Over the last decade, the introduction of microarray technology has had a profound impact on gene expression research. The publication of studies with dissimilar or altogether contradictory results, obtained using different microarray platforms to analyze identical RNA samples, has raised concerns about the reliability of this technology. The MicroArray Quality Control (MAQC) project was initiated to address these concerns, as well as other performance and data analysis issues. Expression data on four titration pools from two distinct reference RNA samples were generated at multiple test sites using a variety of microarray-based and alternative technology platforms. Here we describe the experimental design and probe mapping efforts behind the MAQC project. We show intraplatform consistency across test sites as well as a high level of interplatform concordance in terms of genes identified as differentially expressed. This study provides a resource that represents an important first step toward establishing a framework for the use of microarrays in clinical and regulatory settings.

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.

Salmonellosis caused by Salmonella enterica serovar Newport is a major global public health concern, particularly because S. Newport isolates that are resistant to multiple drugs (MDR), including thirdgeneration cephalosporins (MDR-AmpC phenotype), have been commonly isolated from food animals. We analyzed 384 S. Newport isolates from various sources by a multilocus sequence typing (MLST) scheme to study the evolution and population structure of the serovar. These were compared to the population structure of S. enterica serovars Enteritidis, Kentucky, Paratyphi B, and Typhimurium. Our S. Newport collection fell into three lineages, Newport-I, Newport-II, and Newport-III, each of which contained multiple sequence types (STs). Newport-I has only a few STs, unlike Newport-II or Newport-III, and has possibly emerged recently. Newport-I is more prevalent among humans in Europe than in North America, whereas Newport-II is preferentially associated with animals. Two STs of Newport-II encompassed all MDR-AmpC isolates, suggesting recent global spread after the acquisition of the bla CMY-2 gene. In contrast, most Newport-III isolates were from humans in North America and were pansusceptible to antibiotics. Newport was intermediate in population structure to the other serovars, which varied from a single monophyletic lineage in S. Enteritidis or S. Typhimurium to four discrete lineages within S. Paratyphi B. Both mutation and homologous recombination are responsible for diversification within each of these lineages, but the relative frequencies differed with the lineage. We conclude that serovars of S. enterica provide a variety of different population structures.Salmonellosis is a major global cause of diarrheal and extraintestinal disease in humans and animals (66). Salmonella enterica subspecies enterica (referred to herein as S. enterica) has been subdivided serologically into Ͼ1,500 serovars (35), but we focus on S. enterica serovar Newport (S. Newport) here because over the last decade it has been a very common cause of human salmonellosis in both the United States and Europe (13,16,27). Furthermore, multidrug-resistant S. Newport isolates that are also resistant to extended-spectrum cephalosporins (MDR-AmpC) have now been reported from several countries (3,24,36) and are a serious problem among both food animals and humans (17,25,36,48,67). MDR-AmpC isolates are resistant to ␤-lactams, including third-generation cephalosporins, aminoglycosides, tetracyclines, sulfonamides and chloramphenicol (12, 36). Resistance to ␤-lactams is caused by plasmids carrying the ampC gene bla CMY-2 , which encodes the CMY-2 ␤-lactamase (11,65).Most of our current understanding of the population structure of S. enterica relies on a series of seminal publications from R. K. Selander's group in the 1990s. These publications showed that some serovars consisted of monophyletic groups-so-called clonal groupings-but many other serovars confounded isolates from multiple lineages and were therefore polyphyletic (5,51,55,56). More recent studie...

Antimicrobial resistant strains of bacteria are an increasing threat to animal and human health. Resistance mechanisms to circumvent the toxic action of antimicrobials have been identified and described for all known antimicrobials currently available for clinical use in human and veterinary medicine. Acquired bacterial antibiotic resistance can result from the mutation of normal cellular genes, the acquisition of foreign resistance genes, or a combination of these two mechanisms. The most common resistance mechanisms employed by bacteria include enzymatic degradation or alteration of the antimicrobial, mutation in the antimicrobial target site, decreased cell wall permeability to antimicrobials, and active efflux of the antimicrobial across the cell membrane. The spread of mobile genetic elements such as plasmids, transposons, and integrons has greatly contributed to the rapid dissemination of antimicrobial resistance among several bacterial genera of human and veterinary importance. Antimicrobial resistance genes have been shown to accumulate on mobile elements, leading to a situation where multidrug resistance phenotypes can be transferred to a susceptible recipient via a single genetic event. The increasing prevalence of antimicrobial resistant bacterial pathogens has severe implications for the future treatment and prevention of infectious diseases in both animals and humans. The versatility with which bacteria adapt to their environment and exchange DNA between different genera highlights the need to implement effective antimicrobial stewardship and infection control programs in both human and veterinary medicine.

In the United States, multidrug-resistant phenotypes of Salmonella enterica serotype Newport (commonly referred to as MDR-AmpC) have emerged in animals and humans and have become a major public health problem. Although pulsed-field gel electrophoresis (PFGE) is the current "gold standard" typing method for Salmonella, multilocus sequence typing (MLST) may be more relevant to investigations exploring evolutionary and population biology relationships. In this study, 81 Salmonella enterica serotype Newport isolates from humans, food animals, and retail foods were examined for antimicrobial susceptibility and characterized using PFGE and MLST of seven genes, aroC, dnaN, hemD, hisD, purE, sucA, and thrA. Forty-nine percent of the isolates were resistant to nine or more of the tested antimicrobials. Salmonella isolates displayed resistance most often to sulfamethoxazole (57%), streptomycin (56%), tetracycline (56%), ampicillin (52%), and ceftiofur (49%) and, to a lesser extent, to kanamycin (19%), trimethoprim-sulfamethoxazole (17%), and gentamicin (11%). A total of 43 PFGE patterns were generated using XbaI, indicating a genetically diverse population. The largest PFGE cluster contained isolates from clinically ill swine, cattle, and humans. MLST resulted in 12 sequence types (STs), with one type encompassing 62% of the strains. Ten new sequence types and one novel allele type were identified. Furthermore, MLST typing showed that strains closely related by PFGE clustered in major STs, whereas more distantly related strains were separated into two clusters by PFGE. The results of this study demonstrated that the MLST scheme employed here clustered S. enterica serovar Newport isolates in distinct molecular populations, and strain discrimination was enhanced by combining PFGE, antimicrobial susceptibility, and MLST results.

Salmonella isolates were recovered from a monthly sampling of chicken breasts, ground turkey, ground beef, and pork chops purchased from selected grocery stores in six participating FoodNet sites (Connecticut, Georgia, Maryland, Minnesota, Oregon, and Tennessee) in 2002 and an additional two sites in 2003 (California and New York). In 2002 and 2003, a total of 6,046 retail meats were examined, including 1,513 chicken breasts, 1,499 ground turkey samples, 1,522 ground beef samples, and 1,502 pork chops. Retail meat samples tested increased to 3,533 in 2003 as compared to 2,513 in 2002. Overall, six percent of 6,046 retail meat samples (n = 365) were contaminated with Salmonella, the bulk recovered from either ground turkey (52%) or chicken breast (39%). Salmonella isolates were serotyped and susceptibility tested using a panel of 16 antimicrobial agents. S. Heidelberg was the predominant serotype identified (23%), followed by S. Saintpaul (12%), S. Typhimurium (11%), and S. Kentucky (10%). Overall, resistance was most often observed to tetracycline (40%), streptomycin (37%), ampicillin (26%), and sulfamethoxazole (25%). Twelve percent of isolates were resistant to cefoxitin and ceftiofur, though only one isolate was resistant to ceftriaxone. All isolates were susceptible to amikacin and ciprofloxacin; however, 3% of isolates were resistant to nalidixic acid and were almost exclusive to ground turkey samples (n = 11/12). All Salmonella isolates were analyzed for genetic relatedness using pulsed-field gel electrophoresis (PFGE) patterns generated by digestion with Xba1 or Xba1 plus Bln1. PFGE fingerprinting profiles showed that Salmonella, in general, were genetically diverse with a total of 175 Xba1 PFGE profiles generated from the 365 isolates. PFGE profiles showed good correlation with serotypes and in some instances, antimicrobial resistance profiles. Results demonstrated a varied spectrum of antimicrobial resistance and PFGE patterns, including several multidrug resistant clonal groups among Salmonella isolates, and signify the importance of sustained surveillance of foodborne pathogens in retail meats.

ᰔFlorfenicol (FFC) has recently been approved by the Food and Drug Administration for the treatment of several bacterial diseases of cultured fish species in the United States, including enteric septicemia of catfish (ESC) caused by Edwardsiella ictaluri. The FFC-resistant E. ictaluri strain (M07-1) described herein was isolated from a moribund catfish obtained from the Thad Cochran National Warmwater Aquaculture Research Center (Stoneville, MS) in May of 2007 and was confirmed to be E. ictaluri by 16S rRNA gene sequencing (6). Fish showing signs of ESC were examined for FFC-resistant E. ictaluri because losses due to ESC persisted in this population despite FFC treatment. To characterize the resistance properties of this strain, conjugative transfer experiments were performed as described previously (14) using FFC (30 g/ml) for selection. The antimicrobial susceptibilities of M07-1, the corresponding FFCresistant transconjugant, and the isogenic parent strain (Table 1) were quantified using standard microdilution assays (3, 4, 11), demonstrating that the resistance phenotype observed in strain M07-1 was self-transmissible, conferring resistance to FFC, chloramphenicol, tetracycline, streptomycin, ampicillin, amoxicillin-clavulanic acid, ceftiofur, and cefoxitin, as well as decreased susceptibility to trimethoprim-sulfamethoxazole and ceftriaxone. Resistance transfer correlated to a 150-kb plasmid (referred to hereinafter as pM07-1), suggesting the presence of a multidrug resistance plasmid in this isolate (data not shown). PCR analysis (15) followed by sequencing confirmed that E. ictaluri M07-1 and its multidrugresistant (MDR) transconjugant harbored the floR gene.E. ictaluri plasmid pM07-1 also conferred resistance to ceftiofur and concomitant decreased susceptibility to ceftriaxone, implying the presence of the bla CMY-2 beta-lactamase gene. Recent data indicate a rapid increase in the dissemination of cephalosporin resistance linked to self-transmissible MDR plasmids harboring bla 16). Plasmid-borne bla CMY-2 genes in bacteria isolated from food animals, retail meats, and humans in the United States have been identified previously (1, 2, 5, 8, 16) but have not been detected before in bacteria associated with U.S. aquaculture and were not expected, as there are currently no cephalosporin antimicrobials approved for use in U.S. aquaculture. Therefore, PCR analysis using previously described primers specific to bla CMY-2 was performed to verify the presence of this gene in E. ictaluri

Major concerns surround the use of antimicrobial agents in farm-raised fish, including the potential impacts these uses may have on the development of antimicrobial-resistant pathogens in fish and the aquatic environment. Currently, some antimicrobial agents commonly used in aquaculture are only partially effective against select fish pathogens due to the emergence of resistant bacteria. Although reports of ineffectiveness in aquaculture due to resistant pathogens are scarce in the literature, some have reported mass mortalities in larvae caused by resistant to trimethoprim-sulfamethoxazole, chloramphenicol, erythromycin, and streptomycin. Genetic determinants of antimicrobial resistance have been described in aquaculture environments and are commonly found on mobile genetic elements which are recognized as the primary source of antimicrobial resistance for important fish pathogens. Indeed, resistance genes have been found on transferable plasmids and integrons in pathogenic bacterial species in the genera ,, ,, and . Class 1 integrons and IncA/C plasmids have been widely identified in important fish pathogens ( spp., spp., spp., spp., and spp.) and are thought to play a major role in the transmission of antimicrobial resistance determinants in the aquatic environment. The identification of plasmids in terrestrial pathogens ( serotypes, , and others) which have considerable homology to plasmid backbone DNA from aquatic pathogens suggests that the plasmid profiles of fish pathogens are extremely plastic and mobile and constitute a considerable reservoir for antimicrobial resistance genes for pathogens in diverse environments.