A comparison of antimicrobial regimen outcomes and antibiogram development in microbial keratitis: a prospective cohort study in Alexandria, Egypt.

INTRODUCTION: Antimicrobial resistance in microbial keratitis has not been previously explored in Alexandria. We aim to recommend effective therapies through identification of etiological agents, determination of antimicrobial susceptibilities, and comparing outcomes of empiric topical antimicrobials. METHODS: In this 2022 prospective cohort conducted in Alexandria Main University Hospital cornea clinic, antimicrobial susceptibilities of isolated microorganisms from corneal scrapings were detected and antibiograms were developed. Bacterial (BK), fungal (FK), or mixed fungal/bacterial keratitis (MFBK) patients on empiric regimens were compared for ulcer healing, time-to-epithelialization, best-corrected visual acuity, interventions, and complications. RESULTS: The prevalent microorganisms in 93 positive-cultures were coagulase-negative staphylococci (CoNS, 30.1%), Pseudomonas aeruginosa (14%), and Aspergillus spp. (12.9%). CoNS were susceptible to vancomycin (VAN, 100%) and moxifloxacin (MOX, 90.9%). Gram-negative bacteria showed more susceptibility to gatifloxacin (90.9%) than MOX (57.1%), and to gentamicin (GEN, 44.4%) than ceftazidime (CAZ, 11.8%). Methicillin-resistance reached 23.9% among Gram-positive bacteria. Fungi exhibited 10% resistance to voriconazole (VRC). Percentages of healed ulcers in 49 BK patients using GEN + VAN, CAZ + VAN and MOX were 85.7%, 44.4%, and 64.5%, respectively (p = 0.259). Their median time-to-epithelialization reached 21, 30, and 30 days, respectively (log-rank p = 0.020). In 51 FK patients, more ulcers (88.9%) healed with natamycin (NT) + VRC combination compared to VRC (39.1%) or NT (52.6%) (p = 0.036). Their median time-to-epithelialization was 65, 60, and 22 days, respectively (log-rank p < 0.001). The VRC group required more interventions (60.9%) than NT + VRC-treated group (11.1%) (p = 0.018). In 23 MFBK patients, none healed using NT + CAZ + VAN, while 50% healed using VRC + CAZ + VAN (p = 0.052). Regimens had comparable visual outcomes and complications. CONCLUSION: Based on the higher detected susceptibility, we recommend empiric MOX in suspected Gram-positive BK, gatifloxacin in Gram-negative BK, and GEN + VAN in severe BK. Due to better outcomes, we recommend NT + VRC in severe FK. TRIAL REGISTRATION: ClinicalTrials.gov identifier, NCT05655689. Registered December 19, 2022- Retrospectively registered, https://clinicaltrials.gov/ct2/show/NCT05655689?cond=NCT05655689.&draw=2&rank=1.

Repurposing carvacrol, cinnamaldehyde, and eugenol as potential anti-quorum sensing agents against uropathogenic Escherichia coli isolates in Alexandria, Egypt.

BACKGROUND: Urinary tract infections represent one of the most frequent hospital and community-acquired infections with uropathogenic Escherichia coli (UPEC) being the main causative agent. The global increase in the emergence of multidrug-resistant (MDR) UPEC necessitates exploring novel approaches. Repurposing natural products as anti-quorum sensing (QS) agents to impede bacterial virulence is gaining momentum nowadays. Hence, this study investigates the anti-QS potentials of carvacrol, cinnamaldehyde, and eugenol against E. coli isolated from urine cultures of Egyptian patients. RESULTS: Antibiotic susceptibility testing was performed for 67 E. coli isolates and 94% of the isolates showed MDR phenotype. The usp gene was detected using PCR and accordingly, 45% of the isolates were categorized as UPEC. Phytochemicals, at their sub-inhibitory concentrations, inhibited the swimming and twitching motilities of UPEC isolates, with eugenol showing the highest inhibitory effect. The agents hindered the biofilm-forming ability of the tested isolates, at two temperature sets, 37 and 30 °C, where eugenol succeeded in significantly inhibiting the biofilm formation by > 50% at both investigated temperatures, as compared with untreated controls. The phytochemicals were shown to downregulate the expression of the QS gene (luxS) and critical genes related to motility, asserting their anti-QS potential. Further, the combinatory activity of the phytoproducts with five antibiotics was assessed by checkerboard assay. The addition of the phytoproducts significantly reduced the minimum inhibitory concentrations of the antibiotics and generated several synergistic or partially synergistic combinations, some of which have not been previously explored. CONCLUSIONS: Overall, carvacrol, cinnamaldehyde, and eugenol could be repurposed as potential anti-QS agents, which preferentially reduce the QS-based communication and attenuate the cascades of gene expression, thus decreasing the production of virulence factors in UPEC, and eventually, subsiding their pathogenicity. Furthermore, the synergistic combinations of these agents with antibiotics might provide a new perspective to circumvent the side effects brought about by high antibiotic doses, thereby paving the way for overcoming antibiotic resistance.

Genomic characterization of colistin-resistant Klebsiella pneumoniae isolated from intensive care unit patients in Egypt.

BACKGROUND: Egypt has witnessed elevated incidence rates of multidrug-resistant Klebsiella pneumoniae infections in intensive care units (ICUs). The treatment of these infections is becoming more challenging whilst colistin-carbapenem-resistant K. pneumoniae is upsurging. Due to the insufficiently available data on the genomic features of colistin-resistant K. pneumoniae in Egypt, it was important to fill in the gap and explore the genomic characteristics, as well as the antimicrobial resistance, the virulence determinants, and the molecular mechanisms of colistin resistance in such a lethal pathogen. METHODS: Seventeen colistin-resistant clinical K. pneumoniae isolates were collected from ICUs in Alexandria, Egypt in a 6-month period in 2020. Colistin resistance was phenotypically detected by modified rapid polymyxin Nordmann/Poirel and broth microdilution techniques. The isolates susceptibility to 20 antimicrobials was determined using Kirby-Bauer disk diffusion method. Whole genome sequencing and bioinformatic analysis were employed for exploring the virulome, resistome, and the genetic basis of colistin resistance mechanisms. RESULTS: Out of the tested K. pneumoniae isolates, 82.35% were extensively drug-resistant and 17.65% were multidrug-resistant. Promising susceptibility levels towards tigecycline (88.24%) and doxycycline (52.94%) were detected. Population structure analysis revealed seven sequence types (ST) and K-types: ST383-K30, ST147-K64, ST17-K25, ST111-K63, ST11-K15, ST14-K2, and ST525-K45. Virulome analysis revealed yersiniabactin, aerobactin, and salmochelin siderophore systems in ˃ 50% of the population. Hypervirulence biomarkers, iucA (52.94%) and rmpA/A2 (5.88%) were detected. Extended-spectrum ß-lactamase- and carbapenemase-producers accounted for 94.12% of the population, with blaCTX-M-15, blaNDM-5, and blaOXA-48 reaching 64.71%, 82.35%, and 82.35%, respectively. Chromosomal alterations in mgrB (82.35%) were the most prevailing colistin resistance-associated genetic change followed by deleterious mutations in ArnT (23.53%, L54H and G164S), PmrA (11.76%, G53V and D86E), PmrB (11.76%, T89P and T134P), PmrC (11.76%, S257L), PhoQ (5.88%, L322Q and Q435H), and ArnB (5.88%, G47D) along with the acquisition of mcr-1.1 by a single isolate of ST525. CONCLUSIONS: In this study, we present the genotypic colistin resistance mechanisms in K. pneumoniae isolated in Egypt. More effective antibiotic stewardship protocols must be implemented by Egyptian health authorities to restrain this hazard and safeguard the future utility of colistin. This is the first characterization of a complete sequence of mcr-1.1-bearing IncHI2/IncHI2A plasmid recovered from K. pneumoniae clinical isolate belonging to the emerging high-risk clone ST525.

Pathogenicity Islands in Uropathogenic Escherichia coli Clinical Isolate of the Globally Disseminated O25:H4-ST131 Pandemic Clonal Lineage: First Report from Egypt.

Uropathogenic Escherichia coli (UPEC) is the main etiological agent of urinary tract infections (UTIs). The pathogenesis of UTIs relies upon UPEC's acquisition of virulence determinants that are commonly inserted into large chromosomal blocks which are termed 'pathogenicity islands' (PAIs). In this study, we investigated the virulence-associated genes embedded in the chromosome of a UPEC Egyptian strain, EC14142. Additionally, we present a detailed characterization of the PAIs in the EGY_EC14142 chromosome. The isolate displayed a multidrug-resistant phenotype, and whole genome sequencing indicated that it belonged to the globally disseminated O25:H4-ST131 pandemic lineage and the H30-Rx clade. EGY_EC14142 carried genes that are responsible for resistance to aminoglycosides, fluoroquinolones, extended-spectrum ß-lactams, macrolides, folate pathway antagonists, and tetracyclines. It encoded five PAIs with a high similarity to PAI II536, PAI IV536, PAI V536, PAI-536-icd, and PAIusp. The genome analysis of EGY_EC14142 with other closely related UPEC strains revealed that they have a high nucleotide sequence identity. The constructed maximum-likelihood phylogenetic tree showed the close clonality of EGY_EC14142 with the previously published ST131 UPEC international isolates, thus endorsing the broad geographical distribution of this clone. This is the first report characterizing PAIs in a UPEC Egyptian strain belonging to the globally disseminated pandemic clone O25:H4-ST131.

Genomic Characterization of International High-Risk Clone ST410 Escherichia coli Co-Harboring ESBL-Encoding Genes and blaNDM-5 on IncFIA/IncFIB/IncFII/IncQ1 Multireplicon Plasmid and Carrying a Chromosome-Borne blaCMY-2 from Egypt.

The accelerated dispersion of multidrug-resistant (MDR) Escherichia coli due to the production of extended-spectrum ß-lactamases (ESBLs) or AmpC enzymes has been noted in Egypt, presenting a serious treatment challenge. In this study, we investigate the prevalence of ESBLs and AmpC enzymes among 48 E. coli isolates collected from patients with urinary tract infections admitted to a teaching hospital in Alexandria. Phenotypic and genotypic methods of detection are conducted. Isolates producing both enzymes are tested for the mobilization of their genes by a broth mating experiment. Whole genome sequencing (WGS) is performed for isolate EC13655. The results indicate that 80% of the isolates are MDR, among which 52% and 13% were ESBL and AmpC producers, respectively. Conjugation experiments fail to show the mobilization of blaCMY-2 in EC13655, which was chosen for WGS. In silico analysis reveals that the isolate belongs to a ST410-H24Rx high-risk clone. It coharbors the ESBL-encoding genes blaCTX-M-15, blaTEM-1, blaOXA-1 and blaNDM-5 on an IncFIA/IncFIB/IncFII/IncQ1 multireplicon plasmid. The chromosomal location of blaCMY-2 is detected with a flanking upstream copy of ISEcp1. This chromosomal integration of blaCMY-2 establishes the stable maintenance of the gene and thus, necessitates an imperative local surveillance to reduce further spread of such strains in different clinical settings.

Whole Genome Characterization of the High-Risk Clone ST383 Klebsiella pneumoniae with a Simultaneous Carriage of blaCTX-M-14 on IncL/M Plasmid and blaCTX-M-15 on Convergent IncHI1B/IncFIB Plasmid from Egypt.

Recently, Egypt has witnessed the emergence of multidrug-resistant (MDR) Klebsiella pneumoniae, which has posed a serious healthcare challenge. The accelerated dissemination of blaCTX-M genes among these MDR K. pneumoniae, particularly blaCTX-M-14 and blaCTX-M-15, have been noted. In this study, we investigated the occurrence of blaCTX-M-IV among K. pneumoniae recovered from the laboratory of a major hospital in Alexandria. The 23 tested isolates showed an MDR phenotype and the blaCTX-M-IV gene was detected in ≈22% of the isolates. The transformation of plasmids harboring blaCTX-M-IV to chemically competent cells of Escherichia coli DH5α was successful in three out of five of the tested blaCTX-M-IV-positive isolates. Whole genome sequencing of K22 indicated that the isolate belonged to the high-risk clone ST383, showing a simultaneous carriage of blaCTX-M-14 on IncL/M plasmid, i.e., pEGY22_CTX-M-14, and blaCTX-M-15 on a hybrid IncHI1B/IncFIB plasmid, pEGY22_CTX-M-15. Alignment of both plasmids revealed high similarity with those originating in the UK, Germany, Australia, Russia, China, Saudi Arabia, and Morocco. pEGY22_CTX-M-15 was a mosaic plasmid that demonstrated convergence of MDR and virulence genes. The emergence of such a plasmid with enhanced genetic plasticity constitutes the perfect path for the evolution of K. pneumoniae isolates causing invasive untreatable infections especially in a country with a high burden of infectious diseases such as Egypt. Therefore there is an imperative need for countrywide surveillances to monitor the prevalence of these superbugs with limited therapeutic options.

Genomic Insights into a Colistin-Resistant Uropathogenic Escherichia coli Strain of O23:H4-ST641 Lineage Harboring mcr-1.1 on a Conjugative IncHI2 Plasmid from Egypt.

The reintroduction of colistin, a last-resort antibiotic for multidrug-resistant pathogens, resulted in the global spread of plasmid-mediated mobile colistin resistance (mcr) genes. Our study investigated the occurrence of colistin resistance among Escherichia coli isolated from patients with urinary tract infections admitted to a teaching hospital in Egypt. Out of 67 isolates, three isolates were colistin-resistant, having a minimum inhibitory concentration of 4 µg/mL and possessing the mcr-1 gene. A double mechanism of colistin resistance was detected; production of mcr-1 along with amino acid substitution in PmrB (E123D and Y358N) and PmrA (G144S). Broth mating experiments inferred that mcr-1 was positioned on conjugative plasmids. Whole-genome sequencing of EC13049 indicated that the isolate belonged to O23:H4-ST641 lineage and to phylogroup D. The mcr-1-bearing plasmid corresponded to IncHI2 type with a notable similarity to other E. coli plasmids previously recovered from Egypt. The unbanned use of colistin in the Egyptian agriculture sector might have created a potential reservoir for the mcr-1 gene in food-producing animals that spread to humans. More proactive regulations must be implemented to prevent further dissemination of this resistance. This is the first characterization of mcr-1-carrying IncHI2:ST4 plasmid recovered from E. coli of a clinical source in Egypt.

Resensitization of Fluconazole-Resistant Urinary Candida spp. Isolates by Amikacin through Downregulation of Efflux Pump Genes.

The contribution of fluconazole-resistant Candida spp. isolates to urinary tract infections in Egypt has become a nationwide problem. A recent approach to overcome such disaster is combining conventional antifungals with non-antifungals. This study investigated the interaction of amikacin with fluconazole against resistant Candida strains isolated from the urine culture of patients admitted to Alexandria Main University Hospital. Among the collected Candida spp. isolates, 42.9% were resistant to fluconazole with MICs ranging between 128 and 1,024 µg/ml. The resistance-modifying activity of amikacin (4,000 µg/ml) was studied against fluconazole-resistant isolates where amikacin sensitized 91.7 % of resistant Candida spp. isolates to fluconazole with a modulation factor ranging between 32 and 256. The rhodamine efflux assay was performed to examine the impact of amikacin on efflux pump activity. After 120 minutes of treatment, amikacin affected the efflux pump activity of the isolates tested with a percentage of reduction in the fluorescence intensity of 8.9%. Quantitative real-time PCR was applied to assess the amikacin effect on the expression of the efflux pump genes MDR1, CDR1, and CDR2. The downregulatory effect of amikacin on the expression of the studied genes caused a percentage of reduction in the expression level ranging between 42.1 and 94%. In conclusion, amikacin resensitized resistant Candida spp. isolates to fluconazole and could be used in combination in the management of candiduria with a higher efficiency or at lower administration doses. To the best of our knowledge, this is the first study evaluating the enhancement of fluconazole activity in combination with amikacin against Candida spp.The contribution of fluconazole-resistant Candida spp. isolates to urinary tract infections in Egypt has become a nationwide problem. A recent approach to overcome such disaster is combining conventional antifungals with non-antifungals. This study investigated the interaction of amikacin with fluconazole against resistant Candida strains isolated from the urine culture of patients admitted to Alexandria Main University Hospital. Among the collected Candida spp. isolates, 42.9% were resistant to fluconazole with MICs ranging between 128 and 1,024 µg/ml. The resistance-modifying activity of amikacin (4,000 µg/ml) was studied against fluconazole-resistant isolates where amikacin sensitized 91.7 % of resistant Candida spp. isolates to fluconazole with a modulation factor ranging between 32 and 256. The rhodamine efflux assay was performed to examine the impact of amikacin on efflux pump activity. After 120 minutes of treatment, amikacin affected the efflux pump activity of the isolates tested with a percentage of reduction in the fluorescence intensity of 8.9%. Quantitative real-time PCR was applied to assess the amikacin effect on the expression of the efflux pump genes MDR1, CDR1, and CDR2. The downregulatory effect of amikacin on the expression of the studied genes caused a percentage of reduction in the expression level ranging between 42.1 and 94%. In conclusion, amikacin resensitized resistant Candida spp. isolates to fluconazole and could be used in combination in the management of candiduria with a higher efficiency or at lower administration doses. To the best of our knowledge, this is the first study evaluating the enhancement of fluconazole activity in combination with amikacin against Candida spp.

In Vitro and In Vivo Activity of Zabofloxacin and Other Fluoroquinolones Against MRSA Isolates from A University Hospital in Egypt.

The widespread of infections caused by methicillin-resistant Staphylococcus aureus (MRSA), has necessitated the search for alternative therapies; introduction of new agents being a suggestion. This study compares the in vitro and in vivo activities of zabofloxacin, a novel fluoroquinolone, with moxifloxacin, levofloxacin and ciprofloxacin against clinical isolates of MRSA from patients hospitalized in the Alexandria Main University hospital; a tertiary hospital in Alexandria, Egypt, where zabofloxacin has not been yet introduced. The strains tested showed the highest percentage of susceptibility to zabofloxacin (61.2%) among the tested fluoroquinolones with the most effective MIC50 and MIC90 (0.25 and 2 µg/ml, respectively). Time-kill curve analysis revealed a rapid bactericidal activity of zabofloxacin after 6 h of incubation with a quinolone-resistant isolate and complete killing when tested against a quinolone-sensitive isolate with inhibition of regrowth in both cases. PCR amplification and sequencing of QRDRs in selected strains revealed the following amino acid substitutions: Ser-84→Leu in GyrA, Ser-80→Phe in GrlA and Pro-451→Ser in GrlB. The in vivo studies demonstrated that zabofloxacin possessed the most potent protective effect against systemic infection in mice (ED50: 29.05 mg/kg) with lowest count in the dissected lungs (3.66 log10 CFU/ml). The histopathological examination of lung specimens of mice treated with zabofloxacin displayed least congestion, inflammation, oedema and necrosis with clear alveolar spaces and normal vessels. In conclusion, zabofloxacin was proved to possess high in vitro and in vivo efficacy encompassing its comparators and could be considered as a possible candidate for the treatment of infections caused by MRSA. To our knowledge, this is the first study evaluating the in vitro and in vivo activity of zabofloxacin against Egyptian MRSA clinical isolates.The widespread of infections caused by methicillin-resistant Staphylococcus aureus (MRSA), has necessitated the search for alternative therapies; introduction of new agents being a suggestion. This study compares the in vitro and in vivo activities of zabofloxacin, a novel fluoroquinolone, with moxifloxacin, levofloxacin and ciprofloxacin against clinical isolates of MRSA from patients hospitalized in the Alexandria Main University hospital; a tertiary hospital in Alexandria, Egypt, where zabofloxacin has not been yet introduced. The strains tested showed the highest percentage of susceptibility to zabofloxacin (61.2%) among the tested fluoroquinolones with the most effective MIC50 and MIC90 (0.25 and 2 µg/ml, respectively). Time-kill curve analysis revealed a rapid bactericidal activity of zabofloxacin after 6 h of incubation with a quinolone-resistant isolate and complete killing when tested against a quinolone-sensitive isolate with inhibition of regrowth in both cases. PCR amplification and sequencing of QRDRs in selected strains revealed the following amino acid substitutions: Ser-84→Leu in GyrA, Ser-80→Phe in GrlA and Pro-451→Ser in GrlB. The in vivo studies demonstrated that zabofloxacin possessed the most potent protective effect against systemic infection in mice (ED50: 29.05 mg/kg) with lowest count in the dissected lungs (3.66 log10 CFU/ml). The histopathological examination of lung specimens of mice treated with zabofloxacin displayed least congestion, inflammation, oedema and necrosis with clear alveolar spaces and normal vessels. In conclusion, zabofloxacin was proved to possess high in vitro and in vivo efficacy encompassing its comparators and could be considered as a possible candidate for the treatment of infections caused by MRSA. To our knowledge, this is the first study evaluating the in vitro and in vivo activity of zabofloxacin against Egyptian MRSA clinical isolates.

In vitro activity of tigecycline and comparators against gram-negative bacteria isolated from a tertiary hospital in Alexandria, Egypt.

The emergence of infections caused by multidrug-resistant Gram-negative bacteria, in particular Pseudomonas aeruginosa, Acinetobacter baumannii, and Klebsiella pneumoniae, has necessitated the search for alternative therapy by either introducing new agents or renewing interest in old agents. This study compares the in vitro activity of tigecycline (TIG), recently introduced to Egyptian market, to other potentially active antimicrobials as Colistin (COL), imipenem (IPM), levofloxacin (LEV), and piperacillin/tazobactam (PIP/TAZ) against 67 Gram-negative clinical isolates obtained from El- Meery Hospital in Alexandria, Egypt. El-Meery Hospital is a 1,500-bed tertiary teaching hospital where TIG has not been previously used. Based on MIC(90)s, TIG was found to be a comparator to IPM and COL (MIC(90)= 8 µg/ml). LEV and PIP/TAZ were less active than TIG exhibiting high MIC(90)s. TIG inhibited 100% of Escherichia coli and K. pneumoniae and 60% of Ps. aeruginosa and A. baumannii isolates. In time-kill studies against IPM-resistant isolates, TIG showed bactericidal activity after 6 hours of contact against the Enterobacteriaceae isolates and after 3 hours for the tested Ps. aeruginosa isolates at 4× and 8× MIC. Against A. baumannii, TIG exerted a bacteriostatic effect. TIG demonstrated variable ability to suppress biofilm formation affecting mainly E. coli and A. baumannii isolates. These results point TIG to be a promising agent in treatment of infections caused by strains for which adequate therapy has been limited. As far as we know, this is the first report evaluating the in vitro activity of TIG against Egyptian clinical isolates.