Characterization and pathogenicity of multidrug-resistant coagulase-negative Staphylococci isolates in chickens / Caracterización y patogenicidad de aislados de estafilococos coagulasa negativos multirresistentes en pollos

The pathogenic potential of vancomycin and methicillin-resistant coagulase-negative Staphylococci (VMRCoNS) on Egyptian poultry farms has received little attention. Therefore, this study aims to study the prevalence of CoNS in imported poultry flocks and commercial poultry farms, evaluate the presence of virulence and antibiotic resistance genes (sea, seb, sec, sed, see, and mecA), and assess their pathogenicity in broiler chicks. Seven species were identified among 25 isolates, such as 8 S. gallinarum, 5 S. saprophyticus, 5 S. chromogens, 3 S. warneri, 2 S. hominis, 1 S. caprae, and 1 S. epidermidis. All isolates were resistant to clindamycin, doxycycline, vancomycin, methicillin, rifampicin, and penicillin. The mecA gene was confirmed in 14 isolates, while the sed gene was revealed in seven isolates. Commercial 1-day-old Ross broiler chicks were divided into eight groups of three replicates (10 birds/group): group Ӏ was negative control; groups (П, Ш, IV, V, VI, VII, and VIII) were subcutaneously inoculated with 108 CFUml−1 of S. hominis, S. caprae, S. epidermidis, S. gallinarum, S. chromogens, S. warneri, and S. saprophyticus, respectively. Groups VIII and V had mortality rates of 100% and 20%, respectively, with no evidence of mortalities in the other groups. The highest re-isolation of CoNS species was recorded in groups VII, VIII, and V. Postmortem and histopathological examination revealed the common presence of polyserositis in the internal organs, and hepatic and myocardial necrosis in groups IV, V, and VI. These findings revealed the pathogenic potential of CoNS, so special attention must be directed toward their public health impact.(AU)

Efficacy of ceftiofur N-acyl homoserine lactonase niosome in the treatment of multi-resistant Klebsiella pneumoniae in broilers.

In this study, the efficiency of the ceftiofur N-acyl homoserine lactonase niosome against multi-resistant Klebsiella pneumoniae in broilers was evaluated. Fifty-six K. pneumoniae isolates previously recovered from different poultry and environmental samples were screened for the ahlK gene. The lactonase enzyme was extracted from eight quorum-quenching isolates. The niosome was formulated, characterized, and tested for minimal inhibitory concentration (MIC) and cytotoxicity. Fourteen-day-old chicks were assigned to six groups: groups Ӏ and П served as negative and positive controls, receiving saline and K. pneumoniae solutions, respectively. In groups Ш and IV, ceftiofur and niosome were administrated intramuscularly at a dose of 10 mg/kg body weight for five consecutive days, while groups V and VI received the injections following the K. pneumoniae challenge. Signs, mortality, and gross lesions were recorded. Tracheal swabs were collected from groups П, V, and VI for counting K. pneumoniae. Pharmacokinetic parameters were evaluated in four treated groups at nine-time points. The niosome was spherical and 56.5 ± 4.41 nm in size. The viability of Vero cells was unaffected up to 5 × MIC (2.4 gml-1). The niosome-treated challenged group showed mild signs and lesions with lower mortality and colony count than the positive control group. The maximum ceftiofur serum concentrations in treated groups were observed 2 h following administration. The elimination half-life in niosome-treated groups was longer than that reported in ceftiofur-treated groups. This is the first report of the administration of N-acyl homoserine lactonase for the control of multi-resistant K. pneumoniae infections in poultry.

Antibiofilm activity of a lytic Salmonella phage on different Salmonella enterica serovars isolated from broiler farms / Actividad antibiopelícula de un fago lítico de Salmonella en diferentes serovares de Salmonella enterica aislados de granjas de pollos de engorde

Bacteriophages have been mainly used in treating infections caused by planktonic bacterial cells in the veterinary sector. However, their applications as antibiofilm agents have received little attention. Accordingly, a previously isolated Salmonella infecting Siphoviridae phage was investigated for host range against 15 Salmonella enterica isolates (S. Cape, S. Gallinarum, 4 S. Enteritidis, 3 S. Montevideo, S. Uno, S. Oritamerin, S. Belgdam, S. Agona, S. Daula, and S. Aba) recovered from the litters of commercial broiler farms. All S. enterica isolates were examined for their biofilm activity using a microtiter plate assay and for adrA, csgD, and gcpA genes using conventional PCR. The phage efficacy against established biofilms produced by the selected seven S. enterica isolates (S. Gallinarum, S. Enteritidis, S. Montevideo, S. Uno, S. Oritamerin, S. Belgdam, and S. Agona) was assessed using microtiter plate assay and reverse transcriptase real-time PCR over different incubation times of 5 and 24 h. All S. enterica isolates were strong biofilm formers. Moreover, the phage effectively reduced the biofilm activity of the established S. enterica biofilms in the microtiter plate assay using the independent sample t-test (P < 0.050). Furthermore, the relative expression levels of csgD, gcpA, and adrA genes in the biofilm cells of S. enterica isolate after phage treatment were significantly up-regulated to variable degrees using the independent sample t-test (P < 0.050). In conclusion, the present study revealed the potential use of Salmonella phage in reducing established biofilms produced by S. enterica serovars isolated from broiler farms.(AU)

Characterization and pathogenicity of multidrug-resistant coagulase-negative Staphylococci isolates in chickens.

The pathogenic potential of vancomycin and methicillin-resistant coagulase-negative Staphylococci (VMRCoNS) on Egyptian poultry farms has received little attention. Therefore, this study aims to study the prevalence of CoNS in imported poultry flocks and commercial poultry farms, evaluate the presence of virulence and antibiotic resistance genes (sea, seb, sec, sed, see, and mecA), and assess their pathogenicity in broiler chicks. Seven species were identified among 25 isolates, such as 8 S. gallinarum, 5 S. saprophyticus, 5 S. chromogens, 3 S. warneri, 2 S. hominis, 1 S. caprae, and 1 S. epidermidis. All isolates were resistant to clindamycin, doxycycline, vancomycin, methicillin, rifampicin, and penicillin. The mecA gene was confirmed in 14 isolates, while the sed gene was revealed in seven isolates. Commercial 1-day-old Ross broiler chicks were divided into eight groups of three replicates (10 birds/group): group Ӏ was negative control; groups (П, Ш, IV, V, VI, VII, and VIII) were subcutaneously inoculated with 108 CFUml-1 of S. hominis, S. caprae, S. epidermidis, S. gallinarum, S. chromogens, S. warneri, and S. saprophyticus, respectively. Groups VIII and V had mortality rates of 100% and 20%, respectively, with no evidence of mortalities in the other groups. The highest re-isolation of CoNS species was recorded in groups VII, VIII, and V. Postmortem and histopathological examination revealed the common presence of polyserositis in the internal organs, and hepatic and myocardial necrosis in groups IV, V, and VI. These findings revealed the pathogenic potential of CoNS, so special attention must be directed toward their public health impact.

Antibiofilm activity of a lytic Salmonella phage on different Salmonella enterica serovars isolated from broiler farms.

Bacteriophages have been mainly used in treating infections caused by planktonic bacterial cells in the veterinary sector. However, their applications as antibiofilm agents have received little attention. Accordingly, a previously isolated Salmonella infecting Siphoviridae phage was investigated for host range against 15 Salmonella enterica isolates (S. Cape, S. Gallinarum, 4 S. Enteritidis, 3 S. Montevideo, S. Uno, S. Oritamerin, S. Belgdam, S. Agona, S. Daula, and S. Aba) recovered from the litters of commercial broiler farms. All S. enterica isolates were examined for their biofilm activity using a microtiter plate assay and for adrA, csgD, and gcpA genes using conventional PCR. The phage efficacy against established biofilms produced by the selected seven S. enterica isolates (S. Gallinarum, S. Enteritidis, S. Montevideo, S. Uno, S. Oritamerin, S. Belgdam, and S. Agona) was assessed using microtiter plate assay and reverse transcriptase real-time PCR over different incubation times of 5 and 24 h. All S. enterica isolates were strong biofilm formers. Moreover, the phage effectively reduced the biofilm activity of the established S. enterica biofilms in the microtiter plate assay using the independent sample t-test (P < 0.050). Furthermore, the relative expression levels of csgD, gcpA, and adrA genes in the biofilm cells of S. enterica isolate after phage treatment were significantly up-regulated to variable degrees using the independent sample t-test (P < 0.050). In conclusion, the present study revealed the potential use of Salmonella phage in reducing established biofilms produced by S. enterica serovars isolated from broiler farms.

Bacteriophage mediated control of necrotic enteritis caused by C. perfringens in broiler chickens.

In Egypt, little attention has been paid to the isolation and application of C. perfringens phages for treating necrotic enteritis at the farm level. This study aims to evaluate the efficiency of the podovirus C. perfringens phage in treating necrotic enteritis in broiler chickens. Accordingly, C. perfringens phage was isolated from cecal samples of apparently healthy chickens and characterized by transmission electron microscopy, thermal stability test, and pH stability test. Commercial 14-day-old Arbor Acres broiler chickens were allocated to three groups: group Ӏ received BHI broth and assigned as a negative control, group П served as a positive control group that was challenged with C. perfringens via oral gavage for four successive days, and group Ш was administrated six phage doses on several occasions after oral gavage challenge with C. perfringens. Daily clinical symptoms and mortality were recorded. At three-time intervals, necrotic enteritis lesions were scored. Cecal samples were examined for re-isolation and counting of C. perfringens. The isolated C. perfringens phage was a podovirus with an icosahedral head diameter of 78.7 nm and a short non-contractile tail length of 22.2 nm. It remained stable for 60 min at 30 °C and 50 °C at pH values of 2, 4, 8, and 10. The phage-treated group (Ш) showed mild gross lesions with a lower mortality rate and reduced colony-forming units than the positive control group (П). The findings revealed that the isolated C. perfringens phage effectively treated experimental necrotic enteritis in broiler chickens.

Detection of Quorum Sensing N-Acyl-Homoserine Lactone Molecules Produced by Different Resistant Klebsiella pneumoniae Isolates Recovered from Poultry and Different Environmental Niches.

This study aimed to detect and identify the N-acyl-homoserine lactones molecules (AHLs) produced by different resistant Klebsiella pneumoniae isolates recovered from poultry and environmental samples using a modified validated high-performance liquid chromatography method. A total of 56 K. pneumoniae isolates were recovered, investigated for their antibiotic susceptibility, and screened for AHLs production using the Agrobacterium tumefaciens NTL4 biosensor system and a validated high-performance liquid chromatography method. The results revealed the detection of different short- and long-chain AHLs molecules among 39 K. pneumoniae isolates recovered from poultry and environmental samples. All environmental isolates produced nine peaks with retention times for C4-HSL, C6-HSL, C12-HSL, C8-HSL, C14-HSL, C8-oxo-HSL, C10-HSL, C6-oxo-HSL, and C7-HSL. The most quantifiable AHL signal molecules in poultry isolates were C4-HSL, C6-HSL, and C12-HSL. No statistical correlation between the AHL-producing ability of K. pneumoniae isolates and antibiotic resistance was reported. To the best of our knowledge, this study provides the first detailed report on the detection and identification of AHLs in K. pneumoniae isolates recovered from poultry and environmental samples. Furthermore, it provides a new insight available tool other than LC-MS/MS for detection and identification of AHL molecules.

Effect of peppermint oil and its microemulsion on necrotic enteritis in broiler chickens.

BACKGROUND AND AIM: Clostridium perfringens is one of the multiple drug-resistant intestinal pathogens causing necrotic enteritis disease, leading to great economic losses in poultry farms. This study aimed to evaluate the potential use of peppermint oil and its microemulsion (ME) as an alternative to antibiotics to control necrotic enteritis in broiler chickens. MATERIALS AND METHODS: Peppermint oil ME formulation (15% oil/water) was prepared and characterized by zeta potential, Fourier transform infrared, high-resolution transmission electron microscopy, and liquid chromatography-mass spectrometry (LC-MS/MS). The minimal inhibitory concentrations of the peppermint oil and its ME were investigated. A total of 80 commercial one day old Arbor Acres broiler chickens were randomly assigned to four groups of 20 birds each. The four groups were the negative control, positive control, peppermint oil (0.5 mL/mL water/10 days old), and its ME (0.25 mL/mL water/10 days old) groups. C. perfringens was orally provided at concentration of 1×108 CFU/mL on days 14, 15, and 16. Clinical signs and mortality were observed daily. Growth performance, gross lesions and cecal samples were investigated and examined on days 21, 28, and 35. RESULTS: Peppermint oil ME formulation has a polydispersity index, zeta potential and droplet size of 0.234, -24 mV±4.19, and 29.96±1.56 nm, respectively. LC-MS/MS analysis of oil and ME revealed common presence of phenolic compounds such as rosmorinic (360.31 g/mol), chlorogenic acid (354.31 g/mol), hesperidin (610.56 g/mol), and luteolin 7-O-ß-glucuronide (462.1 g/mol). The treated groups with peppermint oil and ME showed lower lesions, mortality and colony-forming units in addition to higher growth performance (p < 0.05) compared to the positive control group. CONCLUSION: Our study suggests the potential efficacy of peppermint oil and ME in the reduction of necrotic enteritis lesions and C. perfringens count.