Background: Fungal keratitis (FK) is a serious pathogenic condition usually associated with significant ocular morbidity. Natamycin (NAT) is the first-line and only medication approved by the Food and Drug Administration for the treatment of FK. However, NAT suffers from poor corneal penetration, which limits its efficacy for treating deep keratitis. Purpose: The objective of this work was to prepare NAT solid lipid nanoparticles (NAT-SLNs) to achieve sustained drug release and increased corneal penetration. Methods: NAT-SLNs were prepared using the emulsification-ultrasonication technique. Box–Behnken experimental design was applied to optimize the effects of independent processing variables (lipid concentration [X1], surfactant concentration [X2], and sonication frequency [X3]) on particle size (R1), zeta potential (ZP; R2), and drug entrapment efficiency (EE%) (R3) as responses. Drug release profile, ex vivo corneal permeation, antifungal susceptibility, and cytotoxicity of the optimized formula were evaluated. Results: The optimized formula had a mean particle size of 42 r.nm (radius in nanometers), ZP of 26 mV, and EE% reached ~85%. NAT-SLNs showed an extended drug release profile of 10 hours, with enhanced corneal permeation in which the apparent permeability coefficient (Papp) and steady-state flux (Jss) reached 11.59×10-2 cm h-1 and 3.94 mol h-1, respectively, in comparison with 7.28×10-2 cm h-1 and 2.48 mol h-1 for the unformulated drug, respectively. Antifungal activity was significantly improved, as indicated by increases in the inhibition zone of 8 and 6 mm against Aspergillus fumigatus ATCC 1022 and a Candida albicans clinical isolate, respectively, and minimum inhibitory concentration values that were decreased 2.5-times against both of these pathogenic strains. NAT-SLNs were found to be non-irritating to corneal tissue. NAT-SLNs had a prolonged drug release rate, that improved corneal penetration, and increased antifungal activity without cytotoxic effects on corneal tissues. Conclusion: Thus, NAT-SLNs represent a promising ocular delivery system for treatment of deep corneal keratitis.

Inappropriate use of antibiotics in clinical settings is thought to have led to the global emergence and spread of multidrug-resistant pathogens. The goal of this study was to investigate the prevalence of genes encoding aminoglycoside resistance and plasmid-mediated quinolone resistance among clinical isolates of Klebsiella pneumoniae. All K. pneumoniae isolates were phenotypically identified using API 20E and then confirmed genotypically through amplification of the specific K. pneumoniae phoE gene. All isolates were genotyped by the enterobacterial repetitive intergenic consensus polymerase chain reaction technique (ERIC-PCR). Antibiotic susceptibility testing was done by a modified Kirby-Bauer method and broth microdilution. All resistant or intermediate-resistant isolates to either gentamicin or amikacin were screened for 7 different genes encoding aminoglycoside-modifying enzymes (AMEs). In addition, all resistant or intermediate-resistant isolates to either ciprofloxacin or levofloxacin were screened for 5 genes encoding the quinolone resistance protein (Qnr), 1 gene encoding quinolone-modifying enzyme, and 3 genes encoding quinolone efflux pumps. Biotyping using API 20E revealed 13 different biotypes. Genotyping demonstrated that all isolates were related to 2 main phylogenetic groups. Susceptibility testing revealed that carbapenems and tigecycline were the most effective agents. Investigation of genes encoding AMEs revealed that acc(6′)-Ib was the most prevalent, followed by acc(3′)-II, aph(3′)-IV, and ant(3′′)-I. Examination of genes encoding Qnr proteins demonstrated that qnrB was the most prevalent, followed by qnrS, qnrD, and qnrC. It was found that 61%, 26%, and 12% of quinolone-resistant K. pneumoniae isolates harbored acc(6′)-Ib-cr, oqxAB, and qebA, respectively. The current study demonstrated a high prevalence of aminoglycoside and quinolone resistance genes among clinical isolates of K. pneumoniae.

Background and Objective: The emergence of carbapenem-resistant K. pneumoniae (CRKP) continues to escalate and is alarming because of the emergence of pan drug-resistant strains. The objective of this study was to investigate the existence of 12 carbapenemase genes among CRKP clinical isolates. Methods: Ninety-six Klebsiella spp. clinical isolates were collected. The isolates were identified phenotypically and genotypically. These isolates were screened for susceptibility to 24 different antibiotics. The modified Hodge test (MHT) and the Carba Nordmann/Poirel (NP) test were used to phenotypically screen carbapenem-resistant strains for carbapenemase production. Phenotypic characterization of carbapenemases was performed using the combined disk synergy test (CDST). Additionally, the presence of 12 carbapenemase genes in CRKP isolates was investigated. The DNA sequence of bla_NDM and bla_GES genes was determined. The BOX-PCR technique was used to determine the clonal relationship between CRKP isolates. Results: All carbapenem-resistant isolates were related to K. pneumoniae. Susceptibility testing showed that 19.79% (19/96) of the collected isolates were carbapenem-resistant. Of the CRKP isolates, 68.42% (13/19) tested positive for the MHT and Carba NP test. CDST showed that 42.11% (8/19), 63.16% (12/19), 47.37% (9/19), and 73.68% (14/19) of the CRKP isolates tested positive for the inhibitory effect of clavulanic acid, sulbactam, phenylboronic acid, and tazobactam, respectively, while 84.21% (16/19) and 68.42% (13/16) tested positive for the inhibitory effect of EDTA and mercaptopropionic acid, respectively. It was found that 10.53% (2/19) of the isolates tested positive for the inhibitory effect of sodium chloride. Molecular investigation of carbapenemases showed that 26.32% (5/19), 73.68% (14/19), 21.05% (4/19), 10.53% (2/19), and 5.26% (1/19) of the isolates tested positive for bla_NDM, bla_OXA-48, bla_OXA-181, bla_OXA-51, and bla_OXA-23, respectively. None of the isolates tested positive for bla_OXA-40 and bla_OXA-58. Two allelic variants of bla_NDM (bla_NDM-1 and bla_NDM-25) were detected. BOX-PCR revealed high clonal relatedness between CRKP isolates. Conclusion: MHT was more sensitive than Carba NP test for evaluating carbapenemase production and class D carbapenemase genes were the most prevalent of the 12 carbapenemase genes that were evaluated.

Background: Quinolones are among the most effective antibiotics against Pseudomonas spp. Several chromosomal and/or plasmid-mediated quinolone-resistance mechanisms have been found in Pseudomonas. Plasmid-mediated quinolone-resistance (PMQR) is mediated by quinolone-resistance (QNR) proteins, modifying enzymes or efflux pumps. Only a few previous studies examined the prevalence of quinolone-resistance in the Kingdom of Saudi Arabia (KSA) and showed it is increasing. Mechanisms of quinolone-resistance among Pseudomonas spp. in the KSA; examined herein; have not been extensively studied. Methods: Ninety-two Pseudomonas isolates were collected and their resistance to seven different types of quinolones was determined by the microbroth dilution method. PMQR mechanisms were examined using a PCR screen to identify six PMQR genes including qnrA, qnrB, qnrD, qnrS, aac(6´)-Ib-cr, and qepA. Clonal relatedness of the quinolone-resistant isolates was determined by ERIC-PCR. Results: Of the isolates, 42.4% (39/92) were resistant to 1-7 of the tested quinolones. Gemifloxacin resistance was the lowest (28.3%) while resistance to the other six quinolones were ≥ 35%. The most common biotype among the 39 quinolone-resistant isolates was resistance to the seven tested quinolones (26/39; 66.7%). qnrD, qnrS, and aac(6´)-Ib-cr were found in 31 (79.5%), 31 (79.5%) and 28 (71.8%) of the 39 isolates, respectively, and all three genes together were found in 22 of the 39 isolates (56.4%). qnrA, qnrB, and qepA were not detected in any of the isolates and two isolates did not harbor any of the six tested genes. The isolates showed 38 different ERIC profiles and only two isolates (Pa16 and Pa17) had an identical profile. Conclusion: This is the first description of PMQR mechanisms among clinical Pseudomonas isolates from the KSA, which appears to be mainly mediated by qnrD, qnrS, and aac(6´)-Ib-cr.

The extensive use of β-lactam antibiotics has led to emergence and spread of extended-spectrum β-lactamases (ESBLs). This study was conducted to investigate the prevalence of 7 different ESBL genes ( bla _TEM , bla _SHV , bla _CTX-M , bla _VEB , bla _PER , bla _GES , and bla _OXA-10 ) and O25b-ST131 high-risk clone among 61 clinical isolates of Escherichia coli . Also, one broad-spectrum β-lactamase ( bla _OXA-1 ) was investigated. This study was also constructed to evaluate iodometric overlay method in detection of ESBL production. Phenotypic identification of E. coli isolates using API 20E revealed 18 distinct biotypes. DNA fingerprinting using enterobacterial repetitive intergenic consensus polymerase chain reaction (ERIC-PCR) differentiated all isolates into 2 main phylogenetic groups with 60 distinct genetic profiles. Elevated values of minimal inhibitory concentration (MIC) ₅₀ and MIC ₉₀ for third- and fourth-generation cephalosporins were observed. Phenotypic tests revealed that 85.24% of isolates were ESBL producers. The incidence rates of bla _TEM , bla _SHV , bla _CTX-M , bla _GES , bla _OXA-1 , and bla _OXA-10 among E. coli ESBL producer phenotype were 69.23%, 25%, 96.15%, 3.85%, 11.54%, and 48%, respectively. On the other hand, bla _VEB and bla _PER were not detected. Sequencing of bla _TEM and bla _SHV revealed that bla _TEM-214 and bla _SHV-11 were the most prevalent variants. Group characterization of bla _CTX-M revealed that bla _CTX-M-1 was the most prevalent group of bla _CTX-M family. It was found that 30.77% of E. coli ESBL producers belonged to O25b-ST131 clone harboring bla _CTX-M-15 . This study concluded that iodometric overlay method was 100% sensitive in detection of ESBL production. To our knowledge, this is the first Egyptian study that declares the emergence of E. coli O25b-ST131 harboring bla _GES .

Background and objective: Acinetobacter baumannii (A. baumannii) is a common nosocomial pathogen, which developed multi-drug-resistance to different classes of antibiotics including carbapenems. This study examined ten common carbapenemase genes among 32 carbapenem-resistant A. baumannii clinical isolates recovered from Taif, Saudi Arabia. Methods: Isolates were phenotypically identified to the genus level by Vitek^®2 and API 20NE^®. The species level was confirmed by the amplification of bla_OXA-51. The susceptibility for 21 different antibiotics was performed by Vitek 2 and modified Kirby-Bauer method. Isolates were genetically screened for 10 carbapenemases. Phylogenetic relatedness between isolates was determined by ERIC-PCR. Results: Genotypically identified A. baumannii represented 100% of the total phenotypically identified Acinetobacter spp. All the carbapenem-resistant isolates were sensitive to polymyxin B and colistin. Among the other antibiotics, ampicillin/sulbactam and tigecycline were the most effective agents. 90.8% of the isolates were resistant to all ten investigated β–lactams. bla_OXA-51, bla_IPM, bla_NDM and bla_OXA-23 were detected in 100%, 87.5%, 62.5% and 59.4% of isolates, respectively. Also, bla_VIM and bla_OXA-40 were less prevalent and were detected in 9.3% and 3.1% of the isolates, respectively. In addition, bla_KPC, bla_OXA-48, bla_OXA-58, bla_OXA-181 were not detected in any isolate. The A. baumannii isolates were categorised into ten genotypes on the basis of the detected carbapenemase genes and ERIC-PCR revealed a remarkable clonal diversity among these isolates. Conclusion: Class A and class D carbapenemase genes were the most commonly detected among carbapenem resistant A. baumannii (CRAB) clinical isolates.