Kuang Sheng Yeh

A Strain of Pseudomonas sp. Isolated from Piggery Wastewater Treatment Systems with Heterotrophic Nitrification Capability in Taiwan

A high concentration of NH4+ in piggery wastewater is major problem in Taiwan. Therefore, in our study, we isolated native heterotrophic nitrifiers for piggery wastewater treatment. Heterotrophic nitrifier AS-1 was isolated and characterized from the activated sludge of a piggery wastewater system. Sets of triplicate crimp-sealed serum bottles were used to demonstrate the heterotrophic nitrifying capability of strain AS-1 in an incubator at 30°C. All serum bottles contained 80 mL medium, and the remainder of the bottle headspace was filled with pure oxygen. The experimental results showed that 2.5 ± 0.2 mmol L−1 NH4+ was removed by 58 hours, and, eventually, 1.5 ± 0.5 mmol L−1 N2 and 0.2 ± 0.0 mmol L−1 N2O were produced. The removal rate of NH4+ by the strain AS-1 was 1.75 mmol NH4+ g cell−1 h−1. This strain was then identified as Pseudomonas alcaligenes (97% identity) by sequencing its 16S rDNA and comparing it with other microorganisms. Thus, strain AS-1 displays high promise for future application for in situ NH4+ removal from piggery wastewater.

Staphylococcus aureus Isolated from Pork and Chicken Carcasses in Taiwan: Prevalence and Antimicrobial Susceptibility

Staphylococcus aureus is a cause of many diseases in both humans and animals. This pathogen is also a major target in the screening of slaughterhouse carcasses to monitor hygienic conditions during slaughter. During 2004 to 2006, S. aureus was recovered from 8.8% (38 of 430), 11.3% (77 of 680), and 4.3% (13 of 300) of pork carcass samples, respectively, collected at 53 slaughterhouses in Taiwan. During 2003 to 2005, it was recovered from 0.3% (1 of 305), 0.4% (1 of 260), and 7.8% (31 of 395) of rinse fluids from chicken carcasses, respectively, collected at 17 meat processing plants. The minimum dilution method was used to determine antimicrobial susceptibility (MICs) of these strains (n = 103) as well as those collected from pork and chicken carcasses (n = 104) in a previous study beginning in 2000. All 207 strains were sensitive to nitrofurantoin and vancomycin. Over 50% were resistant to clindamycin (MIC that inhibited 90% of strains [MIC90] = 32 microg/ml) and tetracycline (MIC90 = 64 microg/ml). The percentages resistant to methicillin (oxacillin), chloramphenicol, erythromycin, and tylosin were 19.4% (40 of 207), 18.8% (39 of 207), 23.2% (48 of 207), and 20.8% (43 of 207) with MIC90s of 8, 64, > or = 64, and > or = 128 microg/ml, respectively. The methicillin-resistant S. aureus (MRSA) strains exhibited resistance to more antibiotics than did the methicillin-susceptible strains, and 87.5% (35 of 40) of the MRSA strains carried the mecA gene sequence. Since MRSA infections have become a public health concern in both communities and hospitals, testing for the presence of MRSA in animal carcasses during slaughtering operations is warranted.

Mutations in the Salmonella enterica serovar Choleraesuis cAMP-receptor protein gene lead to functional defects in the SPI-1 Type III secretion system.

Salmonella enterica serovar Choleraesuis (Salmonella Choleraesuis) causes a lethal systemic infection (salmonellosis) in swine. Live attenuated Salmonella Choleraesuis vaccines are effective in preventing the disease, and isolates of Salmonella Choleraesuis with mutations in the cAMP-receptor protein (CRP) gene (Salmonella Choleraesuis ∆crp) are the most widely used, although the basis of the attenuation remains unclear. The objective of this study was to determine if the attenuated phenotype of Salmonella Choleraesuis ∆crp was due to alterations in susceptibility to gastrointestinal factors such as pH and bile salts, ability to colonize or invade the intestine, or cytotoxicity for macrophages. Compared with the parental strain, the survival rate of Salmonella Choleraesuis ∆crp at low pH or in the presence of bile salts was higher, while the ability of the mutant to invade intestinal epithelia was significantly decreased. In examining the role of CRP on the secretory function of the Salmonella pathogenicity island 1 (SPI-1) encoded type III secretion system (T3SS), it was shown that Salmonella Choleraesuis ∆crp was unable to secrete the SPI-1 T3SS effector proteins, SopB and SipB, which play a role in Salmonella intestinal invasiveness and macrophage cytotoxicity, respectively. In addition, caspase-1 dependent cytotoxicity for macrophages was significantly reduced in Salmonella Choleraesuis ∆crp. Collectively, this study demonstrates that the CRP affects the secretory function of SPI-1 T3SS and the resulting ability to invade the host intestinal epithelium, which is a critical element in the pathogenesis of Salmonella Choleraesuis.

PCR amplification of the Salmonella typhimurium fimY gene sequence to detect the Salmonella species

This study evaluated the suitability of fimY gene amplification by PCR as an effective means of detecting Salmonella species. Although fimY gene of Salmonella typhimurium is involved in regulating type 1 fimbrial expression, the amino acid sequence of FimY shares very little homology with other known prokaryotic proteins in the GenBank database. Therefore, fimY is a promising target gene to detect the presence of Salmonella species. Herein, two primers internal to the fimY gene of S. typhimurium are used to investigate the distribution of the fimY homologous sequence among 45 Salmonella serovars and 20 non-Salmonella species by using PCR. Experimental results indicated that only Salmonella species possessed the fimY homologous sequence, subsequently generating the specific 526-bp DNA fragments. The sensitivity of the fmY-specific primer set was demonstrated on a Salmonella-free swab sample from a pork carcass surface, which was then artificially contaminated with different concentrations of S. typhimurium. A combining of pre-enrichment step in buffered peptone water and PCR amplification of fimY allowed the detection of S. typhimurium at the concentration of 3.4 x 10(0) CFU/ml from the swab sample. With an additional enrichment step in Rappaport-Vassiliadis (RV) broth, this procedure can also detect pork carcass surface naturally contaminated with Salmonella species in a slaughterhouse. Results in this study demonstrate that fimY is unique to Salmonella species and is an appropriate PCR target for detecting these microorganisms.

Epidemiologic Relationship between Fluoroquinolone-Resistant Salmonella enterica Serovar Choleraesuis Strains Isolated from Humans and Pigs in Taiwan (1997 to 2002)

ABSTRACT The emergence of ciprofloxacin-resistant Salmonella enterica serovar Choleraesuis in recent years has become an important public health issue in Taiwan. The resistant strains that cause human infections are considered to be from pigs. In this study, we characterized 157 swine and 42 human Salmonella serovar Choleraesuis isolates by pulsed-field gel electrophoresis (PFGE) and drug susceptibility testing to investigate the epidemiologic relationship among the isolates. By PFGE analyses, two major clusters (clusters GA and GB) were identified. Isolates in cluster GA were of both human and swine origins, while those in cluster GB were from pigs only. Among the various genotypes identified, genotype gt-1a was the most prevalent, which was found in 71% (30 of 42) and 48% (76 of 157) of human and swine isolates, respectively. The susceptibility tests for the 106 gt-1a isolates identified 44 susceptibility profiles and showed that 73% of human isolates and 34% of swine isolates were resistant to three fluoroquinolones (ciprofloxacin, enrofloxacin, and norfloxacin). Our findings indicate that a clonal group of Salmonella serovar Choleraesuis may have been circulating in human and swine populations in Taiwan for years and that the fluoroquinolone-resistant Salmonella serovar Choleraesuis strains most likely evolved from a gt-1a clone that emerged in 2000 and that then caused widespread infections in humans and pigs. Nevertheless, it is still debatable whether those Salmonella infections in humans are caused by isolates derived from pigs, on the basis of the higher fluoroquinolone and other antimicrobial resistance percentages in human isolates than in pig isolates.

One-year (2003) nationwide pork carcass microbiological baseline data survey in Taiwan.

From January through December 2003, swab samples from 1,650 pork carcasses were collected from 39 slaughter plants in Taiwan. These samples were analyzed for the prevalence of indicator microorganisms and specific pathogens. Viable aerobic bacteria, total coliforms, and Escherichia coli were recovered from 100, 95.3, and 87.5% of these carcasses, respectively. Of those carcasses that harbored bacteria, the mean aerobic plate, total coliform, and Escherichia coli counts were 4.0, 0.6, and 0.1 log CFU/cm2, respectively. Staphylococcus aureus, Clostridium perfringens, Campylobacter jejuni, Campylobacter coli, Listeria monocytogenes, and Salmonella were recovered from 4.8, 0.3, 13.8, 0.7, and 1.7 of 1,038 carcasses, respectively. E. coli O157:H7 was not detected from any carcass. When positive for a specific pathogen, the mean carcass concentration was 0.57 log CFU/cm2 for S. aureus, 0.66 most probable number (MPN)/cm2 for C. jejuni and C. coli, and 0.18 MPN/cm2 for Salmonella. The findings of this study will help provide a reference for establishing hygienic standards and a criterion for evaluating the effects of slaughtering operations in Taiwan.

FimZ binds the Salmonella typhimurium fimA promoter region and may regulate its own expression with FimY

The FimZ protein, an activator of FimA production in Salmonella typhimurium, acts in conjunction with FimY to facilitate the expression of type 1 fimbriae. The predicted amino acid sequence of FimZ suggests that this protein may be a DNA‐binding protein related to BvgA, a sensory regulator of virulence gene expression in Bordetella pertussis. Purification of FimZ following overexpression of the protein by a strong inducible promoter and gel mobility shift assays confirm that FimZ is a 25‐kDa polypeptide that binds to the promoter region of fimA. The region of DNA protected from DNase I digestion by FimZ binding is located between 47 and 98 nucleotides upstream from the fimA transcription initiation site. This region possesses a pair of 7‐base pair tandem repeats, of which at least one is necessary for FimZ binding. One copy of the 7‐base pair sequence is also located in the fimZ promoter region. In addition, expression from a fimZ‐lacZ reporter construct confirms that FimZ plays a role in its own expression. Both FimZ and FimY are required for high‐level expression of FimZ, which suggests that these two fimbrial proteins are involved in regulating both FimA and FimZ.

Transmission of Salmonella between swine farms by the housefly (Musca domestica).

The domestic pig is an important source of human salmonellosis, and houseflies are potential mechanical vectors of foodborne Salmonella pathogens. In 2005, we recovered 144 Salmonella isolates from flies and swine stool samples from 11 farms in Taoyuan County and Hsin Chu County (northwestern Taiwan). A total of 71.5% of the isolates were resistant to at least three antibiotics. There were a total of 14 serotypes, and 8 of these serotypes were present in both flies and swine stool samples. Some multidrug-resistant Salmonella strains coming from different swine farms were found to have identical pulsed-field gel electrophoresis (PFGE). Among four common serotypes, we identified 18 PFGE patterns, 8 of which were present in flies and swine stools. The similarity in PFGE profiles between isolates from swine and flies in different farms indicate the potential of flies to serve as a vector for transmission.

Molecular characterization of enrofloxacin resistant Actinobacillus pleuropneumoniae isolates.

Enrofloxacin (ER) resistant Actinobacillus pleuropneumoniae strains emerged in Taiwan in 2002. The mechanism of ER resistance in A. pleuropneumoniae has not yet been reported. A total of 48 A. pleuropneumoniae isolates were obtained from the lungs of pigs with pleuropneumonia in Taiwan between September 2007 and April 2008. Twenty-nine isolates were found to be resistant to enrofloxacin. To understand the mechanisms of A. pleuropneumoniaes resistance to ER, enrofloxacin susceptibility of the isolates along with the mutations of the quinolone resistance-determining region (QRDR) of DNA gyrase and topoisomerase IV, qnr genes were analyzed. Enrofloxacin resistant isolates were found to carry at least one mutation in the QRDR of gyrA, leading to amino acid changes at codon 83 or 87. Efflux pump inhibitor (Phe-Arg-beta-naphthylamide) decreased enrofloxacin minimum inhibitory concentration 2-16-fold, suggesting participation of efflux in ER resistance. Plasmid mediated quinolone resistance genes qnr were not detected in these isolates. In conclusion, enrofloxacin resistance of A. pleuropneumoniae may be linked to multiple target gene mutations and active effluxs.

Comparison between VIDAS automatic enzyme-linked fluorescent immunoassay and culture method for Salmonella recovery from pork carcass sponge samples

VIDAS Salmonella (VIDAS-SLM) is an automated system that uses the enzyme-linked fluorescent assay method to detect Salmonella species. This study evaluated the efficacy of the VIDAS-SLM method in detecting Salmonella species in pork carcass sponge samples gathered from 10 slaughter plants in Taiwan. Two hundred fifty-seven pork carcass sponge samples were screened by the VIDAS-SLM method and by the culture method in parallel. While 18 sponge samples were found to test positive by both methods, the VIDAS-SLM method detected four additional positive samples for which the culture method failed to recover Salmonella. The specificity of the VIDAS-SLM method was found to be 0.98, and its sensitivity was 1.0, since no false-negative results occurred. Artificially inoculated Salmonella at concentrations as low as 5.0 x 10(0) CFU/ml was detected in the heat-inactivated sponge sample in the presence or absence of 5.0 x 10(4) CFU of Citrobacter freundii per ml. Thus, the VIDAS-SLM method is a rapid screening method and a potential alternative to the time- and labor-intensive culture method.