Identification and characterization of MDR virulent Salmonella spp isolated from smallholder poultry production environment in Edo and Delta States, Nigeria.

Salmonella is responsible for some foodborne disease cases worldwide. It is mainly transmitted to humans through foods of animal origin through the consumption of poultry products. The increased international trade and the ease of transboundary movement could propel outbreaks of local origin to translate into severe global threats. The present study aimed to characterize Salmonella serovars isolated from poultry farms in Edo and Delta States, Nigeria. A total of 150 samples (faecal, water and feed) were collected from ten poultry farms between January and August 2020 and analyzed for Salmonella characterization using standard bacteriological and molecular methods. Salmonella serovars identified include: Salmonella Enteritidis [n = 17 (39.5%)], Salmonella Typhimurium [n = 13 (30.2%)] and other Salmonella serovars [n = 13 (30.2%)]. All Salmonella serovars were cefotaxime and ampicillin resistant. The presence of the invA gene ranged from 9(69.2%) to 15(88.2%). The spvC gene ranged from 2(14.4%) to 10(58.8%). All Salmonella serovars had sdiA gene. The Salmonella isolates produced some extracellular virulence factors (such as protease, lipase, ß-hemolytic activity, and gelatinase), while 13(30.2%) of the overall isolates formed strong biofilms. In conclusion, the detection of multiple antibiotic-resistant Salmonella serovars in faecal sources, which also exhibited virulence determinants, constituted a public health risk as these faecal samples have the potential as manure in the growing of crops. These pathogens can be transmitted to humans nearby and through poultry products, resulting in difficult-to-treat infections and economic loss.

Gastrointestinal Parasites of Dogs in Egypt: An Update on the Prevalence in Dakahlia Governorate and a Meta-Analysis for the Published Data from the Country.

Since the last survey on gastrointestinal (GIT) parasites infecting dogs in Dakahlia governorate, Egypt, was published 40 years ago, the present study detected various GIT parasites in feces of 78 stray dogs in this governorate. Twenty-one dogs (35.9%) had eggs/oocysts of eight different parasites including Toxocara canis (19.2%), Toxascaris leonina (2.6%), hookworms (1.3%), Taenia species (5.1%), Dipylidium caninum (2.6%), Cystoisospora canis (5.1%), Cystoisospora ohioensis (2.6%), and Neospora caninum-like oocysts (1.3%). These results were combined in various meta-analyses with findings of all published surveys on GIT parasites of dogs in Egypt to underline the potential parasitic zoonoses from dogs in the country. Feces and/or gastrointestinal tracts of 19,807 dogs from various Egyptian governorates, but particularly Cairo, have been microscopically tested in 182 datasets published between 1938 and 2022, revealed during our systematic database search. Toxocara canis, interestingly, displayed a twofold higher pooled prevalence (24.7%) when compared to the published global pooled prevalence for T. canis, indicating that dogs represent a major risk for toxocariasis in humans from Egypt. Dipylidium caninum (25.4%) as well as various Taenia species (17.1%) also displayed high pooled prevalences. On the contrary, lower pooled prevalence was estimated for the most important zoonotic taeniid "Echinococcus granulosus" (2.4%) as well as for hookworms (1.8%) in comparison to what has been published from other countries in the region. Relatively high prevalences were estimated for three protozoa detected in dogs and are common to infect children in Egypt; Cryptosporidium (5.5%), Giardia (7.4%), and Entamoeba histolytica (9.8%). In general, the pooled prevalence estimated for various parasites detected in dogs from Egypt has decreased in the recent years, sometimes by as much as one-fifth, but this great decline is statistically insignificant, which should alert the veterinary and public health authorities to continue their efforts for controlling these parasites in a "One Health" approach.

Prevalence and Antimicrobial Resistance of Virulent Listeria monocytogenes and Cronobacter sakazakii in Dairy Cattle, the Environment, and Dried Milk with the In Vitro Application of Natural Alternative Control.

This study aims to detect the prevalence and antimicrobial resistance of Listeria monocytogenes and Cronobacter sakazakii in three dairy households and dried milk from different suppliers, and evaluate the antimicrobial effect of rose water, rose, and orange essential oils. In total, 360 samples were collected from cattle, the environment, and dried milk (n = 30). Antimicrobial activity was evaluated with twofold microtube dilution and the time-kill method. L. monocytogenes was identified in all households (13.3%) with a prevalence in the range of 5.8-17.5%, while C. sakazakii was identified in one household (5.3%). The former and latter pathogens were highly isolated from the feces at 20% and 2.5% and bedding at 12.5% and 1.6%, respectively. L. monocytogenes was isolated only from milk at 7.5%, but C. sakazakii was not detected in either milk or dried milk. L. monocytogenes strains were screened for virulence genes (iap, hylA, and actA). All strains were positive for the iap gene, while for hlyA and actA, the percentages were (35.4% 16.6%, respectively). L. monocytogenes strains showed high resistance against sulfamethoxazole-trimethoprim (100%), followed by gentamicin, penicillin, and imipenem (95.8%, 95.8%, and 91.6%, respectively). All C. sakazakii strains were susceptible to all tested antibiotics. The bactericidal activity of orange oil was the strongest, appeared after 1 h for both pathogens, followed by rose oil and then rose water.

Molecular characterisation of methicillin-resistant and methicillin-susceptible Staphylococcus aureus clones isolated from healthy dairy animals and their caretakers in Egypt.

The purpose of this study was to describe the clonal diversity of Staphylococcus aureus strains derived from healthy dairy cattle and buffaloes as well as their close contact caretakers from the Nile Delta region, Egypt during 2019 and 2020, and to determine their antimicrobial resistance genotypes and virulence determinants. The study included 360 samples (120 from each, dairy cattle, buffaloes and their contact caretakers) collected from eight smallholding dairy herds.The samples included udder skin swabs, composite milk samples and rectal swabs (40 samples each of bovines) and nasal swabs, hand swabs and stool specimens (40 samples each of caretakers). S. aureus were isolated by classical techniques and characterised using the DNA microarray technology. A total of 62 methicillin-resistant (MRSA) and 130 methicillin-sensitive (MSSA) S. aureus isolates were identified. MRSA carriage rate ranged between 2.5% - 15% (Mean: 10%) in dairy cattle, 5% - 15% (9.2%) in dairy buffaloes and 27.5% - 37.5% (30.8%) among the caretakers. Nine different clonal lineages of MRSA (including CC22, CC152, CC5, CC30, CC88, CC45, CC121, CC97, and CC15), and six clonal lineages of MSSA (CC97, CC50, CC188, CC361, CC15 and CC1278) were inferred. The study demonstrated, for the first time, a high clonal diversity of multi-drug resistant S. aureus clones (particularly CC152-MRSA-V, CC30-MRSA-IV, CC121-MRSA-V, CC15-MRSA-V, CC97-MRSA-PseudoSCCmec, CC361-MSSA and CC1278-MSSA) which colonise dairy cattle and buffaloes as well as their caretakers particularly in Damietta villages that located at the northern Mediterranean coast of Egypt. The findings highlight the potential dynamics of humans and animals' S. aureus strains which may represent a health threat for both populations. The complete absence of the lukM/lukF-P83 genes in the recovered isolates indicated that all recovered cattle isolates (except for CC97) were descendants of human lineages and that these replaced the original cow lineages. Hence, a recommendation was given to farm owners to review their hygiene regimen to help minimize the microbiological risks for both populations.

Direct identification and molecular characterization of zoonotic hazards in raw milk by metagenomics using Brucella as a model pathogen.

Metagenomics is a valuable diagnostic tool for enhancing microbial food safety because (i) it enables the untargeted detection of pathogens, (ii) it is fast since primary isolation of micro-organisms is not required, and (iii) it has high discriminatory power allowing for a detailed molecular characterization of pathogens. For shotgun metagenomics, total nucleic acids (NAs) are isolated from complex samples such as foodstuff. Along with microbial NAs, high amounts of matrix NAs are extracted that might outcompete microbial NAs during next-generation sequencing and compromise sensitivity for the detection of low abundance micro-organisms. Sensitive laboratory methods are indispensable for detecting highly pathogenic foodborne bacteria like Brucella spp., because a low infectious dose is sufficient to cause human disease through the consumption of contaminated dairy or meat products. In our study, we applied shotgun metagenomic sequencing for the identification and characterization of Brucella spp. in artificially and naturally contaminated raw milk from various ruminant species. With the depletion of eukaryotic cells prior to DNA extraction, Brucella was detectable at 10 bacterial cells ml-1, while at the same time microbiological culture and isolation of the fastidious bacteria commonly failed. Moreover, we were able to retrieve the genotype of a Brucella isolate from a metagenomic dataset, indicating the potential of metagenomics for outbreak investigations using SNPs and core-genome multilocus sequence typing (cgMLST). To improve diagnostic applications, we developed a new bioinformatics approach for strain prediction based on SNPs to identify the correct species and define a certain strain with only low numbers of genus-specific reads per sample. This pipeline turned out to be more sensitive and specific than Mash Screen. In raw milk samples, we simultaneously detected numerous other zoonotic pathogens, antimicrobial resistance genes and virulence factors. Our study showed that metagenomics is a highly sensitive tool for biological risk assessment of foodstuffs, particularly when pathogen isolation is hazardous or challenging.

Characterisation of S. aureus/MRSA CC1153 and review of mobile genetic elements carrying the fusidic acid resistance gene fusC.

While many data on molecular epidemiology of MRSA are available for North America, Western Europe and Australia, much less is known on the distribution of MRSA clones elsewhere. Here, we describe a poorly known lineage from the Middle East, CC1153, to which several strains from humans and livestock belong. Isolates were characterised using DNA microarrays and one isolate from the United Arab Emirates was sequenced using Nanopore technology. CC1153 carries agr II and capsule type 5 genes. Enterotoxin genes are rarely present, but PVL is common. Associated spa types include t504, t903 and t13507. PVL-positive CC1153-MSSA were found in Egyptian cattle suffering from mastitis. It was also identified among humans with skin and soft tissue infections in Saudi Arabia, France and Germany. CC1153-MRSA were mainly observed in Arabian Gulf countries. Some isolates presented with a previously unknown SCCmec/SCCfus chimeric element in which a mec B complex was found together with the fusidic acid resistance gene fusC and accompanying genes including ccrA/B-1 recombinase genes. Other isolates carried SCCmec V elements that usually also included fusC. Distribution and emergence of CC1153-MRSA show the necessity of molecular characterization of MRSA that are resistant to fusidic acid. These strains pose a public health threat as they combine resistance to beta-lactams used in hospitals as well as to fusidic acid used in the community. Because of the high prevalence of fusC-positive MRSA in the Middle East, sequences and descriptions of SCC elements harbouring fusC and/or mecA are reviewed. When comparing fusC and its surrounding regions from the CC1153 strain to available published sequences, it became obvious that there are four fusC alleles and five distinct types of fusC gene complexes reminiscent to the mec complexes in SCCmec elements. Likewise, they are associated with different sets of ccrA/B recombinase genes and additional payload that might include entire mec complexes or SCCmec elements.

Microarray-based detection of resistance genes in coagulase-negative staphylococci isolated from cattle and buffalo with mastitis in Egypt.

The present study aimed to provide a detailed characterization of coagulase-negative staphylococci (CoNS) isolated from cows and buffaloes with mastitis. The study included seventy-five CoNS isolates (60 came from cattle and 15 from buffaloes) originating from 68 individual quarters of 67 dairy cows (53 cattle and 14 buffaloes). The animals belonged to five different small holding dairy herds (n = 140 cows) that show clinical or subclinical mastitis. CoNS isolates were phenotypically characterized using MALDI-TOF-MS and were further genotypically characterized by microarray-based assays. Furthermore, the antimicrobial susceptibility of CoNS strains which carried the mecA gene was examined by broth microdilution. The occurrence of CoNS in the respective five herds was 10.5%, 14.7%, 14.8%, 12.8%, and 9.9%, with an average of 12.4%. Six different CoNS species were identified: S. sciuri (n = 37; 30 from cattle and 7 from buffaloes), S. chromogenes (n = 14; 8 from cattle and 6 from buffaloes), S. haemolyticus (n = 10; nine from cattle and one buffalo), S. xylosus (n = 10; nine from cattle and one buffalo), S. hyicus (n = 2), S. warneri (n = 1), and unidentified CoNS (n = 1). Twenty percent (20%) of CoNS isolates (17.3% of cattle origin) carried at least one antimicrobial resistance gene, while 4% of the isolate including two isolates of S. haemolyticus and one S. warneri of cattle origin carried the mecA gene and were phenotypically identified as methicillin-resistant strains. The genes detected were blaZ (16%), followed by tet(K) (8%), aacA-aphD (4%), aphA3 (2.6%), msr(A) (2.6%), [far1 (2.6%), and fusC (2.6%)], sat (2.6%), and cat (1.3%) conferring resistance to penicillin, tetracycline, gentamicin, neomycin/kanamycin, erythromycin, fusidic acid, streptothricin, and chloramphenicol, respectively. The majority of investigated CoNS strains displayed considerably low prevalence of resistance genes, while resistance to more than three antibiotics was found in S. haemolyticus and S. warneri. Implementing effective preventive measures is, therefore, important for limiting the transmission of CoNS, rather than using antibiotics to control mastitis in bovines.

Phenotypes, antibacterial-resistant profile, and virulence-associated genes of Salmonella serovars isolated from retail chicken meat in Egypt.

AIM: The present study was designed to investigate the occurrence and distribution of Salmonella serotypes in chicken meat samples, and to explore the susceptibility of the strains to antimicrobials, as well as their virulence-associated genes. MATERIALS AND METHODS: Two-hundred retail chicken meat samples from different shops, as well as 25 stool specimens from retail shop workers, were included in the study. The collected samples were examined bacteriologically for the presence of salmonellae. Salmonella isolates were serotyped using a slide agglutination test for O and H antigens and were screened for the presence of five virulence genes (stn, pef, inv A , sop B , and avrA) using a uniplex polymerase chain reaction assay and for their susceptibility to 18 antimicrobial agents using the disk diffusion method. RESULTS: Thirty-one Salmonella isolates belonging to 12 different serovars were identified. Salmonella Enteritidis and Salmonella Kentucky were the dominant serovars (22.6% each). Salmonella isolates displayed a high antibiotic resistance against erythromycin, sulfamethoxazole/trimethoprim, doxycycline, cephalexin, cefaclor, tetracycline, polymyxin B, cefuroxime, vancomycin, and streptomycin. All Salmonella isolates exhibited multidrug resistance (MDR) and demonstrated different virulence genes. The majority of Salmonella serovars (87.1%) harbored sopB gene, 54.8% carried avrA and pef genes, while all isolates carried invA and stn genes. CONCLUSION: The presence of virulent MDR Salmonellae in raw chicken meat could allow the possibility of transmission of these resistant serovars to humans. Therefore, strict hygienic measures should be followed on the whole poultry production chain to decrease the potential transmission of Salmonella infection from poultry meat to humans.

Contamination Pathways can Be Traced along the Poultry Processing Chain by Whole Genome Sequencing of Listeria innocua.

Foodborne infection with Listeria causes potentially life-threatening disease listeriosis. Listeria monocytogenes is widely recognized as the only species of public health concern, and the closely related species Listeria innocua is commonly used by the food industry as an indicator to identify environmental conditions that allow for presence, growth, and persistence of Listeria spp. in general. In our study, we analyze the occurrence of Listeria spp. in a farm-to-fork approach in a poultry production chain in Egypt and identify bacterial entry gates and transmission systems. Prevalence of Listeria innocua at the three production stages (farm, slaughterhouse, food products) ranged from 11% to 28%. The pathogenic species Listeria monocytogenes was not detected, and Listeria innocua strains under study did not show genetic virulence determinants. However, the close genetic relatedness of Listeria innocua isolates (maximum 63 SNP differences) indicated cross-contamination between all stages from farm to final food product. Based on these results, chicken can be seen as a natural source of Listeria. Last but not least, sanitary measures during production should be reassessed to prevent bacterial contamination from entering the food chain and to consequently prevent human listeriosis infections. For this purpose, surveillance must not be restricted to pathogenic species.

Microarray-based detection of resistance and virulence factors in commensal Escherichia coli from livestock and farmers in Egypt.

The objective of our study was to provide a molecular analysis using DNA-microarray based assays of commensal E. coli populations from apparently healthy livestock and their attendants to assess the virulence potential as well as multidrug resistance (MDR) genotypes. We randomly collected 132 fecal samples from seemingly healthy smallholder´s food producing animals [buffalo (n = 32) and cattle (n = 50)] as well as from contacting farmers (n = 50). Bacterial isolation and identification were performed using standard protocols, while E. coli isolates were characterized using a DNA microarray system targeting 60 different virulence and 47 antibiotic resistance genes of clinical importance and allowing assignment to most common H and O types. From the fecal samples examined, 47 E. coli isolates were obtained. The array predicted serotypes for 14 out of the 47 E. coli isolates. Six E. coli isolates were identified as STEC since Shiga toxin genes were detected. In summary, 36 different virulence genes were identified; of which, hemL, lpfA and iss were most prevalent. Thirty-four E. coli isolates were found to carry at least one antimicrobial resistance gene. Of these, 20 did exhibit genes allowing strain classification as MDR. More than half of the isolates contained antimicrobial resistance genes associated with beta lactam resistance 27/47 (57.5 %). The 13 remaining isolates did not contain any resistance gene tested with the array. Our study demonstrated the presence of antimicrobial resistance genes and virulence genotypes among commensal E. coli of human and animal sources.