Characterization of probiotics isolated from dietary supplements and evaluation of metabiotic-antibiotic combinations as promising therapeutic options against antibiotic-resistant pathogens using time-kill assay.

BACKGROUND: The global probiotics dietary supplements market size is continuously growing. To overcome probiotics' health concerns, metabiotics are recognized as a safer alternative. Aiming to deal with the escalating antimicrobial resistance, the current work demonstrates synergistic metabiotic-antibiotic combinations against antibiotic-resistant pathogens. METHODS: The probiotic properties of lactic acid bacteria (LAB) strains isolated from 3 commercial dietary supplements were characterized in vitro. The combinations of the cell-free supernatants (CFS) of selected probiotic strains and conventional antibiotics against Staphylococcus aureus and Escherichia coli clinical isolates were evaluated using the time-kill assay. To our knowledge, the current literature lacks sufficient time-kill assay studies revealing the kinetics of such metabiotic-antibiotic combinations against S. aureus and E. coli. RESULTS: Four LAB strains isolated from dietary supplements as well as two reference strains were included in this study. The isolated LAB strains were identified by MALDI-TOF mass spectrometry as follows: P2: Lactobacillus acidophilus, P3: Lactiplantibacillus plantarum, P4: Lacticaseibacillus rhamnosus, and P5: Pediococcus acidilactici. The identification matched with that annotated by the manufacturers, except for P3. The tested strains could resist the acidic environment at pH 3. Excluding P2, the examined strains showed less than 1 log reduction in survivors upon the addition of reconstituted skimmed milk to pepsin at pH 2 and displayed an acceptable tolerance to 0.3% ox-bile. All the strains tolerated pancreatin. The hydrophobicity and autoaggregation capacities ranged between 7-92% and 36-66%, respectively. P2 was excluded owing to its inferior probiotic potential. Although the remaining strains showed excellent growth at 0.2% phenol, their growth was reduced at higher concentrations. L. plantarum and P. acidilactici strains possessed bile salt hydrolysis activity. The time-kill assay revealed promising synergistic activities of the combinations of CFS of L. rhamnosus P4 with either ceftazidime or gentamicin against E. coli and with only ceftazidime against S. aureus, as well as CFS of P. acidilactici P5 and ceftazidime against S. aureus. CONCLUSIONS: Strict identification and evaluation of the probiotic strains incorporated in dietary supplements is crucial to ensure their safety and efficacy. The CFS of probiotics could be utilized to formulate novel biotherapeutics targeting problematic pathogens. However, future in vivo studies are required to evaluate the appropriate treatment regimen.

Phenotypic and molecular characterization of extended spectrum- and metallo- beta lactamase producing Pseudomonas aeruginosa clinical isolates from Egypt.

BACKGROUND: Antimicrobial resistance among Pseudomonas aeruginosa (P. aeruginosa), a leading cause of nosocomial infections worldwide, is escalating. This study investigated the prevalence of extended-spectrum ß-lactamases (ESBLs) and metallo-ß-lactamases (MBLs) among 104 P. aeruginosa clinical isolates from Alexandria Main University Hospital, Alexandria, Egypt. METHODS: Antimicrobial susceptibility testing was performed using agar dilution technique, or broth microdilution method in case of colistin. ESBL and MBL prevalence was assessed phenotypically and genotypically using polymerase chain reaction (PCR). The role of plasmids in mediating resistance to extended-spectrum ß-lactams was studied via transformation technique using plasmids isolated from ceftazidime-resistant isolates. RESULTS: Antimicrobial susceptibility testing revealed alarming resistance rates to carbapenems, cephalosporins, and fluoroquinolones. Using PCR as the gold standard, phenotypic methods underestimated ESBL production while overestimating MBL production. Eighty-five isolates (81.7%) possessed only ESBL encoding genes, among which 69 isolates harbored a single ESBL gene [blaOXA-10 (n = 67) and blaPER (n = 2)]. Four ESBL-genotype combinations were detected: blaPER + blaOXA-10 (n = 8), blaVEB-1 + blaOXA-10 (n = 6), blaPSE + blaOXA-10 (n = 1), and blaPER + blaVEB-1 + blaOXA-10 (n = 1). Three isolates (2.9%) possessed only the MBL encoding gene blaVIM. Three ESBL + MBL- genotype combinations: blaOXA-10 + blaAIM, blaOXA-10 + blaVIM, and blaPER + blaOXA-10 + blaAIM were detected in 2, 1 and 1 isolate(s), respectively. Five plasmid preparations harboring blaVEB-1 and blaOXA-10 were successfully transformed into chemically competent Escherichia coli DH5α with transformation efficiencies ranging between 6.8 × 10 3 and 3.7 × 10 4 CFU/µg DNA plasmid. Selected tested transformants were ceftazidime-resistant and harbored plasmids carrying blaOXA-10. CONCLUSIONS: The study highlights the importance of the expeditious characterization of ESBLs and MBLs using genotypic methods among P. aeruginosa clinical isolates to hinder the development and dissemination of multidrug resistant strains.

Prevalence of different virulence factors and their association with antimicrobial resistance among Pseudomonas aeruginosa clinical isolates from Egypt.

BACKGROUND: Emergence of multi-drug resistant Pseudomonas aeruginosa, coupled with the pathogen's versatile virulence factors, lead to high morbidity and mortality rates. The current study investigated the potential association between the antibiotic resistance and the production of virulence factors among P. aeruginosa clinical isolates collected from Alexandria Main University Hospital in Egypt. We also evaluated the potential of the phenotypic detection of virulence factors to reflect virulence as detected by virulence genes presence. The role of alginate in the formation of biofilms and the effect of ambroxol, a mucolytic agent, on the inhibition of biofilm formation were investigated. RESULTS: A multi-drug resistant phenotype was detected among 79.8% of the isolates. The most predominant virulence factor was biofilm formation (89.4%), while DNase was least detected (10.6%). Pigment production was significantly associated with ceftazidime susceptibility, phospholipase C production was significantly linked to sensitivity to cefepime, and DNase production was significantly associated with intermediate resistance to meropenem. Among the tested virulence genes, lasB and algD showed the highest prevalence rates (93.3% and 91.3%, respectively), while toxA and plcN were the least detected ones (46.2% and 53.8%, respectively). Significant association of toxA with ceftazidime susceptibility, exoS with ceftazidime and aztreonam susceptibility, and plcH with piperacillin-tazobactam susceptibility was observed. There was a significant correlation between alkaline protease production and the detection of algD, lasB, exoS, plcH and plcN; pigment production and the presence of algD, lasB, toxA and exoS; and gelatinase production and the existence of lasB, exoS and plcH. Ambroxol showed a high anti-biofilm activity (5% to 92%). Quantitative reverse transcriptase polymerase chain reaction showed that alginate was not an essential matrix component in P. aeruginosa biofilms. CONCLUSIONS: High virulence coupled with the isolates' multi-drug resistance to commonly used antimicrobials would increase morbidity and mortality rates among P. aeruginosa infections. Ambroxol that displayed anti-biofilm action could be suggested as an alternative treatment option, yet in vivo studies are required to confirm these findings. We recommend active surveillance of antimicrobial resistance and virulence determinant prevalence for better understanding of coregulatory mechanisms.

Promising biotherapeutic prospects of different probiotics and their derived postbiotic metabolites: in-vitro and histopathological investigation.

BACKGROUND: Probiotics and their derived postbiotics, as cell-free supernatants (CFS), are gaining a solid reputation owing to their prodigious health-promoting effects. Probiotics play a valuable role in the alleviation of various diseases among which are infectious diseases and inflammatory disorders. In this study, three probiotic strains, Lactiplantibacillus plantarum, Lacticaseibacillus rhamnosus, and Pediococcus acidilactici, were isolated from marketed dietary supplements. The antimicrobial activity of the isolated probiotic strains as well as their CFS was investigated. The neutralized CFS of the isolated probiotics were tested for their antibiofilm potential. The anti-inflammatory activity of the isolated Lactobacillus spp., together with their CFS, was studied in the carrageenan-induced rat paw edema model in male Wistar rats. To the best of our knowledge, such a model was not previously experimented to evaluate the anti-inflammatory activity of the CFS of probiotics. The histopathological investigation was implemented to assess the anti-inflammatory prospect of the isolated L. plantarum and L. rhamnosus strains as well as their CFS. RESULTS: The whole viable probiotics and their CFS showed variable growth inhibition of the tested indicator strains using the agar overlay method and the microtiter plate assay, respectively. When tested for virulence factors, the probiotic strains were non-hemolytic lacking both deoxyribonuclease and gelatinase enzymes. However, five antibiotic resistance genes, blaZ, ermB, aac(6')- aph(2"), aph(3'')-III, and vanX, were detected in all isolates. The neutralized CFS of the isolated probiotics exhibited an antibiofilm effect as assessed by the crystal violet assay. This effect was manifested by hindering the biofilm formation of the tested Staphylococcus aureus and Pseudomonas aeruginosa clinical isolates in addition to P. aeruginosa PAO1 strain. Generally, the cell cultures of the two tested probiotics moderately suppressed the acute inflammation induced by carrageenan compared to indomethacin. Additionally, the studied CFS relatively reduced the inflammatory changes compared to the inflammation control group but less than that observed in the case of the probiotic cultures treated groups. CONCLUSIONS: The tested probiotics, along with their CFS, showed promising antimicrobial and anti-inflammatory activities. Thus, their safety and their potential use as biotherapeutics for bacterial infections and inflammatory conditions are worthy of further investigation.

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.

Iron Acquisition Proteins of Pseudomonas aeruginosa as Potential Vaccine Targets: In Silico Analysis and In Vivo Evaluation of Protective Efficacy of the Hemophore HasAp.

BACKGROUND: Pseudomonas aeruginosa (PA) is a Gram-negative pathogen responsible for fatal nosocomial infections worldwide. Iron is essential for Gram-negative bacteria to establish an infection. Therefore, iron acquisition proteins (IAPs) of bacteria are attractive vaccine targets. METHODOLOGY: A "Reverse Vaccinology" approach was employed in the current study. Expression levels of 37 IAPs in various types of PA infections were analyzed in seven previously published studies. The IAP vaccine candidate was selected based on multiple criteria, including a high level of expression, high antigenicity, solubility, and conservation among PA strains, utilizing suitable bioinformatics analysis tools. The selected IAP candidate was recombinantly expressed in Escherichia coli and purified using metal affinity chromatography. It was further evaluated in vivo for protection efficacy. The novel immune adjuvant, naloxone (NAL), was used. RESULTS AND DISCUSSION: HasAp antigen met all the in silico selection criteria, being highly antigenic, soluble, and conserved. In addition, it was the most highly expressed IAP in terms of average fold change compared to control. Although HasAp did excel in the in silico evaluation, subcutaneous immunization with recombinant HasAp alone or recombinant HasAp plus NAL (HasAP-NAL) did not provide the expected protection compared to controls. Immunized mice showed a low IgG2a/IgG1 ratio, indicating a T-helper type 2 (Th2)-oriented immune response that is suboptimal for protection against PA infections. Surprisingly, the bacterial count in livers of both NAL- and HasAp-NAL-immunized mice was significantly lower than the count in the HasAp and saline groups. The same trend was observed in kidneys and lungs obtained from these groups, although the difference was not significant. Such protection could be attributed to the enhancement of innate immunity by NAL. CONCLUSIONS: We provided a detailed in silico analysis of IAPs of PA followed by in vivo evaluation of the best IAP, HasAp. Despite the promising in silico results, HasAp did not provide the anticipated vaccine efficacy. HasAp should be further evaluated as a vaccine candidate through varying the immunization regimens, models of infection, and immunoadjuvants. Combination with other IAPs might also improve vaccination efficacy. We also shed light on several highly expressed promising IAPs whose efficacy as vaccine candidates is worthy of further investigation.

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.