Urinary paranitrophenol, a metabolite of methyl parathion, in Thai farmer and child populations.

Human exposure to methyl parathion can be assessed by measuring the concentration of its metabolite paranitrophenol (PNP) in urine. Our biologic monitoring study in Chiang Mai, Thailand, measured PNP and dialkylphosphate metabolites (i.e., dimethylphosphate [DMP] and dimethylthiophosphate [DMTP]) of methyl parathion in urine samples collected from 136 farmers (age 20 to 65 years) and 306 school children (age 10 to 15 years) in 2006. Participants came from two topographically different areas: one was colder and mountainous, whereas the other was alluvial with climate fluctuations depending on the monsoon season. Both children and farmers were recruited from each area. Despite methyl parathion's prohibited use in agriculture in 2004, we detected PNP in >90% of all samples analyzed. We applied a nonparametric correlation test (PNP vs. DMP and DMTP) to determine whether the PNP found in most of the samples tested resulted from exposures to methyl parathion. DMP (Spearman's rho = 0.601 [p = 0.001] for farmers and Spearman's rho = 0.263 [p <0.001] for children) and DMTP (Spearman's rho = 0.296 [p = 0.003] for farmers and Spearman's rho = 0.304 [p<0.001] for children) were positively correlated with PNP, suggesting a common source for the three analytes, presumably methyl parathion or related environmental degradates. Although we found a modest correlation between the metabolites, our findings suggest that despite the prohibition, at least a portion (approximately 25% to 60%) of the PNP detected among farmers and children in Thailand may be attributed to exposure from continued methyl parathion use.

Comparison of molecular typing methods for the differentiation of Salmonella foodborne pathogens.

Bacteria belonging to the genus Salmonella are among the leading causes of foodborne disease of bacterial etiology. These bacteria are also widely disseminated throughout the animal kingdom. The ability to identify the food source from which a human pathogen originated would be of great value in reducing the incidence of foodborne disease and the extent of disease outbreaks due to Salmonella. To date, efforts to identify the origin of these pathogens have centered on phenotypic and genotypic characterization of Salmonella isolates. This review focuses molecular or genotypic techniques that are currently being used for typing, and examines their strengths and weaknesses for determining the source of Salmonella foodborne infections.

Antimicrobial susceptibilities of Vibrio parahaemolyticus and Vibrio vulnificus isolates from Louisiana Gulf and retail raw oysters.

The antimicrobial susceptibilities of 168 Vibrio parahaemolyticus and 151 Vibrio vulnificus isolates recovered from 82 Louisiana Gulf and retail oysters in 2005 and 2006 were determined. Overall, the two vibrios remained susceptible to the majority of antimicrobials tested; reduced susceptibility was detected only in V. parahaemolyticus for ampicillin (81%; MIC > or = 16 microg/ml). Additionally, V. parahaemolyticus displayed significantly higher MICs for cefotaxime, ciprofloxacin, and tetracycline than V. vulnificus.

Comparison of subtyping methods for differentiating Salmonella enterica serovar Typhimurium isolates obtained from food animal sources.

Molecular characterization (e.g., DNA-based typing methods) of Salmonella isolates is frequently employed to compare and distinguish clinical isolates recovered from animals and from patients with food-borne disease and nosocomial infections. In this study, we compared the abilities of different phenotyping and genotyping methods to distinguish isolates of Salmonella enterica serovar Typhimurium from different food animal sources. One hundred twenty-eight S. enterica serovar Typhimurium strains isolated from cattle, pigs, chickens, and turkeys or derived food products were characterized using pulsed-field gel electrophoresis (PFGE), repetitive element PCR (Rep-PCR), multilocus sequence typing (MLST), plasmid profiling, and antimicrobial susceptibility testing. Among the 128 Salmonella isolates tested, we observed 84 Rep-PCR profiles, 86 PFGE patterns, 89 MLST patterns, 36 plasmid profiles, and 38 susceptibility profiles. The molecular typing methods, i.e., PFGE, MLST, and Rep-PCR, demonstrated the best discriminatory power among Salmonella isolates. However, no apparent correlation was evident between the results of one molecular typing method and those of the others, suggesting that a combination of multiple methods is needed to differentiate S. enterica serovar Typhimurium isolates that genetically cluster according to one particular typing method.

Broth microdilution susceptibility testing of Campylobacter jejuni and the determination of quality control ranges for fourteen antimicrobial agents.

Quality control ranges were developed for broth microdilution testing of Campylobacter jejuni ATCC 33560 against 14 antimicrobials. Cation-adjusted Mueller-Hinton broth containing 2.5% laked horse blood was the preferred medium, with incubation in a microaerobic atmosphere of 10% CO(2), 5% O(2), and 85% N(2) at 36 degrees C for 48 h or 42 degrees C for 24 h.

Concentrations of selective metabolites of organophosphorus pesticides in the United States population.

We report population-based concentrations (stratified by age, sex, and composite race/ethnicity variables) of selective metabolites of chlorpyrifos (3,5,6-trichloro-2-pyridinol; TCPY), chlorpyrifos methyl (TCPY), malathion (malathion dicarboxylic acid; MDA), diazinon (2-isopropyl-4-methyl-6-hydroxypyrimidine; IMPY), methyl parathion (para-nitrophenol; PNP), and parathion (PNP). We measured the concentrations of TCPY, MDA, IMPY, and PNP in 1997 urine samples from participants, aged 6-59 years, of the National Health and Nutrition Examination Survey, 1999-2000. We detected TCPY in more than 96% of the samples tested. Other organophosphorus pesticide metabolites were detected less frequently: MDA, 52%; IMPY, 29%; and PNP, 22%. The geometric means for TCPY were 1.77 microg/L and 1.58 microg/g creatinine. The 95th percentiles for TCPY were 9.9 microg/L and 8.42 microg/g creatinine. The 95th percentiles for MDA were 1.6 microg/L and 1.8 microg/g creatinine. The 95th percentiles for IMPY and PNP were 3.7 microg/L (3.4 microg/g creatinine) and 5.0 microg/L (4.2 microg/g creatinine), respectively. Multivariate analyses showed that children aged 6-11 years had significantly higher concentrations of TCPY than adults and adolescents. Similarly, adolescents had significantly higher TCPY concentrations than adults. Although the concentrations between sexes and among composite racial/ethnic groups varied, no significant differences were observed.

Molecular characterization of Staphylococcus intermedius carriage by healthy dogs and comparison of antimicrobial susceptibility patterns to isolates from dogs with pyoderma.

We conducted an epidemiological study of Staphylococcus intermedius using arbitrarily primed PCR (AP-PCR) and antibiograms. One hundred and twenty-five S. intermedius isolates were recovered from the oral cavity and/or cranial hair coat of healthy dogs enrolled in a pet therapy program. Commensal S. intermedius was cultured from 32% of the oral cavity cultures and 13% of the cranial hair coat cultures. We characterized the colonization of the dogs as transient, intermittent, or persistent. For dogs characterized as persistently colonized, 73% of the isolates came from the oral cavity. These isolates were also genotyped by AP-PCR. A single major AP-PCR type was observed in 91% of the dogs (n=22); minor variations were frequently observed in these major types. Antibiograms of these commensal isolates were compared to antibiograms from 97 historical clinical isolates (1988-1992) obtained from cases of canine pyoderma. Resistance was most often observed to penicillin (64% and 55%) and tetracycline (38% and 38%) among the commensal and clinical isolates, respectively. The commensal isolates were significantly less resistant to erythromycin, clarithromycin, clindamycin, and trimethoprim/sulfamethoxazole. Our data suggests that differences in both genotype and antimicrobial susceptibility phenotypes exist among S. intermedius strains isolated from different anatomic sites from the same dog and supports the opportunistic nature of S. intermedius in canine infections.

Identification of antimicrobial resistance and class 1 integrons in Shiga toxin-producing Escherichia coli recovered from humans and food animals.

OBJECTIVES: The objective of this study was to identify antimicrobial resistance and class 1 integrons among Shiga toxin-producing Escherichia coli (STEC). METHODS: Two-hundred and seventy-four STEC recovered from poultry, cattle, swine and humans were characterized by antimicrobial susceptibility testing, screened for the presence of class 1 integrons by PCR, and assayed for integron transfer by conjugation. RESULTS: Ninety-three (34%) of the isolates were resistant to streptomycin, followed by 89 (32%) to sulfamethoxazole, 83 (30%) to tetracycline, 48 (18%) to ampicillin, 29 (11%) to cefalothin, 22 (8%) to trimethoprim/sulfamethoxazole, 18 (7%) to gentamicin, 13 (5%) to chloramphenicol and 10 (4%) to cefoxitin. Class 1 integrons were detected in 43 (16%) of the 274 isolates. The adenyl acetyltransferase gene, aadA, which confers resistance to streptomycin, was identified in integrons from 41 (95%) of these 43 isolates, and the dfrA12 gene, which confers resistance to trimethoprim, was identified in integrons from eight (19%) of the isolates. The sat1 gene, which confers resistance to streptothricin, an antimicrobial that has never been approved for use in the United States, was identified in integrons from three (7%) of the isolates. Transfer of integrons by conjugation between strains of E. coli resulted in transfer of antimicrobial-resistant phenotypes for ampicillin, chloramphenicol, cefalothin, gentamicin, tetracycline, trimethoprim, sulfamethoxazole and streptomycin. CONCLUSIONS: Antimicrobial resistance is common in STEC. Class 1 integrons located on mobile plasmids have facilitated the emergence and dissemination of antimicrobial resistance among STEC in humans and food animals.

Encrusted cystitis secondary to Corynebacterium matruchotii infection in a horse.

A 17-year-old gelding was evaluated because of dysuria, inappetence, and weight loss. Cystoscopy revealed severe mucosal ecchymoses with luminal hemorrhage and accumulations of crystalloid sludge. Analysis of a urine sample revealed isosthenuria, an alkaline pH, pyuria, hematuria, bacteriuria, and numerous calcium carbonate crystals. Histologic examination of bladder mucosa biopsy specimens revealed severe neutrophilic infiltration with mineralization. A diagnosis of encrusted cystitis exacerbated by sabulous urolithiasis was made. A Corynebacterium sp susceptible to penicillin, sulfonamide, and enrofloxacin was cultured from the urine and the bladder mucosa biopsy specimens. The horse was treated with penicillin G potassium, IV, for 5 days, followed by trimethoprim-sulfamethoxazole for 4 weeks. Bladder lavage was performed daily for the first 3 days with a balanced electrolyte solution and dimethyl sulfoxide in an attempt to aid expulsion of necrotic debris and crystalline sludge from the bladder. Molecular phylogenetic analysis based on the 16S rDNA gene sequence was used to identify the isolate and determine its phylogenetic position. Results indicated that the isolate was closely related to Corynebacterium matruchotii. To our knowledge, encrusted cystitis secondary to C matruchotii has not been previously identified in a horse.

A liquid chromatography--tandem mass spectrometry multiresidue method for quantification of specific metabolites of organophosphorus pesticides, synthetic pyrethroids, selected herbicides, and deet in human urine.

The ability to estimate low-dose human exposure to commonly used pesticides often is requested in epidemiologic studies. Therefore, fast and robust methods are necessary that can measure many analytes in the same sample. We have developed a method for high-throughput analysis of 19 markers of commonly used pesticides in human urine. The analytes were seven specific metabolites of organophosphorus pesticides, five metabolites of synthetic pyrethroids, six herbicides or their metabolites, and one insect repellant. Human urine (2 mL) was spiked with stable isotopically labeled analogues of the analytes, enzymatically hydrolyzed, extracted using solid-phase extraction, concentrated, and analyzed using high-performance liquid chromatography-tandem mass spectrometry. The sample was divided into two portions and analyzed on two different mass spectrometers, one using atmospheric pressure chemical ionization (APCI) and the other using turbo ion spray atmospheric pressure ionization (TIS). All analytes except the pyrethroid metabolites were analyzed using APCI. The detection limits for all analytes ranged from 0.1 to 1.5 ng/mL of urine, with the majority (17) below 0.5 ng/mL. The analytical precision for the different analytes, estimated as both the within-day and between-day variation, was 3-14 and 4-19%, respectively. The extraction recoveries of the analytes ranged from 68 to 114%. The throughput, including calibration standards and quality control samples, is approximately 50 samples a day. However, the analysis time with the TIS application is much shorter, and if only pyrethroid metabolite data are of interest, the throughput can be increased to 100-150 samples/day.

Antimicrobial-resistant Salmonella serovars isolated from imported foods.

A total of 187 Salmonella isolates representing 82 serotypes recovered from 4072 imported foods in the year 2000 by the U.S. Food and Drug Administration field laboratories were tested for their susceptibility to 17 antimicrobials of human and veterinary importance. Fifteen (8%) isolates were resistant to at least one antimicrobial, and five (2.7%) were resistant to three or more antimicrobials. Most of the isolates (n=9) exhibited resistance to tetracycline. Four isolates from catfish or tilapia from Taiwan or Thailand also demonstrated resistance to nalidixic acid. These nalidixic acid-resistant Salmonella isolates possessed a point mutation at the Ser83 or Asp87 position in DNA gryase, resulting in amino acid substitutions to phenylalanine, tyrosine, or asparagine. One Salmonella Derby isolated from frozen anchovies imported from Cambodia was resistant to six antimicrobials including ampicillin, amoxicillin/clavulanic acid, chloramphenicol, sulfamethoxazole, tetracycline, and trimethoprim/sulfamethoxazole. Of seven isolates displaying resistance to sulfonamides, only one S. Derby and one Salmonella Agona contained class 1 integrons that were further shown to possess the aadA and pse-1 genes conferring resistance to streptomycin and ampicillin, respectively. This study indicates that antimicrobial-resistant Salmonella are present in imported foods, primarily of seafood origin, and stresses the need for continued surveillance of foodborne zoonotic bacterial pathogens from imported foods entering the United States.

Antimicrobials: modes of action and mechanisms of resistance.

After six decades of widespread antibiotic use, bacterial pathogens of human and animal origin are becoming increasingly resistant to many antimicrobial agents. Antimicrobial resistance develops through a limited number of mechanisms: (a). permeability changes in the bacterial cell wall/membrane, which restrict antimicrobial access to target sites; (b). active efflux of the antimicrobial from the cell; (c). mutation in the target site; (d). enzymatic modification or degradation of the antimicrobial; and (e). acquisition of alternative metabolic pathways to those inhibited by the drug. Numerous bacterial antimicrobial resistance phenotypes result from the acquisition of external genes that may provide resistance to an entire class of antimicrobials. These genes are frequently associated with large transferable extrachromosomal DNA elements called plasmids, on which may be other mobile DNA elements such as transposons and integrons. An array of different resistance genes may accumulate on a single mobile element, presenting a situation in which multiple antibiotic resistance can be acquired via a single genetic event. The versatility of bacterial populations in adapting to toxic environments, along with their facility in exchanging DNA, signifies that antibiotic resistance is an inevitable biological phenomenon that will likely continue to be a chronic medical problem. Successful management of current antimicrobials, and the continued development of new ones, is vital to protecting human and animal health against bacterial pathogens.

Characterization of the predominant anaerobic bacterium recovered from digital dermatitis lesions in three Michigan dairy cows.

Digital dermatitis is a superficial epidermatitis of the feet of cattle. Data from previous work suggest that spirochaetes, Campylobacter spp., and Bacteroides spp. may be important in the disease, but the etiology of this disease is not entirely clear. Tissue samples collected from digital dermatitis lesions in three Holstein-Friesian cows from a Michigan dairy yielded a predominant colony type when incubated anaerobically on blood agar at 35 degrees C for 24-48 h. The isolate was a non-flagellated Gram-negative rod, 7 microM long and <0.5 microM wide; its growth was strictly anaerobic and resulted in slight ss-hemolysis on blood agar; 16S rRNA gene sequence analysis indicated it belonged to the cytophoga-flexibacter-bacteroides phylum. The finding that this bacterium was the predominant anaerobe recovered from digital dermatitis lesions suggests it may be involved in the digital dermatitis disease process.

Antimicrobial resistance of Escherichia coli O26, O103, O111, O128, and O145 from animals and humans.

Susceptibilities to fourteen antimicrobial agents important in clinical medicine and agriculture were determined for 752 Escherichia coli isolates of serotypes O26, O103, O111, O128, and O145. Strains of these serotypes may cause urinary tract and enteric infections in humans and have been implicated in infections with Shiga toxin-producing E. coli (STEC). Approximately 50% of the 137 isolates from humans were resistant to ampicillin, sulfamethoxazole, cephalothin, tetracycline, or streptomycin, and approximately 25% were resistant to chloramphenicol, trimethoprim-sulfamethoxazole, or amoxicillin-clavulanic acid. Approximately 50% of the 534 isolates from food animals were resistant to sulfamethoxazole, tetracycline, or streptomycin. Of 195 isolates with STEC-related virulence genes, approximately 40% were resistant to sulfamethoxazole, tetracycline, or streptomycin. Findings from this study suggest antimicrobial resistance is widespread among E. coli O26, O103, O111, O128, and O145 inhabiting humans and food animals.

Comparison of the Etest and agar dilution for in vitro antimicrobial susceptibility testing of Campylobacter.

The performance of the Etest and agar dilution for in vitro antimicrobial susceptibility of Campylobacter spp. was evaluated using a quality control strain Campylobactor jejuni ATCC 33560, and 81 C. jejuni and 54 Campylobacter coli isolates recovered from retail raw meats. Seven antimicrobial agents: chloramphenicol, ciprofloxacin, doxycycline, erythromycin, gentamicin, nalidixic acid and tetracycline, were tested using the two methods, whereas azithromycin was tested using the Etest only. The correlation between the Etest and agar dilution MICs varied greatly depending on the antimicrobial agents tested. The overall agreement of MICs (+/-1 log(2) dilution) between the two methods was 61.9%, ranging from 21.4% for nalidixic acid to 92.6% for gentamicin. MICs obtained using the Etest were generally lower than those by agar dilution regardless of the species of organism tested. MIC(50) and/or MIC(90) values were at least one dilution lower for the Etest than for agar dilution when testing chloramphenicol, ciprofloxacin, doxycycline, erythromycin and nalidixic acid. Based on the agar dilution MICs, the resistant rate of the 135 Campylobacter isolates was highest for tetracycline (82.2%), followed by doxycycline (78.5%), nalidixic acid (21.5%), ciprofloxacin (20.7%) and erythromycin (17.0%). None of the isolates demonstrated resistance to chloramphenicol or gentamicin. The study indicated that the Etest results were not in complete agreement with the agar dilution test. Although the Etest has been proven to be a satisfactory testing method, its use for Campylobacter susceptibility testing requires further standardization. The study also showed that C. jejuni and C. coli isolates resistant to antimicrobials used for treating campylobacteriosis were common in retail raw meats.

Frequency of isolation and antimicrobial susceptibility patterns of Staphylococcus intermedius and Pseudomonas aeruginosa isolates from canine skin and ear samples over a 6-year period (1992-1997).

Staphylococcus intermedius (S. intermedius) was isolated from 88.6% and 49.4% of skin and ear samples, respectively, during the years 1992 through 1997, and frequency of isolation remained unchanged. More than 95% of all S. intermedius isolates were susceptible to cephalothin and oxacillin, providing support for empirical treatment of canine skin and ear infections with cephalexin. Pseudomonas aeruginosa (P. aeruginosa) was isolated from 7.5% and 27.8% of skin and ear samples, respectively. The frequency of isolation from skin samples increased over the study period. Because of multidrug-resistant profiles for P. aeruginosa isolates, especially for ear isolates, empirical treatment of P. aeruginosa infections is not advisable.

Ciprofloxacin resistance in Campylobacter jejuni evolves rapidly in chickens treated with fluoroquinolones.

Fluoroquinolones are commonly used to treat gastroenteritis caused by Campylobacter species. Domestically acquired fluoroquinolone-resistant Campylobacter infection has been documented recently in the United States. It has been proposed that the increase in resistance is due, in part, to the use of fluoroquinolones in poultry. In separate experiments, the effects of sarafloxacin and enrofloxacin treatment of Campylobacter jejuni-infected chickens on the development of ciprofloxacin resistance were measured. Fecal samples were collected before and after treatment and were cultured for C. jejuni. When enrofloxacin or sarafloxacin was used at US Food and Drug Administration-approved doses in broiler chickens, resistance developed rapidly and persisted in C. jejuni. MICs of ciprofloxacin increased from a base of 0.25 microg/mL to 32 microg/mL within the 5-day treatment time frame. These results show that the use of these drugs in chickens rapidly selects for resistant Campylobacter organisms and may result in less effective fluoroquinolone therapy for cases of human campylobacteriosis acquired from exposure to contaminated chicken.