Antifungal and antibiofilm effects of probiotic Lactobacillus salivarius, zinc nanoparticles, and zinc nanocomposites against Candida albicans from Nile tilapia (Oreochromis niloticus), water and humans.

Introduction: Candida albicans (C. albicans) can form biofilms; a critical virulence factor that provides effective protection from commercial antifungals and contributes to public health issues. The development of new antifungal therapies, particularly those targeting biofilms, is imperative. Thus, this study was conducted to investigate the antifungal and antibiofilm effects of Lactobacillus salivarius (L. salivarius), zinc nanoparticles (ZnNPs) and nanocomposites (ZnNCs) on C. albicans isolates from Nile tilapia, fish wash water and human fish sellers in Sharkia Governorate, Egypt. Methods: A cross-sectional study collected 300 samples from tilapia, fish wash water, and fish sellers (100 each). Probiotic L. salivarius was immobilized with ZnNPs to synthesize ZnNCs. The study assessed the antifungal and antibiofilm activities of ZnNPs, L. salivarius, and ZnNCs compared to amphotericin (AMB). Results: Candida spp. were detected in 38 samples, which included C. albicans (42.1%), C. glabrata (26.3%), C. krusei (21.1%), and C. parapsilosis (10.5%). A total of 62.5% of the isolates were resistant to at least one antifungal agent, with the highest resistance to nystatin (62.5%). However, 75% of the isolates were highly susceptible to AMB. All C. albicans isolates exhibited biofilm-forming capabilities, with 4 (25%) isolates showing strong biofilm formation. At least one virulence-associated gene (RAS1, HWP1, ALS3, or SAP4) was identified among the C. albicans isolates. Probiotics L. salivarius, ZnNPs, and ZnNCs displayed antibiofilm and antifungal effects against C. albicans, with ZnNCs showing significantly higher inhibitory activity. ZnNCs, with a minimum inhibitory concentration (MIC) of 10 µg/mL, completely reduced C. albicans biofilm gene expression. Additionally, scanning electron microscopy images of C. albicans biofilms treated with ZnNCs revealed asymmetric, wrinkled surfaces, cell deformations, and reduced cell numbers. Conclusion: This study identified virulent, resistant C. albicans isolates with strong biofilm-forming abilities in tilapia, water, and humans, that pose significant risks to public health and food safety.

Escherichia coli and Salmonella enterica isolated from Egyptian dairy cattle herds: The prevalence and molecular characteristics.

Background: The pathogens Escherichia coli and Salmonella enterica that caused substantial health problems and financial losses were believed to have originated primarily from Egypt's dairy farms. Aim: The purpose of this study was to ascertain the occurrence of E. coli and S. enterica in three large dairy farms located in the Egyptian governorate of Sharkia. Furthermore, biochemical and serological characteristics of the isolated isolates were described. Further analysis revealed that several E. coli serovars had the genes stx1, stx2, eaeA, and hylA, while invA, stn, and hilA genes were found in several S. enterica serotypes using a multi-plex PCR. Methods: A total of 540 samples of fresh raw cow milk, water, feedstuffs, feces, (108 each), as well as swabs from feeders, milker hands and cattle crushes (36 each ), were gathered and analyzed. Results: The recovery of E. coli from various sampling sources was shown to have an overall prevalence of 62.2% (336/540) in the results. Fecal samples had isolated S. enterica, with a frequency of 0.74% (4/540). The existence of various groups of serovars, such as O26, O44, O55, O78 and O111 for E. coli and Salmonella enteritidis, Salmonella typhimurium and Salmonella inganda for S. enterica was revealed by serological identification of the two species. However, it was discovered that a number of E. coli serovars had much higher percentages of the eaeA and hylA genes as well as shiga-toxin types 1 and 2 (stx1 and stx2). The presence of the invA gene, a diagnostic marker for S. enterica was 100% across all serovars. Salmonella enteritidis possessed both the enterotoxin gene (stn) and the hyper-invasive locus gene (hilA). Salmonella typhimurium had the hilA gene, whereas S. inganda had the stn gene. Conclusion: Escherichia coli and S. enterica recovered in this study have significant genetic risk factors for high pathogenicity and virulence, posing a real threat to dairy population productivity and health, which could spread to the general public through milk.

Cross-sectional analysis of risk factors associated with Mugil cephalus in retail fish markets concerning methicillin-resistant Staphylococcus aureus and Aeromonas hydrophila.

Introduction: Aeromonas hydrophila and methicillin-resistant Staphylococcus aureus (MRSA) are potent bacterial pathogens posing major hazards to human health via consuming fish harboring these pathogens or by cross-contamination beyond the contaminated environment. The aim of this study was to determine risk variables associated with the presence of certain pathogenic bacteria from Mugil cephalus fish in retail markets in Egypt. The virulence genes of A. hydrophila and S. aureus were also studied. Furthermore, the antibiotic sensitivity and multidrug resistance of the microorganisms were evaluated. Methods: In a cross-sectional investigation, 370 samples were collected from mullet skin and muscle samples, washing water, fish handlers, knives, and chopping boards. Furthermore, fish handlers' public health implications were assessed via their response to a descriptive questionnaire. Results: S. aureus and Aeromonas species dominated the investigated samples with percentages of 26.76% and 30.81%, respectively. Furthermore, A. hydrophila and MRSA were the predominant recovered bacterial pathogens among washing water and knives (53.85% and 46.66%, respectively). The virulence markers aerA and hlyA were found in 90.7% and 46.5% of A. hydrophila isolates, respectively. Moreover, the virulence genes nuc and mec were prevalent in 80% and 60% of S. aureus isolates, respectively. Antimicrobial susceptibility results revealed that all A. hydrophila isolates were resistant to amoxicillin and all MRSA isolates were resistant to amoxicillin and ampicillin. Remarkably, multiple drug resistance (MDR) patterns were detected in high proportions in A. hydrophila (88.37%) and MRSA (100%) isolates. The prevalence of Aeromonas spp. and S. aureus had a positive significant correlation with the frequency of handwashing and use of sanitizer in cleaning of instruments. MRSA showed the highest significant prevalence rate in the oldest age category. Conclusion: The pathogenic bacteria recovered in this study were virulent and had a significant correlation with risk factors associated with improper fish handling. Furthermore, a high frequency of MDR was detected in these pathogenic bacteria, posing a significant risk to food safety and public health.

Molecular identification and antiprotozoal activity of silver nanoparticles on viability of Cryptosporidium parvum isolated from pigeons, pigeon fanciers and water.

Cryptosporidium is a protozoan that causes acute gastroenteritis, abdominal pain, and diarrhea in many vertebrate species, including humans, animals and birds. A number of studies have reported the occurrence of Cryptosporidium in domestic pigeons. Thus, this study aimed to identify Cryptosporidium spp. in samples collected from domestic pigeons, pigeon fanciers, and drinking water, as well as to investigate the antiprotozoal activity of biosynthesized silver nanoparticles (AgNPs) on the viability of isolated Cryptosporidium parvum (C. parvum). Samples were collected from domestic pigeons (n = 150), pigeon fanciers (n = 50), and drinking water (n = 50) and examined for the presence of Cryptosporidium spp. using microscopic and molecular techniques. The antiprotozoal activity of AgNPs was then assessed both in vitro and in vivo. Cryptosporidium spp. was identified in 16.4% of all examined samples, with C. parvum identified in 5.6%. The highest frequency of isolation was from domestic pigeon, rather than from pigeon fanciers or drinking water. In domestic pigeons, there was a significant association between Cryptosporidium spp. positivity and pigeon's age, droppings consistency, housing, hygienic and heath conditions. However, Cryptosporidium spp. positivity was only significantly associated with pigeon fanciers' gender and heath condition. The viability of C. parvum oocysts was reduced using AgNPs at various concentrations and storage times in a descending manner. In an in vitro study, the highest reduction in C. parvum count was observed at the AgNPs concentration of 1000 µg/mL after a 24 h contact time, followed by the AgNPs concentration of 500 µg/mL after a 24 h contact time. However, after a 48 h contact time, a complete reduction was observed at both 1000 and 500 µg/mL concentrations. Overall, the count and viability of C. parvum decreased with increasing the AgNPs concentration and contact times in both the in vitro and in vivo studies. Furthermore, the C. parvum oocyst destruction was time-dependent and increased with increasing the contact time at various AgNPs concentrations.

Virulence Determinants and Plasmid-Mediated Colistin Resistance mcr Genes in Gram-Negative Bacteria Isolated From Bovine Milk.

A major increase of bacterial resistance to colistin, a last-resort treatment for severe infections, was observed globally. Using colistin in livestock rearing is believed to be the ground of mobilized colistin resistance (mcr) gene circulation and is of crucial concern to public health. This study aimed to determine the frequency and virulence characteristics of colistin-resistant Gram-negative bacteria from the milk of mastitic cows and raw unpasteurized milk in Egypt. One hundred and seventeen strains belonging to Enterobacteriaceae (n = 90), Pseudomonas aeruginosa (n = 10), and Aeromonas hydrophila (n = 17) were screened for colistin resistance by antimicrobial susceptibility testing. The genetic characteristics of colistin-resistant strains were investigated for mcr-1-9 genes, phylogenetic groups, and virulence genes. Moreover, we evaluated four commonly used biocides in dairy farms for teat disinfection toward colistin-resistant strains. Multidrug-resistant (MDR) and extensive drug-resistant (XDR) phenotypes were detected in 82.91% (97/117) and 3.42% (4/117) of the isolates, respectively. Of the 117 tested isolates, 61 (52.14%) were colistin resistant (MIC >2 mg/L), distributed as 24/70 (34.29%) from clinical mastitis, 10/11 (90.91%) from subclinical mastitis, and 27/36 (75%) from raw milk. Of these 61 colistin-resistant isolates, 47 (19 from clinical mastitis, 8 from subclinical mastitis, and 20 from raw milk) harbored plasmid-borne mcr genes. The mcr-1 gene was identified in 31.91%, mcr-2 in 29.79%, mcr-3 in 34.04%, and each of mcr-4 and mcr-7 in 2.13% of the colistin-resistant isolates. Among these isolates, 42.55% (20/47) were E. coli, 21.28% (10/47) A. hydrophila, 19.12% (9/47) K. pneumoniae, and 17.02% (8/47) P. aeruginosa. This is the first report of mcr-3 and mcr-7 in P. aeruginosa. Conjugation experiments using the broth-mating technique showed successful transfer of colistin resistance to E. coli J53-recipient strain. Different combinations of virulence genes were observed among colistin-resistant isolates with almost all isolates harboring genes. Hydrogen peroxide has the best efficiency against all bacterial isolates even at a low concentration (10%). In conclusion, the dissemination of mobile colistin resistance mcr gene and its variants between MDR- and XDR-virulent Gram-negative isolates from dairy cattle confirms the spread of mcr genes at all levels; animals, humans, and environmental, and heralds the penetration of the last-resort antimicrobial against MDR bacteria. Consequently, a decision to ban colistin in food animals is urgently required to fight XDR and MDR bacteria.

Whole-Genome Sequencing of Gram-Negative Bacteria Isolated From Bovine Mastitis and Raw Milk: The First Emergence of Colistin mcr-10 and Fosfomycin fosA5 Resistance Genes in Klebsiella pneumoniae in Middle East.

Antimicrobial resistance is a major concern in the dairy industry. This study investigated the prevalence, antimicrobial resistance phenotypes, and genome sequencing of Gram-negative bacteria isolated from clinical (n = 350) and subclinical (n = 95) bovine mastitis, and raw unpasteurized milk (n = 125). Klebsiella pneumoniae, Aeromonas hydrophila, Enterobacter cloacae (100% each), Escherichia coli (87.78%), and Proteus mirabilis (69.7%) were the most prevalent multidrug-resistant (MDR) species. Extensive drug-resistance (XDR) phenotype was found in P. mirabilis (30.30%) and E. coli (3.33%) isolates. Ten isolates (four E. coli, three Klebsiella species and three P. mirabilis) that displayed the highest multiple antibiotic resistance (MAR) indices (0.54-0.83), were exposed to whole-genome sequencing (WGS). Two multilocus sequence types (MLST): ST2165 and ST7624 were identified among the sequenced E. coli isolates. Three E. coli isolates (two from clinical mastitis and one from raw milk) belonging to ST2165 showed similar profile of plasmid replicon types: IncFIA, IncFIB, IncFII, and IncQ1 with an exception to an isolate that contained IncR, whereas E. coli ST7624 showed a different plasmid profile including IncHI2, IncHI2A, IncI1α, and IncFII replicon types. ResFinder findings revealed the presence of plasmid-mediated colistin mcr-10 and fosfomycin fosA5 resistance genes in a K. pneumoniae (K1) isolate from bovine milk. Sequence analysis of the reconstructed mcr-10 plasmid from WGS of K1 isolate, showed that mcr-10 gene was bracketed by xerC and insertion sequence IS26 on an IncFIB plasmid. Phylogenetic analysis revealed that K1 isolate existed in a clade including mcr-10-harboring isolates from human and environment with different STs and countries [United Kingdom (ST788), Australia (ST323), Malawi (ST2144), Myanmar (ST705), and Laos (ST2355)]. This study reports the first emergence of K. pneumoniae co-harboring mcr-10 and fosA5 genes from bovine milk in the Middle East, which constitutes a public health threat and heralds the penetration of the last-resort antibiotics. Hence, prudent use of antibiotics in both humans and animals and antimicrobial surveillance plans are urgently required.