Propidium monoazide conventional PCR and DNA sequencing: detection of negative culture bacterial pathogens causing subclinical mastitis.

AIMS: This study was conducted to early detect the negative culture bacterial pathogens causing subclinical mastitis for the fast diagnosis of the disease and the reduction of some milk-transmitted pathogenic bacteria to human consumers. METHODS AND RESULTS: A total of 171 positive California mastitis test (CMT) milk samples collected from asymptomatic dairy cows in Sharkia Governorate, Egypt were examined by conventional bacteriological methods. The obtained results revealed that Streptococcus species (77·2%), followed by Staphylococcus species (48·6%) and Escherichia coli (25·7%) were the most predominant bacterial pathogens isolated from positive culture milk samples, whereas Enterobacter and Pseudomonas species were the lowest ones (1·2%, for each). Herein, 13 (7.6%) negative culture milk samples were subjected to propidium monoazide (PMA) conventional PCR assay, followed by DNA sequencing of purified PCR amplicons. Sequence analysis identified seven different types of negative culture bacterial pathogens comprising as following; 4 Enterococcus hirae, 2 Bacillus cereus, 2 Staphylococcus aureus, 1 Bacillus mycoides, 1 Bacillus subtilis, 1 Enterococcus faecium and 1 Escherichia coli. CONCLUSIONS: All the detected negative culture bacterial pathogens by PMA-PCR assay, followed by DNA sequencing were incriminated in causing subclinical mastitis disease and had serious implications on human public health through consumption of milk contaminated with those recovered bacterial pathogens. SIGNIFICANCE AND IMPACT OF THE STUDY: The used methods could be useful in the routine detection of negative culture bacterial pathogens present in milk and consequently, it will help in the rapid diagnosis of subclinical mastitis disease and the reduction of many milk-transmitted diseases to human.

Impact of single and mixed infections with Escherichia coli and Mycoplasma gallisepticum on Newcastle disease virus vaccine performance in broiler chickens: an in vivo perspective.

AIMS: This study was designed to investigate, in an in vivo setting, the effects of single and combined infections with either Mycoplasma gallisepticum (MG) and/or Escherichia coli on the chicken immune response induced by Newcastle disease virus (NDV) vaccine. METHODS AND RESULTS: Humoral immunity was measured through detection of NDV antibody and anti-NDV IgG titres using haemagglutination-inhibition test and enzyme-linked immunosorbent assay, respectively. In addition, the expression levels of pro-inflammatory cytokines' genes (interleukin (IL) 6, IL4 and interferon (IFN) γ) were analysed using quantitative reverse transcription PCR. Significant (P < 0·05) results in all immunological parameters were detected in the vaccinated noninfected chicken group in comparison with those in groups exposed to bacterial infections. Bacterial infection along with vaccination hampered the NDV antibodies production and reduced the vaccine upregulated cytokine genes. The vaccinated mixed infection group reported lower antibody titres and cytokines expression levels compared to those in the single infection groups. All the previously enhanced immunological parameters reflected the maximum protection post challenge with velogenic viscerotropic NDV in the vaccinated noninfected chicken group. CONCLUSIONS: These findings provide novel insights into the immunosuppression activities of MG and E. coli infection in chickens vaccinated against NDV. SIGNIFICANCE AND IMPACT OF THE STUDY: This study hopes to provide a better insight to the immunosuppressive action of bacterial pathogens in chickens. This will help to improve biosecurity strategies during NDV vaccination in the future.

What is behind phylogenetic analysis of hospital-, community- and livestock-associated methicillin-resistant Staphylococcus aureus?

Methicillin-resistant Staphylococcus aureus (MRSA) has been shown to be the predominant life-threatening pathogen in Egypt. MRSA is a major cause of severe healthcare-associated (HA) infections. During the last decades, the incidence of community-associated (CA) MRSA infections has a complex epidemiology arising from the circulation of different strains in the general population. Moreover, livestock-associated (LA) MRSA emerged recently becomes an emerging threat to public health. Therefore, it is important to illuminate the differences between CA-, HA- and LA-MRSA to shed light on their genetic diversity and evolution. This study presents the first data on analysing the correlation between CA-, LA- and HA-MRSA using antibiogram typing, molecular characteristics and antimicrobial resistance and virulence genes' profiles. Overall, HA-MRSA strains tended to be multidrug resistant and less virulent than both LA- and CA-MRSA strains. Importantly, CA-MRSA strains had a high homology with each of HA- and LA-MRSA. However, no similarity was observed between HA- and LA-MRSA. Our findings suggest that the epidemiological changes in genetic behaviour between HA- and LA-MRSA are due to the presence of CA-MRSA confirming that CA-MRSA has created a public health crisis worldwide.

Genetic basis of resistance waves among methicillin resistant Staphylococcus aureus isolates recovered from milk and meat products in Egypt.

Antimicrobial resistance of methicillin-resistant Staphylococcus aureus (MRSA) poses a serious problem for clinicians worldwide. The present study attempted to evaluate the susceptibility patterns of MRSA to various antimicrobials and the prevalence of inducible clindamycin resistance as well as the relevant antibiotic and antiseptic resistance genes among these isolates. Totally, 40 MRSA isolates were recovered from examined milk and meat product samples (18.60%). Multi-drug resistance (MDR) was remarkably observed among 85% of these isolates. There was a good correlation between phenotypic determination of methicillin, amoxicillin/clavulinic acid and tetracycline resistances and PCR detections of mecA, blaZ and tet(K) genes, respectively, but norA gene was not detected in the four ciprofloxacin resistant isolates. Although, 55% of MRSA expressed resistance to benzalkonium chloride (BC), neither qacA/B nor smr gene was detected. Of 20 isolates exhibiting erythromycin- clindamycin discordant resistance pattern, 8 displayed positive double disk diffusion (D-zone) test denoting inducible macrolide-lincosamide-streptogramin B (MLSB) resistance phenotype with the inducibly expressed erm(A) and erm(C) genes in 87.5% of these isolates. Besides, the remaining 12 isolates showed MS phenotype (resistant to macrolides and type B streptogramins only) with a variety of erm(A), mph(C), msr(A) or a combination of these genes including erm(C). Finally, the constitutive MLSB phenotype with the constitutive expression of erm(A), erm(B) and erm(C) genes was comprised in 2 isolates with higher minimum inhibitory concentration (MIC) values for erythromycin (512 and 1024 µg/ml) and clindamycin (16 and 32 µg/ml). These findings suggested the importance of monitoring the evolution of MRSA resistance.

Protective potency of clove oil and its transcriptional down-regulation of Aeromonas sobria virulence genes in African catfish (Clarias gariepinus L.).

Disease episodes of fish caused by Aeromonas species are moved to the top list of limiting problems worldwide. The present study was planned to verify the in vitro antibacterial activities as well as the in vivo potential values of clove oil and ciprofloxacin against Aeromonas sobria in African catfish (Clarias gariepinus). The in vitro phenotypic virulence activities and the successful amplification of aerolysin and hemolysin genes in the precisely identified A. sobria strain were predictive for its virulence. In the in vivo assay, virulence of A. sobria strain was fully demonstrated based on constituent mRNA expression profile of tested virulence genes and typical septicemia associated with high mortalities of infected fish. Apparent lower mortality rates were correlated well with both decrescent bacterial burden and significant down-regulated transcripts of representative genes in the treated groups with clove oil, followed by ciprofloxacin as a prophylactic use for 15 days (P < 0.0001); however, the essential oil apart from ciprofloxacin significantly enhanced different hematological parameters (P < 0.05). In addition, administration of antibiotic may be considered as a pronounced stress factor in the fish even when it used in the prophylactic dose. In conclusion, medicinal plants-derived essential oils provide a virtually safer alternative to chemotherapeutics on fish, consumers and ecosystems.