Ahmed Samir Khairalla

The rising antimicrobial resistance is particularly alarming for Acinetobacter baumannii, calling for the discovery and evaluation of alternatives to treat A. baumannii infections. Some bacteriophages produce a structural protein that depolymerizes capsular exopolysaccharide. Such purified depolymerases are considered as novel antivirulence compounds. We identified and characterized a depolymerase (DpoMK34) from Acinetobacter phage vB_AbaP_PMK34 active against the clinical isolate A. baumannii MK34. In silico analysis reveals a modular protein displaying a conserved N-terminal domain for anchoring to the phage tail, and variable central and C-terminal domains for enzymatic activity and specificity. AlphaFold-Multimer predicts a trimeric protein adopting an elongated structure due to a long α-helix, an enzymatic β-helix domain and a hypervariable 4 amino acid hotspot in the most ultimate loop of the C-terminal domain. In contrast to the tail fiber of phage T3, this hypervariable hotspot appears unrelated with the primary receptor. The functional characterization of DpoMK34 revealed a mesophilic enzyme active up to 50 °C across a wide pH range (4 to 11) and specific for the capsule of A. baumannii MK34. Enzymatic degradation of the A. baumannii MK34 capsule causes a significant drop in phage adsorption from 95% to 9% after 5 min. Although lacking intrinsic antibacterial activity, DpoMK34 renders A. baumannii MK34 fully susceptible to serum killing in a serum concentration dependent manner. Unlike phage PMK34, DpoMK34 does not easily select for resistant mutants either against PMK34 or itself. In sum, DpoMK34 is a potential antivirulence compound that can be included in a depolymerase cocktail to control difficult to treat A. baumannii infections.

Phage lysins are a new class of enzyme-based antibiotics that increasingly gain interest. Lysins kill cells through rapid degradation of the peptidoglycan layer, resulting in sudden osmotic lysis.

Escherichia coli is a major cause of community‐acquired urinary tract infections (CA‐UTIs). In this study, we investigated the antimicrobial resistance patterns, the distribution of phylogenetic groups, and the prevalence and characteristics of integron-bearing E. coli isolates from outpatients with CA‐UTIs in El-Minia governorate, in Upper Egypt. Out of the 583 urine samples collected, 134 were positive for E. coli, from which the most resistant isolates (n = 80) were selected for further analysis. The majority of these isolates (62.5%, 50/80) showed multidrug resistance profiles. Group B2 was the most predominant phylogenetic group (52.5%), followed by group F (21.25%), Clades I or II (12.5%), and finally isolates of unknown phylogroup (13.75%). Of the 80 isolates, 7 (8.75%) carried class 1 integrons, which contained 3 different types of integrated gene cassettes, including those conferring resistance to streptomycin/spectinomycin, trimethoprim, and some open reading frames of unknown function (gcuF). In conclusion, the types and combinations of the gene cassettes in our study may reflect the specific selective pressures to which the isolates were subjected within the study region, therefore, providing valuable data for future intervention strategies that are precisely tailored to prevent the dissemination of the uropathogenic E. coli strains circulating within Upper Egypt.

The incidence of cancer is increasing worldwide; likewise, the emergence of antibiotic-resistant biofilm-forming pathogens has led to a tremendous increase in morbidity and mortality. This study aimed to evaluate the probiotic properties of bacteriocin-producing Enterococcus sp. with a focus on their anti-biofilm and anticancer activities. Three of 79 Enterococcus isolates (FM43, FM65, FM50) were identified as producers of broad-spectrum bioactive molecules and were molecularly characterized as Enterococcus faecium by 16S rRNA sequencing. Phenotypic and genotypic screening for potential virulence factors revealed no factors known to promote pathogenicity. Treatment with proteinase K resulted in diminished antimicrobial activity; PCR-based screening for bacteriocin genes suggested the presence of both entA and entB genes that encode enterocins A and B, respectively. Maximum antimicrobial activity was detected during the early stationary phase, while activity disappeared after 24 h in culture. Bacteriocins from these isolates were stable at high temperatures and over a wide range of pH. Interestingly, crude supernatants of Ent. faecium FM43 and Ent. faecium FM50 resulted in significant destruction (80% and 48%, respectively; P < 0.05) of Streptococcus mutans ATCC 25175–associated preformed biofilms. Moreover, in vitro cytotoxicity assays revealed that extracts from Ent. faecium isolates FM43, FM65, and FM50 inhibited Caco-2 cell proliferation by 76.9%, 70%, and 85.3%, respectively. Taken together, the multifunctional capabilities of the microbial-derived proteins identified in our study suggest potentially important roles as alternative treatments for biofilm-associated infections and cancer.

The prevalence of extensively and pandrug-resistant Acinetobacter baumannii leaves little or no therapeutic options for treatment for this bacterial pathogen. Bacteriophages and their lysins represent attractive alternative antibacterial strategies in this regard. We used the extensively drug-resistant A. baumannii strain MK34 to isolate the bacteriophage PMK34 (vB_AbaP_PMK34). This phage shows fast adsorption and lacks virulence genes, nonetheless its narrow host spectrum based on capsule recognition limits broad application. PMK34 is a Fri1virus member of the Autographiviridae and has a 41.8 kb genome (50 ORFs), encoding an endolysin (LysMK34) with potent muralytic activity (1499.9 ± 131 U/μM), a typical mesophilic thermal stability up to 55 °C and a broad pH activity range (4-10). LysMK34 has an intrinsic antibacterial activity up to 4.8 and 2.4 log units for A. baumannii and Pseudomonas aeruginosa strains, respectively, but only when a high turgor pressure is present. Addition of 0.5 mM EDTA or application of an osmotic shock after treatment can compensate for the lack of a high turgor pressure. The combination of LysMK34 and colistin results in up to 32-fold reduction of the MIC of colistin, and colistin-resistant strains are re-sensitized in both Mueller-Hinton broth and 50 % human serum. As such, LysMK34 may be used to safeguard the applicability of colistin as a last-resort antibiotic. ImportanceA. baumannii is one of the most challenging pathogens for which development of new and effective antimicrobials is urgently needed. Colistin is a last resort antibiotic and even colistin-resistant A. baumannii strains exist. Here, we present a lysin that sensitizes A. baumannii for colistin and can revert colistin resistance to colistin susceptibility. The lysin also shows a strong, turgor pressure dependent intrinsic antibacterial activity, providing new insights in the mode-of-action of lysins with intrinsic activity against Gram-negative bacteria.

Treatment of community urinary tract infections (UTIs) caused by extended-spectrum β lactamase (ESBL)- producing Escherichia coli (E. coli) is more expensive than treating ESBL-negative opposites. Evaluation of the prevalence of ESBL-production among urinary E. coli isolates is crucial due to its great impact on the choice of proper antimicrobials. Accordingly, the aim of this work was to detect and characterize ESBL-producing E. coli isolated from outpatients with signs of UTIs in Upper Egypt. Urinary E. coli isolates were identified by 16S rRNA and their ESBL-production was confirmed by Modified Double Disc Synergy Test (MDDST) and ESBL- CHROMagar media. Isolates were then subjected to Polymerase Chain Reaction (PCR) for new Clermont phylogrouping, ESBL genes detection and CTX-M typing. The study enrolled 583 patients with clinically diagnosed UTIs. Uropathogens were found in 400 urine samples (68.6%) out of which 134 E. coli isolates were identified. Among the examined uropathogenic E. coli (UPEC), 80 (59.7%) were recognized as ESBL-producers. Greater than half of the ESBL-producers were multi-drug resistant (MDR) (62%). All of them were susceptible to meropenem. Most of the E. coli isolates were distributed in 4 phylogenetic groups: B2 = 42 (52.5%), F = 17 (21.25%) and Clade I or II = 10 (12.5%). The predominant gene types were TEM 60 (75%) and CTX-M gene 45 (56.25%). The CTX-M-1 group was the most prevalent (62.2%), including the CTX-M-15 enzyme, followed by the CTX-M-2 group, CTX-M-8 group and CTX-M-9 group. In conclusion, the results present alarming evidence of a serious spread of ESBL genes in Egypt, especially the epidemiological CTX-M 15, with the potential for the dissemination of MDR UPEC strains in the community.

Antimicrobial resistance is a major health problem, particularly in developing countries like Bangladesh, where there is a paucity of information on resistance patterns and prevalence of antimicrobial determinants. Therefore, the aims of this study were to investigate the prevalence of resistance, including multi-drug resistance (MDR), and the associated genetic determinants in Escherichia coli isolates from cloacal swabs of live broiler chickens in Bangladesh. Altogether, 400 cloacal swabs (200 from Rajshahi and 200 from Dhaka divisions) were randomly collected from individual chickens in 50 broiler farms. E. coli was isolated and identified using conventional bacteriological culture and biochemical methods. The isolates were further confirmed using genus-specific 16S rRNA-targeted polymerase chain reaction (PCR) primers. Antimicrobial susceptibilities and MDR of the isolates against nine different antimicrobial agents (ampicillin, erythromycin, tetracycline, gentamicin, ciprofloxacin, levofloxacin, trimethoprim-sulfamethoxazole, colistin sulphate, and streptomycin) were determined using the Kirby-Bauer disc diffusion method. Resistance determinants of E. coli to ampicillin (bla_TEM), streptomycin (aadA1), erythromycin [ere(A)], trimethoprim (dfrA1), and tetracycline [tet(A), tet(B)] were screened using PCR. Our results showed that all swab samples were positive for E. coli. The isolates were uniformly resistant to ampicillin, tetracycline, streptomycin, ciprofloxacin, erythromycin, and trimethoprim-sulphamethoxazole. The isolates exhibited highest susceptibility to colistin sulphate (73.5%), followed by gentamicin (49%), and levofloxacin (17%). All isolates were resistant to three classes of antibiotics, 204 isolates (51%) were resistant to four classes, and 56 isolates (14%) were resistant to five. The highest prevalence of antimicrobial resistance gene was recorded for tetracycline (tet(A):95.25%; tet(B):95.25%) followed by ampicillin (bla_TEM:91.25%), streptomycin (aadA1:88.25%), erythromycin (ere(A):84.75%), and trimethoprim (dfrA1:65.5%). In conclusion, surveillance for MDR bacteria in poultry is a critical piece of knowledge, which would be useful for optimizing empiric antimicrobial treatments and exploring alternative antimicrobial agents.

Introduction: Although miltefosine is the first line for treatment of leishmaniasis, it could have multiple un-recognized effects if any infection accidentally takes place during therapy. The aim is to precisely evaluate the molecular and biochemical remarks of miltefosine on Toxoplasma gondii accidental infection during miltefosine therapeutic course. Methodology: changes implied by miltefosine daily parenteral administration to Toxoplasma-infected mice, subcutaneously or intraperitoneal, have been investigated. Tumor necrosis factor-Alfa, immunoglobulin G and M, IL-12 and interferon-gamma release assay (IGRA) were measured in the animals’ sera post-miltefosine administration in addition to monitoring Tissue parasite load by measuring the daily changes of copy number of B1 gene using quantitative PCR technique (qPCR). Results: Miltefosine significantly increased inflammatory and immunological markers (TNF-α, IgG and IgM) measured on reference to control untreated group, with a significant increase in the parasite burden and distribution in all tested organs (F = 390.9, df = 9, P < 0.0001), (F = 4478.98, df = 4.75, P< 0.0001) and (F = 247.3, df = 4, P < 0.0001); heart, liver and lung, respectively, using MANOVA. Releasing capability of macrophages significantly increased during the first day of infection, however, it finally declined after seven consecutive doses of miltefosine (t = 7.96, P < 0.001). Conclusion: Miltefosine could not control the pathogenesis and multiplication of accidental Toxoplasma infection. Cumulative low parenteral daily doses of miltefosine (1.5 µM) could inversely affected the normal humoral immunity against toxoplasmosis. Therefore, a periodical screening for accidental Toxoplasma infection during the course of therapy is strongly recommended.