Fluoroquinolone-resistant and extended-spectrum ß-lactamase-producing Escherichia coli from the milk of cows with clinical mastitis in Southern Taiwan.

BACKGROUND/PURPOSE: Escherichia coli is a common pathogen to cause clinical and subclinical mastitis in cows. A total of 57 E. coli isolates from raw milk from cows were characterized genetically and biochemically. METHODS: Extended-spectrum ß-lactamase (ESBL) genes, the mechanism for fluoroquinolone resistance, and variations in virulence genes and genomes of these E. coli isolates were investigated by the antimicrobial susceptibility test, simplex and multiplex polymerase chain reaction (PCR), and pulsed-field gel electrophoresis (PFGE). RESULTS: All E. coli isolates were resistant to cloxacillin (100%) and to a lesser extent (50%) to tetracycline, neomycin, gentamycin, ampicillin, ceftriaxone, cefotaxime (CTX), and ceftazidime (CAZ). Nearly 70% of the isolates were resistant to at least two antimicrobials and 28.1% carried AmpA and AmpC genes simultaneously. The predominant bla gene was blaTEM, followed by blaCMY, blaCTX, blaSHV, and blaDHA. Among the six (10.5%) ESBL-producing E. coli carrying blaCTX-M15, blaCTX-M55, or blaCTX-M14, two isolates 31 of ST410 in the ST23 complex and 58 of ST167 in the ST10 complex were also resistant to ciprofloxacin, enrofloxacin, and levofloxacin, with mutations at codon 83 from serine to leucine and codon 87 from aspartic acid to asparagine in GyrA and at codon 80 from serine to isoleucine in ParC. These isolates were genetically diverse in pulsotype analysis, lacked toxin genes of human pathogenic E. coli and carried mostly the prevalent virulence genes fimH, papGII, and α-hemolysin. CONCLUSION: Lacking virulence genes examined, genetic diverse E. coli isolates are unrelated to human pathogenic E. coli. Enhancing sanitation in milk processing and transportation is needed to eliminate multidrug-resistant (MDR), fluoroquinolone-resistant, and ESBL-producing E. coli isolates.

Mycobacterium tuberculosis and M. bovis infection in Feedlot Deer (Cervus unicolor swinhoei and C. nippon taiouanus) in Taiwan.

BACKGROUND/PURPOSE: Mycobacterium bovis frequently infects wild and farm deer species with tuberculosis. This study investigated mycobacterial infection in two native deer species Cervus unicolor swinhoei (Formosan Sambar, Sambar) and C. nippon taiouanus (Formasan Sika, Sika). METHODS: Based on different sampling sources of 19 intradermal tuberculin test (ITT) Sambar, mycobacterial infection and/or species were detected by acid-fast stain, duplex polymerase chain reaction (PCR) and multiplex nested PCR (mnPCR) methods, traditional mycobacterial culture and gross lesion. Blood samples of 167 Sambar deer and 147 Sika deer were then tested by duplex PCR and mnPCR methods to investigate the prevalence of mycobacterial infection. Sequence variations of these mycobacterial species were analyzed as well. RESULTS: Duplex PCR and mnPCR assays could differentiate between MTBC (M. bovis and M. tuberculosis) and M. avium, as well as between M. bovis and M. tuberculosis, respectively. These PCR methods showed a higher detection rate than traditional culture and matched the gross lesions examined in 19 ITT-examined Sambar. Therefore, the mycobacterial infection in blood samples of 314 deer samples was detected using these PCR methods. Duplex PCR and mnPCR showed an identical prevalence of 16.1% in Sambar and 8.2% in Sika and a significant difference in prevalence between these two deer species. M. bovis and M. tuberculosis were the species detected in feedlot Sambar and Sika. M. tuberculosis was found only and first in Sambar fed in central Taiwan. Sequence analysis revealed diverse genetic variations in M. bovis and M. tuberculosis associated with deer subspecies. CONCLUSION: Multiplex PCR methods were established, and M. bovis and M. tuberculosis were identified in feedlot deer in Taiwan. Sequence variations indicated diverse sources of both mycobacterial species.

Emergence of Salmonella enterica serovar Potsdam as a major serovar in waterfowl hatcheries and chicken eggs.

Salmonellosis is a common food-borne illness in humans caused by Salmonella-contaminated poultry and their products. In hatcheries, 110 Salmonella isolates were identified, mostly from first enrichment, and few from delayed enrichment. The Salmonella prevalence in goose and duck hatcheries was higher when measured by four multiplex PCR methods than by traditional culture (73.8% vs. 44.35%, P < 0.05); 97.3% of 110 isolates were Salmonella Potsdam of serogroup C1 and other isolates were Salmonella Montevideo of C1 and Salmonella Albany of C2. Plasmid and pulsed field gel electrophoresis genetic analysis revealed that isolates from duck hatcheries were more diverse than those from goose hatcheries. In Salmonella Potsdam, host species-specific genotypes were observed in geese for genotypes 3, 4, and 5 and in ducks for genotypes 7, 8, and 9, suggesting that Salmonella Potsdam may evolve into goose- and duck-specific isolates. An examination of 1121 eggs found that only Salmonella Potsdam was identified in 1.8% (7/591) of eggs from chickens fed on the ground, not housed in cages, and in egg content (6/7) as well as eggshell membrane (1/7). In conclusion, Salmonella Potsdam may be a major Salmonella infection in waterfowl and chicken eggs.

Characterization of 13 multi-drug resistant Salmonella serovars from different broiler chickens associated with those of human isolates.

BACKGROUND: Salmonella are frequently isolated from chickens and their products. Prevalent serogroups and serovars of Salmonella as well as their genotypes and antibiograms were determined for cloacal samples from 1595 chickens. To understand the possible serovar and H antigens for transmission between chicken and human, serovars and their H antigens of 164 chicken and 5314 human isolates were compared. RESULTS: Prevalence of Salmonella differed among chicken lines and ages. Chicken and human isolates belonged mainly to serogroup B, C1, C2-C3, D, and E. 13 serovars and 66 serovars were identified for chicken and human isolates respectively. The common serovars for chicken and human isolates were S. Typhimurium, S. Enteritidis, S. Albany, S. Derby, and S. Anatum and shared common H1 antigens "g complex; i; e,h; and z4,z24" and H2 antigens "1 complex and -". In human isolates, H1 antigen "i" and H2 antigen "-" were common in all serogroups. In chicken, antimicrobial susceptibility differed among serogroups, serovars and three counties. All isolates were susceptible to cefazolin and ceftriaxone, but highly resistant to ampicillin, chloramphenicol, flumequine, streptomycin, sulfamethoxazole-trimethoprim, and tetracycline. Except those isolates of serogroup C1 of Chick group and serogroup G, all isolates were multi-drug resistance. Only S. Kubacha, S. Typhimurium, S. Grampian, and S. Mons were resistant to ciprofloxacin and/or enrofloxacin. CONCLUSION: In chicken, prevalent serogroups and serovars were associated with chicken ages, lines and regions; and flouroquinolone-resistant and MDR isolates emerged. H1 antigens "g complex and i" and H2 antigens "1 complex and -" might be important for transmission of Salmonella between chicken and human.

Antimicrobial peptides of an anti-lipopolysaccharide factor, epinecidin-1, and hepcidin reduce the lethality of Riemerella anatipestifer sepsis in ducks.

Antimicrobial peptides (AMPs) are effective against a wide range of microbes, but still no research results have reported their use in duck disease therapy. Riemerella anatipestifer (RA) is a Gram-negative bacterium which infects ducks and causes very significant economic losses. The minimum inhibitory concentrations (MICs) of epinecidin-1 for the tested RA strains ranged 6.25-50microg/ml, those of the SALF55-76 cyclic peptide ranged 12.5-25microg/ml, those of the SALF55-76 linear peptide ranged 6.25-25microg/ml, those of hepcidin TH1-5 ranged 25-400microg/ml, and those of hepcidin TH2-3 ranged 100-400microg/ml. The antimicrobial activities of these peptides were confirmed by transmission electron microscopy which showed that RA disruption of the outer membrane brought about cell death. In addition, pretreatment, co-treatment, and post-treatment with peptides were all effective in promoting a significant decrease in duck mortality and decreasing the number of infectious bacteria. A quantitative RT-PCR was performed to survey levels of gene expressions of Mn superoxide dismutase in the brain, lipoprotein lipase in the liver, and H5 histone in the spleen induced in response to bacterial infection and an injection of the AMPs in experiments with the duck, Cairina moschata. Our results indicated that the rescue of ducks by the peptides and the behavior of the peptides, which was like an enhancer in immunology, may involve regulation of the expressions of these genes. Collectively, these peptides reduced the mortality in ducks during bacterial challenge, suggesting that AMPs have the potential to serve as therapeutic drugs for use against bacterial infectious diseases in ducks.

Hematologic and biochemistry values for black-faced spoonbills (Platalea minor) with and recovering from botulism.

Type C1 botulism outbreaks in Black-faced Spoonbills (Platalea minor) occurred in Taiwan from 2002 to 2003, and hematologic and biochemistry parameters from botulism-paralyzed birds and recovered birds were compared. Values for creatinine and uric acid were higher (P<0.0025) in birds with botulism than in recovered birds. Lower white blood cell counts (P<0.005) and values for alkaline phosphatase, aspartate aminotransferase, alanine aminotransferase, and triglycerides (P<0.025) were observed in recovered birds. Based on these observations, we suggest that hematologic and biochemistry analyses should be performed to assess the health condition of birds recovering from botulism.

Genomic diversity and molecular differentiation of Riemerella anatipestifer associated with eight outbreaks in five farms.

Riemerella anatipestifer causes infectious serositis of ducks and geese. The genomic diversity of R. anatipestifer associated with outbreaks in waterfowls was studied using 24 multidrug-resistant R. anatipestifer isolates collected from the visceral organs of ducks and geese from seven outbreaks in four goose farms and one outbreak in one duck farm. Seven methods were used to differentiate these isolates. Plasmid patterns differed in plasmid number and size, ranging from 2.9 kb to 20 kb, and provided seven profiles. Divergent nucleotide sequences (predominant in 670 to 830 base pairs) of the ompA gene categorized the 24 isolates into three groups based on cluster analysis and polymerase chain reaction-restriction fragment length polymorphism. Repetitive-sequence polymerase chain reaction and pulsed-field gel electrophoresis analysis revealed the highest genotypic variations among the isolates. Genotypes and serotypes differed among farms and within the same farm and even within a single goose. In conclusion, a difference in R. anatipestifer genotypes and serotypes was observed for multiple outbreaks in waterfowls.

Prevalence and characterization of multidrug-resistant (type ACSSuT) Salmonella enterica serovar Typhimurium strains in isolates from four gosling farms and a hatchery farm.

Salmonella enterica serovar Typhimurium strains of phage types DT104 and U302 are often resistant to ampicillin, chloramphenicol, streptomycin, sulfonamides, and tetracycline (the ACSSuT resistance type) and are major zoonotic pathogens. Increased consumption of goose meat may enhance the risk of transferring S. enterica serovar Typhimurium and other enteric pathogens from geese to human due to the consumption of meats from infected geese or improper preparation of meats. Therefore, we characterized S. enterica serovar Typhimurium strains isolated from four goose farms (farms A, B, C, and D) and one hatchery farm (farm E) to determine the epidemic and genetic differences among them. Antibiotic susceptibility tests and multiplex PCR confirmed that 77.6% (52/67) of strains were ACSSuT strains isolated from farms A, C, and E. Antibiotic-susceptible strains were isolated mostly from farm B, and no strain was observed in farm D. All ACSSuT strains harbored a 94.7-kb virulence plasmid and contained one 1.1-kb conserved segment identical to that of Salmonella genomic island 1. Four genotypes were determined among these S. enterica serovar Typhimurium isolates by pulsed-field gel electrophoresis analysis of XbaI-digested DNA fragments. Most isolates (85.29%; 29/34) of major genotype Ib were ACSSuT strains isolated mainly from goslings of farm C and egg membranes of farm E, a hatchery farm, suggesting that S. enterica serovar Typhimurium strains in isolates from goslings might originate from its hatchery, from the egg membranes to the gosling fluff after hatching. Multiple phage types, types 8, 12, U283, DT104, and U302, were identified. In conclusion, geese were a reservoir of diverse multidrug-resistant (type ACSSuT) S. enterica serovar Typhimurium strains, and each farm was colonized with genetically closely related S. enterica serovar Typhimurium strains.