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.

Antibacterial and anti-biofilm activities of probiotic Lactobacillus plantarum against Listeria monocytogenes isolated from milk, chicken and pregnant women.

Listeria monocytogenes (L. monocytogenes) is a foodborne pathogen that poses significant risks to public health and food safety. The present study aimed to identify the presence of Listeria spp. in various samples, including pasteurized milk, chicken fillets, and stool samples from pregnant women in Sharkia Governorate, Egypt. Additionally, the study identified the serotypes, virulence-associated genes, antimicrobial resistance patterns, and biofilm formation in L. monocytogenes isolates. Moreover, the antibacterial and anti-biofilm activity of Lactobacillus plantarum ATCC 14917 (L. plantarum) against L. monocytogenes isolates was investigated. A cross-sectional study was conducted from August 2021 to January 2022 to collect 300 samples of pasteurized milk, chicken fillets, and stool from pregnant women admitted to outpatient clinics of hospitals. The results showed that 32.7% of the samples were positive for Listeria spp., including L. innocua (48.9%), L. monocytogenes (26.5%), L. ivanovii (14.3%), L. grayi (5.1%), and L. welshimeri (5.1%). Among all L. monocytogenes isolates, hlyA, actA, inlC, and inlJ virulence-associated genes were detected. However, the virulence genes plcB, iap, and inlA were found in 10 (38.5%), 8 (30.8%), and 25 (96.2%) isolates, respectively. The L. monocytogenes isolates classified into four serotypes (1/2a, 1/2b, 1/2c, and 4b), with 1/2a and 4b each identified in 30.8% of the isolates, while 1/2b and 1/2c were identified in 19.2% of the isolates. All L. monocytogenes isolates showed 100% resistance to streptomycin, kanamycin, and nalidix acid, and 92.3% of isolates showed gentamicin resistance. However, all isolates were susceptible to ampicillin and ampicillin/sulbactam. Multidrug resistance (MDR) was observed in 20 (76.9%) L. monocytogenes isolates. The biofilm formation ability of 26 L. monocytogenes isolates was evaluated at different incubation temperatures. At 4°C, 25°C, and 37°C, 53.8, 69.2, and 80.8% of the isolates, respectively, were biofilm producers. Furthermore, 23.1% were strong biofilm producers at both 4°C and 25°C, while 34.6% were strong biofilm formers at 37°C. Treating L. monocytogenes isolates with L. plantarum cell-free supernatant (CFS) reduced the number of biofilm-producing isolates to 15.4, 42.3, and 53.8% at 4°C, 25°C, and 37°C, respectively. L. plantarum's CFS antibacterial activity was tested against six virulent, MDR, and biofilm-forming L. monocytogenes isolates. At a concentration of 5 µg/mL of L. plantarum CFS, none of the L. monocytogenes isolates exhibited an inhibition zone. However, an inhibition zone was observed against L. monocytogenes strains isolated from pasteurized milk and pregnant women's stools when using a concentration of 10 µg/mL. Transmission electron microscopy (TEM) revealed that L. plantarum CFS induced morphological and intracellular structural changes in L. monocytogenes. In conclusion, this study identified virulent MDR L. monocytogenes isolates with strong biofilm-forming abilities in food products in Egypt, posing significant risks to food safety. Monitoring the prevalence and antimicrobial resistance profile of L. monocytogenes in dairy and meat products is crucial to enhance their safety. Although L. plantarum CFS showed potential antibacterial and anti-biofilm effects against L. monocytogenes isolates, further research is needed to explore its full probiotic potential.

Interplay between cross sectional analysis of risk factors associated with Toxoplasma gondii infection in pregnant women and their domestic cats.

Toxoplasmosis is a global zoonotic disease caused by Toxoplasma gondii (T. gondii). The primary aim of this study was to identify hygienic and cat management practices that could affect the occurrence of T. gondii in cats and their owners in Sharqia Governorate, Egypt. T. gondii infection was evaluated in 80 pregnant women and 29 domestic cats using Real-time PCR. A questionnaire was administered to obtain information regarding the risk factors associated with T. gondii infection. Blood samples were collected from enrolled pregnant women, and fecal samples were collected from their pet cats. Firth logistic regression model complemented with random forest (RF) analysis was used to evaluate the association of different hygiene and cat management practices with T. gondii infection in pregnant women. T. gondii infection was identified in 27.5% of pregnant women and 17% of domestic cats. Drinking raw milk and contacting stray and pet cats were significantly associated with higher odds of T. gondii infection. The proportion of T. gondii-positive women who ate raw meat (60.0%) was higher than those ate heat-cooked meat (25.3%). Moreover, women who did not wash their hands after contact with cats were 6 times (OR 6.12; CI: 3.03-9.21) more likely to experience T. gondii infection than those washed their hands after cat contact. The RF analysis showed that washing hands constitute a serious yet preventable public health concern that requires targeted, community-specific efforts. Cat owners, particularly pregnant women, need to be aware about the risk of T. gondii infection, while handling cat and pet's owner should be advised to take necessary hygienic measures to prevent its infection.

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.

Antimicrobial Resistance of Salmonella enteritidis and Salmonella typhimurium Isolated from Laying Hens, Table Eggs, and Humans with Respect to Antimicrobial Activity of Biosynthesized Silver Nanoparticles.

Salmonella enterica is one of the most common causes of foodborne illness worldwide. Contaminated poultry products, especially meat and eggs are the main sources of human salmonellosis. Thus, the aim of the present study was to determine prevalence, antimicrobial resistance profiles, virulence, and resistance genes of Salmonella Enteritidis (S. enteritidis) and Salmonella Typhimurium (S. Typhimurium) isolated from laying hens, table eggs, and humans, in Sharkia Governorate, Egypt. The antimicrobial activity of Biosynthesized Silver Nanoparticles (AgNPs) was also evaluated. Salmonella spp. were found in 19.3% of tested samples with laying hens having the highest isolation rate (33.1%). S. Enteritidis) (5.8%), and S. Typhimurium (2.8%) were the dominant serotypes. All isolates were ampicillin resistant (100%); however, none of the isolates were meropenem resistant. Multidrug-resistant (MDR) was detected in 83.8% of the isolates with a multiple antibiotic resistance index of 0.21 to 0.57. Most isolates (81.1%) had at least three virulence genes (sopB, stn, and hilA) and none of the isolates harbored the pefA gene; four resistance genes (blaTEM, tetA, nfsA, and nfsB) were detected in 56.8% of the examined isolates. The AgNPs biosynthesized by Aspergillus niveus exhibit an absorption peak at 420 nm with an average size of 27 nm. AgNPs had a minimum inhibitory concentration of 5 µg/mL against S. enteritidis and S. typhimurium isolates and a minimum bactericidal concentration of 6 and 8 µg/mL against S. enteritidis and S. typhimurium isolates, respectively. The bacterial growth and gene expression of S. enteritidis and S. typhimurium isolates treated with AgNPs were gradually decreased as storage time was increased. In conclusion, this study indicates that S. enteritidis and S. typhimurium isolated from laying hens, table eggs, and humans exhibits resistance to multiple antimicrobial classes. The biosynthesized AgNPs showed potential antimicrobial activity against MDR S. enteritidis and S. typhimurium isolates. However, studies to assess the antimicrobial effectiveness of the biosynthesized AgNPs in laying hen farms are warranted.

Molecular identification of avian influenza virus subtypes H5N1 and H9N2 in birds from farms and live bird markets and in respiratory patients.

BACKGROUND: Avian influenza viruses (AIVs) have been endemic in Egypt since 2006, and the co-circulation of high-pathogenic avian influenza H5N1 and low-pathogenic avian influenza H9N2 subtypes in poultry has been reported; therefore, Egypt is considered a hotspot for the generation of new subtypes and genotypes. We aimed to characterize AIVs circulating on commercial farms and in live bird markets (LBMs) during the winters of 2015 and 2016 in the study area and to identify H5N1 and H9N2 viruses in respiratory patients. METHODS: In total, 159 samples were collected from ducks, pigeons and quails on farms (n = 59) and in LBMs (n = 100) and screened by real-time RT-PCR for H5N1 and H9N2 subtypes. Clinical and postmortem examination was carried out on birds from the farms. Positive H5N1 samples were sequenced and analysed for mutations. Tracheal swabs were also collected from 89 respiratory patients admitted to respiratory hospitals in the same study area. RESULTS: Overall, H5N1 was identified in 13.6% of birds from farms, while it was detected in 17% of birds in LBMs. Subtype H9N2 was only identified from pigeons on farms (6.5%) and LBMs (11.4%). Sequencing of the haemagglutination gene (HA) in nine representative H5N1 isolates revealed a multi-basic amino acid motif at the cleavage site (321-PQGEKRRKKR/GLF-333), which is characteristic of highly pathogenic AIV, in five of our isolates, while the other four isolates showed an amino acid substitution (Q322K) at this cleavage site to make it (321-P K GEKRRKKR/GLF-333). All the isolates belonged to clade 2.2.1.2, and a comparison of HA sequences at the amino acid level showed 98.8-100% homology among the nine isolates, while they showed 94.1-96.1% identity with reference strains and the commonly used vaccine strain in Egypt. Out of 89 respiratory patients, 3.4% were positive for H5N1 and no patients were positive for H9N2. DISCUSSION: Our results indicated the circulation of the endemic H5N1 and H9N2 viruses among poultry in 2015 and 2016. Birds on farms and in LBMs are reservoirs playing a role in the dissemination of the virus and producing a public health risk. The application of proper hygienic measures in farms and LBMs to control the exposure of birds and humans to the source of infection along with continuous monitoring of the circulating viruses will provide information on understanding the evolution of the viruses for vaccine studies.

Molecular Characterization of Cronobacter sakazakii in Egypt, Survival and Thermoresistance at Different Temperatures: A Potential Public Health Risk.

Cronobacter sakazakii has been implicated in causing serious infections in neonates due to consumption of contaminated infant powdered milk. The zoonotic potential of the organism was not clear due to scarce evidence about the role of food animals in the transmission of infection. C. sakazakii was identified in infant powdered milk (n = 100), infant stool (n = 100), and dairy animal feces (n = 100) with the percentages of 1%, 2%, and 4%, respectively. The outer membrane protein A (ompA) gene was characterized in all isolates of different origin, while gene encoding for zinc-metaloprotease (zpx) was only identified in isolates from animal feces. Genotyping of C. sakazakii isolates using enterobacterial repetitive intergenic consensus polymerase chain reaction revealed heterogenicity. The survival and thermotolerance of one potentially virulent C. sakazakii isolate of animal origin were examined at different temperatures. The isolate could survive with a stationary number at refrigeration temperature and the number increased significantly at room temperature after 24 h. The isolate showed thermoresistance when subjected to temperature range from 54°C to 64°C with D values ranged from 13.79 and 4.64 min and z value of 14.42. To the best of our knowledge, this is the first report of C. sakazakii isolation from buffalo feces in Egypt.

Listeria monocytogenes in raw milk, milking equipment and dairy workers: Molecular characterization and antimicrobial resistance patterns.

OBJECTIVES: The aim of this study was to evaluate the genetic relatedness and patterns of antimicrobial resistance amongst L. monocytogenes isolated from raw milk, milking equipment, and hand swabs from workers in dairy farms. METHODS: A total of 300 samples of raw milk, milking equipment, and hand swabs were collected from four dairy farms to examine the presence of Listeria species. Suspected isolates were further identified by VITEK-2 system and Polymerase Chain Reaction (PCR). Antimicrobial susceptibility of the L. monocytogenes isolates was determined, and genotyping analysis was performed by enterobacterial repetitive intergenic consensus-polymerase chain reaction (ERIC-PCR). RESULTS: Listeria spp. was isolated from 79 (26.3%) of the 300 samples, including 29 (36.7%), 32 (40.5%), and 18 (22.8%) isolates found in raw milk, milking equipment, and hand swabs, respectively. L. monocytogenes was the most common isolated (87.3%) species, while the remaining Listeria isolates were L. innocua (12.7%). Among the 69 L. monocytogenes isolates, 42 (60.8%) showed the mutual presence of hlyA, prfA, inlA, and inlB virulence-associated genes. L. monocytogenes isolates from raw milk, milking equipment, and hand swabs showed high genetic relatedness. The potentially virulent L. monocytogenes isolates were most frequently resistance to tetracycline and clindamycin (81%, each) followed by rifampicin (71.4%), whereas, antimicrobial susceptibility was most frequently observed for ampicillin, levofloxacin, moxifloxacin, linezolid, and tigecycline (100%, each). Furthermore, 88% of L. monocytogenes isolates showed multidrug-resistance. CONCLUSIONS: The findings of this study show that the contamination of dairy farms with L. monocytogenes is relatively high, and highlight the emergence of multi-drug resistant L. monocytogenes in dairy farms. However, ampicillin is a good choice for treatment of listeriosis in the study area.

Seroprevalence and risk factors associated with Toxoplasma gondii infection in domestic rabbits and humans.

A cross-sectional study was conducted from September 2014 to December 2015 to determine seroprevalence and potential risk factors associated with Toxoplasma gondii (T. gondii) infection in domestic rabbits and humans in Egypt. In total 290 blood samples were collected from humans (n=140) and slaughtered rabbits (n=150) and were analyzed using ELISA for T. gondii IgM and IgG antibodies. T. gondii IgM and IgG antibodies were detected in, respectively, 9 (6%) and 40 (26.7%) of 150 rabbits raised in Cairo, Qalyubia, and Sharkia Governorates, Egypt. Corresponding overall seroprevalences for human participants were 5.7% and 35.7%, respectively. Rabbit age, management (farm, backyard and pet shop) and the presence of cats at rabbit raising areas were significantly associated with the seroprevalence of T. gondii IgG antibodies. T. gondii IgG antibodies seropositivity in pregnant participants and rabbit butchers were 0.17 and 0.63 times lower than immunocompromised participants, respectively. However, participants who consumed undercooked rabbit meat was 7.59 times higher than participants who consumed meat from other sources. The results indicate that domestic rabbits are a potential source of T. gondii infections in human in Egypt. Thus, dissemination of protective measures is essential, especially for rabbit butchers and immunocompromised individuals.