Hygiene and Zoonoses

This study was conducted to determine the antimicrobial activity of methanol and ethanol extracts
of peels of pomegranate (Punica grana), orange (Citrus siensis) and lemon (Limona taris) against four
foodborne pathogens (Listeria monocytogenes, Salmonella Typhimurium, Escherichia coli and methicillinresistant
Staphylococcus aureus (MRSA) and a food spoilage bacterium (Pseudomonas fluorescens.
Inhibition tests were conducted in vitro using the disc diffusion and minimum inhibition concentration (MIC)
assays with the Bioscreen Microbiology analyzer. The study also evaluated the antimicrobial activity of the
extracts in situ by determining CFU/ml of bacteria recovered from rinsates of chicken skin treated with the
peel extracts and by examining the microflora of treated skin samples using scanning electron microscopy
(SEM). The antimicrobial activity of all extracts, except the pomegranate ethanol extract, were dependent on
the concentration of extract that the bacteria were exposed to during the trials. Treating the inoculated chicken
skin with 5 mg/ml of either the five extracts produced significant (p<0.01) reductions in CFU/ml of MRSA, L.
monocytogenes and P. fluorescens recovered and the MRSA findings were supported by SEM observations.
The antimicrobial activity of peel extracts of pomegranate, orange and lemon indicates that these extracts
may be used as sanitizers to reduce microbial contamination of some foods and processing.

The resurgence of Tuberculosis (TB) around the world has renewed interest in
understanding the epidemiology of TB. This study was conducted to elucidate the light
on molecular diagnostic techniques for identification and differentiation of M. bovis and
M. tuberculosis in comparison to conventional culturing. Twenty three lymph nodes
collected from tuberculin positive cattle tested by tuberculin skin test, suspected
tuberculous cattle (non tuberculin tested) and from apparently healthy animals were
screened for HupB gene of M. bovis by N-S PCR. From human, a total of 24 sputum
samples (16 from clinically diagnosed patients and 8 from apparently healthy persons)
were tested by PncA PCR. Also, 6 apparently healthy sputum samples were screened by
N-S PCR for HupB gene of M. bovis. No M. bovis was isolated from sputum by
conventional culturing method. Using N-S PCR, 618 bp was detected in infected animal
samples. While PncA PCR detected 185 bp fragment from human sputum samples.
PCR results were in concordance to that of conventional culturing on LJ except for
samples that yielding no isolates. One sample from 6 apparently healthy sputa give 618
bp fragment upon screening with N-S PCR. Digestion pattern of HpaII enzyme on M.
bovis and M. tuberculosis PCR products detected 252 and 279 bp fragments,
respectively. In conclusion, this study suggested that PCR offers the potential for the
diagnosis of TB in a few hours and differentiation between mycobacteria by PCR-RFLP
allowing appropriate control, treatment and reduces the emergence of drug resistance.

Foodborne pathogens remain global health problems despite concerted efforts to control the transmission of these microorganisms through food. The resurgence of drug resistant bacteria has renewed interest in developing and testing new sources of antimicrobial agents to control foodborne illness. This study was conducted to determine the antimicrobial activity of extract of Citrus sinensis (sweet orange) against some foodborne pathogenic bacteria such as, Methicillin-resistant Staphylococcus aureus (MRSA), Listeria monocytogenes, Salmonella Typhimurium, E. coli, and the food spoilage bacteria, Pseudomonas fluorescens. The antimicrobial activity of C. sinensis extracts was determined using the agar diffusion method and minimum inhibitory concentration (MIC) of the extract was performed using the Bioscreen C Microbiology Reader. Additionally, antibacterial activity of extract was evaluated on spiked chicken skin using changes in bacterial load and scanning electron microscopy (SEM). The results showed that the average diameter of zones of inhibition of bacteria by C. sinensis extract at a concentration of 100 mg/ml for MRSA, L. monocytogenes, S. Typhimurium, E. coli, and P. fluorescens were 13.8 mm, 25.56 mm, 12.1, 7.8 mm, and 24.3 mm respectively. At a concentration of 10 mg/ml, the average zone of inhibition was 10 mm, 20.96 mm, 8.6 mm, 0 mm, and 20.3 mm for MRSA, L. monocytogenes, S. Typhimurium, E. coli, and P. fluorescens, respectively. However, solutions of 1 mg/ml of extract produced no zones of inhibition against MRSA, S. Typhimurium or E. coli. The MIC concentrations of the extract were 5 mg/ml, 1.25 mg/ml and 1.25 mg/ml for MRSA, L. monocytogenes, and P. fluorescens, respectively. MIC for S. Typhimurium and E. coli was ≥10 mg/ml. The bacterial population for MRSA, L. monocytogenes, and P. fluorescens recovered from rinsates of inoculated chicken skin treated with a solution of 5 mg of extract/ml were reduced from 8 log CFU/ml to 4.5~4.7 log CFU/ml while there was only a 1.1 log CFU/ml reduction in the number of Salmonella and E. coli recovered from the treated skin. Abnormalities of the bacterial cell morphology were detected by SEM as indication of antimicrobial activity of C. sinensis. This study shows the potential of a natural plant extract for use as a sanitizer to control foodborne pathogens in vitro and on poultry skin.

Due to culturability requirements encountered by the conventional isolation of Campylobacter spp.,
rapid molecular techniques for its direct identification from clinical samples are useful. In this study,
Campylobacter spp. DNA from human stool and chicken fecal samples were detected by flagellin gene (flaA)
PCR. A total of 297 samples consisting of 163 adult human stools (102 from diarrheic patients and 61 from
healthy persons) and 134 chicken feces were subjected to flaA PCR. Ten reference strains of Campylobacter
spp. were included in this study as positive controls. Thirteen stool samples (7.98%) from the human fecal
samples and 39 chicken fecal samples (29.1%) yielded the genus specific 1.7 Kb amplicon of
Campylobacter spp. Eight (7.84%) diarrheic human stool specimens out of 102 samples and 5 (8.2%)
apparently healthy human stool specimens out of 61 samples were positive by flaA PCR assay. All the
Campyloacter reference strains examined giving the specific amplicon of 1.7 Kb. The existence of
Campylobacter spp. DNA detected by flaA PCR in poultry and human samples taken from locations of Egypt
highlights the zoonotic potential of Campylobacter. To the best of our knowledge, this is the first report in
Egypt that uses flaA PCR as a rapid screening method for the direct detection of Campylobacter spp. from
human and chicken feces.

This study was undertaken to evaluate the comparison among three different assays: extendedspectrum
beta-lactamases (ESBL) Nordmann/ Dortet/ Poirel (NDP) test, flow cytometric assay and disc
diffusion method for the detection of ESBL production. Sixty clinical isolates of Klebsiella pneumoniae
were isolated from patients’ clinical samples admitted to Suez-Canal University Hospital, Ismailia
Governorate. The percentages of ESBLs producing Klebsiella pneumoniae ranged from 70 to 80% by
ESBL NDP and flow cytometric assays, respectively in comparison to 76.6% by disc diffusion method.
The sensitivity and specificity of the three assays were evaluated and the sensitivity by ESBL NDP and
disc diffusion method was 100%, while by the flow cytometric assay, it was 91.3%. The specificity of
disc diffusion method in detection of ESBLs was 100%, followed by the ESBL NDP test (85.7%) and flow
cytometric assay (77.8%). Kappa testing showed perfect agreement between the ESBL NDP test and
disc diffusion method (kappa=0.9), while flow cytometric assay showed substantial agreement (kappa=
0.7). The ESBL NDP test offers an applicable tool for rapid detection of ESBL-production. Although,
flow cytometric assay is a promising method that might be used in the clinical microbiology laboratory
but there is a need for the experienced personnel along with the device.

This study was conducted to determine the occurrence, antimicrobial resistance profile, β-lactamase encoding genes, and class I integrons (intI) of Salmonella serovars in broiler flocks. A total of 100 diseased chickens (5 samples per bird; cloacal swab, liver, gall bladder, spleen, and intestinal content) were randomly taken from different broiler farms at Dakahliya and Kafrelsheikh Governorates, Egypt, during the period from September through December 2013. Conventional isolation and serotyping, antimicrobial resistance phenotyping, PCR identification of β-lactamase encoding genes and intI were performed. The culturing and serotyping identified 23 (23%) Salmonella isolates from diseased birds that belonged to 13 serotypes. The predominant serovars distinguished in this study were Salmonella Enteritidis, S. Typhimurium , S. Kentucky and S. Infantis that constituted 52.2% (12/23) of all isolates. By antimicrobial resistance testing, 87% (20/23) of isolates exhibited multidrug resistance (MDR; resistance to 5 or more antibiotics) mostly against vancomycin, oxacillin, amoxicillin, erythromycin, and nalidixic acid. For 3rd generation cephalosporins, all the isolates were sensitive to cefoxitin and only 5 (21.7%) isolates displayed resistance to ceftriaxone and cefotaxime. Using PCR, all isolates were negative for blaSHV, blaCTX, blaCMY and blaOXA, while only 5 isolates (21.7%) harbored blaTEM (1080 bp). Variable amplicons with int1 cassette were detected by PCR from only 4 isolates (17.4%). Our findings highlighted the zoonotic potential of Salmonella in broilers with a possibility of antimicrobial resistance gene transmission to humans. Continuous surveillance is required to minimize the risk of human exposure to antimicrobial resistance pathogens from food producing animals.

Introduction: The purpose from this study was to determine phenotypes, intestinal virulence-associated genes, and phylotypic profiling of human diarrheagenic E. coli (DEC) and avian pathogenic E. coli (APEC). Methodology: A total of 108 chicken visceral organs (liver, spleen, heart) from 36 diseased birds (three organs per each bird) and 78 human stool samples (50 diarrheic patients and 28 healthy persons) were randomly collected during the first half of 2015 in the district of Mansoura city, Egypt. Conventional culturing, serotyping, and molecular characterization of virulence genes and phylogroups were performed. Results: Sixty-five (35%) biochemically identified E. coli isolates were detected from chicken visceral (29/108; 26.9%) and human stool samples (36/78; 46.2%). Serotypes O78, O2, and O1 were the most prevalent serotypes (62%) distinguished from APEC isolates, and only two similar serotypes (O119:H4 and O26:H11) were identified from both APEC and DEC isolates. By polymerase chain reaction (PCR), the respective percentages of 100 and 35 with eae and Shiga toxin genes were detected from APEC isolates while 50%, 27.8%, and 19.4% of human DEC isolates harbored eae, stx1, and stx2 genes, respectively. Phylogrouping revealed a significantly higher occurrence of pathogenic phylogroups (D and B2) in APEC (19/29; 65.5%) than in human DEC isolates (8/36; 22.2%). Conclusions: APEC isolates shared serotypes, virulence genes, and phylotypes with human DEC isolates, which is a subsequent potential public health concern. To the best of our knowledge, this is the first report in Egypt that determines virulence gene and phylogroup coexistence between APEC and DEC isolates.

Relatedness between single nucleotide polymorphisms in IL8 and TLR4 genes and digital dermatitis
resistance/susceptibility was investigated in seventy Holstein dairy cows. Animals were assigned
into two groups, affected group (n=35) and resistant group (n=35) based on clinical signs
and previous history of farm clinical records. Blood samples were collected for DNA extraction to
ampliy fragments of 267-bp and 382-bp for IL8 and TLR4 genes, respectively. PCR-DNA sequencing
revealed three SNPs in each of IL8 and TLR4 genes. The identified SNPs associated with digital
dermatitis resistance were C94T, A220G, and T262A for IL8 and C118T for TLR4. However,
the G349C and C355A SNPs in TLR4 gene were associated with digital dermatitis susceptibility.
Chi-square analysis for comparison the distribution of all identified SNPs in both IL8 and TLR4
genes between resistant and affected animals showed no significant variation among the identified
SNPs in IL8 gene. Meanwhile, there was a significant variation in case of TLR4 gene. As a pilot study,
the present results revealed that identified SNPs in IL8 and TLR4 genes can be used as a genetic
marker and predisposing factor for resistance/susceptibility to digital dermatitis in dairy cows.
However, TLR4 gene may be a potential candidate for such disease.

The contribution of camels and their products (meat and offal) as potential reservoirs of foodborne pathogens is far from elucidated. Therefore, the purpose of this study was to investigate the contribution of camels as potential sources of different foodborne agents and to determine their antibiotic resistance profile. For this reason, one hundred apparently healthy camels admitted to the Zagazig abattoir (Sharkia province, Egypt) for slaughtering were studied. Three different specimens were collected from each camel including fresh faeces, raw meat and liver which were processed to determine the presence of different foodborne pathogens using standard bacteriological techniques. Antimicrobial susceptibility testing was also performed using the disc diffusion method. The occurrence of Campylobacter in the faeces, raw meat and liver were 20%, 33% and 15%, respectively, with the most prevalent C. coli (21%) followed by C. jejuni (0.7%) and C. lari (0.7%). Enterococcus spp. was isolat...

AimTo gain deeper insight into the seroprevalence of brucellosis, which remains a zoonotic disease of worldwide public health concern, by reviewing studies from countries including North Africa, the Middle East, and India.MethodsStudies on brucellosis performed in countries that are neighbors or important trading partners of the European Union and on trade animals and their products were analyzed. We reviewed 37 seroprevalence studies on brucellosis published from 1948 to 2009 retrieved from PubMed, Google, and ScienceDirect.ResultsThe set of studies was heterogeneous in the number of samples and laboratory tests used. We included studies from Algeria (n = 1), Egypt (n = 7), India (n = 3), Iran (n = 3), Iraq (n = 1), Jordan (n = 5), Libya (n = 3), Saudi Arabia (n = 3), Syria (n = 1), Turkey (n = 5), and Yemen (n = 2). The total number of animals in these studies was 116 317 (cattle 75 375; buffalo 9644; sheep 10 550; goats 14 447; camels 6301). The prevalence of brucellosis in different animal species varied widely. Representative surveillance data have not recently been published in any of the countries.ConclusionsWars in the Middle East, insufficient preventive measures, the lack of adequate control programs in some countries, as well as uncontrolled animal transportation through “open” borders increased the risk that brucellosis will spread in some regions. New seroprevalence data are needed urgently to evaluate the current situation and for continuous monitoring of necessary control programs.

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 s...

Burkholderia mallei (B. mallei) is the etiological agent of glanders, a highly infectious and zoonotic disease of solipeds. Prompt and accurate detection of B. mallei and diagnosis of glanders is important for both, humans and animals. The present study was designed to evaluate the analytical sensitivity and specificity of a commercially available one-step real-time (RT) PCR assay kit (Genekam Biotechnology AG, Germany) for the detection of B. mallei. Furthermore, the comparative evaluation of the analytical sensitivity of an already published fliP RT-PCR protocol and this kit was made using a real-time PCR (RT-PCR) platform: the Mx3000P TM (Stratagene®, Canada). Diagnostic parameters were assessed with a panel of 20 B. mallei, 20 B. pseudomallei, 15 B. mallei related and 10 clinically relevant non-Burkholderia species. Using the one-step RT-PCR on the Mx3000PTM platform, the limit of detection (LOD) was determined as 10 fg. Applying a modified fliP RT-PCR using the Mx3000P platform...