Center for Veterinary Medicine

Antimicrobial resistance (AMR) is a significant public health threat. With the rise of affordable whole genome sequencing, in silico approaches to assessing AMR gene content can be used to detect known resistance mechanisms and potentially identify novel mechanisms. To enable accurate assessment of AMR gene content, as part of a multi-agency collaboration, NCBI developed a comprehensive AMR gene database, the Bacterial Antimicrobial Resistance Reference Gene Database and the AMR gene detection tool AMRFinder. Here, we describe the expansion of the Reference Gene Database, now called the Reference Gene Catalog, to include putative acid, biocide, metal, stress resistance genes, in addition to virulence genes and species-specific point mutations. Genes and point mutations are classified by broad functions, as well as more detailed functions. As we have expanded both the functional repertoire of identified genes and functionality, NCBI released a new version of AMRFinder, known as AMRFinderPlus. This new tool allows users the option to utilize only the core set of AMR elements, or include stress response and virulence genes, too. AMRFinderPlus can detect acquired genes and point mutations in both protein and nucleotide sequence. In addition, the evidence used to identify the gene has been expanded to include whether nucleotide or protein sequence was used, its location in the contig, and presence of an internal stop codon. These database improvements and functional expansions will enable increased precision in identifying AMR genes, linking AMR genotypes and phenotypes, and determining possible relationships between AMR, virulence, and stress response.

isolates phenotypically tested against various antimicrobial agents. Of 87,679 susceptibility tests performed, 98.4% were consistent with predictions. To assess the accuracy of AMRFinder, we compared its gene symbol output with that of a 2017 version of ResFinder, another publicly available resistance gene detection system. Most gene calls were identical, but there were 1,229 gene symbol differences (8.8%) between them, with differences due to both algorithmic differences and database composition. AMRFinder missed 16 loci that ResFinder found, while ResFinder missed 216 loci that AMRFinder identified. Based on these results, AMRFinder appears to be a highly accurate AMR gene detection system.

In recent years, several authors have argued that placebo-controlled trials are invariably unethical when known effective therapy is available for the condition being studied, regardless of the condition or the consequences of deferring treatment. Some have also disputed the value of placebo-controlled trials in such a setting, asserting that the comparison of new treatment with old treatment is sufficient to establish efficacy and is all that should be of interest. This article considers the ethical concerns about use of placebo controls and describes the limited ability of active-control equivalence (also known as noninferiority) trials to establish efficacy of new therapies in many medical contexts. The authors conclude that placebo-controlled trials are not uniformly unethical when known effective therapies are available; rather, their acceptability is determined by whether the patient will be harmed by deferral of therapy. If patients are not harmed, such trials can ethically be carried out. Furthermore, active-control trials, although valuable, informative, and appropriate in many circumstances, often cannot provide reliable evidence of the effectiveness of a new therapy.

BACKGROUND: Previously, we demonstrated that insulin stimulates production of nitric oxide (NO) in endothelial cells. However, specific insulin-signaling pathways mediating production of NO have not been elucidated. METHODS AND RESULTS: We developed methods for transfection of human umbilical vein endothelial cells (HUVECs) and direct measurement of NO to begin defining insulin-signaling pathways related to NO production. HUVECs were cotransfected with enhanced Green Fluorescent Protein (eGFP) and another gene of interest. Transfection efficiencies >95% were obtained by selecting cells expressing eGFP. Overexpression of insulin receptors in HUVECs resulted in an approximately 3-fold increase in production of NO in response to insulin. In contrast, HUVECs overexpressing a tyrosine kinase-deficient mutant insulin receptor had a dose-response curve similar to that of control cells. Overexpression of inhibitory mutants of either phosphatidylinositol 3-kinase (PI3K) or Akt resulted in nearly complete inhibition of insulin-stimulated production of NO. Overexpression of an inhibitory mutant of Ras had a much smaller effect. CONCLUSIONS: Receptor kinase activity is necessary to mediate production of NO through the insulin receptor. Both PI3K and Akt contribute importantly to this process, whereas the contribution of Ras is small.

Salmonella enterica subspecies enterica is traditionally subdivided into serovars by serological and nutritional characteristics. We used Multilocus Sequence Typing (MLST) to assign 4,257 isolates from 554 serovars to 1092 sequence types (STs). The majority of the isolates and many STs were grouped into 138 genetically closely related clusters called eBurstGroups (eBGs). Many eBGs correspond to a serovar, for example most Typhimurium are in eBG1 and most Enteritidis are in eBG4, but many eBGs contained more than one serovar. Furthermore, most serovars were polyphyletic and are distributed across multiple unrelated eBGs. Thus, serovar designations confounded genetically unrelated isolates and failed to recognize natural evolutionary groupings. An inability of serotyping to correctly group isolates was most apparent for Paratyphi B and its variant Java. Most Paratyphi B were included within a sub-cluster of STs belonging to eBG5, which also encompasses a separate sub-cluster of Java STs. However, diphasic Java variants were also found in two other eBGs and monophasic Java variants were in four other eBGs or STs, one of which is in subspecies salamae and a second of which includes isolates assigned to Enteritidis, Dublin and monophasic Paratyphi B. Similarly, Choleraesuis was found in eBG6 and is closely related to Paratyphi C, which is in eBG20. However, Choleraesuis var. Decatur consists of isolates from seven other, unrelated eBGs or STs. The serological assignment of these Decatur isolates to Choleraesuis likely reflects lateral gene transfer of flagellar genes between unrelated bacteria plus purifying selection. By confounding multiple evolutionary groups, serotyping can be misleading about the disease potential of S. enterica. Unlike serotyping, MLST recognizes evolutionary groupings and we recommend that Salmonella classification by serotyping should be replaced by MLST or its equivalents.

This article provides an overview of the patient-specific and drug-specific variables that can affect drug absorption following oral product administration. The oral absorption of any chemical entity reflects a complex spectrum of events. Factors influencing product bioavailability include drug solubility, permeability, and the rate of in vivo dissolution. In this regard, the Biopharmaceutics Classification System has proven to be an important tool for predicting compounds likely to be associated with bioavailability problems. It also helps in identifying those factors that may alter the rate and extent of drug absorption. Product bioavailability can also be markedly influenced by patient attributes such as the integrity of the gastrointestinal tract, physiological status, site of drug absorption, membrane transporters, presystemic drug metabolism (intrinsic variables), and extrinsic variables such as the effect of food or concomitant medication. Through an awareness of a drug's physicochemical properties and the physiological processes affecting drug absorption, the skilled pharmaceutical scientist can develop formulations that will maximize product availability. By appreciating the potential impact of patient physiological status, phenotype, age, gender, and lifestyle, dosing regimens can be tailored to better meet the needs of the individual patient.

ABSTRACT A total of 825 samples of retail raw meats (chicken, turkey, pork, and beef) were examined for the presence of Escherichia coli and Salmonella serovars, and 719 of these samples were also tested for Campylobacter spp. The samples were randomly obtained from 59 stores of four supermarket chains during 107 sampling visits in the Greater Washington, D.C., area from June 1999 to July 2000. The majority (70.7%) of chicken samples ( n = 184) were contaminated with Campylobacter , and a large percentage of the stores visited (91%) had Campylobacter -contaminated chickens. Approximately 14% of the 172 turkey samples yielded Campylobacter , whereas fewer pork (1.7%) and beef (0.5%) samples were positive for this pathogen. A total of 722 Campylobacter isolates were obtained from 159 meat samples; 53.6% of these isolates were Campylobacter jejuni , 41.3% were Campylobacter coli , and 5.1% were other species. Of the 212 chicken samples, 82 (38.7%) yielded E. coli , while 19.0% of the beef samples, 16.3% of the pork samples, and 11.9% of the turkey samples were positive for E. coli . However, only 25 (3.0%) of the retail meat samples tested were positive for Salmonella . Significant differences in the bacterial contamination rates were observed for the four supermarket chains. This study revealed that retail raw meats are often contaminated with food-borne pathogens; however, there are marked differences in the prevalence of such pathogens in different meats. Raw retail meats are potential vehicles for transmitting food-borne diseases, and our findings stress the need for increased implementation of hazard analysis of critical control point (HACCP) and consumer food safety education efforts.

OBJECTIVE: To review the available evidence evaluating the chemicals in refill solutions, cartridges, aerosols and environmental emissions of electronic cigarettes (e-cigarettes). METHODS: Systematic literature searches were conducted to identify research related to e-cigarettes and chemistry using 5 reference databases and 11 search terms. The search date range was January 2007 to September 2013. The search yielded 36 articles, of which 29 were deemed relevant for analysis. RESULTS: The levels of nicotine, tobacco-specific nitrosamines (TSNAs), aldehydes, metals, volatile organic compounds (VOCs), flavours, solvent carriers and tobacco alkaloids in e-cigarette refill solutions, cartridges, aerosols and environmental emissions vary considerably. The delivery of nicotine and the release of TSNAs, aldehydes and metals are not consistent across products. Furthermore, the nicotine level listed on the labels of e-cigarette cartridges and refill solutions is often significantly different from measured values. Phenolic compounds, polycyclic aromatic hydrocarbons and drugs have also been reported in e-cigarette refill solutions, cartridges and aerosols. Varying results in particle size distributions of particular matter emissions from e-cigarettes across studies have been observed. Methods applied for the generation and chemical analyses of aerosols differ across studies. Performance characteristics of e-cigarette devices also vary across and within brands. CONCLUSIONS: Additional studies based on knowledge of e-cigarette user behaviours and scientifically validated aerosol generation and chemical analysis methods would be helpful in generating reliable measures of chemical quantities. This would allow comparisons of e-cigarette aerosol and traditional smoke constituent levels and would inform an evaluation of the toxicity potential of e-cigarettes.

This paper describes research relating to the major recall of pet food that occurred in Spring 2007 in North America. Clinical observations of acute renal failure in cats and dogs were associated with consumption of wet pet food produced by a contract manufacturer producing for a large number of companies. The affected lots of food had been formulated with wheat gluten originating from China. Pet food and gluten were analyzed for contaminants using several configurations of high-performance liquid chromatography (HPLC) and mass spectrometry (MS), which revealed a number of simple triazine compounds, principally melamine and cyanuric acid, with lower concentrations of ammeline, ammelide, ureidomelamine, and N-methylmelamine. Melamine and cyanuric acid, have been tested and do not produce acute renal toxicity. Some of the triazines have poor solubility, as does the compound melamine cyanurate. Pathological evaluation of cats and dogs that had died from the acute renal failure indicated the presence of crystals in kidney tubules. We hypothesized that these crystals were composed of the poorly soluble triazines, a melamine-cyanuric acid complex, or a combination. Sprague dawley rats were given up to 100 mg/kg ammeline or ammelide alone, a mixture of melamine and cyanuric acid (400/400 mg/kg/day), or a mixture of all four compounds (400 mg/kg/day melamine, 40 mg/kg/day of the others). Neither ammeline nor ammelide alone produced any renal effects, but the mixtures produced significant renal damage and crystals in nephrons. HPLC-MS/MS confirmed the presence of melamine and cyanuric acid in the kidney. Infrared microspectroscopy on individual crystals from rat or cat (donated material from a veterinary clinic) kidneys confirmed that they were melamine-cyanuric acid cocrystals. Crystals from contaminated gluten produced comparable spectra. These results establish the causal link between the contaminated gluten and the adverse effects and provide a mechanistic explanation for how two apparently innocuous compounds could have adverse effects in combination, that is, by forming an insoluble precipitate in renal tubules leading to progressive tubular blockage and degeneration.

The recent advancements in rapid and affordable DNA sequencing technologies have revolutionized diagnostic microbiology and microbial surveillance. The availability of bioinformatics tools and online accessible databases has been a prerequisite for this. We conducted a scientific literature review and here we present a description of examples of available tools and databases for antimicrobial resistance (AMR) detection and provide future perspectives and recommendations. At least 47 freely accessible bioinformatics resources for detection of AMR determinants in DNA or amino acid sequence data have been developed to date. These include, among others but not limited to, ARG-ANNOT, CARD, SRST2, MEGARes, Genefinder, ARIBA, KmerResistance, AMRFinder, and ResFinder. Bioinformatics resources differ for several parameters including type of accepted input data, presence/absence of software for search within a database of AMR determinants that can be specific to a tool or cloned from other resources, and for the search approach employed, which can be based on mapping or on alignment. As a consequence, each tool has strengths and limitations in sensitivity and specificity of detection of AMR determinants and in application, which for some of the tools have been highlighted in benchmarking exercises and scientific articles. The identified tools are either available at public genome data centers, from GitHub or can be run locally. NCBI and European Nucleotide Archive (ENA) provide possibilities for online submission of both sequencing and accompanying phenotypic antimicrobial susceptibility data, allowing for other researchers to further analyze data, and develop and test new tools. The advancement in whole genome sequencing and the application of online tools for real-time detection of AMR determinants are essential to identify control and prevention strategies to combat the increasing threat of AMR. Accessible tools and DNA sequence data are expanding, which will allow establishing global pathogen surveillance and AMR tracking based on genomics. There is however, a need for standardization of pipelines and databases as well as phenotypic predictions based on the data.

A total of 133 Salmonella isolates recovered from retail meats purchased in the United States and the People's Republic of China were assayed for antimicrobial susceptibility, the presence of integrons and antimicrobial resistance genes, and horizontal transfer of characterized antimicrobial resistance determinants via conjugation. Seventy-three (82%) of these Salmonella isolates were resistant to at least one antimicrobial agent. Resistance to the following antibiotics was common among the United States isolates: tetracycline (68% of the isolates were resistant), streptomycin (61%), sulfamethoxazole (42%), and ampicillin (29%). Eight Salmonella isolates (6%) were resistant to ceftriaxone. Fourteen isolates (11%) from the People's Republic of China were resistant to nalidixic acid and displayed decreased susceptibility to ciprofloxacin. A total of 19 different antimicrobial resistance genes were identified in 30 multidrug-resistant Salmonella isolates. The bla(CMY-2) gene, encoding a class A AmpC beta-lactamase, was detected in all 10 Salmonella isolates resistant to extended-spectrum beta-lactams. Resistance to ampicillin was most often associated with a TEM-1 family beta-lactamase gene. Six aminoglycoside resistance genes, aadA1, aadA2, aacC2, Kn, aph(3)-IIa, and aac(3)-IVa, were commonly present in the Salmonella isolates. Sixteen (54%) of 30 Salmonella isolates tested had integrons ranging in size from 0.75 to 2.7 kb. Conjugation studies demonstrated that there was plasmid-mediated transfer of genes encoding CMY-2 and TEM-1-like beta-lactamases. These data indicate that Salmonella isolates recovered from retail raw meats are commonly resistant to multiple antimicrobials, including those used for treating salmonellosis, such as ceftriaxone. Genes conferring antimicrobial resistance in Salmonella are often carried on integrons and plasmids and could be transmitted through conjugation. These mobile DNA elements have likely played an important role in transmission and dissemination of antimicrobial resistance determinants among Salmonella strains.

Antimicrobial resistance in Yersinia pestis is rare, yet constitutes a significant international public health and biodefense threat. In 1995, the first multidrug resistant (MDR) isolate of Y. pestis (strain IP275) was identified, and was shown to contain a self-transmissible plasmid (pIP1202) that conferred resistance to many of the antimicrobials recommended for plague treatment and prophylaxis. Comparative analysis of the DNA sequence of Y. pestis plasmid pIP1202 revealed a near identical IncA/C plasmid backbone that is shared by MDR plasmids isolated from Salmonella enterica serotype Newport SL254 and the fish pathogen Yersinia ruckeri YR71. The high degree of sequence identity and gene synteny between the plasmid backbones suggests recent acquisition of these plasmids from a common ancestor. In addition, the Y. pestis pIP1202-like plasmid backbone was detected in numerous MDR enterobacterial pathogens isolated from retail meat samples collected between 2002 and 2005 in the United States. Plasmid-positive strains were isolated from beef, chicken, turkey and pork, and were found in samples from the following states: California, Colorado, Connecticut, Georgia, Maryland, Minnesota, New Mexico, New York and Oregon. Our studies reveal that this common plasmid backbone is broadly disseminated among MDR zoonotic pathogens associated with agriculture. This reservoir of mobile resistance determinants has the potential to disseminate to Y. pestis and other human and zoonotic bacterial pathogens and therefore represents a significant public health concern.

Many pathogenic and commensal organisms are multidrug resistant due to exposure to various antibiotics. Often, this antimicrobial resistance is encoded by integrons that occur on plasmids or that are integrated into the bacterial chromosome. Integrons are commonly associated with bacterial genera in the family Enterobacteriaceae. We determined that class 1 integrases were present in approximately 46% of the isolates from the family Enterobacteriaceae; class 2 integrases were present only among Escherichia coli and Salmonella isolates. Seven percent of veterinary isolates were positive for class 3 integrase by DNA-DNA hybridization but could not be confirmed to be positive by PCR. None of the veterinary isolates possessed the class 4 integrase gene. The distribution of these integrase genes was variable within the members of the family Enterobacteriaceae when some or all integrase classes were absent from a particular genus. There was also considerable variability in the distribution of these integrases within a species, depending on the animal host. Unlike the class 1 integrases, the other integrase class, intI2, appears to be more restricted in its distribution among the members of the family Enterobacteriaceae. There is also considerable variability in the distribution of the class 1 integrases within E. coli strains isolated from different food animals. The class 1 integrases are the most widely disseminated of the four classes among the members of the family Enterobacteriaceae from both the clinical and normal flora of animals. This is the first report to closely examine the distribution of class 2 integrases in members of the family Enterobacteriaceae isolated in the United States.

Laboratory-based in vitro antimicrobial susceptibility testing is the foundation for guiding anti-infective therapy and monitoring antimicrobial resistance trends. We used whole-genome sequencing (WGS) technology to identify known antimicrobial resistance determinants among strains of nontyphoidal Salmonella and correlated these with susceptibility phenotypes to evaluate the utility of WGS for antimicrobial resistance surveillance. Six hundred forty Salmonella of 43 different serotypes were selected from among retail meat and human clinical isolates that were tested for susceptibility to 14 antimicrobials using broth microdilution. The MIC for each drug was used to categorize isolates as susceptible or resistant based on Clinical and Laboratory Standards Institute clinical breakpoints or National Antimicrobial Resistance Monitoring System (NARMS) consensus interpretive criteria. Each isolate was subjected to whole-genome shotgun sequencing, and resistance genes were identified from assembled sequences. A total of 65 unique resistance genes, plus mutations in two structural resistance loci, were identified. There were more unique resistance genes (n = 59) in the 104 human isolates than in the 536 retail meat isolates (n = 36). Overall, resistance genotypes and phenotypes correlated in 99.0% of cases. Correlations approached 100% for most classes of antibiotics but were lower for aminoglycosides and beta-lactams. We report the first finding of extended-spectrum β-lactamases (ESBLs) (blaCTX-M1 and blaSHV2a) in retail meat isolates of Salmonella in the United States. Whole-genome sequencing is an effective tool for predicting antibiotic resistance in nontyphoidal Salmonella, although the use of more appropriate surveillance breakpoints and increased knowledge of new resistance alleles will further improve correlations.

Histopathology is the study of the structural manifestations of disease at the light-microscopic level. The microscopic examination of a tissue specimen is an evaluation of a 2-dimensional image of a complex 3-dimensional

INTRODUCTION: Electronic cigarette (e-cigarette) use is increasing rapidly, and the impact on youth is unknown. We assessed associations between e-cigarette use and smoking intentions among US youth who had never smoked conventional cigarettes. METHODS: We analyzed data from the nationally representative 2011, 2012, and 2013 National Youth Tobacco Surveys of students in grades 6-12. Youth reporting they would definitely not smoke in the next year or if offered a cigarette by a friend were defined as not having an intention to smoke; all others were classified as having positive intention to smoke conventional cigarettes. Demographics, pro-tobacco advertisement exposure, ever use of e-cigarettes, and ever use of other combustibles (cigars, hookah, bidis, kreteks, and pipes) and noncombustibles (chewing tobacco, snuff, dip, snus, and dissolvables) were included in multivariate analyses that assessed associations with smoking intentions among never-cigarette-smoking youth. RESULTS: Between 2011 and 2013, the number of never-smoking youth who used e-cigarettes increased 3-fold, from 79,000 to more than 263,000. Intention to smoke conventional cigarettes was 43.9% among ever e-cigarette users and 21.5% among never users. Ever e-cigarette users had higher adjusted odds for having smoking intentions than never users (adjusted odds ratio = 1.70, 95% confidence interval = 1.24-2.32). Those who ever used other combustibles, ever used noncombustibles, or reported pro-tobacco advertisement exposure also had increased odds for smoking intentions. CONCLUSION: In 2013, more than a quarter million never-smoking youth used e-cigarettes. E-cigarette use is associated with increased intentions to smoke cigarettes, and enhanced prevention efforts for youth are important for all forms of tobacco, including e-cigarettes.

Despite the critical role of plasmids in horizontal gene transfer, few studies have characterized plasmid relatedness among different bacterial populations. Recently, a multiplex PCR replicon typing protocol was developed for classification of plasmids occurring in members of the Enterobacteriaceae. Here, a simplified version of this replicon typing procedure which requires only three multiplex panels to identify 18 plasmid replicons is described. This method was used to screen 1,015 Escherichia coli isolates of avian, human, and poultry meat origin for plasmid replicon types. Additionally, the isolates were assessed for their content of several colicin-associated genes. Overall, a high degree of plasmid variability was observed, with 221 different profiles occurring among the 1,015 isolates examined. IncFIB plasmids were the most common type identified, regardless of the source type of E. coli. IncFIB plasmids occurred significantly more often in avian pathogenic E. coli (APEC) and retail poultry E. coli (RPEC) than in uropathogenic E. coli (UPEC) and avian and human fecal commensal E. coli isolates (AFEC and HFEC, respectively). APEC and RPEC were also significantly more likely than UPEC, HFEC, and AFEC to possess the colicin-associated genes cvaC, cbi, and/or cma in conjunction with one or more plasmid replicons. The results suggest that E. coli isolates contaminating retail poultry are notably similar to APEC with regard to plasmid profiles, with both generally containing multiple plasmid replicon types in conjunction with colicin-related genes. In contrast, UPEC and human and avian commensal E. coli isolates generally lack the plasmid replicons and colicin-related genes seen in APEC and RPEC, suggesting limited dissemination of such plasmids among these bacterial populations.

OBJECTIVE: To review the available evidence regarding electronic cigarette (e-cigarette) product characterisation and design features in order to understand their potential impact on individual users and on public health. METHODS: Systematic literature searches in 10 reference databases were conducted through October 2013. A total of 14 articles and documents and 16 patents were included in this analysis. RESULTS: Numerous disposable and reusable e-cigarette product options exist, representing wide variation in product configuration and component functionality. Common e-cigarette components include an aerosol generator, a flow sensor, a battery and a nicotine-containing solution storage area. e-cigarettes currently include many interchangeable parts, enabling users to modify the character of the delivered aerosol and, therefore, the product's 'effectiveness' as a nicotine delivery product. Materials in e-cigarettes may include metals, rubber and ceramics. Some materials may be aerosolised and have adverse health effects. Several studies have described significant performance variability across and within e-cigarette brands. Patent applications include novel product features designed to influence aerosol properties and e-cigarette efficiency at delivering nicotine. CONCLUSIONS: Although e-cigarettes share a basic design, engineering variations and user modifications result in differences in nicotine delivery and potential product risks. e-cigarette aerosols may include harmful and potentially harmful constituents. Battery explosions and the risks of exposure to the e-liquid (especially for children) are also concerns. Additional research will enhance the current understanding of basic e-cigarette design and operation, aerosol production and processing, and functionality. A standardised e-cigarette testing regime should be developed to allow product comparisons.

A total of 361 Escherichia coli O157 isolates, recovered from humans, cattle, swine, and food during the years 1985 to 2000, were examined to better understand the prevalence of antimicrobial resistance among these organisms. Based on broth microdilution results, 220 (61%) of the isolates were susceptible to all 13 antimicrobials tested. Ninety-nine (27%) of the isolates, however, were resistant to tetracycline, 93 (26%) were resistant to sulfamethoxazole, 61 (17%) were resistant to cephalothin, and 48 (13%) were resistant to ampicillin. Highest frequencies of resistance occurred among swine isolates (n = 70), where 52 (74%) were resistant to sulfamethoxazole, 50 (71%) were resistant to tetracycline, 38 (54%) were resistant to cephalothin, and 17 (24%) were resistant to ampicillin. Based on the presence of Shiga toxin genes as determined by PCR, 210 (58%) of the isolates were identified as Shiga toxin-producing E. coli (STEC). Among these, resistance was generally low, yet 21 (10%) were resistant to sulfamethoxazole and 19 (9%) were resistant to tetracycline. Based on latex agglutination, 189 (52%) of the isolates were identified as E. coli O157:H7, among which 19 (10%) were resistant to sulfamethoxazole and 16 (8%) were resistant to tetracycline. The data suggest that selection pressure imposed by the use of tetracycline derivatives, sulfa drugs, cephalosporins, and penicillins, whether therapeutically in human and veterinary medicine or as prophylaxis in the animal production environment, is a key driving force in the selection of antimicrobial resistance in STEC and non-STEC O157.

Campylobacter jejuni strain 81-176 (HS36, 23) synthesizes two distinct glycan structures, as visualized by immunoblotting of proteinase K-digested whole-cell preparations. A site-specific insertional mutant in the kpsM gene results in loss of expression of a high-molecular-weight (HMW) glycan (apparent Mr 26 kDa to > 85 kDa) and increased resolution of a second ladder-like glycan (apparent Mr 26-50 kDa). The kpsM mutant of 81-176 is no longer typeable in either HS23 or HS36 antisera, indicating that the HMW glycan structure is the serodeterminant of HS23 and HS36. Both the kpsM-dependent HMW glycan and the kpsM-independent ladder-like structure appear to be capsular in nature, as both are attached to phospholipid rather than lipid A. Additionally, the 81-176 kpsM gene can complement a deletion in Escherichia coli kpsM, allowing the expression of an alpha2,8 polysialic acid capsule in E. coli. Loss of the HMW glycan in 81-176 kpsM also increases the surface hydrophobicity and serum sensitivity of the bacterium. The kpsM mutant is also significantly reduced in invasion of INT407 cells and reduced in virulence in a ferret diarrhoeal disease model. The expression of the kpsM-dependent capsule undergoes phase variation at a high frequency.