New Insights into Listeria monocytogenes Antimicrobial Resistance, Virulence Attributes and Their Prospective Correlation.

Listeriosis is one of the most common foodborne diseases caused by Listeria monocytogenes (L. monocytogenes). A poor prognosis has been recorded for the invasive listeriosis, especially neurolisteriosis. In several countries throughout the world, foodborne infections with L. monocytogenes exceeded the legal safety limits in animal sourced foods. Therefore, we decided to investigate the variability, virulence and antimicrobial resistance profiles of this pathogen. Both phenotypic and genotypic methods were used for identifying L. monocytogenes isolates and confirming their virulence profiles. The antimicrobial resistances and their correlation analysis with the existence of virulence genes were detected. Additionally, sequencing and phylogenetic analysis based on L. monocytogenes inlA and inlB genes were undertaken. The prevalence rate (11.9%) and the resistance profiles of L. monocytogenes were shocking. The multi-drug resistance (MDR) phenotypes were common among our isolates (64.9%). Fortunately, the resistance phenotypes were always associated with low virulence arrays and the MDR strains possessed low virulence fitness. Herein, the high genotypic and phenotypic diversity of L. monocytogenes isolates and their weak clonality and adaptability highlighted the difficulty in controlling and managing this pathogen. Therefore, it is important to add more restriction guidelines from national authorities on the consumption of ready to eat foods.

Review on Bovine Tuberculosis: An Emerging Disease Associated with Multidrug-Resistant Mycobacterium Species.

Bovine tuberculosis is a serious infectious disease affecting a wide range of domesticated and wild animals, representing a worldwide economic and public health burden. The disease is caused by Mycobacteriumbovis and infrequently by other pathogenic mycobacteria. The problem of bovine tuberculosis is complicated when the infection is associated with multidrug and extensively drug resistant M. bovis. Many techniques are used for early diagnosis of bovine tuberculosis, either being antemortem or postmortem, each with its diagnostic merits as well as limitations. Antemortem techniques depend either on cellular or on humoral immune responses, while postmortem diagnosis depends on adequate visual inspection, palpation, and subsequent diagnostic procedures such as bacterial isolation, characteristic histopathology, and PCR to reach the final diagnosis. Recently, sequencing and bioinformatics tools have gained increasing importance for the diagnosis of bovine tuberculosis, including, but not limited to typing, detection of mutations, phylogenetic analysis, molecular epidemiology, and interactions occurring within the causative mycobacteria. Consequently, the current review includes consideration of bovine tuberculosis as a disease, conventional and recent diagnostic methods, and the emergence of MDR-Mycobacterium species.

Epidemiological, bacteriological and molecular studies on caseous lymphadenitis in sheep of Dakhlia, Egypt.

Caseous lymphadenitis (CLA) is a chronic and insidious disease that mainly affects small ruminants and caused by Corynebacterium pseudotuberculosis (C. pseudotuberculosis). The aims of this research were to identify C. pseudotuberculosis by PCR from pyogenic lesions, to study the phylogenetic analysis of C. pseudotuberculosis and to detect the prevalence based on the detected superficial lesions of CLA in Dakahlia governorate, Egypt. Out of 3471 clinically examined animals, 129 (3.71%) animals were affected with CLA. The isolation rate of C. pseudotuberculosis in abscess of sheep was 45.74% (59/129). Out of 129 samples examined by PCR assay, 63 (48.83%) were positive phospholipase D (PLD) indicated at fragment size 203 bp. This is the first phylogenetic analysis study of C. pseudotuberculosis isolate in Egypt which was isolated from infected sheep. Nucleotide sequence identity data demonstrated that C. pseudotuberculosis PLD gene (MW187942) Dakahlia share homology 99.01%, 98.83 and 98.48% with Zagazig, Egypt (MN867024), Tamil nadu, India (MG720636) and Sudan (MG692441), respectively. In conclusion, this study provided information on the molecular detection and phylogeny of C. pseudotuberculosis in Egypt. Findings of this study can be conducted in other CLA endemic countries with similar animal breeding practices in the Middle East and developing countries.

Abattoir survey of bovine tuberculosis in tanta, centre of the Nile delta, with in silico analysis of gene mutations and protein-protein interactions of the involved mycobacteria.

Bovine tuberculosis is a transboundary disease of high economic and public health burden worldwide. In this study, post-mortem examination of 750 cattle and buffalo in Tanta abattoir, Centre of the Nile Delta, revealed visible TB in 4% of animals and a true prevalence of 6.85% (95% CI: 5.3%-8.9%). Mycobacterial culture, histopathology and RT-PCR targeting all members of M. tuberculosis complex were performed, upon which 85%, 80% and 100% of each tested lesions were confirmed as TB, respectively. Mpb70-targeting PCR was conducted on ten RT-PCR positive samples for sequencing and identified nine Mycobacterium (M.) bovis strains and, interestingly, one M. tuberculosis (Mtb) strain from a buffalo. Bioinformatics tools were used for prediction of mutations, nucleotide polymorphisms, lineages, drug resistance and protein-protein interactions (PPI) of the sequenced strains. The Mtb strain was resistant to rifampicin, isoniazid and streptomycin, and to the best of our knowledge, this is the first report of multidrug resistant (MDR)-Mtb originating from buffaloes. Seven M. bovis strains were resistant to ethambutol and ethionamide. Such resistances were associated with KatG, rpoB, rpsL, embB and ethA genes mutations. Other mutations and nucleotide polymorphisms were also predicted, some are reported for the first time and require experimental work for validation. PPI revealed more interactions than what would be expected for a random set of proteins of similar size and had dense interactions between nodes that are biologically connected, as a group. Two M. bovis strains belonged to BOV AFRI lineage (Spoligotypes BOV 1; BOV 2) and eight strains belonged to East-Asian (Beijing) lineage. In conclusion, visible TB was prevalent in the study area, RT-PCR is the best to confirm the disease, MDR-Mtb is associated with buffalo TB, and mycobacteria of different lineages carry many resistance genes to chemotherapeutic agents used in treatment of human TB constituting a major public health risk.

Characterization of Campylobacter jejuni isolated from dogs and humans using flaA-SVR fragment sequencing in Ismailia, Egypt.

INTRODUCTION: Dogs are known as asymptomatic carriers forCampylobacter jejuni. The number of pet dogs is increasing in Egypt in the last decade. OBJECTIVE: This study aimed to investigate the frequency ofC. jejuni infection in dogs and humans, molecular typing of associated virulence genes, and flaA-SVR gene using sequencing. METHODOLOGY: 152 unpaired fecal swabs from dogs (n = 72) and humans (80) were examined for the presence of C. jejuni and Campylobacter 23S rRNA, and the pathogenicity genes including mapA genes, virB11, flaA, wlaN, iam, tetO, and aadA genes. Sequencing of the flaA- amplicon was also performed for the representative isolates. RESULTS: The isolation rate ofC. jejuni was 20.8 % and 31.2 %, respectively in dogs and humans, and all isolates were tested positive for 23S rRNA and mapA genes. C. jejuni harbor virB11 and wlaN (20 %, 0%), iam (10 %, 20 %), tetO and aadA1 (40 %, both), and flaA (40 %, 20 %) in human and dog strains, respectively. The flaA-SVR sequences revealed high identity between human and dog isolates (94.8 %), but revealed 18 substitutions in the amino acid sequence, and showed that the dog and human C. jejuni were close to strains isolated from human and poultry sources. CONCLUSION: this study demonstrated the comparative sequence analysis ofC. jejuni flaA-SVR fragment in dogs and some Egyptians, which indicated a high identity percentage between them. The results suggest that C. jejuni reservoirs dogs is an alarming public health concern and effective hygienic measures are necessary for house-holding pets to prevent C. jejuni zoonosis in Egypt's community.

Prevalence, Antimicrobial Susceptibility, Virulence and Genotyping of Campylobacter jejuni with a Special Reference to the Anti-Virulence Potential of Eugenol and Beta-Resorcylic Acid on Some Multi-Drug Resistant Isolates in Egypt.

Campylobacter jejuni is the leading cause of foodborne bacterial gastroenteritis in humans worldwide. Contaminated chickens and their products are the main sources of human campylobacteriosis. Therefore, this study aimed to detect the genotypic and virulence genes' profiles of multi-drug resistant (MDR) C. jejuni isolates and to assess the effects of sub-inhibitory concentrations (SICs) of eugenol and beta-resorcylic acid on the virulence of avian MDR C. jejuni isolates. These isolates were clustered together with the human isolates via enterobacterial repetitive intergenic consensus-PCR (ERIC-PCR) fingerprinting. A total of 345 samples were collected from human stool (100) and different chicken (245) samples in Sharkia Governorate, Egypt. Conventional phenotypic methods identified 113 isolates (32.8%) as C. jejuni, and all C. jejuni isolates were MDR and resistant to erythromycin and ampicillin. The genes virB11, wlaN, and flaA were detected in 52%, 36% and 100% strains, respectively. ERIC-PCR yielded 14 profiles and five main clusters. Interestingly, human and chicken C. jejuni isolates were clustered together in ERIC-PCR clusters II-V, which confirmed the genetic relatedness between the isolates from both origins. Beta-resorcylic acid and eugenol inhibited the invasion of C. jejuni isolates to chicken intestinal cells by 41.66-38.19% and 31.94-29.16%, respectively, and minimized the transcription of flaA, virB11, and wlaN genes in the tested isolates by real-time quantitative reverse transcription PCR (qRT-PCR). In essence, eugenol and beta-resorcylic acid are promising natural antimicrobials for minimizing the virulence of MDR C. jejuni in chickens, thereby managing human campylobacteriosis.

Seroprevalence and molecular characterization of Mycobacterium bovis infection in camels (Camelus dromedarius) in the Delta region, Egypt.

AIM: This study aimed to determine the prevalence rates of Mycobacterium infection in camel sera collected before slaughter and gross lesion tissue collected postmortem (PM) using enzyme-linked immunosorbent assay (ELISA), bacteriological culture, and polymerase chain reaction (PCR). In addition, serum samples from humans who had occupational contact with camels were tested by ELISA and sputum sample by culture. MATERIALS AND METHODS: ELISA was performed on serum samples antemortem. In addition, bacteriological culture and PCR were conducted after PM. Tuberculosis infection was identified in humans who had contact with camels using ELISA for serum samples and culture for sputum samples. RESULTS: Tuberculous lesions were detected in 184 of 10,903 camels (1.7%). The ELISA results revealed that of the 184 examined camel serum samples, 124 (67.39%) were positive and all 20 camel serum samples that had no associated tuberculous lesions were negative. Moreover, only one of 48 (2.08%) human serum samples was positive by ELISA. Mycobacterial culture revealed 112 isolates from the 184 examined camel samples (60.87%), while human sputum sample cultures were all negative. PCR analysis identified the mpb70 gene in three of seven randomly tested samples. CONCLUSION: Gene sequencing was performed on two samples and the sequences were submitted to the National Center for Biotechnology Information GenBank (accession numbers MF990289 and MG59479). A phylogenetic tree was constructed based on the partial DNA sequences of the mpb70 gene; the similarity between the isolates was 98.1%. The similarities between the two isolates and the standard strains of Mycobacterium bovis in GenBank were 98.1% and 100%, respectively. Further investigation on the antemortem detection of M. bovis infection in camels is needed to decrease public risk.

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.