Application of MALDI-TOF MS fingerprinting as a quick tool for identification and clustering of foodborne pathogens isolated from food products.

Foodborne pathogens can be associated with a wide variety of food products and it is very important to identify them to supply safe food and prevent foodborne infections. Since traditional techniques are timeconsuming and laborious, this study was designed for rapid identification and clustering of foodborne pathogens isolated from various restaurants in Al-Qassim region, Kingdom of Saudi Arabia (KSA) using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). Sixty-nine bacterial and thirty-two fungal isolates isolated from 80 food samples were used in this study. Preliminary identification was carried out through culture and BD Phoenix™ methods. A confirmatory identification technique was then performed using MALDI-TOF MS. The BD Phoenix results revealed that 97% (67/69 isolates) of bacteria were correctly identified as 75% Enterobacter cloacae, 95.45% Campylobacter jejuni and 100% for Escherichia coli, Salmonella enterica, Staphylococcus aureus, Acinetobacter baumannii, and Klebsiella pneumoniae. While 94.44% (29/32 isolates) of fungi were correctly identified as 77.77% Alternaria alternate, 88.88% Aspergillus niger and 100% for Aspergillus flavus, Penicillium digitatum, Candida albicans and Debaryomyces hansenii. However, all bacterial and fungal isolates were 100% properly identified by MALDI-TOF MS fingerprinting with a score value ≥2.00. A gel view illustrated that the spectral peaks for the identified isolates fluctuate between 3,000 and 10,000 Da. The results of main spectra library (MSP) dendrogram showed that the bacterial and fungal isolates matched with 19 and 9 reference strains stored in the Bruker taxonomy, respectively. Our results indicated that MALDI-TOF MS is a promising technique for fast and accurate identification of foodborne pathogens.

Pathology and molecular diagnosis of paratuberculosis of camels.

Camels are the prime source of meat and milk in many desert regions of the world including Saudi Arabia. Paratuberculosis of camels, locally called Silag, is a serious and invariably fatal disease in the Arabian camel. Six camels were used in this study. Five camels with clinical paratuberculosis were used to study the pathology of the disease and confirm its aetiology. The sixth camel was clinically healthy and used as a control. The camels were examined clinically and bled for haematological and blood chemistry analysis. They were then humanely killed with a high intravenous dose of thiopental sodium (10 mg/kg) for pathological studies as well as obtaining tissues for microbiological and molecular studies. The clinical signs of the disease were emaciation, diarrhoea, alopecia, wry neck and pale mucous membranes. Laboratory diagnosis showed reduced haemoglobin concentration, low haematocrit and high activity of the serum enzyme alanine aminotransferase. Serum creatinine concentration was normal. These results indicated the infected camels were anaemic and the function of their livers was affected. Postmortem examination showed thickened and corrugated intestinal mucosa, enlarged granulomatous mesenteric lymph nodes, miliary and diffuse granulomas in the liver (in four camels), generalized lymph node granulomas (in one camel), splenic granuloma (in one camel) and mediastinal lymph node granuloma (in two camels). Histopathological examination showed diffuse infiltration of macrophages in all organs showing lesions. Ziehl-Neelsen staining of tissue scraping and tissue sections showed masses of acid fast bacilli, except for the spleen. Infection with Mycobacterium avium subsp. paratuberculosis was confirmed by PCR by targeting the IS900 gene.

First report on the isolation of Chlamydia abortus from female dromedary camels with ovarian hydrobursitis.

This study sought to isolate Chlamydia abortus (C. abortus) from camels with ovarian hydrobursitis (OVHB). To accomplish this goal, bursal tissue (n = 5) and bursal fluid (n = 6) samples were collected from 11 female dromedary camels with unilateral OVHB. A quantitative polymerase chain reaction (qPCR) was used for the preliminary detection of C. abortus in the infected samples. For the purpose of isolation, the prepared samples were inoculated into embryonated chicken eggs. Giemsa, Gimenez, and direct immunofluorescence (DIF) staining were used to detect any chlamydial inclusions in the infected yolk sacs. A second qPCR was then performed on the infected yolk sacs. The C. abortus gene was detected in 83.8% of the infected bursal tissue and bursal fluid samples. All the yolk sac smears treated with Giemsa, Gimenez, and DIF staining revealed intracytoplasmic inclusion bodies. Moreover, hemorrhagic patches, massive congestion, macerated yolk sacs, and dwarfism were observed in the infected chicken embryos. The C. abortus gene was also found in 63.6% of the infected yolk sacs. In conclusion, this is the first report of C. abortus isolation from female dromedary camels with OVHB, which represents a key step toward developing a practical vaccine and avoiding fertility problems in female camels.

Antibacterial effects and resistance induction of silver and gold nanoparticles against Staphylococcus aureus-induced mastitis and the potential toxicity in rats.

Staphylococcus aureus (S. aureus) is one of the prevalent mastitis-inducing pathogens worldwide. The resistance of S. aureus to antibiotics is a common issue for dairy farms. Recently, nanoparticles (NPs) have been used for treating antibiotic-resistant bacteria. We therefore aimed to investigate the antimicrobial effect of silver and gold NPs (AgNPs and AuNPs, respectively) and the resistance developed by S. aureus as well as the toxic effects of both NPs in rats. We used 198 S. aureus strains to determine the antibacterial effects of AgNPs and AuNPs. The microdilution method was used to establish the minimum inhibitory concentrations (MICs) of both NPs. To induce resistance, 20 S. aureus strains were passaged 10 times in broth medium with sublethal doses of NPs and an additional 10 times without NPs to examine the stability of resistance. Histopathology was performed after oral administration to the rats with the study doses of 0.25, 0.5, 1, and 2 mg/kg of NPs for 30 days. The MICs of 10-nm AgNPs, 20-nm AgNPs, 10-nm AuNPs, and 20-nm AuNPs against S. aureus were 14.70 ± 1.19 µg/ml, 9.15 ± 0.13 µg/ml, 24.06 ± 2.36 µg/ml, and 18.52 ± 1.26 µg/ml, respectively. Most strains developed strong resistance when treated with 20-nm or 10-nm AgNPs, whereas only two strains were resistant to 10-nm AuNPs and three strains to 20-nm AuNPs. No cross-resistance between NPs and various antibiotics was identified in any of the adapted S. aureus strains. Organ histopathology revealed that 0.25, 0.5, and 1 mg/kg doses of AgNPs and AuNPs were not toxic to rat tissue. In contrast, a higher dose (2 mg/kg) of NPs impaired all organs tested. This study demonstrates the antibacterial effects of NPs. S. aureus strains develop resistance less frequently against AuNPs than AgNPs, and neither AuNPs nor AgNPs was toxic to rats at low doses.

Proteomic characterization and discrimination of Aeromonas species recovered from meat and water samples with a spotlight on the antimicrobial resistance of Aeromonas hydrophila.

Aeromonas is recognized as a human pathogen following ingestion of contaminated food and water. One major problem in Aeromonas identification is that certain species are phenotypically very similar. The antimicrobial resistance is another significant challenge worldwide. We therefore aimed to use mass spectrometry technology for identification and discrimination of Aeromonas species and to screen the antimicrobial resistance of Aeromonas hydrophila (A. hydrophila). A total of 150 chicken meat and water samples were cultured, and then, the isolates were identified biochemically by the Vitek® 2 Compact system. Proteomic identification was performed by MALDI-TOF MS and confirmed by a microchannel fluidics electrophoresis assay. Principal component analysis (PCA) and single-peak analysis created by MALDI were also used to discriminate the Aeromonas species. The antimicrobial resistance of the A. hydrophila isolates was determined by Vitek® 2 AST cards. In total, 43 samples were positive for Aeromonas and comprised 22 A. hydrophila, 12 Aeromonas caviae (A. caviae), and 9 Aeromonas sobria (A. sobria) isolates. Thirty-nine out of 43 (90.69%) Aeromonas isolates were identified by the Vitek® 2 Compact system, whereas 100% of the Aeromonas isolates were correctly identified by MALDI-TOF MS with a score value ≥2.00. PCA successfully separated A. hydrophila, A. caviae and A. sobria isolates into two groups. Single-peak analysis revealed four discriminating peaks that separated A. hydrophila from A. caviae and A. sobria isolates. The resistance of A. hydrophila to antibiotics was 95.46% for ampicillin, 50% for cefotaxime, 45.45% for norfloxacin and pefloxacin, 36.36% for ceftazidime and ciprofloxacin, 31.81% for ofloxacin and 27.27% for nalidixic acid and tobramycin. In conclusion, chicken meat and water were tainted with Aeromonas spp., with a high occurrence of A. hydrophila. MALDI-TOF MS is a powerful technique for characterizing aeromonads at the genus and species levels. Future studies should investigate the resistance of A. hydrophila to various antimicrobial agents.

Performance of MALDI biotyper compared with Vitek™ 2 compact system for fast identification and discrimination of Staphylococcus species isolated from bovine mastitis.

This study was designed to evaluate the ability of MALDI Biotyper (MBT) compared with Vitek™ 2 compact system for accurate identification of Staphylococcus aureus (S. aureus) and coagulase-negative staphylococci (CNS) strains and discriminate methicillin-sensitive S. aureus (MSSA) from methicillin-resistant S. aureus (MRSA). Throughout Al-Qassim region, Saudi Arabia, a total of 198 isolates of S. aureus (132 MSSA and 66 MRSA) and 44 CNS were collected from five dairy farms where the prevalence of staphylococcal mastitis was reported. The results produced by Vitek™ 2 compact system demonstrated that 123/132 MSSA isolates (93.18%), 61/66 MRSA (92.42%), and 37/44 CNS species (84.09%) were correctly identified. However; 130/132 MSSA (98.48%), 64/66 MRSA (96.96%), and 44/44 CNS (100%) were correctly identified by MBT with score ≥2. 00. The principal component analysis (PCA) dendrogram generated by MBT illustrated that the tested isolates were classified into two groups of Staphylococcus species at the distance level of 600. S. aureus isolates were found to be closely related with higher peak intensities in the mass of 3,993 Da, 4,121 Da and 5,845 Da were detected in MRSA, whereas, that were lost in MSSA. CONCLUSION: This study verified that MBT is an alternative powerful tool for precise identification and discrimination of Staphylococcus species.

Chimpanzee Adenovirus Vaccine Provides Multispecies Protection against Rift Valley Fever.

Rift Valley Fever virus (RVFV) causes recurrent outbreaks of acute life-threatening human and livestock illness in Africa and the Arabian Peninsula. No licensed vaccines are currently available for humans and those widely used in livestock have major safety concerns. A 'One Health' vaccine development approach, in which the same vaccine is co-developed for multiple susceptible species, is an attractive strategy for RVFV. Here, we utilized a replication-deficient chimpanzee adenovirus vaccine platform with an established human and livestock safety profile, ChAdOx1, to develop a vaccine for use against RVFV in both livestock and humans. We show that single-dose immunization with ChAdOx1-GnGc vaccine, encoding RVFV envelope glycoproteins, elicits high-titre RVFV-neutralizing antibody and provides solid protection against RVFV challenge in the most susceptible natural target species of the virus-sheep, goats and cattle. In addition we demonstrate induction of RVFV-neutralizing antibody by ChAdOx1-GnGc vaccination in dromedary camels, further illustrating the potency of replication-deficient chimpanzee adenovirus vaccine platforms. Thus, ChAdOx1-GnGc warrants evaluation in human clinical trials and could potentially address the unmet human and livestock vaccine needs.

In vivo expression of GFP transgene delivered via a replicating feline leukemia virus.

We previously described replication-competent feline leukemia virus (FeLV) vectors with high-level and stable expression of a green fluorescent protein (GFP) or a suicide transgene in cell cultures in vitro. Considering that FeLV might potentially be used to deliver therapeutic genes in vivo, we first evaluated the expression of the GFP gene introduced in cats by the FeLV, Rickard subgroup A (FRA) construct. Eight newborn kittens were either inoculated with pFRA-GFP plasmid DNA intradermally, or challenged intraperitoneally with FRA-GFP-infected feline fibroblasts. During a 12-week observation period, five cats were shown to be progressively viremic. Quantitative PCR and RT-PCR analyses of plasma and tissue samples from these cats showed that GFP was retained in FeLV DNA or RNA to a variable degree, ranging from 0.002 to 27.890%. Tissue DNA samples were analyzed by PCR for the status of GFP and the env-transgene complex. While the proviruses carrying the GFP transgene were shown to be minor species, all tissues, however, retained the full-length GFP transgene. Despite the occurrence of predominant species with various deletions in the viral genome, approximately 1-3% of the total cell population was GFP-positive in the lymphoid tissues as visualized by laser confocal microscopy. Co-localization of immunofluorescent cells indicated that CD3-positive T cells, dendritic cells and macrophages were the major targets for GFP expression. These findings on the detectable in vivo expression of GFP for as long as a period of 3 months could be viewed positively for contemplating a therapeutic strategy for control of FeLV infection in the cats.

Genome-wide analysis of the emerging infection with Mycobacterium avium subspecies paratuberculosis in the Arabian camels (Camelus dromedarius).

Mycobacterium avium subspecies paratuberculosis (M. ap) is the causative agent of paratuberculosis or Johne's disease (JD) in herbivores with potential involvement in cases of Crohn's disease in humans. JD is spread worldwide and is economically important for both beef and dairy industries. Generally, pathogenic ovine strains (M. ap-S) are mainly found in sheep while bovine strains (M. ap-C) infect other ruminants (e.g. cattle, goat, deer), as well as sheep. In an effort to characterize this emerging infection in dromedary/Arabian camels, we successfully cultured M. ap from several samples collected from infected camels suffering from chronic, intermittent diarrhea suggestive of JD. Gene-based typing of isolates indicated that all isolates belong to sheep lineage of strains of M. ap (M. ap-S), suggesting a putative transmission from infected sheep herds. Screening sheep and goat herds associated with camels identified the circulation of this type in sheep but not goats. The current genome-wide analysis recognizes these camel isolates as a sub-lineage of the sheep strain with a significant number of single nucleotide polymorphisms (SNPs) between sheep and camel isolates (∼1000 SNPs). Such polymorphism could represent geographical differences among isolates or host adaptation of M. ap during camel infection. To our knowledge, this is the first attempt to examine the genomic basis of this emerging infection in camels with implications on the evolution of this important pathogen. The sequenced genomes of M. ap isolates from camels will further assist our efforts to understand JD pathogenesis and the dynamic of disease transmission across animal species.