Extensive Drug-Resistant Salmonella enterica Isolated From Poultry and Humans: Prevalence and Molecular Determinants Behind the Co-resistance to Ciprofloxacin and Tigecycline.

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.

Virulence Determinants and Plasmid-Mediated Colistin Resistance mcr Genes in Gram-Negative Bacteria Isolated From Bovine Milk.

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.

Whole-Genome Sequencing of Gram-Negative Bacteria Isolated From Bovine Mastitis and Raw Milk: The First Emergence of Colistin mcr-10 and Fosfomycin fosA5 Resistance Genes in Klebsiella pneumoniae in Middle East.

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 fosA5 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 xerC 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.

Isolation, genotyping and subtyping of single Cryptosporidium oocysts from calves with special reference to zoonotic significance.

The ability of the small-subunit ribosomal RNA (SSU rRNA) based nested PCR and Restriction Fragment Length Polymorphism (PCR-RFLP) to identify and genotype a single Cryptosporidium oocyst isolated from bovine faecal samples was evaluated in this study. In addition, subtyping was carried out by sequencing the 60 kDa glycoprotein (gp60) gene from the same single oocyst. Faecal samples were collected from 40 pre-weaned calves (5-20 days old) from 7 dairy farms located in 3 different counties within the Finger Lakes region of Upstate New York. All the samples were microscopically positive for Cryptosporidium spp. A total of 400 Cryptosporidium oocysts (10 single oocysts from each calf sample) were individually isolated and analyzed using a nested PCR targeting the SSU rRNA gene. The SSU rRNA gene was amplified in 324 (81%) individual oocysts. All SSU rRNA amplified individual oocysts DNA was genotyped using PCR-RFLP. C. parvum was the only identified species; 107 single oocysts generated PCR products from the A gene, 18 generated PCR products from the B gene and 199 generated PCR products from both. Sequence analysis of the gp60 gene in 99 individual oocysts revealed the presence of only subtype IIaA15G2R1 with 99.4-100% and 99.1-100% identity of nucleotides and amino acids, respectively. These sequences were identical (100%) in oocysts from 35 calves and exhibited mutations in the non-repeat region of the gp60 gene in those of 5 other calves. The examination of DNA from individual oocysts with genotyping and subtyping tools provides methodology to more clearly define the genetic characteristics of Cryptosporidium spp. on farms and within individual animals.

New Approach for Controlling Snail Host of Schistosoma mansoni, Biomphalaria alexandrina with Cyanobacterial Strains-Derived C-Phycocyanin.

Schistosomiasis is one of the major communicable diseases of public health and socioeconomic importance in the developing world. It is a waterborne disease in which Biomphalaria alexandrina snails are known to be the intermediate molluscan host for Schistosoma mansoni: the causative agent of human intestinal schistosomiasis. Therefore, snail control is one of the cornerstones of schistosomiasis control programs. Several methods have been used to eliminate snail hosts. One of these methods is chemical molluscicides, which have undesirable effect to nontarget organisms. Consequently, the search for biologically derived molluscicides to complement the use of synthetic molluscicides is a top priority. In this concern, this study is the first to evaluate the molluscicidal potency of Cyanobacterial Phycocyanin (C-PC) as a virtually untapped source. Laboratory assessment of three freshwater Cyanobacterial strains: Anabaena oryzae SOS13, Nostoc muscorum SOS14, and Spirulina platensis SOS13-derived C-Phycocyanin as a biocontrol agent against freshwater mollusks; B. alexandrina snails were performed. Also, the safety of tested C-PC on nontarget organisms (Tilapia fish) was assessed. Results reveal that C-PC extracted from all tested Cyanobacteria strains showed a promising molluscicidal activity (the mortality rate was 100% at 100 µg/mL concentration). Out of the examined strains, A. oryzae SOS13 phycocyanin was found to be the most potent strain (LC50 and LC90 were 38.492 and 49.976 µg/mL, respectively). Moreover, C-PC extracts from all tested strains have been found to be safe to Tilapia fish as the survival rate was 100% at the effective molluscicidal concentrations. We can conclude that C-PC extracts are the first promising microbial biopesticides for the control of freshwater B. alexandrina snails.

Molecular Screening and Genotyping of Cryptosporidium Species in Household Dogs and In-Contact Children in Egypt: Risk Factor Analysis and Zoonotic Importance.

Cryptosporidiosis is a parasitic zoonosis implicated in severe diarrhoea in pets and humans. This study aimed to determine the prevalence and genotypes of Cryptosporidium spp. in household dogs and in-contact children, and the risk factors associated with infection in children in Sharkia Province, Egypt. Fecal samples of 100 children (2-12 years old) and 50 dogs (3 months-1 year old) were randomly collected from both rural (children: n = 85, dogs: n = 40) and urban (children: n = 15, dogs: n = 10) households. Initial parasite detection was done by light microscopy, while, genotyping was based on molecular diagnostic assays. The overall prevalence of Cryptosporidium spp. infection in children was 35% using microscopy and 14% using nested polymerase chain reaction (PCR). In dogs, it was 34% using microscopy and 24% using nested PCR. Cryptosporidium spp. from children were identified as distinct genotypes, with the predominance of human genotype I (Cryptosporidium hominis) over the zoonotic genotype II (Cryptosporidium parvum). Moreover, only zoonotic genotype II (C. parvum) was identified in dog samples. The significant risk factors associated with the prevalence of Cryptosporidium infection in children were the presence of diarrheal episodes during time of survey, improper disposal of garbage, and dog feces and contact with other livestock (p ≤ 0.05). This study concluded that the existence of C. parvum in children and dogs residing the same households confirm the zoonotic transmission and its public health significance. Also, the study recommended the necessity of hygienic disposal of dog feces and preventing direct contact of dogs with other livestock.