene. Acanthamoeba isolates were assessed for the presence of bacterial or fungal endosymbionts using molecular assays, PCR and fluorescence in situ hybridization (FISH).ResultsThe mean age of the patients was 33 years (SD ± 17.4; 95% CI 22.5 to 43.5 years). Six (46.2%) cases had AK associated risk factors; four patients had ocular trauma and two were contact lens wearers. A. culbertsoni (6/13, 46.2%) was the most common species, followed by A. polyphaga and A. triangularis. Most of the isolates (12/13) belonged to genotype T4 and one was a T12; three sub-clusters T4A, T4B, and T4F were identified within the T4 genotype. There was no significant association between Acanthamoeba types and clinical outcomes. Eight (61.5%) isolates harboured intracellular bacteria and one contained Malassezia restricta. The presence of intracellular microbes was associated with a higher proportion of stromal infiltrates (88.9%, 8/9), epithelial defect (55.6%, 5/9) and hypopyon (55.6%, 5/9) compared to 50% (2/4), 25% (1/4) and 25% (1/4) AK cases without intracellular microbes, respectively.ConclusionsGenotype T4 was the predominant isolate in southern India. This is the second report of T12 genotype identified from AK patient in India, which is rarely reported worldwide. The majority of the Acanthamoeba clinical isolates in this study harboured intracellular microbes, which may impact clinical characteristics of AK.

Kinetics and minimal inhibitory concentrations of ceftiofur in tear film following extended-release parenteral administration (Excede®) in dogs.

PurposeDescribe the pharmacokinetics of extended-release parenteral ceftiofur (Excede®) in canine tear film and compare these concentrations to minimal inhibitory concentrations (MICs) of ceftiofur against common ocular pathogens in dogs.MethodSix dogs of various breeds were enrolled. Disruption of blood-tear barrier was achieved with histamine-induced conjunctivitis to ensure clinical relevance of the results. Each dog received a single subcutaneous injection of 5 mg/kg Excede®, followed by tear collection with Schirmer strips at times 0, 0.25, 0.5, 1, 2, 4, 8, 12, 24, 36, 48, 72, 96, 120, 144, 168, 192, 216 and 240 h. Drug quantification was performed with liquid chromatography-mass spectrometry. MICs were determined for Staphylococcus pseudintermedius, Streptococcus canis and Pseudomonas aeruginosa by assessing bacterial growth (n = 10 per bacterial species) in the presence of ceftiofur at increasing concentrations.ResultsBlood-tear barrier breakdown provided tear film concentrations of ceftiofur 3.2–28.9-fold higher than in the contralateral healthy eye (n = 1 dog, pilot experiment). In all six dogs, ceftiofur concentrations in tears varied from 2.3 to 637.5 ng/mL and were detectable up to 10 days (240 h) after subcutaneous injection. However, tear levels always remained below MICs for common ocular isolates (≥640 ng/mL).ConclusionsCeftiofur reached the tear compartment (for up to 10 days) after a single parenteral injection, however tear concentrations were extremely variable and too low to be effective against common bacterial pathogens in dogs. Further studies with different ceftiofur dosage or other long-acting injectable antibiotics are warranted.

Genomics of Staphylococcus aureus Strains Isolated from Infectious and Non-Infectious Ocular Conditions.

Staphylococcus aureus is a major cause of ocular infectious (corneal infection or microbial keratitis (MK) and conjunctivitis) and non-infectious corneal infiltrative events (niCIE). Despite the significant morbidity associated with these conditions, there is very little data about specific virulence factors associated with the pathogenicity of ocular isolates. A set of 25 S. aureus infectious and niCIEs strains isolated from USA and Australia were selected for whole genome sequencing. Sequence types and clonal complexes of S. aureus strains were identified by using multi-locus sequence type (MLST). The presence or absence of 128 virulence genes was determined by using the virulence finder database (VFDB). Differences between infectious (MK + conjunctivitis) and niCIE isolates from USA and Australia for possession of virulence genes were assessed using the chi-square test. The most common sequence types found among ocular isolates were ST5, ST8 while the clonal complexes were CC30 and CC1. Virulence genes involved in adhesion (ebh, clfA, clfB, cna, sdrD, sdrE), immune evasion (chp, esaD, esaE, esxB, esxC, esxD), and serine protease enzymes (splA, splD, splE, splF) were more commonly observed in infectious strains (MK + conjunctivitis) than niCIE strains (p = 0.004). Toxin genes were present in half of infectious (49%, 25/51) and niCIE (51%, 26/51) strains. USA infectious isolates were significantly more likely to possess splC, yent1, set9, set11, set36, set38, set40, lukF-PV, and lukS-PV (p < 0.05) than Australian infectious isolates. MK USA strains were more likely to possesses yent1, set9, set11 than USA conjunctivitis strains (p = 0.04). Conversely USA conjunctivitis strains were more likely to possess set36 set38, set40, lukF-PV, lukS-PV (p = 0.03) than MK USA strains. The ocular strain set was then compared to 10 fully sequenced non-ocular S. aureus strains to identify differences between ocular and non-ocular isolates. Ocular isolates were significantly more likely to possess cna (p = 0.03), icaR (p = 0.01), sea (p = 0.001), set16 (p = 0.01), and set19 (p = 0.03). In contrast non-ocular isolates were more likely to possess icaD (p = 0.007), lukF-PV, lukS-PV (p = 0.01), selq (p = 0.01), set30 (p = 0.01), set32 (p = 0.02), and set36 (p = 0.02). The clones ST5, ST8, CC30, and CC1 among ocular isolates generally reflect circulating non-ocular pathogenic S. aureus strains. The higher rates of genes in infectious and ocular isolates suggest a potential role of these virulence factors in ocular diseases.

Polymicrobial biofilms of ocular bacteria and fungi on ex vivo human corneas

Microbes residing in biofilms confer several fold higher antimicrobial resistances than their planktonic counterparts. Compared to monomicrobial biofilms, polymicrobial biofilms involving multiple bacteria, multiple fungi or both are more dominant in nature. Paradoxically, polymicrobial biofilms are less studied. In this study, ocular isolates of Staphylococcus aureus, S. epidermidis and Candida albicans, the etiological agents of several ocular infections, were used to demonstrate their potential to form mono- and polymicrobial biofilms both in vitro and on human cadaveric corneas. Quantitative (crystal violet and XTT methods) and qualitative (confocal and scanning electron microscopy) methods demonstrated that they form polymicrobial biofilms. The extent of biofilm formation was dependent on whether bacteria and fungi were incubated simultaneously or added to a preformed biofilm. Additionally, the polymicrobial biofilms exhibited increased resistance to different antimicrobials compared to planktonic cells. When the MBECs of different antibacterial and antifungal agents were monitored it was observed that the MBECs in the polymicrobial biofilms was either identical or decreased compared to the monomicrobial biofilms. The results are relevant in planning treatment strategies for the eye. This study demonstrates that ocular bacteria and fungi form polymicrobial biofilms and exhibit increase in antimicrobial resistance compared to the planktonic cells.

Detection of Wzm gene in staphylococcus aureus ocular isolates to find out about biofilm formation and its link to antibiotic resistance

Objectives:This study's purpose is to find out what detection of Wzm gene by PCR-based investigation in staphylococcus aureusisolates isolated from patients with eye infectionsto identify biofilm formation and thecorrelation with antibiotic resistance. Methods and Materials: 160 eye swabs specimenswere collected from iraq hospitals, including Ibn Al-Haitham Teaching Eye Hospital inBaghdad, Al-Imam Al-Sadiq Hospital andHilla Teaching Hospital in Babylon.Distributedaccording to types of ocular infections 119Conjunctivitis32Blepharitis (Eyelids Infection)7 Keratitis 2Dacryocystitis. These samples were collected between November 2021 and March 2022. S. aureus isolates were identified by inoculated on manitol salt agar, blood agar for primary screening of Staphylococcus spp. and then confirmed by biochemical tests and Vitek2 Compact System, and by specific primer for 16S rDNA gene of Staphylococcus spp.CLSI-2021were used to investigate bacterial antibiotic susceptibility testing, microtiter plates andWzm gene for biofilm production. Results:Among theculture-positive growth consist of 57(41.007%) bacterial isolates of staphylococcus aureusand all (57) isolates showedthe percentage of antibioticresistant (100%) to Cefoxitin, (96.49%)toPencillin G, (94.73%) to Oxacillin while the lowest resistant percentages were found with Nitrofurantoin (1.75%). PCR has been used to investigate Wzm gene for biofilm production instaphylococcus aureus and the results showed that 39(68.42%) has Wzm gene.