Background: Emergence of multi-drug resistantPseudomonas 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 amongP. aeruginosaclinical 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,lasBandalgDshowed the highest prevalence rates (93.3% and 91.3%, respectively), whiletoxAandplcNwere the least detected ones (46.2% and 53.8%, respectively). Significant association oftoxAwith ceftazidime susceptibility,exoSwith ceftazidime and aztreonam susceptibility, andplcHwith piperacillin-tazobactam susceptibility was observed. There was a significant correlation between alkaline protease production and the detection ofalgD,lasB,exoS,plcHandplcN; pigment production and the presence ofalgD,lasB,toxAandexoS; and gelatinase production and the existence oflasB,exoSandplcH. 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 inP. aeruginosabiofilms.Conclusions: High virulence coupled with the isolates’ multi-drug resistance to commonly used antimicrobials would increase morbidity and mortality rates amongP. aeruginosainfections. 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.

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.

Recently, Egypt has witnessed the emergence of multidrug-resistant (MDR) Klebsiella pneumoniae, which has posed a serious healthcare challenge. The accelerated dissemination of bla_CTX-M genes among these MDR K. pneumoniae, particularly bla_CTX-M-14 and bla_CTX-M-15, have been noted. In this study, we investigated the occurrence of bla_CTX-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 bla_CTX-M-IV gene was detected in ≈22% of the isolates. The transformation of plasmids harboring bla_CTX-M-IV to chemically competent cells of Escherichia coli DH5α was successful in three out of five of the tested bla_CTX-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 bla_CTX-M-14 on IncL/M plasmid, i.e., pEGY22_CTX-M-14, and bla_CTX-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.

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, andPediococcus 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 isolatedLactobacillusspp., 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 isolatedL. plantarumandL. rhamnosusstrains 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, andvanX, 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 testedStaphylococcus aureusandPseudomonas aeruginosaclinical isolates in addition toP. aeruginosaPAO1 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.

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 β-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.

The accelerated dispersion of multidrug-resistant (MDR) Escherichia coli due to the production of extended-spectrum β-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 bla_CMY-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 bla_CTX-M-15, bla_TEM-1, bla_OXA-1 and bla_NDM-5 on an IncFIA/IncFIB/IncFII/IncQ1 multireplicon plasmid. The chromosomal location of bla_CMY-2 is detected with a flanking upstream copy of ISEcp1. This chromosomal integration of bla_CMY-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.