Streptococcus agalactiae

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.

The presence of a sufficient number of target molecules is critical in detecting conferred fluorescence of probe-target hybrids and permits direct visualization of intact organisms by epifluorescence microscopy. This direct approach to visualization of organisms using fluorescence in situ hybridization (FISH) simultaneously provides information on phylogenetic relationships of organisms, their spatial distribution in the sample matrix, their relative abundance, and their relative physiologic activity. An incubator, a water bath, and an epifluorescence microscope are essential, with the microscope being most critical for successful FISH performance. The main goal in designing specific probes is to find a suitable and unique region within the 16S or 23S rRNA that permits discrimination of target from nontarget organisms. Once a probe has been designed and confirmed to be specific for the motif desired by using the appropriate databases, it must be evaluated against a series of reference organisms. In case of a low number of ribosomes, the simultaneous application of two fluorescence-monolabeled probes targeting two different regions of rRNA with the same specificity would theoretically result in a twofold amplification of signal intensity when applied to the test samples. The chapter talks about expanded techniques combined with FISH. FISH, based upon either DNA or PNA oligonucleotide probes, is a rapid diagnostic method capable of challenging traditional culture techniques for the direct and accurate identification not only of nonfastidious pathogens but also of fastidious, slow-growing, and difficult-to-cultivate organisms.

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.

Microbial interactions involve sensing and responding to chemical signals or cues that are released from cells. In the early stages of plaque accumulation on freshly cleaned teeth, most interactions occur between the initial colonizers such as Streptococcus spp., Actinomyces spp., and Veillonella spp. However, mature dental plaque biofilms may contain up to 200 different phylotypes of bacteria, resulting potentially in almost 20,000 different pairwise interactions. This chapter focuses on just one of these interactions: that between two initial colonizers of oral biofilms, Streptococcus gordonii and Actinomyces oris. Interbacterial binding between oral bacteria has been investigated extensively in vitro using coaggregation assays. In these experiments, pure cultures of genetically distinct bacteria are mixed in test tubes and interactions are scored based on the extent of clumping (coaggregation). Coaggregation is used to model biofilm communities containing S. gordonii and A. oris and investigate gene regulation in mixed-species cultures. A powerful application of postgenomic technologies is the analysis of gene expression using DNA microarrays. The first genome-level analysis of S. gordonii-A. oris interactions employed microarrays to identify genes in S. gordonii that were regulated in response to coaggregation with A. oris. This study demonstrated that S. gordonii specifically activates a set of genes in response to cell-cell contact (coaggregation) with A. oris. In theory, microarrays and other genome-based expression technologies can be used to investigate cell-cell contact-induced gene regulation in any bacterial species for which the genome sequence is known.

Due to the importance of the cephems, it is essential to classify them so as to allow their optimal use. The cephems are characterized by an azetidinone ring, to which is attached an unsaturated hexacycle. Five classes of cephems may be distinguished in terms of the nature of the moiety: cephalosporins, cephamycins and cephabacins, oxa-1-cephems, carba-1-cephems, and miscellaneous. The dosage and dosing frequency of a cephalosporin are the result of a balance between its antibacterial activity and its pharmacokinetic profile. The molecules all have the same antibacterial spectrum, which principally includes gram-negative bacilli and gram-negative and gram-positive cocci, except for S. aureus. The carbacephem-type molecules are asymmetrical, and the substituents on the β-lactam moiety are in the cis position; the cis stereochemistry of the carbacephems is less stable thermodynamically than the trans stereochemistry. The antibacterial activity RWJ-54428 was designed to overcome methicillin resistance in methicillin-resistant S. aureus (MRSA) strains and ampicillin resistance in E. faecium by penicillin-binding proteins (PBP) alterations. In all species, total clearance was slower and the apparent elimination half-life was longer than that of RWJ- 54428. The antistaphylococcal activities of RWJ-333442 in comparison with RWJ-54428, vancomycin, and quinupristindalfopristin against S. aureus Smith were investigated in a sepsis model (Swiss-Webster mice) and were also investigated for S. aureus 076, a methicillin-resistant strain. A synergistic activity was shown to be species dependent in combination with streptomycin against E. faecalis.

Mupirocin is active against gram-positive cocci: it has moderate activity against gram-positive bacilli such as Listeria monocytogenes and Erysipelothrix rhusiopathiae; it is inactive against corynebacteria and Bacillus anthracis. It is inactive against Chlamydia spp. It is active against Staphylococcus aureus, whether the strains are susceptible or resistant to penicillin G, tetracyclines, erythromycin A, fusidic acid, lincomycin, chloramphenicol, or methicillin. Mupirocin has good activity against human and animal mycoplasmas. As mupirocin is highly serum protein bound, the in vitro activity is reduced in the presence of human serum. Mupirocin alters protein synthesis by blocking the incorporation of isoleucine into the peptide during synthesis. Since 1985, when mupirocin was introduced into clinical practice, two types of resistant strains have been described: those for which the MICs are between 8 and 256 µg/ml and those for which the MICs are 512 µg/ml. Extensive use of mupirocin has resulted in the emergence of resistant strains of S. aureus in the United Kingdom. Local application of mupirocin yields in situ concentrations of 20,000 mg/kg. Skin and skin structure infections are very common, but the use of topical antibiotics or other antibacterial agents is limited by a number of factors: the complexity of the anatomy of the skin, the bacterial ecology of the skin, and the nature of the microorganisms involved in these infections. Mupirocin eradicates methicillin-resistant strains of S. aureus colonizing the skin and produces an improvement in the healing of skin wounds.

The chapter mainly focuses on peptidyl deformylase (PDF) inhibitors. Bacterial peptidyl deformylase (PDF) belongs to subfamily 3, with at least two other members: thermolysin and matrix metalloproteases (MMPs), namely, matricins and/or metzincins. Two classes of aminopeptidases are described: MAP-1 and MAP-2. MAP-1 and MAP-2 have similar specificities, with methionine cleavage depending on the nature of the second amino acid. MAP-2 is inhibited by fumagillin and TNP-470. Bacterial PDFs are small monomers composed of about 160 to 200 amino acids with few variations in the lengths of their N- and C-terminal extremities. Deformylase inhibitors can be classified in three groups: natural compounds, hydroxamate derivatives, and miscellaneous compounds. The hydroxamic moiety is crucial for the antibacterial activity, but the pseudopeptide backbone can be altered, such as the methionine-like structure. In addition to the antibacterial activity, it has been shown that actinonin inhibits several aminopeptidases, such as human seminal alanyl aminopeptidase, as well as tumor growth. A series of β-sulfonyl and β-sulfinyl hydroxamic acid derivatives has been shown to be potent PDF inhibitors with in vitro antibacterial activity. A macrocyclic deformylase peptide inhibitor was reported. The improved affinity for PDF is probably due to the rigidity introduced by cyclization. Some compounds exhibited good activity against Bacillus subtilis, Haemophilus influenzae, and Moraxella catarrhalis but weak activity against Streptococcus pneumoniae; they were inactive against Staphylococcus aureus.