Phenotypic and genotypic characterization of erythromycin-resistant Staphylococcus aureus isolated from bovine subclinical mastitis in Egypt.

Background and Aim: Subclinical mastitis (SCM) caused by erythromycin-resistant Staphylococcus aureus is a significant disease in lactating animals. Therefore, it is crucial to understand the genetic factors contributing to erythromycin resistance in S. aureus. This study aimed to estimate the prevalence of S. aureus in milk from subclinical mastitic cattle and buffaloes and tank milk samples as identified by probe-based real-time polymerase chain reaction (PCR) and the genotypic assessment of macrolide and erythromycin resistance profiles, as well as to analyze the phylogenetic relatedness of our local isolates of S. aureus. Materials and Methods: In total, 285 milk samples were analyzed using the California mastitis test to detect SCM. Milk samples were cultured on different specific Staphylococcus media. The presence of S. aureus was confirmed by Gram staining, the catalase and coagulase tests, the detection of hemolytic activity, DNase agar testing, and biofilm activity in Congo red medium. The genotypic identification of S. aureus (nuc) was performed. The determinants of erythromycin (ermA, ermB, ermC, and ermT) and macrolide resistance (msrA) were screened in all isolates. DNA sequencing of our local isolates of S. aureus was used to analyze their phylogenetic relatedness. Moreover, histopathological examination of tissue specimens of mammary gland was performed. Results: The S. aureus positivity rates were 36.4%, 48.8%, and 63.6% in cattle, buffalo, and bulk tank milk, respectively. Probe-based real-time PCR molecularly confirmed all 62 S. aureus isolates. Thirty-one isolates were subjected to PCR to create profiles of their genotypic erythromycin resistance. ermA, ermB, ermC, and ermT were present in 5 (8%), 26 (41.9%), 18 (29%), and 15 (24.1%) S. aureus isolates, respectively. Moreover, msrA was found in three (4.8%) strains. Eight PCR products were produced using standard PCR for DNA sequencing. Multiple sequence alignment, phylogenetic tree construction, and analysis of nuc in S. aureus revealed a high degree of homology (100%) with S. aureus strains isolated from milk in cases of bovine mastitis in India and Kenya. Histological analysis of udder tissues revealed extensive aggregation of mononuclear inflammatory cells in the interstitial connective tissue, primarily lymphocytes, and macrophages. Conclusion: This study showed a high prevalence of erythromycin resistance in S. aureus isolates. This information is vital for controlling mastitis and the spread of resistance genes between bacterial strains and hosts. Moreover, the probe-based real-time PCR approach is helpful for the rapid screening of S. aureus isolates and the consequent efficient treatment and control of S. aureus mastitis.

In vivo experimentally study the effect of Nigella Sativa silver nanoparticles for treatment of salmonella species causing diarrhea in ruminants.

Salmonellais one of the main etiological agents of infectious diarrhea in large and small ruminants but emergence of multidrug-resistant (MDR) strains faster rate than previously, leads to develop of MDR strains among animals needs different alternative therapeutic strategies. Our study was aimed to evaluate the effects of Nigella sativa silver nanoparticles (NS AgNPs) on specific pathogen-free (SPF) Wister rats. Nigella sativa silver nanoparticles were prepared and confirmed their formation by optical observations, UV-Vis spectroscopy, transmission electron microscopy (TEM), and scanning electron microscopy (SEM). Rats in group G2 were infected experimentally with Salmonella spp and treated with ciprofloxacin orally for duration of 6 days at a dose rat 10 mg/kg. On the other hand, rats in group G1 were infected with salmonella and treated for 20 days with NS AgNPs in oral dose of (10 mg/kg rats), and the results were compared to control groups G3 which received bacterial infection without treatment and G4 control negative. The results of optical observation, UV-Vis spectroscopy, TEM, and SEM revealed typical characteristics of prepared NS AgNPs. Liver, kidney function biomarkers, hematologic analysis, and histological examination the tissues of liver, kidney, and stomach of rat's model improved that NS AgNPs has antimicrobial effect and has the ability to decrease the inflammatory reaction caused by Salmonella spp infection. The results of our study indicate that NS AgNPs are effective in controlling MDR Salmonella spp in vivo without causing any adverse effects. Moreover, our findings suggest that reducing the use of antimicrobials could be a key factor in the fight against antimicrobial resistance and can provide valuable insights into identifying the most appropriate treatment strategies to tackle this issue effectively in the future.

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.

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 Toxoplasma gondii propagated in specific pathogen free embryonated chicken egg, for diagnosis of toxoplasmosis in equids and human.

Toxoplasma gondii (T. gondii) is a worldwide distribution infects a wide variety of mammals, including humans. The present study aimed to detect the efficacy of soluble and whole T. gondii antigens propagated in specific pathogen-free of embryonated chicken egg (SPF-ECE) used to improve the potency of serological assays for diagnosis of toxoplasmosis in equids and human. Total of 220 serum samples from 170 equids (90 donkeys and 55 horses and 25 mules) and 50 humans were collected from different governorates in Egypt during the period from October 2017 to March 2018. Crude T. gondii tachyzoites antigens from low or high passages propagated in mice or SPF-ECE was used for modifying some serological tests. The experiment showed that the mortality rate of T. gondii for 103 and 104 low passages were 6/8 (75%) and 7/8 (88%) dead embryos but, lower mortality rate in high passage T. gondii were 4/8 (50%) and 5/8 (63%) dead embryos, respectively. No mortality or inflammatory signs were observed in control of negative groups. In equids sera were examined by S-ELISA using soluble T. gondii antigen propagated in SPF-ECE showed the highest positive results 26 (28.8%), followed by LAT 37 (22%) and MAGPT 36 (21.17%). While, W-ELISA and IFAT used whole T. gondii antigen prepared in SPF-ECE were 35 (20.58%) and 28 (19.41%) showed highly positive results than the same test used the whole antigen prepared in mice. The highest seroprevalence of T. gondii in human and donkeys were 19/50 (38%). and 26/90 (28.88%), more than mules were 6/25 (24%) and horses were 9/55 (16.3%) examined by S-ELISA respectively. SPF-ECE is considered an appropriate experimental model for isolation and propagation of T. gondii tachyzoites, and their soluble antigens used in serological tests (S-ELISA, LAT, and MAGPT) have sensitivity and specificity more than the whole antigen and provided reliable diagnostic tools for detection of toxoplasmosis in human and equids.

Comparative study between serological and molecular methods for diagnosis of toxoplasmosis in women and small ruminants in Egypt.

Routine serological diagnosis of toxoplasmosis provides high sensitivity, but specificity varies depending on the test used; false-positive results (IgM) have been reported. Blood samples were collected from 88 women (59 pregnant and 29 nonpregnant) and 86 contact animals (62 sheep and 24 goats) at El Fayoum Governorate during the period from October 2005 to December 2006. All collected samples were tested for Toxoplasma gondii infection by serological tests (ELISA IgM & IgG and Sabin-Feldman dye test) and polymerase chain reaction (PCR). Results revealed specific IgG in 45.8% and 41.4%, IgM in 30.5% and 24.2%, and positive Sabin-Feldman dye test in 23.7% and 17.2% in pregnant and nonpregnant women, respectively. Positive PCR products were detected in 32.2% and 27.6% in pregnant and nonpregnant women, respectively. Regarding animals, positive ELISA IgG and PCR were detected in 98.4% and 67.7% of sheep and 41.7% and 25.0% of goats, respectively. It was concluded that serological tests can detect higher rate of toxoplasmosis than PCR, so ELISA combined with the PCR technique is a recommended tool for accurate diagnosis of toxoplasmosis.