Molecular characterization of tetracycline and vancomycin-resistant Enterococcus faecium isolates from healthy dogs in Egypt: a public health threat.

BACKGROUND: Vancomycin-resistant enterococci (VRE) are among the most common causative pathogens for nosocomial infections worldwide. Moreover, strains of VRE have been isolated from several domestic livestock in Egypt. METHODS: This study examined if healthy dogs are a potential source of VRE infection by isolating and characterizing Enterococcus faecium strains from stool samples on a morphological basis and biochemical activities. Subsequently, it was confirmed by genotypic characterization using polymerase chain reaction (PCR), followed by the detection of antibiotic resistance genes, virulence determinants, and genes contributing to enterocin production by PCR. Furthermore, the phylogenetic relationships among vanB and tetL genes were analyzed. RESULTS: All ten fecal samples were identified as E. faecium and confirmed by PCR. In addition, 90% of the isolates tested were positive for the virulence genes gelE and esp, and all the isolates tested were positive for the antibiotic resistance genes tetL and vanB. Only three of the five enterocin genes examined were detected. Ent As-48, bacteriocin 31, and Ent L50 were identified in 100%, 80%, and 60% of the samples, respectively. CONCLUSION: Dogs should be regarded as a reservoir of E. faecium that carries vancomycin resistance and virulence determinants that may affect public health in Egypt, considering a "One Health" task force approach to restrict their spread.

Phenotypic diagnosis and genotypic identification of Bacillus cereus causing subclinical mastitis in cows.

Background and Aims: Bovine mastitis is a disease that affects dairy cows and impacts the global dairy industry. Bacillus spp. can infect the mammary gland during lactation, intramammary treatment, or dry cow therapy. This study aimed to isolate and identify Bacillus spp. in raw milk samples from cows with subclinical mastitis from dairy farms in Beheira, Giza, Alexandria, and Menoufia Governorate, Egypt. We also investigated their antibiotic sensitivity and detected the enterotoxigenic and antibiotic resistance genes. Materials and Methods: A total of 262 milk samples (15-20 ml each) were examined microscopically, biochemically, and phenotypically. A polymerase chain reaction was used for genotypic identification and detecting antibiotic-resistance and enterotoxigenic genes. Antibiotic sensitivity was tested using the agar well diffusion test. Results: Bacillus cereus was identified in 47.7% of samples. Nhe and hblD enterotoxin genes were found in 93.64% (103/110) and 91.82% (101/110) of the samples, respectively. Tetracycline and ß-lactam antibiotic-resistance genes were present in 0% (0/110) and 98.18% (108/110), respectively, of the samples. All isolates were resistant to cefepime, cefixime, and oxacillin, while they were susceptible to amoxicillin-clavulanic, chloramphenicol, ampicillin/sulbactam, and levofloxacin. Conclusion: These results highlight the need to promote awareness regarding B. cereus, the most common pathogen causing mastitis in Egyptian dairy cows. We also emphasized that antibiotic misuse during mastitis is a potential public health threat.

Genotypic characterization of mecA gene and antibiogram profile of coagulase-negative staphylococci in subclinical mastitic cows.

Background and Aim: Coagulase-negative staphylococci (CNS) are becoming the major cause of clinical and subclinical bovine mastitis around the world. This study aims to estimate the prevalence, antibiogram, and frequency of the methicillin-resistant (MR) (mecA) gene in CNS collected from cows with subclinical mastitis. Materials and Methods: Thirty-four milk samples were collected from 20 cows. Fifteen subclinical mastitis samples (~44.12%) were identified as CNS isolates. The Vitek2 compact system method was employed for the identification of the species. Furthermore, antibiotic sensitivity tests were performed against 10 different antibiotics for CNS strains. The mecA gene from isolated CNS was detected by conventional polymerase chain reaction (PCR). Results: Staphylococcus haemolyticus was the most predominant isolated species with an incidence of 33.3% (5/15 isolates), followed by 26.7% for Staphylococcus sciuri and Staphylococcus vitamins (4/15 isolates), and 13.3% for Staphylococcus vitulinus (2/15 isolates), respectively. The highest resistance rates were determined to be 40% (6/15 isolates) against penicillin and oxacillin (OX), 33.3% (5/15 isolates) against clindamycin, 13% (2/15 isolates) against chloramphenicol, amoxicillin, and erythromycin, and 5% (1/15 isolates) against ciprofloxacin, respectively. The results revealed that the isolates were resistant to one or more antimicrobial agents, with five isolates displaying multiple antimicrobial resistance. Furthermore, the results exhibit that all CNS isolates had the mecA gene at 310 bp with a 100% frequency. Moreover, for detecting MR isolates, there are significant discrepancies between phenotypic and genotypic approaches, and only 6/15 CNS isolates phenotypically demonstrated OX resistance. Conclusion: The results emphasize the necessity of frequent monitoring of phenotypic and genotypic profiles of CNS isolates to ensure effective control measures and the prevention of multidrug resistance strain evolution.

Determination of extended-spectrum ß-lactamase-producing Klebsiella pneumoniae isolated from horses with respiratory manifestation.

Background and Aim: The World Health Organization considers multidrug-resistant (MDR) Klebsiella pneumoniae a major global threat. Horses harbor commensal isolates of this bacterial species and potentially serve as reservoirs for human MDR bacteria. This study investigated antimicrobial resistance in horses caused by extended-spectrum ß-lactamase (ESBL)-producing K. pneumoniae. Materials and Methods: One hundred fifty-nine nasal swab samples were collected from horses with respiratory distress not treated with cefotaxime and erythromycin. Biochemical and serological identification was performed on all samples. Polymerase chain reaction (PCR) was used to detect 16S-23S ITS, mucoviscosity-associated gene (magA), uridine diphosphate galacturonate 4-epimerase gene (uge), and iron uptake system gene (kfu), bla TEM, bla SHV, and bla CTX genes. Sequence analysis and phylogenetic relatedness of randomly selected K. pneumoniae isolates carrying the bla TEM gene were performed. Results: Ten isolates of Klebsiella spp. were obtained from 159 samples, with an incidence of 6.28% (10 of 159). Based on biochemical and serological identification, K. pneumoniae was detected in 4.4% (7 of 159) of the samples. Using PCR, all tested K. pneumoniae isolates (n=7) carried the 16S-23S ITS gene. By contrast, no isolates carried magA, uge, and kfu genes. The bla TEM gene was detected in all test isolates. Moreover, all isolates did not harbor the bla SHV or bla CTX gene. Sequence analysis and phylogenetic relatedness reported that the maximum likelihood unrooted tree generated indicated the clustering of the test isolate with the other Gram-negative isolate bla TEM. Finally, the sequence distance of the bla TEM gene of the test isolate (generated by Lasergene) showed an identity range of 98.4-100% with the bla TEM gene of the different test isolates. Conclusion: The misuse of antimicrobials and insufficient veterinary services might help generate a population of ESBL-producing K. pneumoniae in equines and humans, representing a public health risk.

Vancomycin resistant Streptococcus equi subsp. equi isolated from equines suffering from respiratory manifestation in Egypt.

BACKGROUND AND AIM: Upper respiratory tract infections are common in horses and can be caused by a variety of pathogens, mainly Streptococcus equi subsp. equi, which are a significant equine pathogen causing major health issues as well as financial losses to the equine industry. This study aimed to determine the prevalence of Streptococcal bacteria in equines in Egypt, and characterize vancomycin-resistant S. equi subsp. equi phenotypically and genotypically. MATERIALS AND METHODS: S. equi subsp. equi was isolated from internal nares of horses. All strains were confirmed by polymerase chain reaction-based detection of Streptococcus genus-specific 16S rRNA, sodA and seeI genes. Antibiotic susceptibility was determined phenotypically using the disk diffusion method. Genotypic detection of antibiotic resistance genes was performed by analyzing as b-lactamase resistance (blaZ), tetracycline resistance (tetK), vancomycin resistance (vanA), and chloramphenicol resistance (fexA). RESULTS: Eight streptococcal isolates were confirmed as S. equi subsp. equi. The genotypic characterization of antibiotic resistance showed resistance to vanA and tetK, with a frequency of 87.5% and 12.5%, respectively, while the frequency of sensitivity was 100% for blaz gene and fexA gene. CONCLUSION: In this study, we assessed vancomycin-resistant S. equi subsp. equi from equines suffering from respiratory manifestation in Egypt.

Oral nutritional supplements (ONSs) for cirrhotic patients undergoing liver resection assessed by ultrasound measurement of rectus femoris and anterior tibialis muscles thickness. Randomized clinical trial.

PURPOSE: We evaluated the effects of postoperative administration of (ONSs) on the liver function and the outcome of cirrhotic patients using ultrasound (US) assessment of rectus femoris (RF) and anterior tibialis (AT) muscles. PATIENTS AND METHODS: Forty-three malnourished adult hepatic patients who underwent major liver resections were recruited in this study. In the conventional diet (CD) group, the patients took water at postoperative day (POD) 0 and routine soft diet starting from POD1. In the ONS group, a commercially elemental diet was started from POD1 for 7 days postoperatively, with a target endpoint of 35-40 kcal/kg and 1.2-1.5 g/kg of protein per day. US assessment of the RF and AT muscles was done preoperatively and at POD3 and 7, including anterior-posterior (AP) diameter, lateral-lateral (LL) diameter, and cross-sectional area (CSA). Muscles' echogenicity was defined by the Heckmatt scale. The outcome of the patients was also recorded. RESULTS: Consumption of ONS preserved the measured RF and AT characteristics (AP and LL diameters and CSA) in the ONS group at POD3 and 7 compared to the CD group. Heckmatt scale was significantly increased at POD3 and 7 in the CD group compared to the ONS group. Both total protein and albumin levels at POD3 and 7 were significantly lower in the CD group compared to the ONS group [P = (0.02, 0.03) and (0.05, 0.04), respectively]. Serum phosphate was significantly lower at POD7 in the ONS group than the CD group (p = 0.04). There were significant decreases in the ICU stay and time of passing flatus (h) in the ONS group comparing with the CD group (P = 0.045 and P = 0.00, respectively). CONCLUSIONS: ONS maintains muscle mass and echogenicity of RF and AT along with better liver function and intestinal function recovery.

Therapeutic effect of biosynthetic gold nanoparticles on multidrug-resistant Escherichia coli and Salmonella species isolated from ruminants.

BACKGROUND AND AIM: Multidrug-resistant (MDR) pathogenic microorganisms have become a global problem in ruminants as a result of the intensive use of antibiotics, causing the development of resistance among gut microbiota. The antibiotic-resistant microorganisms can be transferred from diseased animals to humans. This study aimed to determine the prevalence of MDR Escherichia coli and Salmonella spp. isolated from cattle, buffaloes, sheep, and goats suffering from respiratory signs, diarrhea, and mastitis and to screen the antibiotic sensitivity of selected isolated bacteria. It also detected antibiotic-resistance genes by polymerase chain reaction (PCR), produced green gold nanoparticles (AuNPs) using plant extracts (Artemisia herba-alba and Morus alba), and evaluated the antimicrobial activities of these biosynthesized nanoparticles on selected pathogens (E. coli and Salmonella spp.). MATERIALS AND METHODS: MDR E. coli and Salmonella spp. were investigated using fecal samples (n=408), nasal swabs (n=358), and milk samples (n=227) of cattle, buffaloes, sheep, and goats with or without clinical signs, including respiratory manifestations, pneumonia, diarrhea, and mastitis, from different governorates in Egypt. E. coli and Salmonella spp. were isolated and identified on selective media, which were confirmed by biochemical reactions and PCR. Antimicrobial susceptibility testing against 10 commonly used antibiotics was performed using the Kirby-Bauer disk diffusion method. Antibiotic resistance genes blaTEM, blaSHV, blaOXA , and bla CTX-M were detected by PCR. The antibacterial effect of the biosynthesized AuNPs was evaluated by MIC and well diffusion assay. The biosynthesized AuNPs were also characterized by ultraviolet-visible spectrophotometry and transmission electron microscopy (TEM). RESULTS: Among all fecal samples, the prevalence of E. coli was 18.4% (183/993) and that of Salmonella spp. was 16.7% (66/408), as determined by cultural and molecular tests. All isolates of E. coli and Salmonella spp. were 100% resistant to ampicillin (AM) and amoxicillin and highly resistant to cefoxitin and AM-sulbactam. The total rate of resistance genes in E. coli was 61.2% (112/183), while that in Salmonella was 63.6% (42/66) for pathogens isolated from ruminants with respiratory manifestations, pneumonia, diarrhea, and mastitis. Among the resistance genes, blaTEM had the highest prevalence rate in E. coli (25.9%, 21/81) while blaSHV had the lowest (9.8%, 8/81) in fecal swabs. AuNPs were successfully synthesized using aqueous leaf extract of A. herba-alba and M. alba as bioreducing agents. TEM analysis showed particle size of 10-42 nm for A. herba-alba and M. alba AuNPs. The biosynthesized AuNPs showed antibacterial activity against MDR E. coli and Salmonella spp. CONCLUSION: Rapid and accurate diagnostic methods are the cornerstone for effective treatment to reduce the risk of antimicrobial-resistant pathogenic microorganisms. This is particularly important for overcoming the increasing rate of MDR in ruminants with respiratory manifestations, pneumonia, diarrhea, and mastitis. This can be complemented by the development of AuNPs synthesized in an environmentally friendly manner AuNPs using natural plant extracts for the treatment of antibiotic-resistant microorganisms.

Evaluation of specific and non-specific immune response of four vaccines for caseous lymphadenitis in sheep challenged.

BACKGROUND: Caseous lymphadenitis (CLA) is a serious disease affects sheep and goat, caused by Corynebacterium pseudotuberculosis. Due to it is non-treatable disease, so the effective preventive vaccines are considered a significant way to combat the disease. All strains of C. pseudotuberculosis have several virulence factors that associated with their cell invasion, survival, and proliferation such as phospholipase D (PLD), outer lipid coat, and secreted proteases. AIM: The present study was directed to perform a comparative innate and acquired immune response assessment of different four vaccine formulas to evoke protection against induced (CLA) challenge in sheep. MATERIALS AND METHODS: Negative ELISA (free of CLA) 15 local breed male (Balady) sheep were divided into five groups, each has received a different vaccine while the control has received saline buffer. The first vaccine composed of toxoid PLD alone the second composed of toxoid PLD with bacterin (formalinkilled bacteria), the third vaccine composed of toxoid PLD plus covaccine 8, while the fourth one composed of toxoid PLD plus locally produced polyvalent clostridial vaccine. The specific immune response was evaluated through lymphocyte proliferation assay using ELISA BrdU kit, while the non-specific response was estimated by superoxide anion production and lysozyme activity assays. RESULTS: The study revealed that PLD toxoid could evoke the highest specific immune response, showing a stimulation index (9.12%). On the other hand, combined toxoid ↱PLD with bacterin followed by PLD toxoid showed a significant increase in the non-specific innate immune response. CONCLUSION: The present study indicated that the toxoid PLD alone vaccine was most efficient and provided innate and acquired immune response in animals against CLA.