Prevalence of antimicrobial-resistant bacteria in conventional vs. organic livestock farms in Egypt: a cross-sectional comparative study.

The silent pandemic of antimicrobial resistance (AR) has been on the rise for the past decades. It is essential to determine the burden of AR in animal farms that spreads leading to human exposure. A total of 100 samples including soil, litter, animal excreta, and wastewater were collected from seven conventional and one organic farm in Egypt. The prevalence of extended-spectrum ß-lactamase-producing Escherichia coli (ESBL-producing E. coli), fluoroquinolone-resistant E. coli, fluoroquinolone-resistant Salmonella, and vancomycin-resistant enterococci (VRE) was determined in studied farms. Conventional farms had a higher prevalence of antimicrobial-resistant bacteria than the organic farm (73.81% vs. 18.75%, P < .001). In conventional farms 21.43% of samples yielded mixed isolates; however, in the organic farm, only single isolates of ESBL-producing E. coli were detected. The most prevalent ESBL-production gene was blaTEM (82.14%), followed by blaCTX-M (48.22%), and blaSHV (19.64%), either alone or in combination with another gene. The most prevalent fluoroquinolone-resistance genes were qnrS (82.69%) and qnrB (42.30%), either alone or in combination with another gene(s). A total of five VRE isolates harbored vanA gene (83.33%), none carried vanB gene, and one isolate was negative for both genes. The studied conventional livestock farms had significantly higher rates of serious AR threats than the organic farm.

In vitro synergistic activity of colistin and teicoplanin combination against multidrug-resistant Acinetobacter spp.

Drug combinations may have a crucial role in treating infections due to multidrug resistant Acinetobacter spp. One suggested combination is colistin with teicoplanin. The effect of colistin on Acinetobacter spp. outer membrane can permit teicoplanin to its target in the cell wall. The aim of this study was to evaluate the synergistic activity of colistin and teicoplanin combination against 29 multidrug resistant isolates of Acinetobacter spp. The antimicrobial activity of colistin alone and in combination with teicoplanin was assessed using MIC and time-kill assays. The combination of 1 mg/l colistin and 10 mg/l teicoplanin showed in vitro synergism against all tested Acinetobacter isolates except one (Acinetobacter lowffii). The combination of 1 mg/l colistin and 10 mg/l teicoplanin was bactericidal at 6 h against 100% of Acinetobacter baumannii isolates with no bacterial regrowth at 24 h. The same combination was bactericidal against three out of seven non-baumannii Acinetobacter isolates. The increased concentration of teicoplanin (20 mg/l) was synergistic but still not bactericidal against the four remaining isolates. The combination of colistin and teicoplanin was synergistic against all tested Acinetobacter spp It is therefore recommended that clinical trials are conducted to clarify the therapeutic potential of the combination.

Bilayered Medium for Rapid Isolation of Mycobacterium tuberculosis.

BACKGROUND: A bilayered medium (BLM) seemed to indicate a rapid recovery time from Mycobacterium tuberculosis (M. tuberculosis) for the patients in question, namely, 48 hours. METHOD: Sputum specimens collected from 112 patients with clinically suspected pulmonary tuberculosis were subjected to Ziehl-Neelsen staining and inoculation on Löwenstein-Jensen medium (LJM) and BLM. RESULTS: BLM grew all the 36 (100%) smear positive samples, while LJM grew only 20 (55.5%). On the first 2 days of incubation, BLM grew M. tuberculosis colonies in 14 specimens and in 36 specimens after 3 to 6 days. From the seventh day until the end of the incubation period, BLM grew M. tuberculosis in only 9 specimens. The mean (SD) time for detection on BLM was 6 (5) days, whereas on LJM, it was 22 (12) days. CONCLUSIONS: BLM is more sensitive than LJM in positive and negative smears. It was also much faster than other methods in detecting the presence of M. tuberculosis.

Injured coliforms in swimming pools : How big a threat?

BACKGROUND: A swimming pool is an important leisure facility, but it can harbor injured cells creating potential health hazards. Disinfection of swimming pools can cause bacterial injury, when cells are exposed to a suboptimal concentration of disinfectants. Possible pathogenic bacteria can enter into an injured state, for example, Escherichia coli, Klebsiella spp, and Enterococcus faecalis. Injured bacteria can retain their pathogenicity and virulence and they may recover causing diseases. AIM: To assess the presence of injured coliforms in swimming pools based on differential plating media. MATERIALS AND METHODS: This study compared the difference in recovery of coliforms between two differential media, one designed for recovery of injured coliforms (HiCrome ECC selective agar and the other is CHROMagar ECC). A total of 120 samples were collected from 10 semi-public swimming pools with sporadic distribution around Alexandria, Egypt, and included in this study. Five pools were used for swim training, 4 were used for both training and recreational swimming and one was used for children only. RESULTS: The recovery medium (HiCrome ECC selective agar) detected 1.47 and 2.54 times total coliforms and E. coli, respectively, as CHROMagar ECC. The compliance of samples per fresh water swimming pool Egyptian standard in total coliforms and E. coli fell from 54.10% when examined by CHROMagar ECC medium to 35% by HiCrome ECC selective medium. CONCLUSION: The current findings may not be universal to all swimming pools but may be applicable to ones where the physicochemical properties of their water induce coliform injury. Results suggest that the use of media that detect injured yet viable coliforms will give a more sensitive and representative guidance about the quality of examined water and will assist in the treatment and decontamination of swimming pools.

Enhanced neutralising antibody response to bovine viral diarrhoea virus (BVDV) induced by DNA vaccination in calves.

DNA vaccination is effective in inducing potent immunity in mice; however it appears to be less so in large animals. Increasing the dose of DNA plasmid to activate innate immunity has been shown to improve DNA vaccine adaptive immunity. Retinoic acid-inducible gene I (RIG-I) is a critical cytoplasmic double-stranded RNA pattern receptor required for innate immune activation in response to viral infection. RIG-I recognise viral RNA and trigger antiviral response, resulting in type I interferon (IFN) and inflammatory cytokine production. In an attempt to enhance the antibody response induced by BVDV DNA in cattle, we expressed BVDV truncated E2 (E2t) and NS3 codon optimised antigens from antibiotic free-plasmid vectors expressing a RIG-I agonist and designated either NTC E2t(co) and NTC NS3(co). To evaluate vaccine efficacy, groups of five BVDV-free calves were intramuscularly injected three times with NTC E2t(co) and NTC NS3(co) vaccine plasmids individually or in combination. Animals vaccinated with our (previously published) conventional DNA vaccines pSecTag/E2 and pTriExNS3 and plasmids expressing RIG-I agonist only presented both the positive and mock-vaccine groups. Our results showed that vaccines coexpressing E2t with a RIG-I agonist induced significantly higher E2 antigen specific antibody response (p<0.05). Additionally, E2t augmented the immune response to NS3 when the two vaccines were delivered in combination. Despite the lack of complete protection, on challenge day 4/5 calves vaccinated with NTC E2t(co) alone or NTC E2t(co) plus NTC NS3(co) had neutralising antibody titres exceeding 1/240 compared to 1/5 in the mock vaccine control group. Based on our results we conclude that co-expression of a RIG-I agonist with viral antigen could enhance DNA vaccine potency in cattle.

Evidence for human norovirus infection of dogs in the United kingdom.

Human noroviruses (HuNoVs) are a major cause of viral gastroenteritis, with an estimated 3 million cases per year in the United Kingdom. HuNoVs have recently been isolated from pet dogs in Europe (M. Summa, C.-H. von Bonsdorff, and L. Maunula, J Clin Virol 53:244-247, 2012, http://dx.doi.org/10.1016/j.jcv.2011.12.014), raising concerns about potential zoonotic infections. With 31% of United Kingdom households owning a dog, this could prove to be an important transmission route. To examine this risk, canine tissues were studied for their ability to bind to HuNoV in vitro. In addition, canine stool samples were analyzed for the presence of viral nucleic acid, and canine serum samples were tested for the presence of anti-HuNoV antibodies. The results showed that seven different genotypes of HuNoV virus-like particles (VLPs) can bind to canine gastrointestinal tissue, suggesting that infection is at least theoretically possible. Although HuNoV RNA was not identified in stool samples from 248 dogs, serological evidence of previous exposure to HuNoV was obtained in 43/325 canine serum samples. Remarkably, canine seroprevalence for different HuNoV genotypes mirrored the seroprevalence in the human population. Though entry and replication within cells have not been demonstrated, the canine serological data indicate that dogs produce an immune response to HuNoV, implying productive infection. In conclusion, this study reveals zoonotic implications for HuNoV, and to elucidate the significance of this finding, further epidemiological and molecular investigations will be essential.

Serological evidence for multiple strains of canine norovirus in the UK dog population.

Noroviruses are associated with intestinal disease in humans, cows, pigs, mice, and, more recently, dogs. In 2007, the first canine norovirus (CNV) was identified and characterized in Italy. Subsequent studies have identified CNV in stools of dogs from Portugal, Greece, and the United States. To investigate the prevalence of CNV in the UK dog population, 228 canine stool samples were screened for CNV by qPCR, and 396 serum samples were screened for anti-CNV antibodies. qPCR of RNA extracted from canine stool samples did not reveal any CNV-positive samples, based on samples collected from diarrhoeic and control dogs in 2012-2013. CNV virus-like particles to three different CNV strains were produced using recombinant baculoviruses and a seroprevalence screen undertaken. Anti-CNV antibodies were identified at significant levels in canine serum; 38.1% of samples collected between 1999-2001 and 60.1% of samples collected in 2012-2013 were seropositive. The increase in seroprevalence over time (p<0.001) suggests that the CNV strains screened for are becoming more widespread. Variation in seroprevalence to different CNV strains was also identified. Two-thirds of the dogs were seropositive to a single strain, whereas the remaining third were seropositive to two or three of the strains analysed. This study has provided the first evidence that CNV is present in the UK, with seroprevalence identified to multiple circulating strains. This warrants further study and increased awareness of this recently discovered canine virus.

A phylogenetic analysis of Bovine Viral Diarrhoea Virus (BVDV) isolates from six different regions of the UK and links to animal movement data.

Bovine Viral Diarrhoea Virus (BVDV) is a pestivirus which infects cattle populations worldwide and is recognised as a significant source of economic loss through its impact on health and productivity. Studies investigating the molecular epidemiology of BVDV can give invaluable information about the diversity of viral strains present in a population and this, in turn, can inform control programs, drive vaccine development and determine likely infection sources. The current study investigated 104 viral isolates from forty farms across the UK. Through phylogenetic and nucleotide sequence analysis of the 5'UTR and Npro regions of the isolates investigated, it was determined that BVDV 1a was the predominant sub-genotype. However, BVDV 1b, 1e and 1i were also identified and, for the first time in the UK, BVDV 1d. Through analysis of animal movement data alongside the phylogenetic analysis of these BVD isolates, it was possible to link animal movements to the viral isolates present on several premises and, for the first time, begin to elucidate the routes of viral transmission. With further work, this type of analysis would enable accurate determination and quantification of the true biosecurity risk factors associated with BVDV transmission.

A pestivirus DNA vaccine based on a non-antibiotic resistance Escherichia coli essential gene marker.

Antibiotic resistance genes are widely used to produce plasmid DNA vaccines, but risk unwanted exposure to antibiotic residues and the spread of resistance genes. To overcome the limitations of existing selection technologies, we developed an alternative system applying the widely used household biocide triclosan as the selective agent and an endogenous growth essential target gene, fabI, as the plasmid-borne marker in Escherichia coli. The fabI/triclosan system enables efficient, non-antibiotic selection of transformed bacteria, with improved safety and plasmid production features. Here we aimed to evaluate the performance of this non-antibiotic selection system using a plasmid DNA vaccine against bovine viral diarrhoea virus as an example. The new system displayed high-yield plasmid DNA production in a standard E. coli host strain and growth media. Notably, the purified pDNA provided efficient in vitro protein expression and a strong in vivo neutralising antibody response in a mouse model, with measures comparable to that of the parental plasmid DNA based on ampicillin resistance. The fabI/triclosan system requires only low levels of triclosan for selection (1 µM) and residual triclosan in isolated DNA was below the limit of detection (< 20 parts per trillion). The fabI/triclosan selection system provides a simple, non-antibiotic resistance marker for plasmid selection, applicable to DNA vaccines and possibly other recombinant vaccine applications.

The dilemma of widal test - which brand to use? a study of four different widal brands: a cross sectional comparative study.

BACKGROUND: Serodiagnosis of typhoid fever by Widal test based on demonstrating the presence of agglutinins (antibodies) in the serum of an infected patient, against the H (flagellar) and O (somatic) antigens of Salmonella enterica serotype Typhi has been associated with many debates. This is why the aim of this study was to: (i) Compare the diagnostic accuracy of four different commercial kits used to perform Widal test (Remel, BioSystems, Dialab and Biotec). (ii) Compare the sensitivity and specificity of both anti-O and anti-H antibodies. (iii) Compare the validity of single versus paired serum samples with a rising titer for the diagnosis of typhoid fever. METHODS: Duplicate serum samples were obtained from 150 patients clinically diagnosed as typhoid fever patients. Moreover, single serum samples were obtained from 25 patients with febrile diseases other than typhoid fever. All samples were tested using the four different Widal brands and Salmonella Typhi IgM anti-LPS ELISA. RESULTS: -The results of Widal tests differed markedly using the four Widal brands in terms of sensitivity and specificity at three cut-off values of 1/80, 1/160 and 1/320. Remel brand gave the highest sensitivities and the lowest specificities and Dialab brand gave the highest specificities and the lowest sensitivities for both anti-O and anti-H antibodies at the three cut-off values.-Four fold rise in the antibodies titer was not demonstrable among clinically diagnosed typhoid fever patients-H agglutinins were less sensitive and less specific than O agglutinins CONCLUSIONS: -Widal test results showed marked discrepancies using different Widal brands. None of the serum samples of the typhoid fever patients showed four fold rise in the antibody titers. Raised O agglutinins were of slightly greater diagnostic value than raised H agglutinins. SIGNIFICANCE AND IMPACT OF STUDY: Widal test done sequentially using two brands could be of value in typhoid fever diagnosis. Single serum sample could be used for typhoid fever diagnosis relying on anti O titer.

TUBEX Test Versus Widal Test In The Diagnosis Of Typhoid Fever In Kafr El -Shekh, Egypt.

BACKGROUND: The value of the Widal test for the diagnosis of typhoid fever has been debated for as many years as it has been available. TUBEX test is a serological test which was stated to have the advantages of the Widal test without its controversies. The aim of this study was to evaluate TUBEX test versus the Widal test regarding sensitivity and specificity for the diagnosis of typhoid fever in an endemic area like Kafr El -Shekh, Egypt. MATERIALS AND METHODS: Serum samples were collected from typhoid (n=91) and febrile non-typhoid patients (n=25) and used to evaluate the performance of both Widal and TUBEX tests in diagnosis of typhoid fever using IgM anti-LPS ELISA as a reference test. RESULTS: TUBEX test had sensitivity, specificity, accuracy, positive predictive value and negative predictive value of 74.6%, 75%, 74.7%, 89.2% and 58% respectively. Widal test had higher results. CONCLUSION AND RECOMMENDATION: TUBEX test results are not superior to Widal test. TUBEX has a very serious shortcoming regarding its color scoring system. We do not recommend the use of TUBEX test for diagnosis of typhoid fever in Egypt as Widal test which is the test commonly used in diagnosis gave better performance.

The zoonotic potential of rotavirus.

Rotaviruses are generally species-specific, but cross-species transmission is possible, as has been demonstrated experimentally. Several case studies have indicated infection of humans by animal rotaviruses. Comparison of genetic sequences of human and animal rotaviruses often reveals close identity. Surveillance of circulating rotaviruses in the human population has revealed the presence of several uncommon genotypes. Many of these have been found in domestic animals, and it is possible that they arose in the human population through zoonotic transmission. The low incidence of uncommon strains would suggest that such transmission, or at least the establishment of an animal rotavirus or a human/animal reassortant virus in the human population, does not happen with any great frequency. However, many millions of people will be exposed year on year to animal rotaviruses. This happens within farming communities, and potentially to visitors to the countryside. There may be some measure of environmental contamination through livestock excrement. This exposure may not result in high levels of infection, but some infection could occur. There may be a continual input of rotavirus strains or sequences into the human population from the animal population albeit at a very low level.