Gram-Positive Pathogens, Third Edition

In the same way arms, legs, hair, and fur are used in higher species for their survival in the environment, bacteria use surface appendages for similar purposes. Surface molecules in bacteria range from complex structures, such as flagella that propel the organism in aqueous environments, to less sophisticated polysaccharides and proteins. All of these molecules benefit the organism for survival in a hostile environment, such as the waters of a rushing stream, the blood of an infected animal, the surface of an object, or the surface of a mucosal epithelium. Although it was previously believed that bacteria were simple single-cell organisms with little complexity, it is now apparent that they are highly evolved, advanced particles that possess a wide array of surface molecules that manipulate the organism in its environment. For human pathogens, surface molecules have been finely tuned to allow adherence and colonization of host surfaces, invasion of cells, evasion of the host’s immune response, and persistence in infected tissues.

Streptococcus pyogenes has evolved a variety of both surface-bound and extracellular factors that alter the inflammatory response and impair phagocytic clearance of the bacteria. The more than 150 genotypes use both similar and different strategies at the biochemical level to colonize their host and avoid protective defenses. These are reviewed elsewhere ( 5 ). Intracellular invasion is dependent on at least two classes of surface proteins, the M proteins ( 6 , 7 ) and fibronectin (Fn)-binding proteins ( 3 , 8 ). The M proteins serve many functions in the pathogenesis of S. pyogenes, including resistance to phagocytosis, adherence, and intracellular invasion ( 5 , 9 ). The Fn-binding protein F (PrtF) and the allelic variant SfbI are also adhesins and invasins, produced by 50 to 60% of the M genotypes. The function of these proteins in the context of intracellular invasion is described below.

In her early studies of group A streptococci (Streptococcus pyogenes), Rebecca Lancefield noted an association between virulence and a distinctive appearance of the bacterial colonies on solid media. Isolates that were highly virulent for mice and that grew well in fresh human blood typically formed large colonies with a translucent, liquid appearance (mucoid) or an irregular, collapsed appearance (matte). By contrast, avirulent isolates that grew poorly in human blood formed compact, opaque colonies (glossy). Strains that grew as mucoid or matte colonies usually produced large amounts of M protein, which Lancefield gave the designation “M” because of this association with colony morphology ( 1 , 2 ). Later work by Armine Wilson demonstrated that the mucoid or matte appearance of such strains was in fact due to elaboration of capsular polysaccharide, not M protein ( 3 ). Wilson showed that the mucoid or matte colony type was converted to a nonmucoid or glossy colony by growth on medium containing hyaluronidase which digested the hyaluronic acid capsule, whereas growth on medium containing trypsin, which digested M protein, had no such effect. Furthermore, while many strains that produced abundant capsular polysaccharide also were rich in M protein, expression of the two surface products was not always linked; certain mucoid or highly encapsulated strains produced little or no M protein, and certain strains rich in M protein produced little or no capsule ( 1 , 4 ).

Streptococcus pyogenes (i.e., the group A Streptococcus) is a human-restricted and versatile bacterial pathogen that produces an impressive arsenal of both surface-expressed and secreted virulence factors. S. pyogenes exists primarily as an asymptomatic colonizer of the skin and mucous membranes of the nasopharynx, and despite being universally sensitive to β-lactam antibiotics in vitro, this bacterium continues to generate significant morbidity and mortality on a global scale. Human diseases induced by S. pyogenes usually occur as relatively uncomplicated manifestations such as pharyngitis and skin infections, but it may also cause more problematic diseases including erysipelas and scarlet fever. In addition, S. pyogenes can cause devastating invasive diseases including puerperal sepsis, bacteremia, necrotizing fasciitis, and streptococcal toxic shock syndrome (TSS). This bacterium is further recognized as a very important cause of postinfection sequelae including acute rheumatic fever and rheumatic heart disease, acute glomerulonephritis, and potentially, pediatric autoimmune neuropsychiatric disorders associated with streptococcal infections. Although surface-expressed virulence factors are clearly vital for colonization, establishment of infection, and the development of disease, the secreted virulence factors are likely the major mediators of tissue damage and toxicity seen during active infection. The collective exotoxin arsenal of S. pyogenes is rivaled by few bacterial pathogens and includes extracellular enzymes, membrane active proteins, and a variety of toxins that specifically target both the innate and adaptive arms of the immune system, including the superantigens; however, despite their role in S. pyogenes disease, each of these virulence factors has likely evolved with humans in the context of asymptomatic colonization and transmission. In this article, we will focus on the biology of the true secreted exotoxins of the group A Streptococcus, as well as their roles in the pathogenesis of human disease.

Streptococcus pyogenes (the group A streptococcus) is remarkable in terms of the large number of very different diseases it can cause in humans. These range from superficial and self-limiting diseases of the pharynx (e.g., pharyngitis, commonly known as strep throat) and skin (impetigo) to infections that involve increasingly deeper layers of tissue and are associated with increasing degrees of destruction of tissue (e.g., erysipelas, cellulitis, necrotizing fasciitis, and myositis). The organism has the ability to spread rapidly through tissue and to penetrate into the vasculature to cause lethal sepsis. Other diseases result from the production of toxins that spread through tissue or spread systemically from a site of local infection (scarlet fever and toxic shock syndrome). Still other diseases are the result of an immunopathological response on the part of the host that is triggered by a streptococcal infection. These diseases include rheumatic fever, acute glomerulonephritis, certain types of psoriasis, and potentially even some forms of obsessive-compulsive syndrome disorder (pediatric autoimmune neuropsychiatric disorders associated with streptococcal infections).

Cross-reactive antigens are molecules on the group A streptococci that mimic host molecules and during infection or immunization induce an autoimmune response against host tissues, leading to the autoimmune group A streptococcal sequelae ( 1 – 25 ). “Molecular mimicry” is the term used to describe immunological cross-reactivity between the host and bacterial antigens. Immunological cross-reactions between streptococcal and host molecules have been identified involving antibodies or T cells that react with streptococcal components and tissue antigens ( 6 , 7 , 22 , 24 , 25 ). The advent of monoclonal antibodies (mAbs) and T cell clones/hybridomas has greatly advanced the identification of the host and streptococcal antigens responsible for immunological cross-reactions associated with immunization, infection, and autoimmune sequelae. The identification of cross-reactive antigens in group A streptococci and important in our understanding of the pathogenesis of autoimmune sequelae, such as rheumatic fever including carditis ( 8 , 11 ) and Sydenham chorea ( 7 ), which may follow group A streptococcal infection ( 3 ).

The adherence to and invasion of eukaryotic cells are the main strategies pathogenic bacteria use for colonization, evasion of immune defense, survival, and causing disease in their mammalian hosts. Most Gram-negative bacteria use pili to achieve adherence and invasion ( 1 ), while only a few Gram-positive species are equipped with these structural elements ( 2 , 3 ). Hence, Gram-positive pathogens express specific cell surface components called adhesins, or MSCRAMM (microbial surface components recognizing adhesive matrix molecules), which directly or indirectly facilitate the adherence of the bacteria to host tissues, thereby mediating not only colonization but also invasion of microbes ( 4 , 5 ). Bacterial adhesins of several Gram-positive pathogens, including Staphylococcus aureus, Streptococcus pyogenes, and Streptococcus pneumoniae, are of utmost importance to enable their attachment to adhesive glycoproteins of the host extracellular matrix (ECM) as well as their adherence to host epithelial or endothelial cells, utilizing this strategy also to trigger immune evasion mechanisms ( 5 – 7 ).

Bacteria must adhere to, colonize, and invade the target tissue while evading host protective immune responses to be successful pathogens. A pathogen sequentially engages (adheres to) its surface-exposed adhesins with complementary host receptors to successfully colonize and infect the targeted cell. Group A Streptococcus (Streptococcus pyogenes) is one of the most successful pathogens and is a model organism used to understand various aspects of pathogenesis. The latter may be related to toxins, horizontal gene transfer, mammalian protein binding, cellular invasion, intracellular proliferation, immune evasion, innate immunity, programmed cell death, and/or autoimmunity. S. pyogenes displays a remarkable tissue tropism for the human pharynx and the skin, causing a variety of diseases ( 1 – 3 ). Initial S. pyogenes infection can easily be treated with penicillin, and there is no known emergence of penicillin-resistant strains. Despite important advances in hygiene and modern methods of prevention, S. pyogenes still ranks among the 10 deadliest human pathogens, causing an estimated more than 500,000 deaths annually ( 4 , 5 ).

Streptococcus pyogenes (Lancefield group A) is a human pathogen responsible for a wide range of diseases, the most common of which are nasopharyngeal infections (or strep throat) and impetigo. More than 25 million cases of group A streptococcal infections occur each year in the United States at a cost of over $1 billion to the public in addition to losses in productivity. In recent years, an increase in streptococcal toxic shock and invasive infections (particularly, necrotizing fasciitis) has been reported with certain strains of group A streptococci, resulting in rapid fatalities in up to 30% of the cases ( 1 , 2 ). Since there have been no reports of penicillin resistance in group A streptococci, streptococci-related diseases can be successfully treated with this antibiotic; however, with erythromycin, the second drug of choice for these bacteria, resistance is currently observed. Before treatment, group A streptococcal infections are usually associated with fever, significant discomfort, and generalized lethargy mostly due to the wide range of toxic substances secreted. About 3% of individuals with untreated or inadequately treated streptococcal pharyngitis develop rheumatic fever and rheumatic heart disease, a sequela of the streptococcal infection resulting in cardiac damage, particularly of the mitral valve. While in the United States outbreaks of rheumatic fever are usually sporadic and infrequent, affecting only local areas of the country ( 3 ), in developing countries as many as 1% of school-age children are estimated to have rheumatic heart disease ( 4 ). Because of this, and the concern that penicillin-resistant strains may appear, there is a strong incentive to develop a safe and effective vaccine against group A streptococcal pharyngitis.

The year 2015 was the centenary of the discovery of bacteriophages (phages) by Frederick Twort ( 1 ), and the first associations of phages with Streptococcus pyogenes (group A streptococcus) were made by Cantacuzene and Boncieu in 1926 ( 2 ) and by Frobisher and Brown in 1927 ( 3 ), both groups showing that the “scarlatina toxin” (erythrogenic toxin, pyrogenic exotoxin A) could be passed to a new strain after exposure to a sterile filtrate from a toxigenic isolate. Additional studies by Evans in the 1930s and 1940s documented the prevalence of group A streptococcal phages, linked them with virulence, and provided early additional evidence for lysogeny ( 4 – 8 ). More studies followed of both lysogenic and lytic streptococcal phages ( 9 – 15 ), but the link between lysogeny and the transmission of the erythrogenic toxin was not made until the landmark study by Zabriskie in 1964 ( 16 ); the phage-encoded toxin structural gene (speA) was subsequently identified by Weeks and Ferretti ( 17 , 18 ) and independently by Johnson and Schlievert ( 19 ). The history and contributions of these and other early workers to our knowledge of the bacteriophages of S. pyogenes are covered elsewhere ( 20 ). Following these studies, a significant body of research has shown that phages contribute to the success and virulence of their host streptococci by acting as vectors for genes encoding virulence factors and through their ability to disseminate streptococcal genes from one cell to another through transduction. Since group A streptococci are not thought to be normally competent for transformation, except possibly under certain conditions ( 21 ), and since Hfr-type conjugal transfer of chromosomal genes has not been observed, it is easy to argue that the bacteriophages of S. pyogenes play a major role in the genetics and molecular biology of this pathogen.

The group A streptococcus (GAS; Streptococcus pyogenes) is a free-living organism. Its ecological niche appears to be quite narrow, and its only known biological host of import is humans. There may be occasional or very rare natural infections in non-human primates and other mammals ( 1 , 2 ).

With an uncanny ability to interpret results obtained with a handful of immunologic techniques that would be considered simplistic and insensitive by today’s standards, Rebecca Lancefield and her coworkers in the 1930s set the stage for decades of research on Streptococcus agalactiae, also known as group B Streptococcus (GBS). In contrast to group A Streptococcus, which was isolated mainly from humans, the first strains of GBS studied by Lancefield were isolated from cows with mastitis or from “normal milk.” At that time, GBS was a well-known cause of bovine mastitis and a concern mainly of the dairy industry and veterinary medicine ( 1 ).

Streptococcus agalactiae, also known as group B Streptococcus (GBS), was first differentiated from other streptococci by Rebecca Lancefield in the 1930s after it was isolated from milk and cows with bovine mastitis ( 1 ). Lancefield described GBS colonization of the vaginal tract of asymptomatic women, but human pathogenicity was not described until 1938 when three reports of fatal postpartum infection were published ( 2 , 3 ). Invasive GBS disease was rarely identified in humans until the 1960s, when increasing reports of adult and neonatal invasive infections were published ( 4 – 7 ). The incidence of invasive GBS disease continues to increase, and it remains a significant pathogen among both infants and adults.

The genus Streptococcus comprises more than 70 species (some talk of more than 100) classified into eight phylogenetic groups, with Lancefield group C streptococci (GCS) and group G streptococci (GGS) being discovered in four of them, including one unknown group. GCS and GGS carry the immunedeterminant residues N-acetylgalactosamine and rhamnose, respectively, on the oligosaccharide side chains of their cell wall carbohydrate antigens. These organisms are distributed in both humans and animals. They are isolated as opportunistic commensals from the skin, nose, throat, vagina, and gastrointestinal tract but may also be associated with clinically important infections of these sites and with hospital outbreaks. Serious diseases caused by human GCS and GGS often resemble those due to their closest genetic relatives, the group A streptococci (GAS, carrying the immunodominant N-acetylglucosamine side chains of their cell wall carbohydrate antigens), and include septicemia, pharyngitis, cellulitis, otitis media, septic arthritis, meningitis, infective endocarditis, multiple organ abscesses, necrotizing fasciitis, and toxic shock syndrome ( 1 – 11 ). At one time, the genetic relationships of GCS and GGS strains were complicated and incompletely resolved due to the diversity of species within the serogroups and the uncertainties in assigning species names, particularly to the human-specific strains now classed with the phylogenetic Anginosus group ( 12 ). The current classification ( Table 1 ) arrived at by numerous early ( 13 – 33 ) and more recent ( 34 – 43 ) studies relies on habitats, pathogenicity properties, physiological characteristics, and relationships of informational macromolecules, with serological grouping being useful for differentiating infraspecific biotypes. Before the advent of large-scale DNA sequencing and gene cloning technologies, GCS and GGS had rarely been subjected to genetic studies. However, studies of the host range of bacteriophages isolated from GAS, GCS, and GGS, and of their transducing potential, provided evidence for intergroup phage reactions and intergroup transduction between strains belonging to different Lancefield groups, thus amending the original notion of the strict group specificity of streptococcal phage-host interactions and emphasizing the importance of lateral gene transfer for the evolution of GCS and GGS ( 44 , 45 ). An attempt to elucidate the genetic relationships between GAS, GCS, and GGS based on the nucleotide sequence of selectively neutral housekeeping genes supported events of interspecies gene transfer that predominantly occurred from GAS donors to GCS and GGS recipients ( 46 ). In S. dysgalactiae subsp. equisimilis, recently reviewed studies show that both mobile genetic elements and integrative conjugative elements are involved in horizontal gene transfer, with the latter playing the prominent role in the evolution of this subspecies ( 47 ). This applies also to the interspecies transfer of integrative and conjugative elements (ICEs) between S. salivarius (Salivarius group) and S. parasanguinis (Mitis group; see Table 1 ), in the case of which components of the mobile subfamily ICESt3 element were very recently detected in their conjugation modules at high nucleic sequence identities ( 48 ). The application of recombinant DNA techniques and phylogenomic studies have advanced our understanding of GCS and GGS in diverse areas, and this article concentrates on the molecular genetics and the structure and function of pathogenetically relevant genes and proteins studied at the molecular level in recent years.

The pyogenic streptococci of Lancefield groups C and G were initially recognized as a cause of animal infections long before they were even considered as agents of human disease, even though they are widely distributed in animals and humans. They comprise a heterogeneous complex of streptococcal species that act as causative agents of a spectrum of diseases ranging from mild pharyngitis to skin infection to life-threatening systemic infections associated with high mortality rates. In this article we provide an overview of the various group C and group G streptococcal species, the diseases they cause, and the major pathogenicity factors that contribute to their virulence ( Table 1 ).

Conrary to the homogeneity typical of streptococci belonging to Lancefield groups A (S. pyogenes) and B (S. agalactiae), groups C and G streptococci (GCGS) represent a variety of species that are widely variable in regard to biochemical reactions, hemolytic characteristics, predilection for host species, and clinical illnesses produced in humans and animals. These organisms are found as commensals in the throat, skin, and occasionally the female genitourinary tract, and their epidemiologic patterns and clinical manifestations reflect this distribution.

Since the last revision of this article (2006), a number of important publications have appeared in the literature, the inclusion of which made it necessary to limit the topics of this article to information that has bearing on the biochemical and genetic aspects of covalently linked components of the pneumococcal cell wall. Information on proteins noncovalently attached to the cell wall, cell walls and phase variation (Jing Li and Jing-Ren Zhang, in press), and inflammatory activity of cell walls (Allister J. Loughran, Carlos J. Orihuela and Elaine I. Tuomanen, in press) are reviewed separately.

The presence of what is now recognized as the polysaccharide capsule on the surface of Streptococcus pneumoniae was noted by Pasteur in the first published description of the organism in 1880, and since that time it has been the direct or indirect focus of intensive investigation [reviewed by Austrian ( 1 , 2 )]. Studies during the first three decades of the 20th century demonstrated the existence of multiple capsular serotypes of S. pneumoniae and the fact that antibodies to the capsule conferred type-specific protection against challenge in laboratory animals. The capsular material itself was isolated by Dochez and Avery in 1917 ( 3 ), but the fact that it was immunogenic led them to believe that this “soluble substance of the pneumococcus” was proteinaceous in nature. It was not until 1925 that Avery and colleagues ( 4 , 5 ) demonstrated that the pneumococcal capsule consisted of polysaccharide, the first nonprotein antigen to be recognized.

Streptococcus pneumoniae (the pneumococcus) is a leading cause of otitis media (OM), community-acquired pneumonia, bacteremia, and meningitis. The pneumococcus is a human-specific pathogen which colonizes the nasopharynx and spreads between hosts through aerosols and potentially through the contamination of objects with mucosal secretions if the bacteria is living within a biofilm ( 1 – 3 ). Rates of carriage vary from 5 to 10% of healthy adults to 20 to 40% of healthy children. However, these numbers can vary widely based on where the samples are collected ( 4 – 7 ). Risk factors associated with higher rates of carriage include race (particularly Australian Aboriginals and Native Americans) ( 8 – 12 ), infancy ( 13 , 14 ), season, with higher carriage during winter months ( 13 ), and crowded areas such as childcare centers, with estimates suggesting that 40 to 60% of children who attend childcare are colonized ( 15 ). Duration of colonization decreases with age and varies from 2 weeks to 4 months ( 14 , 16 , 17 ). The introduction of pneumococcal conjugate vaccines has reduced carriage rates for serotypes covered by the vaccine, while nonvaccine serotypes have emerged to occupy this empty niche ( 18 ). Nasopharyngeal colonization is usually asymptomatic ( 19 ).

Phase variation, a type of phenotypic variation, refers to reversible, ON-and-OFF production of a molecule, structure, or complex characteristic ( 1 ). Since Andrewes’ description of “diphasic salmonellas” ( 2 ), the first report of phase variation in bacteria, phase variation has been discovered in many bacteria, particularly pathogens. Our current understanding of the genetic and molecular basis of phase variation is almost entirely based on the studies of virulence determinants. Examples include variable generation in surface proteins ( 3 – 5 ), fimbriae/pili ( 6 ), flagella ( 7 – 9 ), capsule ( 10 , 11 ), modification of lipopolysaccharides and lipooligosaccharides ( 12 – 14 ), and colony morphology ( 15 – 17 ). This type of phenotypic variation in pathogenic bacteria permits the selection of variants with optimal adaption or fitness in an individual host or distinct host environment. Phase variation is particularly common in Gram-negative pathogens, such as Escherichia coli, Salmonella typhimurium, Haemophilus influenzae, Campylobacter jejuni, and pathogenic Neisseria ( 18 ).

The first complete genome sequence of the pneumococcal type 4 strain TIGR4 was published in 2001. To date (February 2018), GenBank hosts 37 complete pneumococcal genomes and more than 8,000 draft genome sequences. The genetics of Streptococcus pneumoniae began with the discovery of bacterial transformation by Griffith in 1928 and subsequent identification of DNA as the genetic material by Avery, MacLeod, and McCarty in 1944 ( 1 ). Many of the S. pneumoniae strains commonly used in genetic studies are derivatives of the clinical strains obtained by Avery in 1916 ( Table 1 ) ( 2 – 4 ).

With the discovery of the pneumococcus in 1881, it became apparent that this Gram-positive pathogen was a major cause of serious and often fatal pneumonia ( 1 ). It is also a major cause of meningitis and otitis media in children. Pneumococci are the largest cause of community-acquired pneumonia in the developed world. According to the Centers for Disease Control and Prevention, in the United States the combined rates of invasive pneumococcal diseases has shown a decline since the introduction of the conjugate vaccines, with rates among the elderly declining from 59/100,000 in 1998 to 23/100,000 in 2015 and rates among children under 5 years of age falling from 95/100,000 in 1998 to 9/100,000 in 2016 (https://www.cdc.gov/pneumococcal/surveillance.html). The rate of meningitis in children in the United States is about 4 cases per 100,000 children, with a fatality rate of about 15% ( 2 ). In the developing world pneumococci are an important cause of childhood deaths due to bacterial respiratory infection following viral disease. The World Health Organization suggests that globally, such infections have been reported to have killed an estimated over 500,000 children in 2008 (http://www.who.int/immunization/monitoring_surveillance/burden/estimates/Pneumo_hib/en/). Recently, about one-third to one-half of pneumococci recovered from humans in the United States have been found to be at least partially resistant to penicillin, and penicillin-resistant strains are frequently also resistant to other common antibiotics ( 3 ). The rise of antibiotic resistance among pneumococci has already complicated treatment, especially of meningitis ( 4 ), and threatens to greatly increase the morbidity and mortality caused by pneumococci unless new control measures are developed.

Enterococci are hardy, Gram-positive cocci that are common residents of the gastrointestinal tracts of nearly all land animals, including humans. While they are a core member of the microbiome, they are also capable of causing a variety of severe infections, most often among antibiotic-treated hospitalized patients with perturbed intestinal microbiota. Here, we present an overview of the pathogenicity of enterococci and discuss the most prominent features of this hospital-associated pathogen.

Interest in enterococcal genetics began with three landmark discoveries: (i) identification of the first conjugative plasmids whose transfer systems are induced by an identifiable signal ( 1 ), (ii) identification of the first “transposons” capable of intercellular (conjugative) transposition ( 2 , 3 ), and (iii) the acquisition of vancomycin resistance ( 4 , 5 ). Since the most prevalent antibiotic resistance genes are located on plasmids and transposons, early work on enterococcal genetics focused heavily on mobile genetic elements (MGEs). Examination of the complete sequence of a vancomycin-resistant clinical isolate of Enterococcus faecalis, V583, reaffirmed the importance of MGEs in the evolution of this species, revealing that over a quarter of the genome consists of mobile and/or exogenously acquired DNA ( 6 ). Further genome sequencing has revealed extensive variability in the proportion of horizontally acquired DNA in E. faecalis strains, with the presence of clustered regularly interspaced short palindromic repeats (CRISPR) with CRISPR-associated (Cas) protein (CRISPR-Cas) loci playing an important role in regulating MGE acquisition ( 7 , 8 ). More recent genomic analysis has revealed that MGEs are important drivers of genome diversity and evolution in Enterococcus faecium as well ( 9 ).

Warm, moist, and rich in nutrients, the oral cavity provides an ideal environment for colonization by a community of bacteria, fungi, protozoa, archaea, and viruses, often in the form of a complex structure called biofilm or plaque ( 1 ). In addition, several microbial niches exist within the oral cavity (e.g., cheek, gingiva, teeth, tongue, palate) that vary in nutrient content, pH, oxygen tension, and shear force due to salivary flow and mastication. These physico-chemical characteristics select for suitable microorganisms for each oral niche such that the microbial compositions can differ greatly from one site to another. Saliva is the biological fluid of the oral cavity, and its microbial composition is a collection of bacteria that have shed from various oral niches. As an easily accessible body fluid, saliva can be used in assessment of general oral health, as the disappearance or emergence of certain taxa can indicate health or disease ( 2 , 3 ). While biofilms from oral soft tissues, such as the tongue, cheeks, and palate, and the supragingival biofilm (plaque on tooth surfaces above the gum line) are constantly bathed in saliva and subjected to an array of environmental fluctuations, the subgingival plaque (plaque on tooth surfaces below the gum line) is a unique niche with less abrupt environmental fluctuations. In particular, with the gum serving as a physical barrier, the oxygen tension and shear forces are reduced, and gingival crevicular fluid (serum) serves as a major nutrient source ( 4 ).

In 1924, J. Clarke isolated an organism from carious lesions and called it Streptococcus mutans, because he thought the oval-shaped cells observed were mutant forms of streptococci ( 1 ). However, it was in the late 1950s when S. mutans gained widespread attention within the scientific community, and by the mid-1960s, clinical and animal-based laboratory studies depicted S. mutans as an important etiologic agent in dental caries ( 2 ). The natural habitat of S. mutans is the human oral cavity, more specifically, the dental plaque, a multispecies biofilm formed on hard surfaces of the tooth. It has been largely accepted that the cariogenic potential of S. mutans resides in three core attributes: (i) the ability to synthesize large quantities of extracellular polymers of glucan from sucrose that aid in the permanent colonization of hard surfaces and in the development of the extracellular polymeric matrix in situ, (ii) the ability to transport and metabolize a wide range of carbohydrates into organic acids (acidogenicity), and (iii) the ability to thrive under environmental stress conditions, particularly low pH (aciduricity) ( 3 ). While S. mutans does not act alone in the development of dental caries, studies from several laboratories have convincingly demonstrated that S. mutans can alter the local environment by forming an extracellular polysaccharide (EPS)-rich and low-pH milieu, thereby creating a favorable niche for other acidogenic and aciduric species to thrive. As a human pathogen, S. mutans is also implicated in subacute bacterial endocarditis, a life-threatening inflammation of heart valves, while a subset of strains has been linked to other extraoral pathologies such as cerebral microbleeds, IgA nephropathy, and atherosclerosis.

Our view of oral streptococci has largely been influenced by the approach taken in the last century to identify etiologic agents of disease. As a consequence, beneficial aspects of streptococcal colonization of the oral cavity were initially overlooked. The first comprehensive analysis of the resident oral microbiota was accomplished in 2005 ( 1 ), and with this, a new picture began to emerge. With the availability of high-throughput sequencing techniques and an increased sensitivity in analysis methods, the presence of a defined microbiome associated with oral health has been shown ( 2 ). Alongside this, “omics” techniques have revealed that prevalent oral diseases such as caries and periodontal disease are polymicrobial in nature and the result of microbial dysbiosis ( 3 , 4 ). Even more striking, the metabolic output of these mixed microbial communities seems to be more relevant than their precise microbial composition ( 4 ). This is also reflected by the fact that the severity of caries and periodontal disease is heavily influenced by the synergistic interactions of the individual members of the polymicrobial consortium, including metabolic cross-feeding and interspecies signaling with transcriptional adjustment to the metabolic output. Thus, the ecological context of the microbial community seems to be of importance to understanding oral health and disease development. As a consequence, polymicrobial diseases cannot be explained by the behavior of one bacterial species and certainly cannot be treated like diseases that follow Koch’s postulates ( 5 – 7 ). Novel approaches to combat oral polymicrobial diseases should therefore focus on the bacterial community that is present in the healthy oral cavity. Since oral streptococci are abundant during initial colonization of the tooth ( 8 , 9 ), their function is to provide a favorable environment for incorporation of later species and to support accretion of the mature oral biofilm, which in general has a health-protecting function ( 10 , 11 ).

Lactococci have been used for centuries in dairy fermentation. These Gram-positive, generally nonpathogenic, nonmotile, and nonsporulating bacteria are members of the Streptococcaceae family, which includes food, commensal, and virulent species ( Fig. 1 ). Lactococcus lactis is a relatively simple bacterium, with a 2.4-Mbp genome. Many of its functions of interest are nonredundant, which facilitates functional genetic studies of nonessential genes. Lactococci are presumed to be devoid of virulence factors (although isolated cases of L. lactis as the infectious agent in human and bovine infections have been reported [ 1 , 2 ]). The goal of this article is to confront previous and current information in different areas of lactococcal genetics, keeping in mind the relevance of findings to related bacteria, especially pathogens. Work on pathogens has long focused on surface and secreted virulence factors, while work on lactococci has gone deeper in characterizing basic metabolic properties, nutrient uptake, and survival. Genes in basic metabolic pathways (e.g., respiration, metal homeostasis, amino acid metabolism) are now known to be essential not only for fitness but also for virulence. Numerous Lactococcus researchers who shifted their focus to pathogens have contributed to this understanding. The overall nonvirulence of lactococci has also been useful in determining how metabolic and virulence factors participate in bacterial “everyday life” outside the animal host. Our deep knowledge of L. lactis physiology has led to new concepts and general findings, for example by (i) establishing the bases for dialog between Firmicutes, (ii) providing concrete in vivo data on the biomedical or probiotic potential of recombinant and wild-type lactic acid bacteria (LAB) ( 3 , 4 ), and (iii) establishing the existence of an inverse correlation between bacterial mRNA concentration and stability ( 5 ).

The genome of a bacterial cell encodes all of the potential functions of that cell and how they respond to environments ( 1 ). The first staphylococcal genomes to be sequenced in full were landmark achievements in 2001 ( 2 ), allowing the first overview and predictions of the metabolic, regulatory, and virulence potential of Staphylococcus aureus. As more genomes were sequenced in the next few years, different regions of the genomes were discovered that were core and conserved, often including essential genes, as well as regions that were variable, nonessential, or mobile and allow the cell to adapt to new environments ( 3 – 7 ). Now that thousands of genomes are available, we understand the constant flux that staphylococcal genomes are under. Change occurs in small nucleotide variations (SNVs) or by horizontal gene transfer (HGT), and evolution occurs when these changes provide a selective advantage to the bacterial cell and become fixed in subpopulations. Particular variants adapt to evolving habitats, new environments, and stresses, leading to the spread and expansion of successful clones that continue to evolve.

As has been the case for other bacterial genera, studies of the genetic basis of staphylococcal pathogenicity and antimicrobial resistance highlighted the presence of determinants not uniformly represented in the genomes of all staphylococcal strains. Although not a fundamental requirement for survival per se, such accessory DNA usually encodes functions that are advantageous in a particular environmental niche. The extent of the accessory component of the genome has been revealed by comparative analysis of whole Staphylococcus aureus genome sequences, which indicate that it can constitute in excess of 20% of a strain’s genetic makeup ( 1 – 5 ). It is clear that the acquisition, maintenance, and dissemination of accessory functions have been central to the ongoing success of staphylococci as pathogens. These processes are underpinned by interactions between mobile genetic elements, such as insertion sequences and transposons that mediate intracellular movement of DNA, and plasmids, bacteriophage and integrative and conjugative elements (ICEs) that facilitate intercellular DNA mobility.

The diversity of the Staphylococcus aureus species is mainly determined by mobile genetic elements, many of which are prophages or phage-related genomic islands. Strain evolution as a result of short- and long-term adaptation to diverse environments is tightly linked to phages. Many phages carry accessory genes coding for staphylococcal virulence factors, which are important for the success of certain S. aureus clonal complexes (CCs). Second, phages support the induction, packaging, and transfer of genomic islands ( 1 , 2 ). This topic is reviewed elsewhere. Third, phage-mediated transduction is an efficient way to transfer not only extrachromosomal mobile elements, such as plasmids, but also chromosomal markers (albeit with lower efficiency). S. aureus is thought not to be naturally competent, so that recombination and horizontal gene transfer are mostly phage mediated and, to a lesser extent, conjugative. Here, we will first give a brief overview of previously used methods to classify S. aureus phages. Then we will mainly focus on the impact of temperate phages on the evolution of the bacterial host.

Once upon a time, bacteria, like other organisms, were considered to have stable genomes with essentially constant overall composition that unequivocally defined each species. The bacterial genome was initially seen to consist of a single very large circular DNA molecule (chromosome) containing all of the organism’s genes ( 1 ). In retrospect, the proof that prophages were physically integrated into the chromosomal DNA provided the first inkling that this view was incorrect: discrete, variable, and mobile genetic units could exist within the bacterial chromosome. The profundity of this observation, however, was not immediately realized, since phages were seen as parasitic invaders that had found integration as a way to achieve stable intracellular inheritance. It was soon realized, however, that not only temperate phages but also the F plasmid and, as it was then thought, the ColE1 plasmid could reversibly integrate into the host cell’s chromosome and that there were also short DNA segments (insertion sequences) that could be found in various locations ( 2 ). These observations led to the idea of discrete elements of genomic variability superimposed on the overall constancy of the chromosomal genophore; indeed, by 1996 the intergeneric transfer of mobile genetic elements had been documented so widely that David Summers was led to suggest, a bit whimsically, that all bacteria are but a single megaspecies, cohered by a vast marketplace of exchangeable genetic units ( 3 ).

The end point of bacterial respiration is the production of ATP as it uses oxygen, or other molecules, to accept electrons while oxidizing dinucleotides ( Fig. 1 ). Because multiple cell wall biosynthetic enzymes require ATP ( 1 ), lowering ATP levels reduces the rate of growth of Staphylococcus aureus, resulting in small colonies. Some small colony variants (SCVs) have been found to be defective in respiration ( 2 ). Under glucose-rich conditions, ATP is produced via glycolysis without using oxygen, wherein glucose metabolism ends with the production of lactate ( 3 ). Under aerobic conditions, once glucose and other simple sugars are exhausted, pyruvate is further metabolized to acetate/acetyl-CoA, which enters the tricarboxylic acid (TCA) cycle (also called the Krebs cycle), and the chemical energy is fed into dinucleotides such as NADH and reduced flavin adenine dinucleotide (FADH2). Under anaerobic conditions, pyruvate is mainly metabolized to lactate ( 3 , 4 ). In addition to the oxidation of carbohydrates, the metabolism of amino acids and lipids also results in the production of acetyl-CoA ( 3 ). As acetyl-CoA is oxidized to CO2 and H2O, there is a simultaneous reduction of NAD+ and FAD+ to NADH and FADH2, respectively. Ultimately, these reduced dinucleotides must be reoxidized because many reactions in the cell, e.g., dehydrogenases, require NAD(P)+ for their activity.

Regulatory RNAs have been identified in many bacteria and in pathogenic bacteria such as Staphylococcus aureus, where they play major roles in the regulation of virulence or the synthesis of metabolic proteins, besides transcriptional factors and two-component systems ( 1 – 4 ). Most of them are noncoding RNAs (sRNAs), but some of them express small peptides. Certain sRNAs, acting in cis, are situated at the 5′ untranslated regions (UTRs) of mRNAs and act as sensors of metabolites, tRNA, or environmental stimuli (e.g., temperature, pH) or are situated at the 3′ UTR. In contrast, the genes encoding sRNAs, which act in trans, sit on the opposite strand of the regulated mRNA or at genomic locations distant from the mRNAs they regulate. Cis-encoded sRNAs, also called antisense RNAs (asRNAs), are fully complementary to their targets. In contrast, trans-encoded sRNAs share only partial complementarity, and as a consequence, they can regulate many mRNAs. Most of them are encoded mainly in the core genome, while a few of them are localized within mobile elements, pathogenic islands, or plasmids. In this review, we will focus on the most recent mechanisms of RNA regulation discovered in S. aureus and how regulatory RNAs are part of sophisticated networks that allow the bacteria to adapt quickly to their environment or survive in their host.

Ever since the recognition of the cell wall in the late 1940s to early 1950s as a unique anatomical component of all eubacterial cells, Staphylococcus aureus has often served as the Gram-positive model in wall-related studies. One of the first demonstrations that bacterial cell walls can be isolated as physical entities with the size and shape of the whole bacterium was with S. aureus. It was in penicillin-treated S. aureus that the UDP-linked amino sugar-containing wall precursor peptides were discovered, providing the first insights into the unique building blocks of cell wall biosynthesis. The history of interest in the staphylococcal cell wall also reflects the history of success and failure of the antibiotic era. The clue that eventually led to the discovery of penicillin (and later to the autolytic enzymes) was provided by the lysis of staphylococcal colonies in the vicinity of a mold contaminant on an agar plate in Fleming’s laboratory. Elucidation of the mode of action of several important antibiotics in the 1960s and 1970s has been intimately linked to studies of the biosynthesis of staphylococcal cell walls. This included studies of the mode of action of penicillin and other β-lactam antibiotics as specific inhibitors (acylating agents) directed against the active site of penicillin binding proteins (PBPs)—transpeptidases with or without an additional transglycosylase function—which catalyze terminal stages in the assembly of the bacterial cell wall. It was mainly from studies of S. aureus and from parallel studies of Escherichia coli that by the early 1980s a coherent picture emerged of the biosynthetic pathway that leads to the formation of the lipid-linked disaccharide pentapeptide, which with some structural variations, is the universal building block of cell wall peptidoglycan in both Gram-positive and Gram-negative bacteria. Reviews and references summarizing various aspects of studies of the cell walls of staphylococci up to the late 1980s are available ( 1 , 2 ).

The cell wall envelope of Staphylococcus aureus is a surface organelle with anchored proteins, teichoic acids, and polysaccharides that enable bacteria to promote specific interactions with their environment ( 1 ). During colonization and invasion of their mammalian hosts, secreted and cell wall-anchored surface proteins fulfill a wide spectrum of functions, including bacterial adhesion to host cells or tissues, evasion of innate host defenses, and diversions of host adaptive immune responses ( 2 ). Secreted proteins trafficking in and out of the cell wall envelope promote both the persistent colonization of mammalian hosts and the pathogenesis of specific disease states that are characteristically associated with S. aureus ( 2 ).

Cell wall-anchored (CWA) proteins are characterized by the presence of a sorting signal at the C-terminus which is responsible for coupling the protein covalently to peptidoglycan. The surface of Staphylococcus aureus is decorated with up to 24 CWA proteins. The precise number depends on the strain and the growth conditions. The repertoire of CWA proteins expressed by S. aureus is limited and many have evolved to perform important interactions with the host. They can be categorized into distinct structural and functional groups ( Fig. 1 , Table 1 ). Several are multifunctional due to the proteins comprising distinct domains which recognize different ligands, while for others a single domain is capable of binding different ligands by different mechanisms.

The Gram-positive bacterium Staphylococcus aureus is considered a commensal bacterium because roughly one-third of the human population is colonized by it without developing disease ( 1 ). Colonization occurs in the human nose, whereby the host nasal microbiota plays a major role in promoting or inhibiting S. aureus colonization ( 2 ). Despite the fact that S. aureus is considered a commensal, nasal carriage of S. aureus is linked to bacteremia ( 3 ). The bacterium may cause a range of infections, from cellulitis and superficial skin disease to abscesses, bacteremia, sepsis, endocarditis, and pneumonia ( 4 ). Moreover, S. aureus has been shown to adapt in its interaction with humans by increasing resistance against methicillin and is currently a leading cause of human bacterial disease worldwide. Methicillin-resistant S. aureus (MRSA) was identified in the 1960s as a nosocomial pathogen, when hospitalized patients showed distinct risk factors for acquisition ( 5 ). The prevalence of methicillin resistance among nosocomial S. aureus isolates increased from 2.1% in 1975 to 35% in 1991 ( 6 ). MRSA epidemiology changed in the 1990s when infections of healthy individuals outside hospitals were reported. These strains, with increased virulence, were the first reports of community-acquired MRSA ( 7 , 8 ). Now, community-acquired MRSA has been reported as the leading cause of bacterial infections in the bloodstream, skin, soft tissue, and lower respiratory tract in developed countries ( 9 ). As a consequence, research interest in the pathophysiology of S. aureus has increased.

Staphylococcus aureus is a highly successful pathogen that colonizes ∼30% of the population asymptomatically, but it is also capable of causing infections ranging from mild skin and soft tissue infections to invasive infections, such as sepsis and pneumonia ( 1 ). When S. aureus infects the host, it produces many virulence factors that promote the manipulation of the host’s immune responses while ensuring bacterial survival. These virulence factors include secreted toxins (exotoxins), which represent approximately 10% of the total secretome ( 2 ). While there are over 40 known exotoxins produced by these bacteria, many of them have similar functions and have high structural similarities. Closer examination of these seemingly redundant exotoxins revealed that each has unique properties. Exotoxins fall into three broad groups based on their known functions: cytotoxins, superantigens (SAgs), and cytotoxic enzymes ( Table 1 ). Cytotoxins act on the host cell membranes, resulting in lysis of target cells and inflammation. Superantigens mediate massive cytokine production and trigger T and B cell proliferation. Secreted cytotoxic enzymes damage mammalian cells. Collectively, these exotoxins modulate the host immune system and are critical for S. aureus infections.

Staphylococcus aureus is a Gram-positive commensal bacterium and opportunistic pathogen. The main sites of colonization are the skin and mucous membranes, and approximately 30% of the healthy adult population are colonized by S. aureus ( 1 ). Although S. aureus is primarily a commensal microbe, it has the potential to cause a wide range of diseases that can vary considerably in severity. The most common problems are skin infections, and some of the most severe are bloodstream infections, endocarditis, osteomyelitis, and necrotizing fasciitis ( 2 ). To survive and adapt to different environmental niches, S. aureus has evolved an intricate regulatory network to control virulence factor production in both a temporal and host location manner ( 3 ). The regulatory machinery and virulence factors are known as accessory genes, since they are not essential for normal growth. These accessory factors are used to establish dominance in the host and contribute to the pathogenicity of S. aureus, and they include cell surface components and proteins directly released into the extracellular environment. The functions of these molecules include adherence to host cells, evasion of host defenses, nutrient degradation, and acquisition. These accessory genetic elements are encoded directly on the chromosome and on mobile elements that include phages, plasmids, and pathogenicity islands.

Of the many species of staphylococci, Staphylococcus aureus has evolved to be far and away the most virulent. This stems largely from the horizontal acquisition of virulence determinants that are all coordinately regulated by several transcription factors covered elsewhere (e.g., AgrA, SaeR, Rot, and SarA) ( 117 ). In parallel, a separate set of transcription factors act in concert to control the expression of enzymes in various metabolic pathways (e.g., CcpA, CcpE, CodY, RpiRc, SrrA, and Rex). Of note, recent findings have demonstrated that many of these metabolic transcriptional regulators both directly and indirectly influence the functions of the virulence gene regulators. Moreover, many of the virulence gene regulators also affect the expression of key metabolic pathways. Thus, S. aureus has intertwined transcriptional control of virulence and metabolism. In addition, several groups have published findings that shed light on the direct contributions of S. aureus metabolism to disease outcomes. Different sets of metabolites are available at different infection/colonization sites, thereby necessitating unique metabolic pathways. For instance, the skin surface has abundant lactate, urea, and certain amino acids, but carbohydrates are relatively scarce. This is in contrast to deep tissue sites where S. aureus would have access to serum glucose. In addition to metabolite availability, other environmental variables differ among infection sites, such as pH, oxygen tension, and the presence of robust inflammation. For instance, the skin surface is oxygenated and acidic (pH ∼5.5) with minimal inflammation, whereas deep tissue infection sites become hypoxic, are relatively well buffered (pH ∼6.5), and are almost always accompanied by an immune cell infiltrate. These contrasting environmental factors constrain and alter the metabolic landscape in S. aureus, requiring significant adaptation to cause infection/colonization. This chapter highlights recent findings in this area and outlines current knowledge about the linking of virulence with metabolism in this important human pathogen.

Over the last decades, biofilms have gained more and more recognition as the common mode of growth that microorganisms adapt in nature ( 1 ). Furthermore, many types of human infection have been found to progress with the involvement of biofilms or originate from biofilm-associated primary infections ( 2 ). Second only to the Gram-negative Pseudomonas aeruginosa, staphylococci have been the focus of biofilm researchers. As common colonizers of the human skin, staphylococci are the most frequent sources of biofilm infections on surgically implanted indwelling medical devices. These include serious infections, including endocarditis and prosthetic joint infections (PJIs), and may lead to life-threatening conditions, such as sepsis ( 3 – 5 ).

Fulminant infections are not clearly defined in the field of staphylococcal infections. According to Wikipedia, “fulminant” is a descriptor, especially used in the field of medicine, for any event or process that occurs suddenly and escalates quickly and is intense and severe to the point of lethality, i.e., it has an explosive character. The word comes from Latin fulminare, to strike with lightning. For staphylococcal infections in which, despite the fact that Staphylococcus aureus is considered a commensal, progression may be devastating in many circumstances, it appears pertinent to add to this definition the notion of an unexpected event leading to a “thunderstorm in a quiet blue sky.” Hence, fulminant staphylococcal infection indicates an explosive, intense, and very severe infection occurring in a patient whose previous condition and antecedent would never have suggested any anticipation of life-threatening development. Considering this definition, fulminant is an adjective that could be associated with several staphylococcal infections, including necrotizing pneumonia, necrotizing fasciitis, and to some extent toxic shock syndrome and infective endocarditis (IE). In the three former diseases, toxin production plays a major role, whereas in the latter (fulminant presentation of IE), association with any particular toxinic profile has never been demonstrated.

The human body offers several distinct niches for specific microbiomes, variable consortia of bacterial communities. The skin, for instance, is primarily colonized by members of the genera Propionibacterium (now Cutibacterium), Corynebacterium, and Staphylococcus ( 1 ). A similar pattern of genera is found in the human nose, which is regarded as a transition zone from the dry skin to the moist, mucoid airways ( 2 ). This rather confined area, more specifically, the region from the anterior nasal vestibule to the posterior nasopharyngeal cavity, is the favored colonization site of Staphylococcus aureus. While S. aureus is a member of the normal nasal microbiome in ca. 30% of the human population, it can also become an aggressive, life-threatening pathogen ( 3 ). Nasal carriage is a major risk factor for S. aureus infections. Accordingly, S. aureus is eradicated from the nose in at-risk patients by treatment with the antibiotic mupirocin. Interestingly, a significant percentage of humans seem never to be colonized by S. aureus, for reasons that are currently unknown. Increasing evidence suggests that the composition of the nasal microbiome is an important factor for the exclusion of S. aureus from the nose ( 2 ).

The genus Staphylococcus currently comprises 81 species and subspecies (https://www.dsmz.de/bacterial-diversity/prokaryotic-nomenclature-up-to-date/prokaryotic-nomenclature-up-to-date.html), and most members of the genus are mammalian commensals or opportunistic pathogens that colonize niches such as skin, nares, and diverse mucosal membranes. Several species are of significant medical or veterinary importance. Staphylococcus pseudintermedius ( 1 ) is a leading cause of pyoderma in dogs and is considered to be a significant reservoir of antimicrobial resistance factors for the genus ( 2 , 3 ). S. pseudintermedius is very similar to Staphylococcus intermedius and can be distinguished from other coagulase-positive staphylococci by positive arginine dihydrolase and acid production from β-gentiobiose and d-mannitol ( 4 ) or by using a multiplex-PCR approach targeting the nuclease gene nuc ( 5 ). Staphylococcus saprophyticus is the second leading cause of uncomplicated urinary tract infections ( 6 ). While Staphylococcus epidermidis is a normal component of the epidermal microbiota, it is a leading cause of biofilm contamination of medical devices ( 7 ). The most promiscuous and most significant human pathogenic staphylococcal species is Staphylococcus aureus, which is the causal agent of a variety of disease symptoms that can range from cosmetic to lethal manifestations. S. aureus is distinguished from most members of the genus by its abundant production of secreted coagulase, an enzyme which converts serum fibrinogen to fibrin and promotes clotting. However, the S. intermedius group and some strains of Staphylococcus lugdunensis have coagulase activity ( 5 , 8 , 9 ).

Staphylococcus aureus gives rise to a variety of infections, and treatment of these relies on antibiotics. A number of antibiotics are used to treat staphylococcal infections ( Table 1 ), and they target major bacterial processes including cell wall synthesis, translation, transcription, and DNA synthesis. However, resistance to antibiotics is a growing problem, and treatment failures are associated with enormous human and medical costs. Antibiotic resistance arises by several different mechanisms, such as altered drug targets, enzymatic drug inactivation, increased efflux of antimicrobial compounds, and altered drug accessibility ( 1 ), and the spread of resistance is aided by a multitude of mobile genetic elements (reviewed in 2 – 4 ). Although resistance has been observed for essentially all compounds, individual strains resistant to all drugs have not appeared. Yet resistance still poses treatment challenges, as exemplified by vancomycin. Fully resistant strains of which are rare, but the more common intermediately resistant (VISA) strains are associated with more severe infections and longer duration of treatment compared to susceptible strains despite the fact that they only display a minor increase in the MIC of vancomycin. This highlights the problem that we only have limited knowledge of how resistance genes and mutations affect the overall biology of resistant strains and of the impact of resistance on pathogenesis. In this article, we focus on the biology of antibiotic resistance in S. aureus and on the behavior of resistant strains, and we conclude with a description of some of the new therapeutic approaches that in the future may become treatment options for infections with antibiotic-resistant staphylococci.

Most adult humans have high levels of circulating antibodies against many staphylococcal antigens, indicative of prior subclinical infections, but these antibodies are generally not protective, and clinically significant infection with S. aureus fails to provide protective immunity. Multiple vaccines have been developed for the prevention of S. aureus infections, but none were proven efficacious in the human trials reviewed in references 1 – 4 . All of the vaccine candidates functioned well in animal models, mostly murine models, but also in rabbits and primates. The reliance on murine models can be related to the extensive data available about murine immunity. Based on the large number of failures, a reasonable conclusion is that murine immunity and human preventive immunity against S. aureus are significantly different. The divergence of human and murine immunity has been detailed in the recent literature ( 1 – 6 ).

Staphylococcus aureus is one of the most important human pathogens, causing a variety of diseases, including skin and soft tissue infections, osteomyelitis, endocarditis, surgical site infections, pneumonia, and sepsis. In recent decades, the treatment of staphylococcal infections has become increasingly difficult as the prevalence of multidrug-resistant strains continues to rise. Penicillin-resistant S. aureus emerged in the 1940s, followed by the appearance of methicillin-resistant S. aureus (MRSA) in 1961 ( 1 , 2 ). Subsequent introduction of new antibiotics has been followed by reports of resistance ( 3 ). With increasing mortality rates and medical costs associated with MRSA and other drug-resistant strains, there is an urgent need for alternative therapeutic options ( 4 ). Therefore, considerable effort has been put forth to identify and develop novel S. aureus treatment strategies as alternatives to conventional antibiotics.

Listeria monocytogenes is a Gram-positive motile facultative anaerobe that inhabits a broad ecologic niche ( 1 – 3 ). With selective media it can be readily isolated from soil, water, and vegetation, including raw produce designated for human consumption without further processing ( 4 , 5 ). Newer chromogenic media may offer some advantages in the detection of contaminated foodstuffs ( 6 , 7 ). Surface contamination of meat and vegetables is relatively common, with up to 15% of these foods harboring the organism. In addition, the organism is a transient inhabitant of both animal and human gastrointestinal tracts ( 8 – 10 ), and intermittent carriage suggests frequent exposure. The gut is the source for the organism in invasive listeriosis when it occurs, and the virulence factor ActA may promote carriage ( 11 ). The organism is psychrophilic and enjoys a competitive advantage against other Gram-positive and Gram-negative microorganisms in cold environments, such as refrigerators. It may also be amplified in spoiled food products, particularly when spoilage leads to increased alkalinity. Feeding of spoiled silage with a high pH has resulted in epidemics of listeriosis in sheep and cattle ( 12 ).

Mammalian hosts have multiple, redundant ways to rapidly respond to the presence of infection. These innate immune mechanisms are triggered within minutes to hours after infection. Although they are not microbe specific, they do contribute to limiting exponential growth of L. monocytogenes and may even reduce bacterial dissemination to secondary infection sites. The role of these rapid immune responses can be clearly demonstrated by examining bacterial loads in various inbred strains of laboratory mice, with CFU burdens differing by 100- to 1,000-fold within the first few days of infection ( 1 , 2 ). This difference has been attributed largely to multiple innate immune mechanisms, and both the prototypic susceptible strain (BALB/c) and highly resistant C57BL/6 mice can induce robust CD8+ T cell responses and eventually clear sublethal doses of L. monocytogenes. Weak or delayed innate immunity allows L. monocytogenes to replicate exponentially for several days and thus is a major determinant of disease severity. The innate immune responses generated during the first few days of infection are also critical for proper induction of the adaptive immune mechanisms that lead to immunological memory ( 3 – 5 ).

The move from soil to cytosol requires the interplay of L. monocytogenes factors that promote survival in the gut, bacterial invasion, phagosomal escape, replication and movement within the cytosol, and spread to adjacent cells ( Fig. 1 ). Bacterial gene products contributing to many key aspects of host infection continue to be identified, and new factors and novel functions often emerge ( 1 , 2 ). Among the cast of well-known players are surface proteins that promote bacterial attachment to and invasion of nonprofessional phagocytic cells, such as the internalins InlA and InlB as well as Lap and InlP (which appears to be specific for placental invasion) ( 3 – 5 ). Following cell entry, L. monocytogenes escapes from host cell vacuoles via the secretion of the pore-forming cytolysin listeriolysin O (LLO) and two phospholipases, a phosphatidylinositol-specific phospholipase C (PI-PLC) encoded by plcA and a broad-range phospholipase C (PC-PLC) encoded by plcB ( 6 ). Entry into the cytosol requires metabolic adaptation as L. monocytogenes shifts from glycolysis to the oxidative pentose phosphate pathway and replicates by scavenging phosphorylated sugars, glycerol, lipoic acid, branched-chain amino acids, and peptides from conquered host cells ( 7 ). Bacteria spread to neighboring cells by usurping actin polymerization as a motile force, a process dependent upon expression of the bacterial surface protein ActA ( 8 ). The breaking and entering of L. monocytogenes into adjacent cells is further facilitated by InlC, which relieves cortical tension to allow the extension of membrane protrusions ( 9 ). Escape from the double membrane vacuoles formed as a result of L. monocytogenes cell-to-cell spread is once again dependent upon the activities of LLO, PC-PLC, and PI-PLC. Protein secretion of a variety of factors thus promotes bacterial survival and life within host cells, and central to the secretion of active protein products is the presence of the secretion chaperone PrsA2, located at the bacterial membrane-cell wall interface, where it contributes to the folding and activity of translocated polypeptide chains ( 10 ). L. monocytogenes is thereby able to maintain a complex and multifunctional protein arsenal to increase bacterial survival and replication within mammalian host cells.

Listeria monocytogenes is a facultative intracellular pathogen that has the capacity to actively invade and multiply within mammalian cells. Intracellular replication of L. monocytogenes within mononuclear cells was noted in the 1926 publication by Murray and colleagues reporting on this bacterial pathogen for the first time ( 1 ). In the 1960s, the seminal work of Mackaness that identified the main actors of cellular immunity against bacterial intracellular pathogens took advantage of the L. monocytogenes intracellular lifestyle as a model ( 2 ). In the late 1980s and early 1990s, major L. monocytogenes virulence factors involved in bacterial adaptation to intracellular life were molecularly characterized ( 3 – 7 ) and the precise stages of the L. monocytogenes intracellular life-cycle were morphologically identified ( 8 , 9 ). Since then, cellular effectors involved in the infection process have been also identified and characterized ( 10 – 12 ). In this article, we review the molecular mechanisms driving L. monocytogenes adaptation to the mammalian host cell intracellular environment.

The bacterial pathogen Listeria monocytogenes is well adapted to life in the soil and on vegetation as a saprophyte (i.e., extracellular environments) and to life in the cytosol of mammalian cells as a pathogen (i.e., intracellular environment). These environments differ greatly in their metabolite repertoires (e.g., carbon, sulfur, and nitrogen sources) and abundance (e.g., amino acid availability). Therefore, L. monocytogenes requires niche-specific adaptations to support growth and uses metabolic cues to trigger virulence mechanisms, such as the master virulence regulator, PrfA ( 1 ). The complete metabolic potential of L. monocytogenes has been inferred bioinformatically from genomic ( 2 – 5 ) and transcriptomic studies ( 1 , 6 – 8 ), as well as experimentally using both defined media as a test for auxotrophy ( 9 – 12 ) and more recently by isotopologue metabolomics ( 13 , 14 ). While these studies have certainly taught us much about L. monocytogenes’ metabolic potential, it is important to note that, given the wide array of environments L. monocytogenes is capable of inhabiting, our understanding of its metabolic potential and how strain to strain variation might affect this potential, is incomplete.

The microorganisms constituting the Bacillus cereus group are Gram-positive low-GC-content bacteria belonging to the phylum Firmicutes. The group of spore-forming, aerobic, facultative anaerobic, rod-shaped bacteria comprises at least eight closely related species: B. anthracis, B. cereus, B. thuringiensis, B. mycoides, B. pseudomycoides, B. weihenstephanensis, B. cytotoxicus, and B. toyonensis ( 1 ). With the exception of B. cytotoxicus, which is the most divergent of the group, with a chromosome of 4.085 Mb ( 2 ), the genomes of the B. cereus group species are highly conserved, with sizes of 5.2- to 5.9-Mb and very similar 16S rRNA gene sequences.

Notably, different mechanisms exist between these organisms for forming and germinating spores. Some of these differences reflect phylogenetic differences between the Clostridiaceae (represented by C. perfringens and C. botulinum) and the Peptostreptococcaceae (represented by C. difficile) ( 1 ). Other differences reflect genetic diversity within each species ( 2 – 4 ). C. botulinum is the most divergent, being divided into four metabolic groups (groups 1 to IV) that effectively represent different species despite their shared production of botulinum toxin ( 5 ).

The Clostridiaceae family (and within it the Clostridium genus) consists of an extremely diverse group of primarily Gram-positive bacteria that have traditionally been grouped together based on their anaerobic growth requirements and their ability to produce heat-resistant endospores. Historically, the members of the genus were very dissimilar, such that the genus lacked phylogenetic coherence, with over 200 species, at least 35 of which cause disease in humans and animals ( 1 ). Two recent studies have proposed a phylogenetic reorganization of the clostridia and subsequently changed the name of Clostridium difficile to Peptoclostridium difficile ( 2 ) and Clostridioides difficile ( 3 , 4 ). However, the earlier name (P. difficile) did not comply with the Internal Journal of Systematic and Evolutionary Microbiology Bacterial Code and was rejected ( 3 ). Also recently, Clostridium sordellii has been reclassified as Paeniclostridium sordellii ( 5 ). For simplicity, in this article pathogenic members of the genera Clostridium, Clostridioides, and Paeniclostridium will all be referred to as pathogenic clostridia.

The earliest DNA-based studies of the clostridia investigated the guanine + cytosine (G+C) content of DNA and measured hybridization as an indication of the relatedness of strains ( 1 – 3 ). Since those early “genome” studies, a range of other DNA-based typing methods, such as pulsed field gel electrophoresis, amplified fragment-length polymorphism analysis, 16S rRNA gene sequence comparisons, multilocus variable-number tandem-repeat analysis, and multilocus sequence typing (MLST), have been used to classify, group, and differentiate clostridial isolates ( 4 ). However, these methods have now been largely superseded. With the rapid advances in DNA sequencing technologies and the subsequent drop in the price and effort required to acquire whole-genome sequence (WGS) data, the comparative analysis of strains is now commonly based on core genome single-nucleotide polymorphisms (SNPs), gene content analysis, MLST on a large group of genes, or average nucleotide identity (ANI) across whole genomes. Whole-genome analysis is the tool of choice for comparing isolates and informs our understanding of the pathogenic mechanisms that are deployed by the clostridia, the epidemiology of disease outbreaks, and the evolutionary histories of the organisms. Analysis of WGS allows the identification of metabolic pathways or the lack thereof (e.g., amino acid metabolism and substrate degradation pathways) and thus indicates how clostridia are adapted to their lifestyles within the ecological niches that they occupy. The quality of WGS information available in sequence databases is variable, so some caution is needed in how it is interpreted when investigating genome structure and absolute gene content. Complete closed genomes are particularly valuable and can be used as scaffolds around which the more usual draft assemblies can be made. Like WGS data from all species, the clostridial WGS annotations need to undergo continual refinement as gene functions are experimentally confirmed and extended. Commonly, a quarter to a third of the open reading frames identified in genomes encode putative proteins of unknown function. Whole-genome analysis provides a much better tool for taxonomic classification than the traditional biochemical, phenotypic, and limited molecular characterization methods (e.g., 16S rRNA gene sequencing) that were previously applied.

Many clostridial species are ubiquitous in the environment and in the intestinal tracts of birds, fish, and mammals. Commensal species are often carried asymptomatically within a host. However, if the immune status of the host is compromised, due to either age, illness or a change in diet, disease can result from toxigenic strains. Alternatively, some clostridial species or strains don’t require predisposing factors. They can cause disease simply if they gain entry into the host either through damage to the skin or through the gastrointestinal tract, often via poorly prepared or incorrectly stored food. These clostridia then overgrow and cause cell and tissue damage. Diseases mediated by the clostridial species discussed in this review are predominantly mediated by potent protein toxins, many of which are located extrachromosomally. These toxins have diverse mechanisms of action and include pore-forming cytotoxins, phospholipases, metalloproteases, ADP-ribosyltransferases and large glycosyltransferases. This review focuses on these toxins and the elements that carry the toxin structural genes. For ease of discussion it has been structured on a bacterial species-specific basis.

Clostridium perfringens is a Gram-positive, spore-forming rod with ubiquitous environmental distribution, including a presence in soil and sewage ( 1 – 3 ). Given its anaerobic nature, it is unsurprising that C. perfringens is also a component of the normal gastrointestinal (GI) tract microbiota of humans and other animals ( 1 , 2 , 4 ). Additionally, this bacterium is an important cause of intestinal and histotoxic infections in humans and other animals ( 2 , 5 , 6 ).

Clostridioides difficile is a Gram-positive anaerobic, spore-forming bacterium and the most common identifiable infectious agent of nosocomial antibiotic-associated diarrhea (AAD) ( 1 , 2 ). This bacterium is also linked to several life-threatening syndromes in humans, including pseudomembranous colitis and toxic megacolon ( 1 , 2 ). Disease symptoms associated with C. difficile infection (CDI), including diarrhea, fluid loss, and inflammation, result from the production and activity of two exotoxins, toxin A (TcdA) and toxin B (TcdB) ( 2 ). These toxins disrupt the Rho family of GTPases within the host cell, which eventually results in cell rounding and death ( 3 , 4 ). C. difficile was originally discovered by Hall and O’Toole as Bacillus difficilis during their 1935 study of neonatal fecal microbiota and was named “difficilis” due to the difficulty of its cultivation and isolation ( 5 ). At the time of its initial isolation, C. difficile was not considered pathogenic, with recognition of C. difficile as a human pathogen occurring roughly 40 years after its initial discovery ( 5 ). With time, identification of the obligately anaerobic nature of this organism ultimately resulted in reclassification from B. difficilis to Clostridium difficile. More recently, with an update to the classification of the clostridia, it was first suggested that C. difficile be reclassified as Peptoclostridium difficile ( 6 ) and, more recently, to Clostridioides difficile ( 7 ). However, as recently highlighted in Smits et al. ( 8 ), there is reluctance for reclassification, because there currently exists a large knowledge base surrounding Clostridium difficile, in addition to a strong public awareness which could be lost upon reclassification. Despite these reservations, Clostridioides difficile has now been officially approved by the International Journal of Systematic and Evolutionary Microbiology and International Committee on Systematics of Prokaryotes and has begun to be used more commonly ( 9 – 11 ).

Many pathogenic clostridial species cause potentially fatal soft tissue infections in humans and animals because of their ability to produce extracellular protein toxins ( 1 , 2 ). The ability of these pathogens to produce spores that are resistant to environmental stress is an important factor in the epidemiology of these diseases. Disease pathogenesis involves the growth of the clostridial pathogen in the tissues and extensive tissue destruction, which is the result of the action of extracellular toxins. Typical histotoxic clostridial diseases include human gas gangrene or myonecrosis and blackleg in cattle. Although there are several clostridial species that are responsible for these syndromes ( Table 1 ), this review will focus on histotoxic infections caused by Clostridium perfringens and Clostridium septicum, primarily because these are the species that have been the subject of the most extensive molecular and functional studies.

Because of the importance of mycobacterial diseases such as tuberculosis and leprosy, mycobacteriophages have long been studied, with the first ones being isolated in the 1940s ( 1 – 3 ). At that time, it was recognized that bacteriophages display particular profiles of specificity for their bacterial hosts, nearly always distinguishing between bacteria in different genera and sometimes distinguishing between strains of the same bacterial species ( 4 – 7 ). Mycobacteriophages thus presented a plausible means of identifying (i.e., typing) clinical isolates by scoring for phage sensitivity profiles, with the prospects of obtaining data considerably faster than standard protocols that required extensive growth of the host. Mycobacterium tuberculosis—the causative agent of human tuberculosis—has a remarkably slow growth rate (24-hour doubling time), so phage typing can speed up diagnosis by several weeks. This general concept of exploiting the relatively rapid propagation of mycobacteriophages has been a common theme in their subsequent development ( 8 – 10 ).

Mycobacterium tuberculosis, the etiologic agent of tuberculosis (TB), remains a significant global public health burden ( 1 ). In 2016, there were 10.4 million new TB cases reported globally and nearly 1.7 million TB-related deaths ( 1 ). Understanding the host response to M. tuberculosis infection is a key aspect of efforts to eradicate TB through the development of effective vaccines and immune therapeutics. M. tuberculosis is an intracellular pathogen transmitted via inhalation of aerosolized, bacteria-containing droplets. Innate immune cells in the lungs, primarily macrophages, dendritic cells, monocytes, and neutrophils, readily phagocytose M. tuberculosis and are the earliest defenders against the pathogen. The transformation of bacteria-containing phagosomes into acidified, antimicrobial compartments is a central tenet of defense against M. tuberculosis. In this regard, the production of interferon-γ (IFN-γ), which can activate infected myeloid cells and inhibit bacterial replication, is a well-known antimycobacterial contribution by adaptive immune cells such as CD4 and CD8 T cells. Despite pressures from host immunity, M. tuberculosis is able to persist in the host. M. tuberculosis infection results in hallmark lesions called granulomas, which are initially aggregates of infected and uninfected myeloid cells circumscribed by a lymphocytic cuff. The granuloma is thought to prevent bacterial dissemination to extrapulmonary sites but can also become a niche for long-term bacterial persistence. M. tuberculosis has evolved myriad strategies to evade and subvert immune responses to persist within a host, and it is becoming increasingly clear that the immune response to M. tuberculosis infection involves contributions from a wide variety of innate and adaptive immune cells. A clearer understanding of the complex cross talk between M. tuberculosis and host immunity is essential for the development of efficacious TB vaccines. Despite being developed nearly a century ago, Mycobacterium bovis bacillus Calmette-Guérin (BCG), an attenuated strain of M. bovis, remains the only licensed vaccine against TB. Vaccination with BCG provides protection against severe forms of disseminated TB in children but has variable efficacy in preventing pulmonary disease in children and adults ( 2 – 4 ). However, the immunological basis for the poor efficacy of BCG remains unclear. Moreover, long-held concepts regarding the nature of desired immune responses in an ideal TB vaccine, namely, the induction of antigen-specific CD4 T cells producing IFN-γ, are being updated to reflect the expanding knowledge of host immunity to M. tuberculosis infection gathered from animal models and human cohort studies. Advances in imaging and single-cell technologies combined with high-throughput approaches and systems-based analyses are providing more information on the immune response to M. tuberculosis infection at increasingly higher resolutions. As our understanding of the host response to M. tuberculosis infection grows, opportunities to leverage knowledge of the immunology of M. tuberculosis infection toward improving therapeutics and vaccines for TB are increasing.

The bacterial cell envelope, defined as the structure that surrounds the cytosol, is critical for bacterial physiology, because many crucial processes take place in this compartment. The cell wall contributes to the bacterial shape, mechanical resistance of the cells, and their protection against hostile environments. In addition, the envelope constituents are involved in cell division, transport of molecules (solutes and ions) and macromolecules (proteins), cell motility, adhesion, and protection, being in direct contact with the environment etc. The genus Mycobacterium belongs to the Corynebacteriales order, which also includes corynebacteria, nocardia, rhodococci, and other related microorganisms. Mycobacteria are probably the most successful microorganisms to parasitize animals and humans. Among the more than 200 valid species described to date in the genus Mycobacterium, only three are strict pathogens for humans: Mycobacterium tuberculosis (the Koch’s bacillus), M. leprae, and M. lepromatosis. Tuberculosis still represents a major public health problem worldwide, remaining one of the leading causes of death from an infectious agent; about one-quarter of the world’s population is infected by the Koch’s bacillus and thus susceptible to develop the disease. In addition, two-thirds of mycobacteria species are opportunistic pathogens for humans, and all mycobacteria produce granulomatous lesions in experimental animals with large enough inoculum ( 1 ). Most mycobacterial pathogenic species grow very slowly (e.g., generation times of 24 hours and 13 days for the tubercle and leprosy bacilli, respectively), whereas saprophytes are relatively rapid growers (e.g., doubling time of 6 hours for the widely used genetically tractable M. smegmatis).

Bacterial psychology is a challenging field. Like many psychologists, Jacob impressed the bacterium with our own value system. And part of that is defining what it is to be alive. As microbiologists, we generally define bacterial life as the ability to form progeny, to become two bacteria through cell division. Like all reproductive processes, bacterial division uses a combination of mechanics and mystery. Here, we will focus on the mechanics (and mystery) of cell division in acid-fast mycobacteria, a genus that includes the major human pathogens Mycobacterium tuberculosis and Mycobacterium leprae.

The notion that metabolic capacity might correlate with lifestyle has largely been confirmed by comparative genomics analyses of different bacterial species: free-living bacteria tend to have larger, more complex genomes than pathogens and symbionts ( 1 ), with obligate intracellular organisms often representing the extreme limit of small genome size ( 2 ). This correlation exists also in Mycobacterium, a genus which includes environmental organisms as well as a select group of animal and human pathogens—primarily of the Mycobacterium tuberculosis complex (MTBC) ( 3 )—whose genome sizes and compositions relate broadly to habitat and host range ( 4 , 5 ). Most notable among these from a public health perspective is M. tuberculosis, the causative agent of tuberculosis (TB), an airborne infectious disease which continues to result in over 10 million new cases and more than 1.5 million deaths globally each year ( 6 ). This is despite the widespread use of a neonatal anti-TB vaccine ( 7 ) and the existence of an effective combination chemotherapy ( 8 ), the insufficiency of which points to systemic failures in TB diagnosis and treatment ( 9 , 10 ), as well as the complexities encountered in eliminating an organism that has coevolved with its human host ( 11 ) and, throughout modern history, has proven adept at exploiting urbanization ( 12 ), economic disparity ( 13 , 14 ), societal upheaval ( 15 ), and incarceration ( 16 – 18 ) in maintaining a global prevalence characterized by high-burden endemic regions ( 6 ).

Mycobacteria are Actinobacteria, which is a phylum of high-GC Gram-positive bacteria. Among the wide range of Mycobacterium spp. are several important pathogens. Most notable of these is Mycobacterium tuberculosis, the causative agent of tuberculosis (TB). Although the number of TB cases and deaths show a declining trend in the past decade, the World Health Organization recently warned that “current actions and investments in research are falling far short” (http://www.who.int/mediacentre/news/releases/2016/tuberculosis-investments-short/en/). Over 1.3 million deaths and up to 10.0 million new infections were attributed to M. tuberculosis in 2017, which makes M. tuberculosis the most deadly infectious agent in the world, surpassing HIV and the malaria parasite ( 1 ). One issue facing efforts to control TB is that the live attenuated bacillus Calmette-Guérin (BCG) vaccine strain is not able to provide lifelong protection against M. tuberculosis ( 2 ). Another problem is the increased incidence of infections caused by multidrug-resistant M. tuberculosis strains. Consequently, major research efforts focus on understanding M. tuberculosis pathogenesis and physiology to advance the development of new anti-TB vaccines and antibiotics.

Over the past 4 decades, perhaps no other infectious disease has been as successfully studied as diphtheria ( 1 , 2 ). Indeed, the study of diphtheria toxin established the structure-function paradigm for the study of other toxins in the bacterial protein toxin field. Moreover, when coupled with the molecular genetic study of the iron-activated regulatory element, DtxR, that controls the expression of diphtheria toxin, we now have a detailed understanding of the entire tox genetic system, from the regulation of expression to the molecular mechanism of diphtheria toxin action. In this article, we review the development of our current understanding of diphtheria toxin, from its structure-function relationships to its mechanism of entry into the eukaryotic cell cytosol, the molecular mechanism of transition metal ion activation of DtxR and its regulation of tox expression, and finally, the protein engineering of diphtheria toxin for the development of highly potent and selective cell-surface receptor-targeted fusion protein toxins for the treatment of human diseases.