DNA Microarray Analysis

This chapter focuses on biofilm formation by the pathogenic fungus Candida albicans. Biofilms are surface-associated microbial communities surrounded by an extracellular matrix. The chapter examines the steps of biofilm formation, from the genes known to function in C. albicans biofilm development, the cell-cell communication within the biofilm, the environmental responses that contribute to biofilm formation, the drug resistance of biofilms, and experimental techniques used to study biofilms. Cell wall genes and adhesins provide mechanisms that promote biofilm initiation, and the transcription factors that regulate their expression couple biofilm initiation with internal and external signals. The study of transcription factors has laid the framework for gene regulatory networks. While much is now known, detailed testing of other known adherence factors, cell wall proteins, transcription factors, kinases, and others is required to identify more genes involved in biofilm formation, and the elucidation of upstream regulation of these factors will allow for greater insights into the signaling events important for biofilm development. Quorum sensing governs functions as diverse as bioluminescence and virulence. It has a vital role in bacterial biofilm dynamics, and its role in C. albicans biofilms is now beginning to be understood. While major challenges lie ahead to further refine the gene regulatory networks and correlate them to in vivo results, it is nonetheless an exciting time for the field of C. albicans biofilm formation.

Two recent European surveys of patient stool samples ranked Arcobacter as the fourth most frequently recovered Campylobacter-like microbe. Aerotolerant campylobacteria (Arcobacter spp.) were first described as occurring in aborted porcine and bovine fetuses. Handling and consuming raw or contaminated poultry meat are acknowledged potential sources of human Arcobacter infection. Due to the phylogenetic relationship between Campylobacter and Arcobacter, it is logical to assume that animal models, virulence factors (including adherence), invasion, cytotoxicity, and toxin production of the two organisms would be similar. Classification of arcobacters as free-living, environmental organisms should be reflected in the gene content of the Arcobacter genomes. The genomes of some Arcobacter species would be predicted to contain niche-related genes, e.g., osmoprotectant genes in A. halophilus and nitrogen fixation genes in A. nitrofigilis. An multilocus sequence typing (MLST) method that could be used to type five Arcobacter species was recently designed. Future MLST and DNA microarray analyses will provide further insights into Arcobacter divergence. MLST analysis has identified putative horizontal gene transfer (HGT) events in Arcobacter. Members of the genus Arcobacter can be generalized as free-living organisms found predominantly in aqueous environments, occasionally associated with food animals, and infrequently associated with humans. The publication of the complete 2.341-Mb Arcobacter genome sequence has facilitated identification of unique virulence factors and genetic markers to monitor its transmission through the food chain and which underlie its distinctive epidemiology.

This review focuses on the cold shock response of Escherichia coli. Change in temperature is one of the most common stresses that an organism encounters in nature. Temperature downshift affects the cell on various levels: (i) decrease in the membrane fluidity; (ii) stabilization of the secondary structures of RNA and DNA; (iii) slow or inefficient protein folding; (iv) reduced ribosome function, affecting translation of non-cold shock proteins; (v) increased negative supercoiling of DNA; and (vi) accumulation of various sugars. Cold shock proteins and certain sugars play a key role in dealing with the initial detrimental effect of cold shock and maintaining the continued growth of the organism at low temperature. CspA is the major cold shock protein of E. coli, and its homologues are found to be widespread among bacteria, including psychrophilic, psychrotrophic, mesophilic, and thermophilic bacteria, but are not found in archaea or cyanobacteria. Significant, albeit transient, stabilization of the cspA mRNA immediately following temperature downshift is mainly responsible for its cold shock induction. Various approaches were used in studies to detect cold shock induction of cspA mRNA. Sugars are shown to confer protection to cells undergoing cold shock. The study of the cold shock response has implications in basic and health-related research as well as in commercial applications. The cold shock response is elicited by all types of bacteria and affects these bacteria at various levels, such as cell membrane, transcription, translation, and metabolism.

The best characterized of the campylobacters is Campylobacter jejuni subsp. jejuni, a common commensal of warm-blooded animals, especially birds. The availability of eight new nonjejuni Campylobacter (NJC) genomes permits genomic comparisons between the NJC group and between the NJC genomes and the C. jejuni subsp. jejuni genomes. This chapter compares and contrasts the genomes of Campylobacter species other than C. jejuni subsp. jejuni. The primary phenotypic marker associated with Campylobacter plasmids is antibiotic resistance. Genes encoding resistance to tetracycline, kanamycin, and chloramphenicol have been identified on Campylobacter plasmids. Campylobacter plasmids fall into two basic groups: cryptic plasmids and megaplasmids. Homopolymeric G: C tracts have been identified in all of the NJC genomes. Several genes within the NJC genomes are frameshifted or have other defects and are considered putative pseudogenes. In Escherichia coli, the conjugative F episome cointegrates into the chromosome via homologous recombination between insertion sequences (IS) elements common to both the chromosome and the F plasmid, resulting in an Hfr strain. Helicobacter strains that possess the Entner-Doudoroff (ED) pathway contain a phosphoglucose isomerase (PGI) distinct from those found in the other Epsilonproteobacteria. A complete oxidative tricarboxylic acid (TCA) cycle is present in C. jejuni subsp. jejuni, as evidenced by the presence of all three subunits of succinate dehydrogenase (encoded by sdhABC). The chapter talks about restriction/modification systems, and virulence/pathogenicity loci. Genomic data for the NJC genomes will lead to new typing methods and perhaps culture methods.

When a bacterial culture growing exponentially at a temperature optimum for its growth is shifted to low temperature, it exhibits cold-shock response. This is irrespective of the preferred optimum growth temperature; thus all types of bacteria such as psychrotrophic, psychrophilic, mesophilic, and thermophilic bacteria possess cellular machinery to elicit this response. Recent global transcript profiling of Escherichia coli cells undergoing cold shock showed that several genes encoding proteins involved in sugar transport and metabolism were induced by cold shock. Cold-shock response of cold-adapted bacteria is similar to that of mesophiles in aspects such as in many cases a lag phase of growth precedes acclimation to low temperature, specific proteins are induced by temperature downshift, membranes undergo adaptive changes, and enzymes are adapted to function at low temperature. One of the main differences in the cold-shock response of these two types of bacteria is the presence of cold acclimation proteins (Caps) in cold-adapted bacteria. The cold-shock response machinery of cyanobacteria is different from that of E. coli. The two main differences are: (i) the absence of CspA homologs and (ii) the presence of desaturases. Desaturases play an important role in cold-shock response of cyanobacteria. With the advent of DNA microarray technology, several groups have carried out global transcript profiling of cold-shock response of different bacteria. Cellular events occurring during cold-shock response are used in applications such as in food and agricultural industry and in research.

The history of biotechnology reveals that scientific discoveries lead to technological advances and these are usually followed by a phase of research and development to find applications for the technologies. The authors are currently in another application phase, where new technologies such as real-time PCR (rtPCR), DNA microarrays, and biosensors are providing very sophisticated tools for use in diagnostics. This chapter therefore includes discussions on these "next-generation technologies," which will have an impact on the way one can test for pathogens and toxins in foods. All identification assays require a pure culture of the unknown bacteria, which is then identified most often by its biochemical characteristics. It is a lengthy, labor-intensive, and media-consuming process. The introduction of miniaturized biochemical kits, which provide a quick biochemical profile of bacteria at a great savings in cost, labor, and time, has simplified the process of bacterial identification and continues to be important in regulatory testing of foods. Many of the new methods use next-generation technologies such as rtPCR, biosensors, and DNA chips, which may be more rapid, more sensitive, and capable of multitarget testing and hence are well suited for use in screening large numbers of samples in compliance or food security surveillance programs. Furthermore, comparative evaluation by with standard methods or validation of rapid methods is critical to document their efficacy in detecting foodborne pathogens and toxins.

This chapter presents an overview on pathogenic organisms. Lan and Reeves describe the evolution of three important groups of enteric pathogens, Salmonella enterica, Escherichia coli (including Shigella), and Yersinia spp. At one time, the genus Salmonella was divided into more than 2,000 species based on surface and flagella antigens, but these are now considered to be one species, S. enterica, which is subdivided into seven subspecies and 2,501 serovars. Yersinia pestis is a recently emerged clone that shares 99.7% identity in 16S ribosomal RNA with Y. pseudotuberculosis and might more properly called Y. pseudotuberculosis Pestis. This clone has acquired additional virulence factors such as Yersinia murine toxin (Ymt) and plasminogen activator (Pla) that allow it to colonize the flea gut, be transmitted to a new host via biting, and disseminate hematogenously from the infected site. Clonal associations probably reflect the historical or prehistorical dissemination of M. tuberculosis with human migrations, similar to the associations that have been established for H. pylori. The great diversity of fungi, among which pathogenic species are dispersed across three phyla, plus the lack of information on virulence factors and the pathogenesis of fungal infections make the promulgation of generalized concepts for the evolution of fungal pathogens extremely difficult, if not impossible. In some cases, such as Legionnaires’ disease, archived clinical specimens may reveal that the disease existed decades ago but appropriate diagnostic techniques did not yet exist.

The combination of molecular characterization of resistant strains with precise identification of the antibiotic resistance gene(s) or mutation(s) and the genetic elements involved in the dissemination of these genes is an effective approach in the control of the spread of antibiotic resistance. Molecular methods also help to determine the location of the gene and to differentiate between horizontal gene transfer and clonal spread. This chapter describes polymerase chain reaction (PCR) and microarray analysis in detail, and specifies applications of these methods in the investigation of antibiotic-resistant strains. Numerous PCR assays for the detection of antibiotic resistance genes have been developed and the development of microarrays for the simultaneous detection of a large number of these genes and the genetic elements involved in their dissemination is in progress. The implementation of molecular methods in routine analysis can be achieved only when it is supported by the proper validation of the methods and the availability of the necessary controls, reference strains, and educated personnel. The molecular characterization of antibiotic-resistant strains can help to identify atypical resistant strains, describe new outbreak strains at an early stage, elucidate the epidemiology of resistant strains at a genotypic level, and explain the processes leading to the selection of resistant and virulent strains. In addition, molecular methods can allow proper risk assessment with respect to the use of antimicrobial substances. If the transcriptional and translational expression of antibiotic resistance genes can be better understood, molecular methods may replace phenotypic measurements.