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Multiple drug resistance (MDR) has gained pronounced attention among Enterobacterales . The transfer of multiple antimicrobial resistance genes, frequently carried on conjugative incompatibility F (IncF) plasmids and facilitating interspecies resistance transmission, has been linked to Salmonella spp. and E. coli in broilers. In Egypt, the growing resistance is exacerbated by the limited clinical efficacy of many antimicrobials. In this study, IncF groups were screened and characterized in drug-resistant Salmonella spp. and E. coli isolated from broilers. The antimicrobial resistance profile, PCR-based replicon typing of bacterial isolates pre- and post-plasmid curing, and IncF replicon allele sequence typing were investigated. Five isolates of E. coli (5/31; 16.13%) and Salmonella spp. (5/36; 13.89%) were pan-susceptible to the examined antimicrobial agents, and 85.07% of tested isolates were MDR and extensively drug-resistant (XDR). Twelve MDR and XDR E. coli and Salmonella spp. isolates were examined for the existence of IncF replicons (FII, FIA, and FIB). They shared resistance to ampicillin, ampicillin/sulbactam, amoxicillin/clavulanate, doxycycline, cefotaxime, and colistin. All isolates carried from one to two IncF replicons. The FII-FIA-FIB+ and FII-FIA+FIB- were the predominant replicon patterns. FIB was the most frequently detected replicon after plasmid curing. Three XDR E. coli isolates that were resistant to 12-14 antimicrobials carried a newly FIB replicon allele with four nucleotide substitutions: C99→A, G112→T, C113→T, and G114→A. These findings suggest that broilers are a significant reservoir of IncF replicons with highly divergent IncF-FIB plasmid incompatibility groups circulating among XDR Enterobacterales. Supporting these data with additional comprehensive epidemiological studies involving replicons other than the IncF can provide insights for implementing efficient policies to prevent the spreading of new replicons to humans.

Antimicrobial resistance poses considerable issues for current clinical care, so the modified use of antimicrobial agents and public health initiatives, coupled with new antimicrobial approaches, may help to minimize the impact of multidrug-resistant (MDR) bacteria in the future. This study aimed to evaluate the antimicrobial, antioxidant, and immunomodulatory activities of Lagenaria siceraria , Thymus vulgaris , and their chitosan nanocomposites against extensive drug-resistant (XDR) Pseudomonas aeruginosa and vancomycin-resistant Staphylococcus aureus (VRSA) using both in vitro and in vivo assays. The in vitro antimicrobial susceptibilities of P. aeruginosa and VRSA strains revealed 100% sensitivity to imipenem (100%). All P. aeruginosa strains were resistant to cefoxitin, cefepime, trimethoprim + sulfamethoxazole, and fosfomycin. However, S. aureus strains showed a full resistance to cefoxitin, amoxicillin, ampicillin, erythromycin, chloramphenicol, and fosfomycin (100% each). Interestingly, all S. aureus strains were vancomycin-resistant (MIC = 32-512 μg/mL), and 90% of P. aeruginosa and S. aureus strains were XDR. The antimicrobial potential of Lagenaria siceraria and Thymus vulgaris nanocomposites with chitosan nanoparticles demonstrated marked inhibitory activities against XDR P. aeruginosa and VRSA strains with inhibition zones' diameters up to 50 mm and MIC values ranging from 0.125 to 1 μg/mL and 1 to 8 μg/mL, respectively. The results of the in vivo approach in male Sprague Dawley rats revealed that infection with P. aeruginosa and S. aureus displayed significant changes in biochemical, hematological, and histopathological findings compared to the negative control group. These values returned to the normal range after treatment by chitosan nanoparticles, either loaded with Lagenaria siceraria or Thymus vulgaris . Real-time quantitative polymerase chain reaction (RT-qPCR) findings presented significant upregulation of the relative expression of the IL10 gene and downregulation of the IFNG gene throughout the experimental period, especially after treatment with chitosan nanoparticles loaded either with Lagenaria siceraria or Thymus vulgaris in comparison to the positive control groups. In conclusion, this is the first report suggesting the use of Lagenaria siceraria and Thymus vulgaris nanocomposites with chitosan nanoparticles as a promising contender for combating XDR P. aeruginosa and VRSA infections as well as a manager for inflammatory situations and oxidative stress-related disorders.

Background: Bacillus cereus ( B. cereus ) biofilm is grown not only on medical devices but also on different substrata and is considered a potential hazard in the food industry. Quorum sensing plays a serious role in the synthesis of biofilm with its surrounding extracellular matrix enabling irreversible connection of the bacteria., Aim: The goal of the current investigation was to ascertain the prevalence, patterns of antimicrobial resistance, and capacity for B. cereus biofilm formation in meat and meat products in Egypt., Methods: In all, 150 meat and meat product samples were used in this study. For additional bacteriological analysis, the samples were moved to the Bacteriology Laboratory. Thereafter, the antimicrobial, antiquorum sensing, and antibiofilm potential of apple cider vinegar (ACV) on B. cereus were evaluated., Results: Out of 150 samples, 34 (22.67%) tested positive for B. cereus . According to tests for antimicrobial susceptibility, every B. cereus isolates tested positive for colistin and ampicillin but negative for ciprofloxacin and imipenem. The ability to form biofilms was present in all 12 multidrug-resistant B. cereus isolates ( n = 12); of these, 6 (50%), 3 (25%), and 3 (25%) isolates were weak, moderate, and strong biofilm producers, respectively. It is noteworthy that the ACV demonstrated significant inhibitory effects on B. cereus isolates, with minimum inhibitory concentrations varying between 2 and 8 μg/ml. Furthermore, after exposing biofilm-producing B. cereus isolates to the minimum biofilm inhibitory concentrations 50 of 4 μg/ml, it demonstrated good antibiofilm activity (>50% reduction of biofilm formation). Strong biofilm producers had down-regulated biofilm genes ( tasA and sipW ) and their regulator ( plcR ) compared to the control group, according to reverse transcriptase quantitative polymerase chain reaction analysis., Conclusion: Our study is the first report, that spotlights the ACV activity against B. cereus biofilm and its consequence as a strong antibacterial and antibiofilm agent in the food industry and human health risk.

Pseudomonas aeruginosa is notorious for its ability to develop a high level of resistance to antimicrobial agents. Resistance-nodulation-division (RND) efflux pumps could mediate drug resistance in P. aeruginosa . The present study aimed to evaluate the antibacterial and anti-efflux activities of cinnamon essential oil either alone or combined with ciprofloxacin against drug resistant P. aeruginosa originated from human and animal sources. The results revealed that 73.91% of the examined samples were positive for P. aeruginosa ; among them, 77.78% were of human source and 72.73% were recovered from animal samples. According to the antimicrobial resistance profile, 48.73% of the isolates were multidrug-resistant (MDR), 9.2% were extensive drug-resistant (XDR), and 0.84% were pan drug-resistant (PDR). The antimicrobial potential of cinnamon oil against eleven XDR and one PDR P. aeruginosa isolates was assessed by the agar well diffusion assay and broth microdilution technique. The results showed strong antibacterial activity of cinnamon oil against all tested P. aeruginosa isolates with inhibition zones' diameters ranging from 34 to 50 mm. Moreover, the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values of cinnamon oil against P. aeruginosa isolates ranged from 0.0562-0.225 µg/mL and 0.1125-0.225 µg/mL, respectively. The cinnamon oil was further used to evaluate its anti-efflux activity against drug-resistant P. aeruginosa by phenotypic and genotypic assays. The cartwheel test revealed diminished efflux pump activity post cinnamon oil exposure by two-fold indicating its reasonable impact. Moreover, the real-time quantitative polymerase chain reaction (RT-qPCR) results demonstrated a significant ( p < 0.05) decrease in the expression levels of MexA and MexB genes of P. aeruginosa isolates treated with cinnamon oil when compared to the non-treated ones (fold changes values ranged from 0.4204-0.7474 for MexA and 0.2793-0.4118 for MexB ). In conclusion, we suggested the therapeutic use of cinnamon oil as a promising antibacterial and anti-efflux agent against drug-resistant P. aeruginosa .

Caseous lymphadenitis (CLA) is a bacterial infection caused by Corynebacterium pseudotuberculosis ( C. pseudotuberculosis ) that affects sheep and goats, leading to abscess formation in their lymph nodes. The present study aimed to isolate and identify C. pseudotuberculosis from CLA in smallholder sheep and goats, and determine the resistance patterns, virulence, and resistance genes of the isolates. Additionally, genotypic and phylogenetic analysis of the isolates was conducted using ERIC-PCR and DNA sequencing techniques. A cross-sectional study examined 220 animals (130 sheep and 90 goats) from 39 smallholder flocks for clinical signs of CLA. Fifty-four (24.54%) animals showed CLA-compatible lesions, confirmed by C. pseudotuberculosis isolation and PCR identification. Sheep had a lower infection rate of CLA (18.46%) compared with goats (33.3%). Antimicrobial susceptibility testing of 54 C. pseudotuberculosis isolates to 24 antimicrobial drugs revealed that they were 100% resistant to bacitracin and florfenicol, while none of the isolates were resistant to norfloxacin. A high resistance rate was observed for penicillin and erythromycin (92.6% each). Interestingly, 16.7% of C. pseudotuberculosis isolates recovered from sheep showed vancomycin resistance. Molecular characterization of C. pseudotuberculosis isolates revealed that PLD , PIP , and FagA virulence genes were present in all examined isolates. However, the FagB , FagC , and FagD genes were detected in 24 (100%), 20 (83%), and 18 (75%) of the sheep isolates, and 26 (87%), 26 (87%), and 18 (60%) of the goat isolates, respectively. The β -lactam resistance gene was present in all isolates. Furthermore, 83% of the sheep isolates carried the aminoglycoside ( aph(3″)-lb ), chloramphenicol ( cat1 ), and bacitracin ( bcrA ) resistance genes. Among the isolates recovered from goats, 73% were found to contain macrolides ( ermX ), sulfonamide ( sul1 ), and bacitracin ( bcrA ) resistance genes. It is worrisome that the glycopeptide ( vanA ) resistance gene was detected in 8% of the sheep isolates as a first report. ERIC-PCR genotyping of 10 multi-drug-resistant C. pseudotuberculosis isolates showed a high similarity index of 83.6% between isolates from sheep and goats. Nucleotide sequence analysis of partial 16S rRNA sequences of C. pseudotuberculosis revealed 98.83% similarity with biovar Ovis of globally available reference sequences on the Genbank database. Overall, our findings might indicate that C. pseudotuberculosis infection in smallholders in Egypt might be underestimated despite the significant financial impact on animal husbandry and potential health hazards it poses. Moreover, this study highlights the importance of implementing a sustainable control strategy and increasing knowledge and awareness among smallholder breeders to mitigate the economic impact of CLA.

In the present study, biologically active compounds such as phenolic-rich extract (PRE), 7S globulin (vicilin), and 11S globulin (legumin) from red kidney bean ( Phaseolus vulgaris L.) seeds were extracted and evaluated as antibacterial agents against multidrug-resistant (MDR) Enterobacterales isolated from both animal and human sources. The overall occurrence rate of Enterobacterales was 43.6%, which significantly differed between animal (38.75%) and human (56.67%) sources. Antimicrobial susceptibility testing revealed that Enterobacterales isolates exhibited full resistance (100%) to amoxicillin-clavulanic acid, followed by ampicillin (75.44%), erythromycin (71.93%), cefoxitin (70.18%), amoxicillin (66.66%), ceftriaxone (64.91%), and trimethoprim/sulfamethoxazole (56.14%). Worthy of note, 97.92% of Enterobacterales isolates were MDR. The total phenolic contents (TPC; 53 ± 2 mg GAE g-1) and total flavonoid contents (TFC; 26 ± 1 mg QE g-1) were recorded. The major phenolic and flavonoid components were catechol (17.63 μg/mL) and hesperidin (11.37 μg/mL), respectively. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed to detect the 7S and 11S globulin's molecular mass. The data revealed that red kidney bean protein isolate (KPI) includes two major portions: 7S and 11S globulins. The bioactive compounds of Phaseolus vulgaris were investigated for their antibacterial activities against Enterobacterales for the first time. The protein component (MIC = 0.125 - 2 μg/mL; 53.85%) and its 7S and 11S globulin subunits (MIC = 0.5 - 2 μg/mL; 30.77% each) were the most potent extracts, whereas the methanolic extract was the least effective one (MIC = 2 μg/mL; 15.38%). The results displayed the potential of protein bioactive compounds as a hopeful candidate for enhancing future medication plans for the treatment of Enterobacterales originating from animal and human sources., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Ebrahim, Abd El-Aziz, Elariny, Shindia, Osman, Hozzein, Alkhalifah and El-Hossary.)

Urinary tract infection (UTI) is one of the most common bacterial infections in the world, which is associated with high morbidity and mortality rates. Enterobacterales species are considered the most causative agent for UTI, especially uropathogenic Escherichia coli (UPEC). Here, we investigated the antibacterial activity of the green fungal metabolite, 6-pentyl α pyrone lactone, alone or in combination with zinc oxide nanoparticles (ZnONPs) against multidrug-resistant Enterobacterales recovered from UTI. The results revealed that 57.27% of human urine samples were positive for Enterobacterales , where E. coli was the most prevalent bacterial pathogen (66.67%). Of note, 98.41% of Enterobacterales isolates were multidrug-resistant (MDR) with multiple antimicrobial resistance (MAR) indices ranged from 0.437 to 1. Fifty percent of the examined isolates were positive for the integrase gene; 60% out of them harbored class 2 integron, whereas the other 40% carried class 1 integrons. The broth microdilution assay ensured that the 6-pentyl-α-pyrone lactone had a reasonable antimicrobial effect against the examined isolates (Minimum inhibitory concentration (MIC) values of 16-32 μg/mL). However, ZnONPs showed a strong antimicrobial effect against the investigated isolates with MIC values ranging from 0.015 to 32 μg/mL. Interestingly, the MICs decreased 5-12 fold and 3-11 fold for 6-pentyl-α-pyrone lactone and ZnONPs, respectively, against examined isolates after their combination. This is the first report suggesting the use of 6-pentyl α pyrone lactone and ZnONPs combination as a promising candidate against MDR Enterobacterales recovered from UTI.

Antimicrobial resistance is a major concern in the dairy industry. This study investigated the prevalence, antimicrobial resistance phenotypes, and genome sequencing of Gram-negative bacteria isolated from clinical ( n = 350) and subclinical ( n = 95) bovine mastitis, and raw unpasteurized milk ( n = 125). Klebsiella pneumoniae , Aeromonas hydrophila , Enterobacter cloacae (100% each), Escherichia coli (87.78%), and Proteus mirabilis (69.7%) were the most prevalent multidrug-resistant (MDR) species. Extensive drug-resistance (XDR) phenotype was found in P. mirabilis (30.30%) and E. coli (3.33%) isolates. Ten isolates (four E. coli , three Klebsiella species and three P. mirabilis ) that displayed the highest multiple antibiotic resistance (MAR) indices (0.54-0.83), were exposed to whole-genome sequencing (WGS). Two multilocus sequence types (MLST): ST2165 and ST7624 were identified among the sequenced E. coli isolates. Three E. coli isolates (two from clinical mastitis and one from raw milk) belonging to ST2165 showed similar profile of plasmid replicon types: IncFIA, IncFIB, IncFII, and IncQ1 with an exception to an isolate that contained IncR, whereas E. coli ST7624 showed a different plasmid profile including IncHI2, IncHI2A, IncI1α, and IncFII replicon types. ResFinder findings revealed the presence of plasmid-mediated colistin mcr - 10 and fosfomycin fosA 5 resistance genes in a K. pneumoniae (K1) isolate from bovine milk. Sequence analysis of the reconstructed mcr-10 plasmid from WGS of K1 isolate, showed that mcr-10 gene was bracketed by xer C and insertion sequence IS26 on an IncFIB plasmid. Phylogenetic analysis revealed that K1 isolate existed in a clade including mcr-10 -harboring isolates from human and environment with different STs and countries [United Kingdom (ST788), Australia (ST323), Malawi (ST2144), Myanmar (ST705), and Laos (ST2355)]. This study reports the first emergence of K. pneumoniae co-harboring mcr - 10 and fosA5 genes from bovine milk in the Middle East, which constitutes a public health threat and heralds the penetration of the last-resort antibiotics. Hence, prudent use of antibiotics in both humans and animals and antimicrobial surveillance plans are urgently required., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Tartor, Abd El-Aziz, Gharieb, El Damaty, Enany, Soliman, Abdellatif, Attia, Bahnass, El-Shazly, Elbediwi and Ramadan.)

The emergence of extensive drug-resistant (XDR) Salmonella in livestock animals especially in poultry represents a serious public health and therapeutic challenge. Despite the wealth of information available on Salmonella resistance to various antimicrobials, there have been limited data on the genetic determinants of XDR Salmonella exhibiting co-resistance to ciprofloxacin (CIP) and tigecycline (TIG). This study aimed to determine the prevalence and serotype diversity of XDR Salmonella in poultry flocks and contact workers and to elucidate the genetic determinants involved in the co-resistance to CIP and TIG. Herein, 115 Salmonella enterica isolates of 35 serotypes were identified from sampled poultry (100/1210, 8.26%) and humans (15/375, 4.00%), with the most frequent serotype being Salmonella Typhimurium (26.96%). Twenty-nine (25.22%) Salmonella enterica isolates exhibited XDR patterns; 25 out of them (86.21%) showed CIP/TIG co-resistance. Exposure of CIP- and TIG-resistant isolates to the carbonyl cyanide 3-chlorophenylhydrazone (CCCP) efflux pump inhibitor resulted in an obvious reduction in their minimum inhibitory concentrations (MICs) values and restored the susceptibility to CIP and TIG in 17.24% (5/29) and 92% (23/25) of the isolates, respectively. Molecular analysis revealed that 89.66% of the isolates contained two to six plasmid-mediated quinolone resistance genes with the predominance of qepA gene (89.66%). Mutations in the gyrA gene were detected at codon S83 (34.62%) or D87 (30.77%) or both (34.62%) in 89.66% of XDR Salmonella . The tet (A) and tet (X4) genes were detected in 100% and 3.45% of the XDR isolates, respectively. Twelve TIG-resistant XDR Salmonella had point mutations at codons 120, 121, and 181 in the tet(A) interdomain loop region. All CIP and TIG co-resistant XDR Salmonella overexpressed ramA gene; 17 (68%) out of them harbored 4-bp deletion in the ramR binding region (T-288/A-285). However, four CIP/TIG co-resistant isolates overexpressed the oqxB gene. In conclusion, the emergence of XDR S. enterica exhibiting CIP/TIG co-resistance in poultry and humans with no previous exposure to TIG warrants an urgent need to reduce the unnecessary antimicrobial use in poultry farms in Egypt., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Abd El-Aziz, Tartor, Gharieb, Erfan, Khalifa, Said, Ammar and Samir.)

A major increase of bacterial resistance to colistin, a last-resort treatment for severe infections, was observed globally. Using colistin in livestock rearing is believed to be the ground of mobilized colistin resistance ( mcr ) gene circulation and is of crucial concern to public health. This study aimed to determine the frequency and virulence characteristics of colistin-resistant Gram-negative bacteria from the milk of mastitic cows and raw unpasteurized milk in Egypt. One hundred and seventeen strains belonging to Enterobacteriaceae ( n = 90), Pseudomonas aeruginosa ( n = 10), and Aeromonas hydrophila ( n = 17) were screened for colistin resistance by antimicrobial susceptibility testing. The genetic characteristics of colistin-resistant strains were investigated for mcr - 1-9 genes, phylogenetic groups, and virulence genes. Moreover, we evaluated four commonly used biocides in dairy farms for teat disinfection toward colistin-resistant strains. Multidrug-resistant (MDR) and extensive drug-resistant (XDR) phenotypes were detected in 82.91% (97/117) and 3.42% (4/117) of the isolates, respectively. Of the 117 tested isolates, 61 (52.14%) were colistin resistant (MIC >2 mg/L), distributed as 24/70 (34.29%) from clinical mastitis, 10/11 (90.91%) from subclinical mastitis, and 27/36 (75%) from raw milk. Of these 61 colistin-resistant isolates, 47 (19 from clinical mastitis, 8 from subclinical mastitis, and 20 from raw milk) harbored plasmid-borne mcr genes. The mcr-1 gene was identified in 31.91%, mcr-2 in 29.79%, mcr-3 in 34.04%, and each of mcr-4 and mcr-7 in 2.13% of the colistin-resistant isolates. Among these isolates, 42.55% (20/47) were E. coli , 21.28% (10/47) A. hydrophila , 19.12% (9/47) K. pneumoniae , and 17.02% (8/47) P. aeruginosa . This is the first report of mcr -3 and mcr -7 in P. aeruginosa . Conjugation experiments using the broth-mating technique showed successful transfer of colistin resistance to E. coli J53-recipient strain. Different combinations of virulence genes were observed among colistin-resistant isolates with almost all isolates harboring genes. Hydrogen peroxide has the best efficiency against all bacterial isolates even at a low concentration (10%). In conclusion, the dissemination of mobile colistin resistance mcr gene and its variants between MDR- and XDR-virulent Gram-negative isolates from dairy cattle confirms the spread of mcr genes at all levels; animals, humans, and environmental, and heralds the penetration of the last-resort antimicrobial against MDR bacteria. Consequently, a decision to ban colistin in food animals is urgently required to fight XDR and MDR bacteria., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Tartor, Gharieb, Abd El-Aziz, El Damaty, Enany, Khalifa, Attia, Abdellatif and Ramadan.)

Mastitis remains a serious problem for dairy animals. The misappropriation of antimicrobial agents helps accelerate resistance, which poses a serious challenge in controlling environmental S. uberis infection. Here, we study the virulence attributes, antimicrobial and biocide resistance, and epidemiological typing of S. uberis recovered from bovine clinical mastitis in dairy farms of diverse hygienic interventions in Egypt. The overall S. uberis infection rate was 20.59%; all were multidrug-resistant (MDR). The sua gene was the most frequent virulence gene (42.02%), followed by pau A (40.57%), cfu (21.73%), skc (20.28%), and opp (11.59%). The erm (B) gene served as the predominant antimicrobial-resistant gene (75.36%), followed by fexA (52.63%) and tet (M), blaZ , and aac(6')aph(2″) genes (46.38% each). Of note, 79.71%, 78.26%, and 18.84% of S. uberis isolates harbored qacED1 , qacC/D , and qacA/B genes, respectively. All analyzed isolates were S. uberis type I by their unique RFLP-PCR pattern. In conclusion, the sustained presence of pauA and sua genes throughout the investigated farms contributes to a better understanding of the bacterium's pathogenicity. Furthermore, MDR coupled with the existence of biocide resistance genes indicates the importance of S. uberis surveillance and the prudent use of antimicrobials in veterinary clinical medicine to avoid the dissemination of antimicrobial resistance.

Aims: This study was applied to evaluate the usefulness of a high-throughput sample preparation protocol prior to the application of quantitative real-time PCR (qPCR) for the early diagnosis of bloodstream and pyogenic infections in humans and animals compared to matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) and classical culture., Methods and Results: Saponin-mediated selective host cell lysis combined with DNase-1 was applied for processing of whole blood and pus clinical samples collected from suspected cases of septicaemia and pyogenic infections in humans and animals. The pre-PCR processing strategy enabled the recovery of microbial cells with no changes in their colony forming units immediately after the addition of saponin. DNase-1 was efficient for removing the DNAs from the host cells as well as dead cells with damaged cell membranes. The metagenomic qPCR and MALDI-TOF MS could identify the bacterial community of sepsis at species level with a concordance of 97·37% unlike the conventional culture. According to qPCR results, Staphylococcus aureus (24·24%) was predominated in animal pyogenic infections, whereas Klebsiella pneumonia (31·81%) was commonly detected in neonatal sepsis., Conclusions: Saponin combined with DNase-1 allowed the efficient recovery of microbial DNA from blood and pus samples in sepsis using qPCR assay., Significance and Impact of the Study: Metagenomic qPCR could identify a broad range of bacteria directly from blood and pus with more sensitivity, higher discriminatory power and shorter turnaround time than those using MALDI-TOF MS and conventional culture. This might allow a timely administration of a prompt treatment., (© 2020 The Society for Applied Microbiology.)

This study investigated the frequency of carbapenem and colistin resistance in ESBL-producing K. pneumoniae (ESBLK) isolates recovered from chickens and their environment, contact farm workers and hospitalized patients in Egypt. Further, the phenotypic and genotypic relationships between the community and hospital-acquired K. pneumoniae isolates in the same geographical area were investigated. From 272 total samples, 37 (13.6%) K. pneumoniae isolates were identified, of which 20 (54.1%) were hypervirulent. All isolates (100%) were multidrug-resistant (MDR) with multiple antibiotic resistance (MAR) indices ranging from 0.19 to 0.94. Colistin-resistant isolates (18.9%) displayed colistin MIC values >2 μg/mL, all harbored the mcr -1 gene. All isolates from patients (13/90, 14.4%), workers (5/22, 22.7%), chickens (9/100, 9%) and the environment (10/60, 16.7%) harbored a single or multiple β-lactamase genes, bla SHV , bla TEM , bla CTX-M1 and bla OXA-1 , often in combination with carbapenemase genes ( bla VIM , bla NDM-1 or bla IMP ; 45.9%), the mcr -1 gene (18.9%) or both (13.5%). Enterobacterial repetitive intergenic consensus (ERIC)-PCR genotyping revealed 24 distinct ERIC types (ETs) with a discrimination index of 0.961. Six ETs showed clusters of identical isolates from chicken and human sources. The increased frequency and genetic relatedness of ESBLK and carbapenemase-producing K. pneumoniae (CPK) from chickens and humans pose a public health threat that urge more prudent use of antimicrobials in chicken farms to avoid the propagation and expansion of both ESBLK and CPK from the chicken sources to humans.

Background: Streptococcus agalactiae (S. agalactiae) is a contagious pathogen of bovine mastitis. It has financial implications for the dairy cattle industry in certain areas of the world. Since antimicrobial resistance increases in dairy farms, natural antimicrobials from herbal origins and nanoparticles have been given more attention as an alternative therapy. Hence, this study reported the antimicrobial and antibiofilm potentials of cinnamon oil, silver nanoparticles (AgNPs), and their combination against multidrug-resistant (MDR) S. agalactiae recovered from clinical bovine mastitis in Egypt., Results: Our findings revealed that 73% (146/200) of the examined milk samples collected from dairy cows with clinical mastitis were infected with Streptococci species. Of these, 9.59% (14/146) were identified as S. agalactiae and categorized as MDR. S. agalactiae isolates expressed four virulence genes (Hyl, cylE, scpB, and lmb) and demonstrated an ability to produce biofilms. Cinnamon oil showed high antimicrobial (MICs ≤0.063 μg /mL) and antibiofilm (MBIC 50  = 4 μg/mL) potentials against planktonic and biofilms of S. agalactiae isolates, respectively. However, AgNPs showed reasonable antimicrobial (MICs ≤16 μg/mL) and relatively low antibiofilm (MBIC 50  = 64 μg/mL) activities against screened isolates. Synergistic antimicrobial or additive antibiofilm interactions of cinnamon oil combined with AgNPs were reported for the first time. Scanning electron microscope (SEM) analysis revealed that biofilms of S. agalactiae isolates treated with cinnamon oil were more seriously damaged than observed in AgNPs cinnamon oil combination. Moreover, reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) showed that cinnamon oil exerted a remarkable down-regulation of pili biosynthesis genes (pilA and pilB) and their regulator (rogB) against S. agalactiae biofilms, meanwhile the AgNPs cinnamon oil combination demonstrated a lower efficacy., Conclusions: This is an in vitro preliminary approach that documented the antibiofilm potential of cinnamon oil and the inhibitory activity of cinnamon oil and its combination with AgNPs against MDR S. agalactiae recovered from clinical mastitis. Further in vivo studies should be carried out in animal models to provide evidence of concept for implementing these alternative candidates in the treatment of dairy farms infected by streptococcal mastitis in the future.

Campylobacter species are common commensals in the gastrointestinal tract of livestock animals; thus, animal-to-human transmission occurs frequently. We investigated for the first time, class 1 integrons and associated gene cassettes among pan drug-resistant (PDR), extensively drug-resistant (XDR), and multidrug-resistant (MDR) Campylobacter species isolated from livestock animals and humans in Egypt. Campylobacter species were detected in 58.11% of the analyzed chicken samples represented as 67.53% Campylobacter jejuni (C. jejuni ) and 32.47% Campylobacter coli ( C. coli ). C. jejuni isolates were reported in 51.42%, 74.28%, and 66.67% of examined minced meat, raw milk, and human stool samples, respectively. Variable antimicrobial resistance phenotypes; PDR (2.55%), XDR (68.94%), and MDR (28.5%) campylobacters were reported. Molecular analysis revealed that 97.36% of examined campylobacters were integrase gene-positive; all harbored the class 1 integrons, except one possessed an empty integron structure. DNA sequence analysis revealed the predominance of aadA (81.08%) and dfrA (67.56%) alleles accounting for resistance to aminoglycosides and trimethoprim, respectively. This is the first report of aacC5-aadA7Δ4 gene cassette array and a putative phage tail tape measure protein on class 1 integrons of Campylobacter isolates. Evidence from this study showed the possibility of Campylobacter -bacteriophage interactions and treatment failure in animals and humans due to horizontal gene transfer mediated by class 1 integrons.

Campylobacter jejuni ( C. jejuni ) are able to colonise and infect domestic poultry and also pose a risk for humans. The aim of this study was to determine the extent of genotypic diversity among C. jejuni isolates recovered from avian and human sources in Egypt. Furthermore, the short variable region (SVR) of flagellin A ( flaA ) gene was analysed for the presence of allelic variants. Our results showed that C. jejuni isolates differ in their capacity to harbour each of the virulence genes alone or when present in various combinations. The flaA gene was detected in all C. jejuni strains and none of the strains had all the studied virulence genes together. When considering C. jejuni strains from the investigated sources, the cdtC gene was the most similar, while the cdtB and iam genes were the most dissimilar. We could identify 13 novel alleles in the analysed strains. The analyses of virulence gene patterns, flaA gene sequences and allelic variants showed that C. jejuni strains from different sources overlapped largely suggesting potential involvement of poultry in transmitting C. jejuni to humans. We also found that the strains isolated from the same host were highly heterogeneous, with chicken strains exhibiting the highest diversity. Moreover, the human strains were clustered closer to chicken ones than to those from pigeon. The results of this study should be taken into consideration when assessing the epidemiology and risk potential of Egyptian C. jejuni not only in poultry, but also in humans., (Copyright © 2019 Abd El-Hamid, Abd El-Aziz, Samir, El-Naenaeey, Abo Remela, Mosbah and Bendary.)

The emergence of multidrug-resistant (MDR) pathogens was reported worldwide. Herein, SHV extended-spectrum β-lactamase (SHV-ESBL) variants detection was investigated in MDR E. coli and K. pneumoniae isolates recovered from human subjects (n = 144), one day-old chicks (n = 36) and broiler clinical samples (n = 90). All examined samples were positive for E. coli (n = 246/270; 91.11%) and Klebsiella pneumoniae (n = 24/270; 8.89%). Antimicrobial susceptibility testing was performed on E. coli and K. pneumoniae. SHV-ESBL producing isolates were defined followed by SHV-ESBL amino acids sequence and proteins structure-function analyses. Phylogenetic analysis of 11 MDR isolates resistant to at least 6 β-lactams was designed to determine their genetic relationship with those previously identified in Egypt. SHV-ESBL variants were detected in 28% and 16% of E. coli and K. pneumoniae isolates, respectively. Among the 11 SHV-ESBL producing isolates, one isolate displayed 100% blaSHV-12 similarity with three point mutations, while the other 10 isolates displayed amino acid substitutions at previously non-reported sites. Amino acid sequence analyses of these 10 isolates displayed 96-100% identity to blaSHV-10 (2 isolates with 3-6 point mutations), blaSHV-18 (one isolate with 4 point mutations), blaSHV-58 (4 isolates with 4-5 point mutations), and blaSHV-91 (3 isolates with 3-7 point mutations). These mutations altered SHV-enzyme pocket dimensions and its binding sites chargeability. The blaSHV phylogeny analysis revealed occurrence of variants in closely related lineages with blaSHV-5 and blaSHV-12 with possibility of blaSHV gene transfer between human and birds. The occurrence of these variants in Egypt could help in epidemiological studies and could explain the emergent resistance to β-lactams., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Introduction: Avian mycoplasmas impose a significant economic burden to the poultry industry. In recent years, macrolide-resistant Mycoplasma gallisepticum have occasionally been encountered in Egypt., Methodology: This study was designed to document the involvement of macrolide-resistant M. gallisepticum in respiratory organs of chickens suffering respiratory problems. Concurrently, an exhaustive molecular characterization of the intrinsic resistance of recovered isolates to macrolides was done., Results: Of 120 chickens showing respiratory problems, 14 (11.67%) M. gallisepticum were isolated and genetically identified; 8 of them were recovered from air sacs, 4 from lungs, and 2 from tracheas. Broth microdilution of all M. gallisepticum isolates showed various degrees of minimum inhibitory concentrations (MICs) against macrolides: erythromycin (0.25-32 µg/mL), tylosin (0.0625-4 µg/mL), and tiamulin (0.031-2 µg/mL). Nucleotide sequencing of domain V (peptidyl transferase region) of the 23S rRNA gene of macrolide-resistant M. gallisepticum isolates revealed transition mutations at positions 2068 and 2069 (corresponding to 2058 and 2059 in Escherichia coli numbering) in an isolate and at position 2067 (corresponding to 2057 in E. coli numbering) in three isolates as hot spots for macrolide resistance. Surprisingly, a transversion mutation at position 2621 (corresponding to 2611 in E. coli numbering) was reported in one of the recovered isolates as a first report., Conclusion: Generation of new mutations is evidence for persistence of M. gallisepticum despite macrolide treatment. Periodic surveys to monitor for the possible appearance of resistant strains are recommended.

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.

Acute respiratory tract infections are leading causes of morbidity in poultry farms allover the world. Six pathogens; Escherichia coli, Mycoplasma gallisepticum, Staphylococcus aureus, Pasteurella multocida, Mannheimia haemolytica and Pseudomonas aeruginosa were involved in respiratory infections in poultry. Herein, conventional identification procedures and polymerase chain reaction (PCR) were applied for detection of the most common respiratory bacterial pathogens in clinical specimens of poultry obtained from 53 Egyptian farms with various respiratory problems and the results were compared statistically. The analyzed data demonstrated a significantly higher rate of detection of the most recovered microorganisms (P<0.05) by PCR comparing to classical culture procedures. Further, multiplex PCR could detect E. coli, M. gallisepticum, S. aureus and Ps. aeruginosa in a single reaction, however, M. haemolytica was reported in a uinplex system. According to PCR results, the most commonly recorded bacterial pathogens in examined poultry farms were E. coli and Ps. aeruginosa (54.71% each), followed by M. haemolylica (35.85%) and M. gallisepticum (20.75%). In conclusion, PCR assay offered an effective alternative to traditional typing methods for the identification and simultaneous detection of the most clinically relevant respiratory pathogens in poultry.

Three kinds of plasmid—mediated quinolone resistance (PMQR) determinants (qnr genes, qepA and aac(6')—Ib—cr) have been discovered and shown to be widely distributed among clinical isolates. To characterize the prevalence of PMQR determinants among AmpC—producing E. coli strains in food—producing animals and animal by—products in Egypt, twenty—nine E. coli strains were tested for their susceptibilities to antimicrobials and screened for PMQR determinants and AmpC Beta lactamases using PCR and plasmid profiling. It was found that qnr genes being detected alone or in combination with qepA or aac(6')—Ib—cr genes in 11 (37.9%) strains comprising 9 for qnrA and only one for both qnrB and qnrS. Moreover, qepA and aac(6')—Ib—cr were detected in 41.38% and 3.45% of E. coli strains, respectively. The ampC β—lactamase genes were detected in 75.86 % of all strains and in 100% and 53.3% of the PMQR determinant—positive and negative strains, respectively. In several cases, plasmid profiling of E. coli strains exhibiting the coexistence of both PMQR determinants and ampC genes on a single plasmid as a first report in Egypt that may contribute to rapid spread and increase in bacterial resistance, which is important to public health concern.