Gram-Positive Cocci

Clinical microbiology testing is crucial for the diagnosis and treatment of community and hospital-acquired infections. Laboratory scientists need to utilize technical and problem-solving skills to select from a wide array of microbial identification techniques. The inquiry-driven laboratory training required to prepare microbiology graduates for this professional environment can be difficult to replicate within undergraduate curricula, especially in courses that accommodate large student cohorts. We aimed to improve undergraduate scientific training by engaging hundreds of introductory microbiology students in an Authentic Large-Scale Undergraduate Research Experience (ALURE). The ALURE aimed to characterize the microorganisms that reside in the healthy human oral cavity—the oral microbiome—by analyzing hundreds of samples obtained from student volunteers within the course. Students were able to choose from selective and differential culture media, Gram-staining, microscopy, as well as polymerase chain reaction (PCR) and 16S rRNA gene sequencing techniques, in order to collect, analyze, and interpret novel data to determine the collective oral microbiome of the student cohort. Pre- and postsurvey analysis of student learning gains across two iterations of the course (2012–2013) revealed significantly higher student confidence in laboratory skills following the completion of the ALURE (p < 0.05 using the Mann-Whitney U-test). Learning objectives on effective scientific communication were also met through effective student performance in laboratory reports describing the research outcomes of the project. The integration of undergraduate research in clinical microbiology has the capacity to deliver authentic research experiences and improve scientific training for large cohorts of undergraduate students.

This tip describes a simple laboratory exercise to assess the microbial contamination of mobile phones, and suggests extension work that enables additional exploration of the topic. At its most basic, it is suitable for the school classroom; more advanced development of the suggested activities are suitable for undergraduate project work.

Current criteria for inclusion in the genus Enterococcus and for the description of new enterococcal species encompass a polyphasic approach resulting from a combination of different molecular techniques (frequently involving DNA-DNA reassociation experiments, 16S rRNA gene sequencing, and whole-cell protein profiling analysis) and phenotypic tests. Several other molecular methods, mostly nucleic acid-based assays, have been used as additional tools to assess the phylogenetic relationships among enterococcal species and to formulate the description of new species, but their use is still limited. Positive catalase testing has also been reported for strains of Enterococcus haemoperoxidus and Enterococcus silesiacus when cultivated on blood-containing agar media. Phenotypic characteristics are used for the identification of Enterococcus species and some physiologically related species of other gram-positive cocci. Serologic tests for detecting antibody responses to different enterococcal antigens have been proposed. In addition to the intrinsic resistance traits, enterococci have acquired different genetic determinants that confer resistance to several classes of antimicrobial agents, including chloramphenicol, tetracyclines, macrolides, lincosamides and streptogramins, aminoglycosides, β-lactams, glycopeptides, quinolones, and even some of the more recently available drugs, such as linezolid, daptomycin, and quinupristin-dalfopristin. Antimicrobial resistance can be classified as either intrinsic or acquired. Molecular methods have been used to detect specific antimicrobial resistance genes and have substantially contributed to the understanding of the spread of acquired resistance among enterococci, especially resistance to vancomycin. However, because of their high specificity, molecular methods do not detect antimicrobial resistance due to mechanisms not targeted by the testing, including emerging resistance mechanisms.

This chapter emphasizes that the Gram stain (performed on 24- to 48-h-old colonies from rich media) and macroscopic morphologies are initial key features for the differentiation of aerobic gram-positive rods. All strains of aerobic gram-positive rods (except the non-rapidly growing mycobacteria) are initially grown on blood agar plates. Catalase activity should be tested with colonies grown on media lacking heme groups. Type of metabolism can be evaluated using oxidative-fermentative media or in cystine Trypticase agar (CTA) medium. Genera which contain strictly anaerobic gram-positive rods may also contain species, or strains within a species, which grow reasonably well aerotolerantly or aerobically. Some gram-positive rods (e.g., Rhodococcus spp. or Dermabacter might be initially misidentified as gram-positive cocci because of their initial gram stain appearance. Molecular approaches to characterize or subtype pathogens are described in the chapter.

The organisms included in this chapter are obligately anaerobic, non-spore-forming, sometimes elongated cocci. The genera Anaerococcus, Anaerosphaera, Finegoldia, Gallicola, Parvimonas, Peptococcus, Peptoniphilus, and Peptostreptococcus, as well as the newly described taxon Murdochiella , are gram-positive, coccobacillary, or, occasionally, coccoid cells. F. magna is the most pathogenic and one of the most frequently isolated gram-positive anaerobic coccal species found in human clinical specimens. Molecular methods, such as nucleic acid probe hybridization and PCR amplification, are not yet standardized or available commercially for the direct demonstration of medically important gram-positive anaerobic cocci from clinical specimens. New information regarding the antimicrobial susceptibilities of gram-positive anaerobic cocci is sparse compared with the information available for other anaerobic species. Bacteria present in pure culture or in large numbers are probably of major importance, as are organisms recovered in multiple cultures and isolated from normally sterile sites. Furthermore, Gram stain results can guide the laboratory in choosing media for optimal recovery of the predicted organisms. The significance of finding anaerobic gram-positive and gram-negative cocci in clinical specimens depends on the specimen and the likelihood that it was contaminated by the microbiota of the skin or mucous membranes. Hence, interpretation of culture results is dependent on the nature and quality of the specimen submitted to the laboratory.

Antimicrobial chemotherapy has played a vital role in the treatment of human infectious diseases since the discovery of penicillin in the 1920s. Hundreds of antimicrobial agents have been developed or synthesized to date, and a broad number and variety of agents are currently available for clinical use. However, the sheer numbers and continuing development of agents make it difficult for clinicians to keep up with progress in the field. This chapter provides an overview of the antibacterial agents currently marketed in the United States, with major emphasis on their mechanisms of action, spectra of activity, important pharmacologic parameters, and toxicities. The major antibacterial action of penicillins is derived from their ability to inhibit a number of bacterial enzymes, namely penicillin-binding proteins (PBPs), that are essential for peptidoglycan synthesis. Cephalosporins are generally very well tolerated. The monobactams are β-lactams with various side chains affixed to a monocyclic nucleus. Since the first aminoglycoside, streptomycin, was introduced, this class of antibiotic has played a vital role in the treatment of serious gram-negative infections. The currently available aminoglycosides are derived from Micromonospora spp. or from Streptomyces spp. Tetracyclines are broad-spectrum bacteriostatic antibiotics with the hydronaphthacene nucleus, which contains four fused rings. Glycylcyclines are a group of semisynthetic tetracycline derivatives containing a glycylamido substitution at position 9. Streptogramins are natural cyclic peptides produced by Streptomyces spp. They are a unique class of antibiotics in which each member is a combination of at least two structurally unrelated components, groups A and B streptogramins.

This chapter describes the currently available methodologies and their interpretation for susceptibility testing of anaerobes. At present, alternative testing methods include limited agar dilution, broth microdilution (for the B. fragilis group), and the Etest gradient strip method. Tigecycline, a glycylcycline derivative of minocycline, was recently introduced for treatment of intra-abdominal and skin and soft tissue infections. Several studies have found very good activity against anaerobes, with rare strains exhibiting tigecycline MICs of >8 µg/ml. Extensive taxonomic changes have occurred within the non-spore-forming gram-positive bacilli, especially with those previously in the genus Eubacterium. It is important to compare the drug-containing plates to the drug-free control plate when reading the tests, as different species of anaerobic bacteria can have very differently appearing spots, ranging from mucoid-opaque, as with the B. fragilis group, to gray-transparent, as with Bacteroides ureolyticus. Broth microdilution MIC determinations require criteria similar to those for the agar dilution procedure for reading end points: the concentration at which the most significant reduction in growth is observed and interpreted as the MIC. Current methodologies allow for accurate surveillance or individual isolate testing by most laboratories. Future studies comparing broth microdilution to the reference agar method and the anticipated development of an improved microdilution system will result in better standardization of the more-user-friendly method and, possibly, more widespread commercial availability.

Nucleic acid sequencing of various bacterial genes and other DNA targets has been used for determining the phylogeny of bacteria and for their identification. A brief overview of nucleic acid sequencing is shown in this chapter. DNA targets have conserved regions flanking variable regions that can be used to differentiate closely related bacterial species. The routine use of sequencing can greatly enhance the ability of the clinical microbiology laboratory to identify bacteria on many levels. Of consideration in the routine use of DNA target sequencing is the need for technical expertise and its cost. The chapter addresses preparation of DNA from pure culture. Certain conventional methods such as latex agglutination assays are quicker, simpler, and less expensive than DNA target sequencing for the identification of beta-hemolytic streptococci. Basic conventional methods perform well in identifying common isolates, such as Bacteroides fragilis group, Peptostreptococcus spp., and most Clostridium spp. DNA target sequencing can provide more accurate identifications, especially since databases from conventional methods often are not current and do not reflect the tremendous genetic diversity within anaerobic taxa. For agents of bioterrorism, i.e., Bacillus anthracis, Brucella spp., Clostridium botulinum, Francisella tularensis, and Yersinia pestis, 16S rRNA sequencing has varying utility. Molecular studies have enhanced our knowledge about the taxonomical diversity among bacteria and allowed better definition of the epidemiology of bacterial infections.

Understanding the technological, microbiological, and biochemical processes that occur during meat, poultry, and fish fermentation is essential for ensuring safe, palatable products. Dry and semidry sausages represent the largest category of fermented meat products, with many present-day processing practices having their origin in the Mediterranean region. Micrococci and staphylococci reduce nitrate to nitrite to generate nitric oxide, which reacts with myoglobin to produce the characteristic cured color of fermented meats. Slightly larger amounts of fermentable carbohydrate should be used in formulas containing higher-pH meats, such as poultry. Mechanically deboned poultry meat is an acceptable meat source for fermented dry sausages when limited to 10% of the meat block, as sausages tend to become soft when larger amounts are used. Thus, the combined effects of low pH, increased acidity, concomitant loss of moisture during drying, reduction of aw, concentration of curing salts, such as sodium chloride and sodium nitrite, bacterial inhibition of spoilage or pathogenic microorganisms, and heat processing (if applied) preserve fermented meat and poultry products against spoilage by inactivating indigenous tissue and bacterial enzymes. Pyruvate-formate lyase generates formate and acetyl Coenzyme A (CoA) from pyruvate and CoA, and the acetyl CoA can be used either as a precursor for substrate-level phosphorylation, for direct reduction to ethanol, or both. This alteration in pyruvate metabolism has been demonstrated in studies where lactobacilli have been shown to shift fermentation patterns and, concomitantly, the amount of potential ATP formed as the growth rate changes.