Mechanisms of bacterial host-to-host transmission

SUMMARY

Bacterial pathogens must navigate complex host environments to thrive, replicate, and ultimately transmit to new hosts. Effective transmission is critical for pathogen propagation and often requires overcoming host defenses and exploiting environmental conditions. The mechanisms used by bacterial pathogens to cause disease have been studied for decades, and numerous virulence factors have been identified and characterized through the use of genetic tools and animal models. While insightful, these discoveries have only scratched the surface of our understanding of disease mechanisms. Even less well understood is how pathogens move from an infected host to colonize and establish infection in a new host. Pathogens can move between hosts via direct and indirect modes, relying on numerous routes, such as respiratory, fecal-oral, direct contact, vector-borne, and vertical transmission. Recent advances in animal models for the study of bacterial transmission have enabled a more accurate recapitulation of transmission between humans. This review summarizes the current knowledge of bacterial transmission factors and animal models of transmission, and how these tools are advancing our understanding of the transmission mechanisms used by bacterial pathogens.

INTRODUCTION

The driving force behind any bacterial pathogen is the need to replicate and thrive in its niche environment. When the environment is a eukaryotic host, pathogens face challenges such as competition for nutrients and host responses that provide selective pressures to spread from an infected host to a naïve host. The dynamic interactions between hosts and bacterial pathogens depend on the ability of the pathogen to acquire a niche in the host, the rate at which the pathogen induces mortality, the degree of host immunity, and the necessity to transmit to a new host. The transmission of bacterial pathogens from one host to another is a complex process and remains one of the least understood facets in the lifecycle of a successful pathogen.

Bacterial pathogens can be transmitted through various routes, broadly categorized as direct, indirect, and vector-borne. Direct transmission, as defined by the Centers for Disease Control (CDC), involves close proximity between an infected individual and a susceptible individual, often through direct contact with the infectious source or reservoir (1) (Fig. 1A). This type of transmission typically occurs quickly and includes skin-to-skin contact, direct droplet transmission via inhalation, and vertical transmission from a mother to child during pregnancy, childbirth, or breastfeeding. By contrast, indirect transmission does not require close proximity to the infectious source (reservoir), and it does not necessarily occur immediately (Fig. 1B). This category includes airborne transmission via aerosols, and vehicle-borne transmission via exposure to contaminated food, water, or soil, as well as contact with fomites, such as contaminated devices or surfaces. Vector-borne transmission (Fig. 1A), which occurs when arthropods such as ticks and fleas transfer the infectious agent from one host to another, can be considered a form of direct or indirect transmission (2). It is important to note that the definitions used to categorize the various modes of transmission are more complex than described here, as few, if any, bacterial pathogens are transmitted through a single mode. In this review, we describe bacterial transmission mechanisms and recent advances in animal models for the study of direct, indirect, and vector-borne transmission, with an emphasis on pathogens that transmit via respiratory and fecal-oral routes. In addition, we delve into the barriers that pathogens must overcome to colonize naïve hosts, common themes of host defense mechanisms, and vaccination as a strategy to mitigate pathogen spread.

Fig 1.

Direct and indirect modes of bacterial transmission. (A) Direct modes of transmission consist of a direct connection between an infected individual and the new host. These include physical contact, droplet spread, vertical transmission, and vector-borne transmission. Direct contact comprises an infected individual and the new host physically touching and often exchanging bodily fluids. Droplet spread refers to large droplets (>5 µm) that travel short distances (3–6 feet) before settling. Vertical transmission occurs when a mother transmits bacteria to the offspring in utero (through the placenta), during birth, or postnatally through breastfeeding. Vector-borne transmission, here categorized as direct, can be considered either direct or indirect. Vector-borne transmission is under the direct mode, but separated as it can also be considered indirect. This form of transmission often utilizes blood-feeding arthropods, such as mosquitoes, ticks, and fleas, which transport pathogens from one host to another. (B) Indirect modes of transmission involve the spread of bacteria without direct human-to-human contact. They can be divided into airborne and vehicle-borne transmission. Airborne transmission of bacteria results from aerosolized bacteria (small droplets < 5 µm) that remain in the air for extended periods of time and travel long distances. Vehicle-borne transmission occurs via the spread of a vehicle or an inanimate object that can carry the bacteria from its reservoir to a susceptible new host. These can be further divided into food-borne and water-borne, spread by contaminated food, water, or soil, or fomites (contaminated surfaces and objects) which include contaminated clothing, medical devices, doorknobs, and various household items.

MODES OF TRANSMISSION

Airborne transmission

Pathogens that cause respiratory tract infections are generally transmitted via airborne particles that can be produced by an infected individual when breathing, talking, sneezing, and coughing (Fig. 1B). These droplets generally consist of saliva, mucus, immune cells, and potential infectious pathogens. Particles vary in size, with the World Health Organization (WHO) defining particle size greater than 5 µm to be considered a droplet, and those less than 5 µm as an aerosol (3). For simplicity, both droplets and aerosols were recently defined as infectious respiratory particles (IRP) (4). However, for consistency with prior transmission studies, we will maintain the traditional terminology where appropriate. Environmental factors such as temperature, humidity, and air flow all contribute to the motion and persistence of IRPs. An infected individual’s immune status can also dictate the number, size, and composition of the particles generated. Higher intensity of coughing and sneezing is likely to increase the volume and infectivity of the expelled droplets (5). Due to gravity, larger droplets fall to the ground within a few feet (1 meter) of the infected individual who expelled them, while smaller droplets last longer in the air. By contrast, the smaller droplets last longer in the air and can even undergo evaporation, becoming even smaller and therefore remaining in the air for extended periods of time. This evaporation can also place considerable osmotic stress on the bacteria, potentially affecting their viability and, in turn, impacting transmission kinetics (6). Studies have suggested that coughing and sneezing can expel a mixture of droplets and aerosols at high velocity, with the aerosols remaining infective for several hours (5, 7). Given this element of time, aerosol/airborne transmission of smaller droplets is considered an indirect mode of transmission, whereas larger droplets, which are rapidly acquired by a susceptible host, are considered a direct mode of transmission.

Skin-to-skin transmission

Many bacterial infections require direct physical contact for transmission between infected and susceptible individuals, with skin-to-skin contact being one of the most common routes. This route of transmission frequently occurs among family members who reside in the same household and includes sexual contact, direct contact with infectious lesions, and transmission between pets and owners. Direct transmission also includes vertical transmission, where a fetus is infected in utero via the placenta, during birth, or shortly after birth (Box 1), and oral or skin lesion contact with brackish water or soil that contain bacterial pathogens among their complex ecosystems.

Box 1. Vertical Transmission .

Vertical transmission occurs between mother and offspring, and can lead to serious neonatal infections. Many bacteria, viruses, and protozoan pathogens are capable of vertical transmission. These include members of the TORCH pathogens group: Toxoplasma gondii, Other (Listeria monocytogenes, HIV etc.), Rubella, Cytomegalovirus and Herpes simplex virus (8). One key example of vertical bacterial transmission is the transmission of group B streptococcus (GBS) from an infected mother to a newborn during vaginal delivery. GBS readily colonizes the mucosal surfaces of the genitourinary and/or GI tract of adult humans. Vertical transmission of GBS from colonized mothers is a common cause of infant infections that can manifest as pneumonia, or more invasive infections such as septicemia and meningitis (9). The current standard of prevention in many countries is intrapartum antibiotic prophylaxis (IAP), which has led to fewer vertical transmission events and thus, reduced development of disease states in infants. Although this antibiotic treatment strategy has significantly lowered infection rates, it has not completely abolished transmission. GBS early-onset disease remains at ~0.25 cases per 1,000 live births annually in the United States, but IAP has no effect on later-onset disease because maternal colonization is reduced but not eliminated by IAP (10, 11). More recently, a murine model of GBS infection and vertical transmission was developed, where IAP treatment reduced disease development in infant mice, thus recapitulating human colonization and transmission dynamics (12). This murine model will be a critical resource for better understanding GBS pathogenesis and in developing additional treatment strategies against GBS infections.

Vehicle-borne transmission

The fecal-oral route is an indirect mode of transmission common among enteric bacterial pathogens, spreading through contaminated food or water and contact with fomites (Fig. 1B). Food and water can become contaminated from a number of sources, and one key source is agricultural runoff from farms (13, 14). Fomites are inanimate objects such as countertops, doorknobs, toys, and medical equipment that become contaminated when infectious material lands on these surfaces through respiratory particles or touch and can serve as transmission conduits to a new host. In general, microbial cells deposited on fomites often enter a metabolically inactive but viable state, allowing them to persist for extended periods (15). The transfer efficiency of bacteria from a fomite surface to a host depends on various factors, including the surface material, hydrophobicity, and moisture content, and the capacity of the pathogen to withstand these conditions.

Vector-borne transmission

Insects and arthropods can act as pathogen reservoirs and function as intermediaries for transmission to human hosts in what is defined as vector-borne transmission (Fig. 1A). Most vector-borne bacterial infections are transmitted by ticks, but some are transmitted by fleas, lice, mites, and chiggers. The WHO lists 27 different diseases caused by vector-borne pathogens, but only seven of these are bacterial and include diseases such as plague, Lyme disease, typhus, spotted fevers, relapsing fevers, and tularemia (16). The CDC lists two additional tick-borne Rickettsial diseases (anaplasmosis, ehrlichiosis) (17), and one that is known to be transmitted by chiggers (scrub typhus) (18). These organisms largely remain treatable with antibiotics and are generally not subsequently transmitted person-to-person but through complex life cycles involving multiple hosts.

Asymptomatic transmission

An important and often underestimated aspect of bacterial transmission is that it can occur from individuals who are not currently experiencing any disease symptoms, adding further complexity to the process. Individuals who harbor a pathogen but do not display outward signs of disease are referred to as carriers. These asymptomatic carriers are often either in the incubation period, which is defined as the time between transmission and the onset of the infectious state, in a latent stage, or in recovery from an infection and still contagious (convalescence). Active carriers are those who are either in the incubation or convalescence periods and either will or have experienced disease symptoms. Conversely, passive carriers are individuals who become inoculated by a sick individual and can then transmit to a naive host individual without ever developing disease symptoms themselves. A classic example of an asymptomatic passive carrier is Mary Mallon, known as “Typhoid Mary,” who carried Salmonella typhi in her gastrointestinal tract and was responsible for several Typhoid fever outbreaks in New York state in the early 20th century (19).

BACTERIAL FACTORS THAT PROMOTE HOST-TO-HOST TRANSMISSION

To facilitate dissemination from one host to another, a critical step likely includes the pathogen successfully navigating and subverting the host response. The specific niche a pathogen occupies within the host dictates the molecular mechanism(s) employed to promote transmission. For example, a pathogen that occupies the gastrointestinal tract is regularly expelled out of the host through the peristalsis process that occurs whether the host is healthy (normal defecation) or not (diarrhea or vomiting), whereas respiratory tract pathogens likely employ inherently different strategies to promote their transmission and elicit expulsion events that do not always occur in healthy hosts (coughing, sneezing). Pathogens also encode specific bacterial factors (discussed below) that contribute to host-to-host transmission.

Investigating host-to-host transmission in a laboratory setting requires animal model systems. Several small animal models have been developed that offer a cost-effective and genetically tractable platform for investigating direct, indirect, and vector-borne modes of transmission (Table 1). Mice in particular have unique characteristics that make them well-suited for transmission studies, such as serving as a natural reservoir for multiple vector-borne pathogens, their co-housing behavior (personal and mutual grooming, huddling), and coprophagic nature. The act of coprophagia itself can be akin to the consumption of contaminated food. The effectiveness of an animal model is contingent upon its ability to recapitulate human disease faithfully, and some models do this better than others. An example would be the infant murine model to study upper respiratory tract colonization, shedding, and transmission of Streptococcus pneumoniae, which successfully replicates human community dynamics for colonization and transmission, such as the events primarily occurring in the young, strain-to-strain variability in S. pneumoniae shedding, and transmission requiring close contact (20). However, even though mice can have a cough and sneeze response to tussive stimuli (21–24), the S. pneumoniae transmission to naïve mice occurred through exposure to contaminated surfaces (25), thus limiting insight into airborne transmission. In the sections below, we highlight these animal models and the transmission factors that have been identified through the application of the models.

TABLE 1.

Animal models for the study of bacterial transmission

Pathogen	Model organism	Primary infection site	Inoculation route	Disease state	Transmission model	Caveats/limitations	Reference(s)
V. cholerae	Infant mice and rabbits	GI tract	Oral gavage	Colonization and diarrhea	None	Only recapitulates the hyperinfective state; no true transmission model to date	(26–29)
	Zebrafish	GI tract	Oral feeding	Colonization	Co-housing naïve and inoculated	Genetic diversity requires larger sample size; differences in gut microbial composition	(30)
C. difficile	Antibiotic-treated mice (C57BL/6)	GI tract	Oral gavage	Colonization	Co-housing naïve and inoculated	May not fully recapitulate human infection	(31, 32)
S. Typhimurium	Mice (129 × 1/svj)	GI tract	Oral gavage	Persistence	Co-housing naïve and inoculated	Defect in inflammatory cell recruitment	(33)
	Zebrafish	GI tract	Oral feeding	Colonization	Co-housing naïve and inoculated	Genetic diversity requires larger sample size; differences in gut microbial composition	(34)
P. aeruginosa	Mice (BALB/c)	GI tract	Tail vein	Bacteremia	Co-housing naïve and inoculated	Transmission only from ingestion of intact fecal pellets from infected mice	(35)
	Mice (C3H/hen)	GI tract	Oral feeding	GI colonization	Co-housing naïve and inoculated	Naïve mice were treated with antibiotics	(36)
Shigella spp.	Zebrafish	GI tract	Oral feeding	Colonization	Co-housing naïve and inoculated	Genetic diversity requires larger sample size; differences in gut microbial composition	(37)
K. pneumoniae	Mice (C57BL/6)	GI tract	Oral feeding	Colonization	Co-housing naïve and inoculated	Technically challenging; mouse to mouse variability in gut colonization	(38)
B. pertussis	Baboons	Lungs		Pneumonia	Co-housing and separate housing	Costly, not genetically tractable, ethical concerns	(39)
B. bronchiseptica	Piglets	Upper & lower respiratory tract	Intranasal	Colonization and pneumonia	Co-housing naïve and inoculated	Costly, not genetically tractable, different from B. pertussis	(40, 41)
	Mice (C3H/hen)	Upper respiratory tract	Intranasal	Colonization	Co-housing naïve and inoculated	More susceptible to infection by gram-negative pathogens	(42)
S. pneumoniae	Infant mice	Upper respiratory tract, nasal passages	Intranasal	Asymptomatic carriage	Co-housing via dam; with and without IAV co-inoculation	May only represent indirect/fomite transmission	(20, 25)
	Ferrets	Nasal passages, lungs	Intranasal	Asymptomatic carriage and pneumonia	Open cage and co-housing; with and without IAV co-inoculation	Cost; ferrets not genetically tractable	(43)
Group B streptocci	Pregnant mice (BALB/c)	Vaginal tract	Intravaginal	Symptomatic offspring	Vertical transmission to offspring	Only hypervirulent strain tested	(12)
Y. pestis	Mice	Lymph node	Flea bite	Bubonic plague	Infected flea feeding on naïve mice	Technical challenges of inoculation	(44–46)
	Flea		Y. pestis in blood meal	GI blockage	Biofilm development	Only models blockage as a proxy for transmission	(44, 47–50)
B. burgdorferii	Mice	Skin/dermis	Tick bite	Immune response (serum positive)	Infected tick feeding on naïve mice	Technical challenges of inoculation and detection	(51–55)
Rickettsia spp.	Guinea pigs, dogs, mice (C3H/hen)	Varies by species	Tick bite	Eschar, fever, edema, bacteremia	Infected tick feeding on naïve animals	Technical challenges of inoculation and detection	(56–59)

Models for transmission of gut pathogens by the fecal-oral route

Vibrio cholerae

V. cholerae is the causative agent of cholera, a human secretory diarrhea with over 3 million cases annually (60). Despite extensive studies employing vertebrate and invertebrate models, a practical animal model for the fecal-oral transmission of V. cholerae remains elusive (26, 27). However, infant mouse models have been developed that approximate transmission dynamics (26). Notably, V. cholerae undergoes changes to a hyperinfectious state during transit through a mammalian host, and mathematical modeling suggests this contributes to the epidemic spread of cholera (61, 62) as acquisition of only a few bacteria from an infected host can cause transmission to a naïve individual. This hyperinfectious state has been successfully replicated in infant mice, where V. cholerae isolated from murine guts exhibited an ID₅₀ of 10–100 colony-forming units (CFU), in contrast to the minimum dose of 500 CFU required to infect mice with V. cholerae grown in laboratory medium (28). Thus, these in vivo and computational studies suggest that passage through the GI tract enhances efficient transmission of V. cholerae. In the natural environment, V. cholerae is known to colonize the GI tract of certain fish species (63), and colonization and transmission of V. cholerae have been demonstrated using zebrafish. In this model, the El Tor biotype efficiently colonized fish intestines following exposure in water (30). Infected fish displayed signs of diarrhea and were able to transmit V. cholerae with high efficiency to naïve fish that then became colonized.

The diarrheal disease is largely caused by cholera toxin (CT), an ADP-ribosylating toxin that mediates stimulation of the host chloride ion efflux channel (64–66), resulting in secretion of ions and water into the intestinal lumen. Insights into V. cholerae pathogenesis using human challenge and animal model studies identified that CT was critical for achieving a high number of bacteria shed from an infected host (29, 67, 68), but is not required for colonization. Another way that CT promotes transmission is through modulation of nutrient availability in the gut. Production of CT correlated with increased heme and other nutrients that selectively provided V. cholerae a growth advantage (69). This suggests that the primary roles for CT are enhancing growth and eliciting diarrhea, which are both necessary for efficient transmission to a new host (68). In addition, the accessory toxin encoded by rtxA actively suppresses production of interleukin-8 (IL-8) from intestinal epithelial cells; IL-8 is a proinflammatory chemokine that is required for neutrophil recruitment (70) and contributes to the clearance of rtxA mutants from the intestine (71). Thus, dampening the host inflammatory response promotes persistent colonization, allowing for continuous shedding and increasing the likelihood of V. cholerae transmission to a naïve host.

Clostridioides difficile

C. difficile is a significant contributor to antibiotic-associated diarrhea in healthcare settings, and its gut-associated pathogenesis has been investigated using small animal models such as Syrian hamsters and mice (31, 72, 73). C. difficile spreads from host to host through metabolically dormant spores (74). Using C57BL/6 mice, Lawley et al. (32) demonstrated fomite-mediated transmission evidenced by successful colonization of the GI tracts of naïve mice following exposure to a contaminated environment that contained as little as five spores/cm² (32, 74). Aerosolization of spores is common in patients with active C. difficile infection (75), suggesting that there is an airborne component to C. difficile transmission. This highlights how pathogens have developed the capability to transmit by multiple routes.

Antibiotic-mediated dysbiosis of the gut microbiota can allow several pathobionts to bloom, resulting in a super-shedder state (Box 2). In mice, a bloom of C. difficile following antibiotic treatment resulted in mucosal damage, including epithelial cell death, disruption of tight junctions, microvillus effacement, and inflammatory cell infiltrate (32). The mucosal damage is attributed to the TcdA and TcdB toxins, which are tightly regulated in part by the transcriptional regulator Spo0A. A Δspo0A strain has increased toxin production that correlates with a more robust inflammatory response. However, this mutant had reduced persistence and a corresponding defect in transmission (76). These data suggested that a regulated and finely tuned inflammatory response is required for persistence within the host, which, in turn, increases the likelihood of transmission (32, 76).

Box 2. Role of super-spreaders and super-shedders in transmission.

Historically, it was assumed that all infected individuals within a population possessed equal likelihood of transmitting an infection to others. This view changed with the observation that a small subset of individuals seemed to account for the majority of transmission events, a phenomenon that came to be known as the 20/80 rule (77, 78). The rule states that 20% of infected individuals are responsible for 80% of secondary infections. These individuals who disproportionately transmit the infection are described as super-spreaders. One of the most well-known historical examples is the case of Mary Mallon, who became known as “Typhoid Mary” and who is estimated to have infected between 51 and 122 individuals in the early 1900s with Salmonella enterica serotype Typhi, the causative agent of typhoid fever. Mallon was responsible for several typhoid outbreaks which resulted in the confirmed deaths of three individuals. She was also the first-ever recognized asymptomatic carrier of an infectious disease. Super-spreaders have been identified for various infectious diseases, including tuberculosis, Ebola, and various coronavirus infections (79–81).

Where super-spreaders describe the disproportionate transmission of disease from hosts to hosts, super-shedder is a term used to describe an individual who produces and releases substantially more infectious units of a disease than the average infected individual (82). There are certain conditions that can influence the development of a super-shedder state. Studies have shown that super-shedding can be influenced by host factors, co-infections, or treatment with antibiotics. For example, carrier mice that shed low levels of Clostridium difficile triggered a highly contagious super-shedder state following antibiotic treatment, with mice shedding 1,000,000-fold more C. difficile in their feces (32). Long-term exposure to antibiotics could prolong this state, resulting in persistent super-shedder mice. Similarly, mice colonized with K. pneumoniae that were treated with antibiotics transiently displayed a super-shedder phenotype, with more than 10⁸ CFU per gram of feces (38). Co-infections can also enhance shedding and transmission rates. Such is the case with Streptococcus pneumoniae and influenza A virus (IAV) co-infections. Both in ferret and mouse models, co-infections with these two pathogens led to increased bacterial shedding and transmission (43, 83). This is due, in part, to the increased mucus flow and is correlated with upper respiratory tract inflammation in response to IAV infection. Super-spreaders and super-shedders are responsible for most transmission events. Thus, control strategies that fail to target these groups result in low efficacy and poor disease control.

Salmonella enterica

In a Salmonella enterica serovar Typhimurium persistence model (33) using 129 × 1/SvJ mice that have altered neutrophil and macrophage responses (84), transmission was observed when a single infected mouse was co-housed with four naïve mice. However, transmission events were noted with a subset of infected mice (27%) that were in a “super-shedder” state (>10⁸ CFU/g feces), which is 100-fold higher than moderate shedders (~10⁶ CFU/g feces) (33). By contrast, no transmission events were observed between moderate shedders and naïve mice, suggesting that a persistently higher level of shedding is required for transmission in this model system. Transmission of Salmonella enterica serovar Typhimurium and Enteritidis (S. Typhimurium and S. Enteritidis) has also been modeled using zebrafish (34). A single infected fish that was colonized in the intestine at 10⁹ CFU transmitted to naïve fish co-housed together. The infected fish was treated with gentamicin to remove any bacteria on the surface of the fish and placed in fresh water before co-housing, indicating that the source of transmitted Salmonella was shedding from the GI tract and that transmission was facilitated by water (34). Both these models suggest that high Salmonella levels in the GI tract are required for robust shedding to overcome the transmission barrier. The zebrafish model has also been used to observe Shigella pathogenesis and transmission. Interestingly, fish housed at 30°C failed to become colonized with Shigella, whereas those housed at 37°C became persistently colonized. In this model, robust colonization was preceded by high shedding of Shigella in the water that manifested as 100% transmission to co-housed naïve fish at 37°C. These data imply that temperature plays an important role in Shigella pathogenesis and transmission (37).

Similar to C. difficile, transmission of S. Typhimurium was primarily observed in mice that developed colitis and the super-shedder state. The colitis was concomitant with an influx of neutrophils and dendritic cells, suggestive of high levels of gut inflammation. Transmission frequency correlated with the level of bacteria in fecal shedding and was dependent on the Salmonella pathogenicity islands 1 and 2 that encode type III secretion systems (T3SS) (33). Thus, T3SS effector proteins may contribute to robust Salmonella gut colonization, shedding, and subsequent transmission by modulating inflammation. Interestingly, mice that were persistently colonized but only moderate shedders became super-shedders following antibiotic treatment and displayed increased inflammation of the gut. These mice remained in the super-shedder state even after removal of the antibiotic, suggesting that both the host response and microbiota components contribute to the development of the super-shedder state and subsequent transmission (33).

Pseudomonas aeruginosa

As an opportunistic pathogen, P. aeruginosa is occasionally present in the healthy human GI tract and can cause systemic infections when host barriers are compromised. Systemic infections with P. aeruginosa are considered as evolutionary “dead-ends.” However, in a mouse (BALB/c) model of bloodstream infection (tail vein), P. aeruginosa spread from the bloodstream to the gallbladder, where they replicated to high numbers (35). Gallbladder bacteria seeded the intestines and were then shed in feces, leading to transmission of P. aeruginosa to uninfected cage-mates. This highlights a complex alternative route of fecal-oral transmission with a role for the gallbladder in spreading P. aeruginosa from the bloodstream to feces during bacteremia, thus promoting transmission to subsequent hosts (35). Whether the gallbladder contributes to transmission in a human host remains to be elucidated. Furthermore, using C3H/HeN mice treated with antibiotics to deplete murine microbiota and inoculated with P. aeruginosa in the water, Kamei et al. (36) demonstrated that both epidemic and non-epidemic P. aeruginosa strains were able to transmit to naïve antibiotic-treated mice. Together, these two studies indicate that P. aeruginosa outbreaks that occur in CF clinics can potentially ensue through the fecal-oral route (85–87).

Klebsiella pneumoniae

Klebsiella pneumoniae is a frequent source of hospital-acquired infections and has been isolated from the GI tract of hospital patients and staff (88, 89). Evidence for a fecal-oral transmission route came from studies using an oral inoculation route in mice in which K. pneumoniae showed persistent colonization of the oropharyngeal space and lower gastrointestinal tract in the absence of antibiotic pretreatment (38). When these infected mice were co-housed with naïve mice, rapid, transient transmission was observed in all the naïve mice. Only ~30% of newly infected mice developed persistent colonization, but this correlates with epidemiological studies (90, 91). When infected mice were treated with antibiotics, they developed a super-shedder phenotype, and rapid transmission to co-housed naïve mice, also on antibiotics, was observed. 90% of these newly infected mice became persistently colonized. Co-housing of infected and naïve mice in a metabolic cage to reduce coprophagia showed no transmission events, further supporting the fecal-oral transmission route for K. pneumoniae. Thus, this transmission model will be useful for improving our understanding of the colonization and transmission dynamics of K. pneumoniae both in the presence of an intact gut microbiota or with antibiotic-mediated gut dysbiosis that is a common occurrence in a hospital setting.

Models for transmission of respiratory pathogens by the aerosol/droplet route

Bordetella spp.

The gram-negative human-adapted pathogen causing whooping cough, Bordetella pertussis, relies on continued transmission between hosts due to the acute nature of the infection. Since small animal models do not adequately recapitulate human disease, Warfel et al. (39) utilized a baboon model and demonstrated that B. pertussis transmits to naïve hosts via respiratory droplets. Co-housing experiments in which naïve baboons were introduced to cages 24 hours after index baboons had been inoculated resulted in transmission to the naïve baboons, implying that transmission occurred through droplets or contact with nasal secretions. Transmission was evidenced by detection of B. pertussis CFU in nasal washes and an increased number of white blood cells in blood samples. In another experiment, naive baboons became infected after being housed in separate cages 7 feet apart from the infected baboons. These data provided experimental evidence of likely airborne transmission, thus demonstrating this transmission mode in the spread of B. pertussis.

Using a swine model, Nicholson et al. (40) demonstrated that piglets infected with Bordetella bronchiseptica, a species related to B. pertussis, were capable of transmission to naïve hosts by both direct and indirect modes of infection. Transmission was dependent on B. bronchiseptica producing a functional T3SS, which modulates the host inflammatory response similarly to that seen in Salmonella (33). The infected piglets showed increased development of nasal lesions and an increased inflammatory response. Surprisingly, both pro- (IL1β, IL-8, and MCP-1) and anti-inflammatory cytokines (IL-10) were increased post-inoculation but then declined over time. Production of IL-10 from peripheral blood mononuclear cells isolated from B. bronchiseptica-infected piglets required the T3SS. However, the T3SS was not directly involved in transmission, but rather, contributed to persistent colonization (40). Persistent colonization would, in turn, increase the likelihood of transmission, and thus, potentially increase the number of transmission events. More recently, a murine model of B. bronchiseptica infection identified Pertactin, an outer membrane autotransporter protein that is also present in B. pertussis and a component of the acellular vaccine, as a transmission determinant (42).

Streptococcus pneumoniae

S. pneumoniae (the pneumococcus) is a gram-positive human-adapted pathogen that can asymptomatically colonize the nasopharynx, particularly in young children. Transmission of S. pneumoniae has been demonstrated using an infant mouse model where index mice were intranasally inoculated. Transmission between infected and uninfected pups was only detected with co-housing, which mimics a daycare center for young children. Considerable variability in shedding and transmission frequency was observed between different S. pneumoniae strains, potentially explaining why certain isolates are more common in the human population, as they may be better adapted for the human environment (20). Transmission of S. pneumoniae to naïve pups was increased when they were co-housed with mice infected with both S. pneumoniae and influenza A virus (IAV), a common occurrence in a community setting, and this was attributed to an IAV-dependent increase in pneumococcal shedding (20). That transmission in these studies required co-housing suggests that the dam served as a fomite source, as both infected and naïve pups interacted with the dam, and indicates that S. pneumoniae can spread by an indirect, non-aerosol mode of transmission (25). A similar IAV-dependent transmission effect was observed using a ferret model (43). Co-infection with S. pneumoniae and IAV resulted in increased pneumococcal density in the upper respiratory tract compared to ferrets inoculated with S. pneumoniae alone, and this increased density facilitated increased transmission to naïve ferrets both when co-housed and when separated. These experiments provided evidence of aerosol-mediated transmission and are thus considered as an example of direct transmission. Transmission to naïve ferrets was reduced or absent from ferrets infected only with S. pneumoniae, indicating the role of IAV co-infection in promoting pneumococcal transmission (43). In these studies, the separation of infected animals from their naïve cohort allowed for distinguishing between fomite or aerosol-mediated transmission. More recently, it was demonstrated that expelled aerosols from S. pneumoniae-IAV co-infected ferrets contained both S. pneumoniae and IAV infectious particles, and the presence of IAV stabilized S. pneumoniae, likely enhancing its ability to transmit to a naïve host (92). S. pneumoniae-IAV co-infections are frequently observed, and these mono- and co-infection transmission models allow us to gain a mechanistic understanding of the transmission dynamics of these two pathogens.

In contrast to studies with V. cholerae that showed enhanced transmission due to bacterial-mediated immune suppression (70, 93, 94), several studies have demonstrated that S. pneumoniae-induced inflammation correlates with a higher number of bacteria being shed from the upper respiratory tract, thus favoring transmission. This inflammatory response is mediated largely by the sole cytolysin in S. pneumoniae, pneumolysin (Ply), and includes increased IL-1β production and subsequent neutrophil recruitment to the upper respiratory tract (25). The ability of Ply to modulate inflammation stems from its pore-forming activity, as a S. pneumoniae mutant producing a Ply variant unable to form pores did not elicit an inflammatory response. This ply mutant did not have altered colonization but displayed reduced shedding in nasal secretions and less efficient host-to-host transmission in the infant mouse model (25). Mice co-infected with IAV and S. pneumoniae showed increased shedding and transmission, and this was associated with increased mucus production and inflammation (83, 95–97). Two independent transposon mutagenesis screens using the ferret and infant mouse models identified pneumococcal factors important for transmission (98, 99). The dlt locus, which encodes enzymes that add D-alanine to lipoteichoic acid (LTA), a component of the cell envelope of gram-positive organisms, was implicated as a transmission determinant (99). The modified LTA has a reduced cell surface charge that protects against host antimicrobial peptides, but, triggers a more robust TLR2-mediated inflammatory response that promotes S. pneumoniae shedding, and, in turn, transmission (99). These results suggest that there is more than a single type of inflammatory response elicited by pneumococcal colonization, but that any mechanisms that enhance inflammation will also increase bacterial shedding and transmission. A distinction to make here is that while inflammation enhances colonization for several gut pathogens by enabling super-shedder formation and overcoming the threshold to transmit, inflammation in the URT increases the number of bacteria expelled without necessarily impacting colonization levels.

Models for transmission of vector-borne pathogens by arthropods

For vector-borne pathogens, transmission factors can be defined as elements that are required to establish infection in a mammalian host when delivered by the arthropod vector, but are not essential for initial colonization of the arthropod. With this strict definition, few transmission factors have been identified and characterized in vector-borne pathogens. In addition, the vector-to-rodent models present numerous technical challenges, and some vector-borne pathogens have limited genetic tools, although they are continually being improved upon. Several recent and thorough reviews have been published, and we direct readers to those sources in the text below.

Yersinia pestis

Yersinia pestis is a vector-borne bacterial pathogen that has a complex life cycle that involves transmission between mammalian hosts and fleas (100). The mammalian host range is wide and includes humans, dogs and cats (domesticated and wild), and rodents. Human-to-human transmission can occur, presumably through respiratory droplets and aerosols, causing primary pneumonic plague (101). Bubonic plague is transmitted via the flea-to-mammalian route, and a flea-mouse model has been used to establish key events and factors that contribute to flea-mediated transmission. Seminal studies from the early 20th century demonstrated that the gastrointestinal tract of fleas infected with Y. pestis becomes blocked, and this blockage contributes to transmission to mammalian hosts (47, 48). Subsequent work with two different species of fleas indicated that the blockage event is not essential for transmission (44, 45). In this model system, fleas are inoculated with Y. pestis using artificial feeders that contain a suspension of Y. pestis in mouse blood at 10⁹ CFU/mL, and mouse skin is stretched over the blood from which the fleas feed. Inoculated fleas are then placed in a chamber with an anesthetized mouse. Transmission to the mouse is determined by the development of plague symptoms and detection of Y. pestis CFU in organs or of antibodies against the F1 antigen (a Y. pestis-specific surface structure) (44). A recent study directly compared mice infected by fleas that were in the early stage of colonization or fully blocked and found distinct differences in disease progression compared to mice inoculated by intradermal or subcutaneous injection, highlighting the utility of the more natural flea bite model (46).

Because of early studies demonstrating that blockage by Y. pestis is a key event for transmission from flea to mammal (47, 48), several investigators have used blockage development as a proxy for modeling transmission. Several bacterial genes were identified to contribute to the blockage development and are presumed to be important for efficient transmission, including genes that promote biofilm development and LPS modification, and others that protect gainst flea defenses (summarized in references 49, 50). Because of the challenges presented by vector-host transmission experiments, an alternative approach used by many groups was to analyze the transcriptomic differences between organisms isolated from vector and mammal, or cultures grown at temperatures indicative of vector (~25°C) and mammalian host (37°C). These have provided valuable information about genes required in each environment and provide a set of candidate genes that may directly contribute to transmission (49, 102).

Borrelia burgdorferi and Rickettsia spp.

Transmitted through ticks, B. burgdorferi is the causative agent of Lyme disease. After overcoming difficulties culturing the organism in vitro, animal models for the study of pathogenesis and transmission were developed using a number of rodent and primate hosts (summarized in reference 103). The most common transmission model involves a tick-to-mouse route, where infected ticks are allowed to feed on naïve mice. Ticks can be inoculated by a number of methods. Artificial methods include capillary inoculation (51), use of mouse skin placed over B. burgdorferi-containing blood (52), and immersing tick larvae in a B. burgdorferi suspension (53). Ticks can be naturally inoculated by feeding on infected mice that were previously inoculated with B. burgdorferi either by subcutaneous or intradermal injection or from infected ticks (54). Mouse-to-tick transmission is determined by enumerating spirochetes by CFU or qPCR (55). Tick-to-mouse transmission is assessed by assaying for antibodies against B. burgdorferi proteins (typically OspA) and enumerating spirochetes by CFU or qPCR.

Members of the genus Rickettsia cause diseases falling into the spotted fever group (SFG), the typhus group (TG), and the transitional group that shares features of both SFG and TG. The species causing typhus are typically transmitted by fleas and lice, and those in the transitional group are transmitted by a variety of vectors, including fleas, lice, mites, and ticks. The species in the SFG are predominantly transmitted by ticks. Among the SFG, Rickettsia rickettsii is the causative agent of Rocky Mountain Spotted Fever, one of the more common rickettsial diseases in the United States (104). R. rickettsii transmission has been modeled using guinea pigs (56, 57) and dogs (58), and mice have been employed with other Rickettsia species (59). Similar to the tick transmission model for B. burgdorferi, ticks are inoculated by feeding on rabbits that were infected by intraperitoneal injection, then allowed to feed on naïve hosts. Infection of guinea pigs is monitored by the development of disease symptoms (105). Because this pathogen is an obligate intracellular organism, the presence and quantification of R. rickettsii in ticks and recipient mammalian host tissues is determined by qPCR (106, 107).

The identification of transmission factors for tick-borne pathogens has been slow. In Rickettsia spp., a combination of challenges presents significant hurdles: the genetic tools are only just beginning to become widely available, they are obligate intracellular organisms, and the tick-to-mouse model is laborious and time-consuming (108–110). Many of these hurdles are reduced but not absent in the study of Borrelia spp. For example, these species are culturable in laboratory-prepared growth media, ticks can be artificially inoculated, and the mouse species can serve as a model mammalian host. Use of the tick-to-mouse model has led to the identification of genes that can be defined as transmission factors, but the function and role of these genes in transmission remain elusive. Only four genes, bba07 (111), bba03 (112), and bba64/bba66 (113, 114), have been identified as important for transmission to mice from ticks, but not required when mice were inoculated by needle injection. However, no specific role for any of these genes has been determined. Several recent and thorough reviews have been written on all aspects of Borrelia biology, and readers are directed to these resources (103, 115–117).

HOW PATHOGENS OVERCOME ENVIRONMENTAL AND HOST-MEDIATED CONSTRAINTS

While most transmission determinants are unique to each pathogen, there are overlapping themes identified through animal models discussed below that connect transmission strategies employed by pathogens that occupy disparate niches within the host.

Overcoming environmental impediments

Transmission from one host to another generally requires transit through a non-host environment, which could be for a short duration or a longer period. The infectious agent must remain viable in this environment to colonize and propagate in another host (Fig. 2). Pathogens such as S. pneumoniae can transmit via fomites, where aerosols or nasal secretions have landed on surfaces. S. pneumoniae was observed to be desiccation-tolerant for weeks and remained infective after rehydration (118). However, when starved for nutrients in saline, S. pneumoniae showed reduced viability, yet the viable cells remained infective for 24 hours (119). The capsular polysaccharide (CPS) of S. pneumoniae was dispensable for desiccation survival, but critical for survival under starvation conditions, with experimental data suggesting that CPS served as a nutrient source (118, 119). These results suggest that S. pneumoniae can cannibalize its CPS sugar moieties to enhance its viability and increase the likelihood of acquisition by a naïve host. Another bacterial feature that enhances survival under desiccation stress is the ability to form bacterial aggregates, which are more stress tolerant than individual bacteria (120). S. pneumoniae surface protein A (PspA) was found to promote survival in a non-host environment. PspA mediates interactions with dying epithelial cells in the upper respiratory tract through surface-bound mammalian (m)GAPDH, allowing S. pneumoniae to form aggregates consisting of hundreds of bacterial cells, bound to host cell debris within expelled nasal secretions (121–123). These PspA-dependent aggregative structures were desiccation-tolerant and remained infective after rehydration, suggesting that aggregate formation allows S. pneumoniae to survive and remain infective outside the host (121).

Fig 2.

Host and environmental factors that influence the success of bacterial infections. The severity and spread of bacterial infections are shaped by a combination of host and environmental factors. Host factors include genetics, age, sex, immune status, ongoing infections or illnesses, and the composition of the host microbiota. Environmental factors, such as sanitation, the quality of food, water and soil, and air quality or ventilation, also play a critical role in transmission dynamics. In addition, the success of bacterial spread depends on the pathogen’s ability to survive in harsh or fluctuating environments. The ability to withstand desiccation, temperature extremes, or resist disinfection methods will promote bacterial persistence and spread. Climate and seasonal variations can further impact bacterial growth, replication, and survival, thereby affecting infection patterns (124).

C. difficile is another organism that can persist for long periods in unfavorable environments outside a host. Unlike other nosocomial pathogens, C. difficile is distinct in that it produces highly infectious spores that are tolerant of desiccation and resistant to disinfectants (125). During antibiotic treatment, the complexity of the gut microbiota is reduced, and simple dietary sugars such as fructose and glucose can become available to C. difficile (126), leading to higher colonization levels of C. difficile in the GI tract and increased spore production (127). Hydrogen peroxide is considered the most effective strategy in completely inactivating C. difficile spores (128), and interestingly, the spores from mice fed a diet high in fructose/glucose were more resistant to hydrogen peroxide treatment than spores from mice fed a ribose-rich diet (126). These findings highlight that C. difficile responds to environmental metabolic cues in the gut, which can impact its transmission efficiency.

Similar to C. difficile, Acinetobacter baumannii has a much higher desiccation and starvation tolerance compared to other organisms. Importantly, A. baumannii was more virulent when rehydrated after desiccation compared to non-desiccated bacteria (129). This increased desiccation resistance phenotype is controlled by the two-component system BfmRS, which is known to control stress response (130). Through a transposon mutagenesis screen, Green et al. (129) identified two hydrophilin proteins (DtpA and DtpB) that contribute to desiccation tolerance by stabilizing other enzymes under desiccation and heat stress, and expression of dtpA in E. coli Nissle enhanced the ability of this organism to colonize mice following desiccation. While transmission was not directly tested in this study, the enhanced desiccation survival conferred by DtpA and DtpB likely contributes to indirect transmission via fomites.

Curiously, some bacterial mutants display increased environmental survival. Compared to a wild-type S. pneumoniae strain, a spxB mutant displayed increased survival following desiccation and increased growth in media lacking carbon and metals, both conditions similar to those outside the host (98). This mutant also displayed higher transmissibility in the infant mouse model compared to the parental strain. spxB encodes pyruvate oxidase, an enzyme that converts pyruvate to acetyl-phosphate and hydrogen peroxide, a toxic byproduct. In the spxB mutant, glycolytic metabolism and other metabolic pathways are altered, likely providing a fitness advantage during desiccation stress (98). Consequently, compared to the parental strain, an spxB mutant was able to survive significantly longer in stationary phase (131). A transposon mutagenesis screen identified genetic determinants in S. pneumoniae that either enhanced or reduced desiccation tolerance. Notably, uvrA, which encodes the enzyme for nucleotide excision repair, was associated with lower desiccation tolerance, while spxB was linked to improved survival and validated earlier findings (132). Subsequently, uvrA showed reduced transmission efficiency in the infant mouse model (132). These results are consistent with desiccation tolerance being a critical step in transmission.

K. pneumoniae can remain viable up to 4 weeks on surfaces. To mitigate the outbreaks in a hospital setting, robust cleaning strategies must be implemented (133). In gram-negative bacteria, MgrB regulates the activity of the PhoPQ two-component system (TCS). Mutations that impact PhoPQ activity have been shown to lead to resistance to colistin, a cationic antibiotic of last resort (134). In K. pneumoniae, Bray et al. (135) demonstrated that an mgrB mutant was resistant to colistin and showed increased survival on a solid surface without any carbon or nitrogen source. In a murine model of transmission, the mgrB mutant showed higher transmission rates to naïve co-housed mice compared to the parental strain. This increase in environmental survival and transmission was linked to altered PhoPQ activity, which likely affected membrane rigidity and morphology of K. pneumoniae (135, 136). This report also raises the specter of antibiotic resistance acquisition concomitant with increased transmissibility.

Modulation of host inflammatory response

Pattern recognition receptors (PRRs) detect pathogens and are a critical first step in the host defense systems (137). As a consequence, pathogens have developed processes to either dampen the host response or exploit it to their advantage (138, 139), allowing them to proliferate within the host and subsequently transmit to a naïve host (Fig. 2). For S. pneumoniae that resides in the URT, shedding and transmission were enhanced by induction of the inflammatory response. S. pneumoniae induces inflammation by several mechanisms, including Toll-like receptor 2 recognizing lipoteichoic acid on the surface of S. pneumoniae (140), platelet-activating factor receptor (PAFr) recognizing S. pneumoniae phosphorylcholine (141), and pneumococcal infection-induced stimulation of type I interferon signaling (IFN-1) through the IFN-1 receptor (Ifnar)(142). Increased levels of the CXCL-1 and CXCL-2 chemokines and the pro-inflammatory cytokine interleukin-1β (IL-1β) were also correlated with increased shedding from the URT. Treatment with dexamethasone reduced expression of CXCL-1, CXCL-2, and IL-1β and significantly reduced pneumococcal shedding from the URT without impacting colonization density, suggesting that inflammation is critical for the robust S. pneumoniae shedding that correlates with transmission (25). For C. difficile, the TcdA and TcdB toxins synergize to induce a strong inflammatory response that leads to severe diarrhea, which likely contributes to transmission (143). Interestingly, loss of the transcriptional regulator Spo0A resulted in increased production of TcdA and TcdB and increased disease severity, suggesting the likelihood of increased transmissibility. However, while the number of vegetative bacteria in feces was similar between the spo0A mutant and the wild type strain, the mice inoculated with the spo0A mutant had undetectable spore levels in the feces. The lack of spores resulted in failure to transmit, highlighting the dependence of Spo0A for spore formation and efficient C. difficile transmission (76). In addition, Spo0A regulates several processes beyond spore formation that could contribute to the transmission process (144).

The super-shedder state is required for efficient host-to-host transmission of S. Typhimurium and pathogenic Escherichia coli (33, 82, 145). It was demonstrated that the cecum and colon of super-shedder mice displayed increased inflammation, mucosal necrosis, collapse, and glandular hyperplasia compared to non-super-shedder mice (33). Similar gut mucosal damage was observed in pig and calf models of S. Typhimurium infection (146, 147). Super-shedder mice and pigs displayed an increase in pro-inflammatory cytokines, including IL-1β, IFN-γ, and chemokines CXCL-1 and CXCL-2, concomitant with an increase in neutrophils and macrophages. Although this T3SS-mediated pro-inflammatory response is critical for the prevention of S. Typhimurium dissemination to deeper tissues, it provides an environment within the gut that fuels the growth of S. Typhimurium through aerobic and anaerobic respiration, allowing it to reach a super-shedder state and spread to naïve hosts (33, 146–149). Another pathogen that utilizes host-mediated inflammation to promote its transmission is B. bronchiseptica. All members of the B. bronchiseptica cluster produce pertactin, an autotransporter required for a robust inflammatory response that is characterized by an influx of neutrophils, NK cells, macrophages, and B cells in the nasal cavities of infected mice. This inflammatory response was correlated with increased mucus production, shedding, and transmission of B. bronchiseptica in the murine model (42). Thus, bacterial factors that contribute to host-mediated inflammation, or that allow bacteria to take advantage of the inflamed environment, play a critical role in pathogen dissemination.

Contrary to the pathogens described above, V. cholerae transmission is enhanced by suppression of the host inflammatory response through the activity of the CT and RtxA toxins (70, 94). This immune suppression promotes persistent colonization and continuous shedding, and thus, increases the likelihood of V. cholerae transmission to a naïve host. Thus, while bacterial toxins increase disease severity by either subverting or enhancing the host response, they also end up contributing to the transmission process by increasing the number of bacteria shed from an infected host.

Bottlenecks in the context of transmission

Transmission involves multiple discrete steps, many of which involve interactions with “protective barriers” within the host that a pathogen has to navigate to successfully colonize and spread to the next susceptible host (Box 3). These include (but are not limited to) the mucus layer, antimicrobial peptides, changes in pH (e.g., stomach versus colon), and competition with microbiota for nutrient resources. These barriers can be considered as bottlenecks (stochastic loss of clonal population) that a pathogen must overcome to establish colonization. As such, a better understanding of bottlenecks could reveal intriguing points of intervention.

Box 3. Host-derived transmission barriers.

Although many bacteria possess the ability to cause serious disease, the human body has at its disposal an arsenal of natural barriers to help prevent or limit infection. These include physical, chemical, microbial, and immune-mediated barriers.

Physical barriers: The skin, considered the largest organ of the human body, is the first line of defense against microorganisms. To initiate infection, bacteria must penetrate the skin or enter through the mucous membranes. Once inside, bacteria encounter additional physical barriers that limit their ability to cause disease. In the gastrointestinal tract, a single layer of epithelial cells covered with a thick mucus layer separates ingested contents and microorganisms, as well as toxic molecules, from the rest of the body. Tight junction proteins hold and seal these cells together, regulating permeability (150). Intestinal peristalsis and the natural sloughing of the intestinal epithelial cells routinely remove microorganisms. In the urinary tract, constant urine flow serves a similar role, washing out microbes. In the respiratory tract, epithelial cells are lined by cilia or microtubule-based hair-like projections that sweep out bacteria entrapped in mucus, a process known as mucociliary clearance (MCC). The blood-brain barrier is an added structure that protects the brain from blood-borne infections. Lastly, the placental barrier during pregnancy restricts microbial access to the developing fetus.

Chemical barriers: Various antimicrobial components can be found throughout the body, adding another layer of protection. Both the gastrointestinal and respiratory tracts contain goblet cells which produce mucus that coats the epithelial cells and can trap bacteria. In addition, tears, cerumen (earwax), and saliva contain enzymes, such as lysozyme, that destroy bacterial cell walls and inhibit bacterial growth (151). Additional sites of the body provide harsh environments with low pH that can denature the enzymes of various pathogens. For instance, the presence of stomach acid alters the pH of the stomach, ranging between pH 1 and 3. Vaginal pH is also slightly acidic, ranging between 3.8 and 4.5, due to the resident acid-producing bacteria.

Microbial barriers: The microbial communities associated with the host act as a barrier for pathogen invasion either through direct or indirect mechanisms, a phenomenon known as colonization resistance (152, 153). Direct mechanisms involve competition for resources and space, the release of secondary bile acids and bacteriocins by resident bacteria, and bacterial contact-dependent killing. As such, many bacteria, including Dolosigranulum pigrum and Citrobacter amalonaticus, have been shown to protect against pathogenic bacteria (154, 155). Indirect mechanisms consist of microbiota-dependent alterations of the environment and stimulation of the host immune system.

Host immunity: If bacteria circumvent all other barriers, the host immune system provides various protective measures to destroy and eliminate pathogens. Phagocytes are specialized cells that can recognize, engulf, and produce reactive oxygen species to destroy many pathogens. The mucosal surfaces secrete immunoglobulin A (IgA), often found as a dimer, which promotes pathogen clearance via immune exclusion. Secretory IgA (SIgA) prevents microbial access to epithelial receptors, causing bacteria to remain trapped in the mucus layer, where they are susceptible to MCC. Many host cells also produce different antimicrobial peptides, such as α- and β-defensins and cathelicidins, which can damage bacterial cell membranes, destroying cell morphology and leading to bacterial death.

Circumventing host barriers: Despite the many barriers present to protect the host from acquisition and establishment of colonization, bacteria have evolved to adapt, evade, escape, or traverse many of these barriers. A de novo response is critical for the diverse environments and barriers a pathogen encounters before it can gain access to its colonization niche. While not comprehensive, the following are some examples of bacterial factors that contribute to the success of a pathogen after it is acquired by a naïve host. For instance, B. pertussis can paralyze the MCC function of the respiratory tract via the induction of nitric oxide-mediated ciliostasis (156). S. pneumoniae produces pneumolysin, which can slow ciliary beating and damage the epithelial cells, and its capsular polysaccharide prevents mucus entrapment (157, 158). Many gram-negative bacteria, including E. coli, K. pneumoniae, and S. enterica, acylate the lipid A component of their lipopolysaccharide (LPS), effectively changing the charge on their surface and repelling cationic antimicrobial peptides (CAMPs). While H. pylori resides primarily attached to the epithelial layer lining the stomach, where the pH is neutral, it uses acid-activated UreI to transport urea into the cell, which is converted to ammonium and allows the bacteria to neutralize the protons entering the periplasm (159, 160). In addition, chemotaxis-based flagellar motility plays a critical role for H. pylori and other pathogens to gain access to the preferred niche environment (161). V. cholerae tightly regulates the expression of the clcA, which encodes an H⁺/Cl⁻ transporter, which together with the CadAB post-acquisition promotes its survival in the acidic environment of the stomach and gains access to the lower GI tract to establish colonization (162, 163).

Bottleneck analyses have been demonstrated for several pathogens through the use of strains with differential markers and, more recently, with a diverse chromosomal barcoded library combined with high-throughput sequencing to allow for the detection of individual clonal populations that survive a particular stressor. However, most of these studies have focused on providing an understanding of bottlenecks in the context of within-host events and not host-to-host transmission (35, 164–167). Recent studies using Staphylococcus aureus and commensal E. coli strains found that host-associated bottlenecks promote rapid accumulation of mutations, and distinct host selective pressures quickly lead to outgrowth of surviving populations with advantageous mutations (168, 169). Within the same host species, Kono et al. (170) used different antibiotic resistance marker-tagged S. pneumoniae to demonstrate an extremely tight bottleneck between donor and recipient hosts, suggesting that transmission requires a high number of shed bacteria from the infected host during S. pneumoniae-IAV coinfection and is a rare event. Antibiotic resistance markers provide limited molecular understanding of infection population dynamics, which can be overcome using a barcoded library with computational analyses. A recent study using a barcoded library in S. pneumoniae (171) showed that only 50% of the inoculated clones were detected in the upper respiratory tract during early colonization stages, and the number of clones decreased further over several weeks of persistence. The use of this library to examine transmission in the infant mouse model revealed that only 11% of the clones inoculated into the index mice were detected in co-housed mice at early time points, and this diversity decreased further as infection progressed. These changes in clonal diversity during persistence in the nasopharynx in both the donor and recipient mice were not due to host immune responses or microbiome composition, but rather through BlpC, a peptide pheromone that acts on the S. pneumoniae blp locus, which encodes bacteriocins that are involved in inter-strain and intra-strain killing. In this colonization scenario, the reduced clonal diversity is due to the selfish blp locus rather than being mediated by the host.

The mucus layer covering epithelial cells acts as a bottleneck that pathogenic bacteria encounter, as it can entrap and subsequently clear bacteria from the mucosal surface (172). Bacterial capsular polysaccharide (CPS) modulates interactions with the host mucus layer. S. pneumoniae strains synthesize and display on their surface over 100 different capsule types (173–175), but only some CPS serotypes are commonly associated with human carriage or disease (176). This suggests a role for CPS in the transmission of S. pneumoniae. Isogenic capsule switch mutants and strains that produced different amounts of capsule demonstrated that capsule type and amount were important S. pneumoniae shedding factors and were determinants that impacted host-to-host transmission (177–179). The reduction in shedding by certain CPS types and mutants that produced lower capsule amounts was attributed to the inability to escape the mucus layer that lines the epithelial layer (177). Similarly, the Bordetella extracellular polysaccharide locus (EPS) was identified in a small screen of mutants to be important for shedding and transmission but did not impact colonization of the upper respiratory tract (180). In addition, testing different inoculum doses revealed that the Δeps mutant required a higher dose than the parental strain to establish colonization of the nasopharynx, suggesting that EPS is not only important for bacterial shedding but also for acquisition by a susceptible host (180). How the EPS locus of Bordetella contributes to robust shedding and transmission to a new host remains to be explored, but as the CPS of multiple pathogens is an important mediator of interaction with the mucus layer (135, 158, 177), it is possible that the Bordetella EPS behaves in a similar fashion. Furthermore, S. pneumoniae uses glycosidases to cleave and metabolize glycans found on mucins that form part of the mucus layer. Similar to CPS, the glycosidase activity allows S. pneumoniae to escape entrapment in the mucus layer and be shed from the host (181). The S. pneumoniae two-component system, YesMN, was shown to contribute to robust pneumococcal shedding and host-to-host transmission. YesMN regulates genes involved in zinc and glycan acquisition and metabolism that play a critical role in escape from entrapment in the human nasal secretions (182). All of these processes that block entrapment inside the host likely contribute directly to bacterial transmission.

The microbial communities associated with the host act as a barrier for pathogen invasion either through direct or indirect mechanisms, a phenomenon known as colonization resistance (152, 153). Salmonella was observed to transmit to naïve mice only when index mice displayed the super-shedder state (33). Through mutations in metabolic genes and diet manipulation, Ruddle et al. (183) showed that the development of the super-shedder phenotype is driven by the ability to metabolize L-arabinose that was liberated from dietary polysaccharides by the enzyme alpha-N-arabinofuranosidase. L-arabinose metabolism fuels Salmonella growth in the GI tract, allowing it to achieve the super-shedder phenotype, and thus increasing the ability to transmit to a susceptible host. In a colitis model, the Salmonella super-shedder phenotype is mediated through T3SS effectors that modulate the resident gut microbiota. An avirulent mutant lacking effectors from T3SS-1 and T3SS-2 (invGsseD) colonizes the GI tract initially as the wild-type strain, but does not elicit an inflammatory response and is unable to modify the gut microbiome, which lead to its eventual outcompetition by the microbiota (184). However, mice that are predisposed to gut inflammation enabled the T3SS mutant to colonize as well as the parental wild-type strain, suggesting that manipulation of gut microbiome through inflammation is critical for robust colonization of Salmonella, which likely, in turn, promotes transmission (184). Furthermore, loss of microbiota complexity can result in pathogens becoming super-shedders (32, 33, 38). For instance, reduced diversity of the gut microbiota from antibiotic treatment or inflammation can lead to depletion of commensal clostridia species that produce butyrate, which serves as a preferred energy source for host colonocytes. Reduced levels of butyrate force colonocytes to switch from fermenting glucose to lactate, which, in turn, increases oxygen availability in the GI tract. S. Typhimurium possesses the enzymes cytochrome bd II-oxidase and nitrate reductase, and their activity allows for aerobic growth and pathogen expansion (super-shedder state), further driving inflammation and facilitating transmission (148).

Another pathogen that modulates host response is Brucella abortus, which is primarily a livestock pathogen but can cause human infections. After initial acquisition by a naïve animal, B. abortus avoids triggering a major host immune response by primarily residing inside macrophages and inhibiting cell death, leading to granuloma formation (185). In pregnant animals, however, B. abortus can infect the trophoblast, a component of the placenta, causing endoplasmic reticulum (ER) stress through its Type IV Secretion System effector proteins (186, 187). This ER stress ultimately results in cell death, with a concomitant strong inflammatory response. This specific infection pattern has been replicated in a murine model, demonstrating that the cell death and inflammation caused by B. abortus leads to abortion (185, 188). The aborted fetus harbors a high concentration of bacteria, which can be transmitted within the herd through direct contact with the fetal material.

VACCINATION AS A STRATEGY TO PREVENT TRANSMISSION

Vaccines against bacterial pathogens have been immensely successful in reducing the disease burden and lowering mortality rates (189), reducing transmission frequency, and providing herd immunity. In 2019, the total number of deaths due to lower respiratory infections declined to about 2.6 million lives lost from 3.1 million lives lost in 2000 (190). S. pneumoniae is the leading etiologic agent of lower respiratory infections and associated deaths (191). Although vaccines against S. pneumoniae have long existed for adults, these vaccines failed to provide protection in the at-risk and high-carriage infant and toddler population. In 2000, the first pneumococcal conjugate vaccine, Prevnar 7, was licensed in the United States and administered to infants. The vaccine was comprised of the capsular polysaccharide (CPS) from the seven most common CPS serotypes conjugated to CRM₁₉₇, a non-toxic recombinant variant of diphtheria toxin. In 2023, the vaccine was updated to include CPS from 20 strains (Prevnar 20). Conjugation to the CRM₁₉₇ protein antigen enables a strong T-cell-dependent response that imparts long-lasting humoral immunity against CRM₁₉₇ and the conjugated CPS (192). These conjugate vaccines have had both direct and indirect effects on the population, resulting in a drastic drop in the incidence of invasive disease caused by serotypes targeted by the vaccines. Surprisingly, the vaccines have also elicited high titers of serotype-specific IgG, which has resulted in a decrease in carriage and transmission frequencies and therefore has imparted protection of unimmunized individuals and the elderly via herd immunity (193). A drawback of widespread use of pneumococcal vaccines is the rise of isolates producing serotypes not protected by the vaccines, highlighting a need for additional preventative measures (194).

Similar to S. pneumoniae, Haemophilus influenzae type b (Hib) is an encapsulated bacterium that can cause pneumonia and meningitis in children, and a successful Hib conjugate vaccine has provided protection against disease development. The polyribosylribitol phosphate (PRP) vaccines that were used in the 1970s did not prevent Hib nasopharyngeal carriage. In the 1980s, PRP vaccines were replaced with Hib conjugate vaccines that did prevent carriage and thus, effectively stopped the Hib transmission chain. These vaccines were implemented into routine infant immunization programs and were successful in reducing disease incidence globally. It is estimated that Hib vaccines prevented as many as 1.2 million deaths between the years 2000 and 2015 (195).

Whole-cell pertussis vaccines (wPV) that target Bordetella pertussis, the causative agent of pertussis (whopping cough), are another example of a successful vaccine. Initially introduced in 1914, the wPV included formalin-inactivated bacteria and toxins. This vaccine was modified in the 1940s to a combination vaccine that provided protection against diphtheria, tetanus, and pertussis (DTP vaccine) (196). These whole-cell vaccines were highly effective in not only preventing disease and controlling bacterial burden but also reducing transmission events. After questions arose over the safety and efficacy of the whole-cell vaccine, an acellular DTaP vaccine comprised of one to five B. pertussis protein components was developed and licensed for use in the 1990s (197). Despite being effective in preventing disease and having fewer adverse side effects, the acellular vaccines have several limitations. The elicited protection wanes over time, and they fail to adequately prevent colonization and subsequent transmission (198, 199). Due to waning immunity, there has been a rise in pertussis cases in older children and adolescents (200). Collectively, vaccines that prevent colonization in addition to disease protection can break the chain of transmission and could be considered a gold standard as they provide both individual and herd immunity.

CONCLUDING REMARKS

Estimates based on statistical modeling suggest approximately 4.95 million deaths are associated with bacterial antimicrobial resistance, and this is predicted to increase to 10 million by 2050 (201). While the majority of the research on combating bacterial infections has focused on strategies that would mitigate disease states, blocking the chain of transmission for bacterial pathogens would significantly reduce the frequency of infections that require treatment. Developing strategies for blocking transmission requires foundational knowledge of the mechanisms used by pathogens for transmission, highlighting the interdependence of basic science and translational research. However, the capacity to identify bacterial factors specifically required for transmission from one host to another has received less focus than identifying factors essential for virulence. This is due, in large part, to the challenges of developing animal models for the study of transmission that appropriately recapitulate human-pathogen interactions.

While tractable animal models can provide valuable insight into the host-to-host transmission process, careful consideration must be made regarding their limitations, such as artificial conditions, applicability to human disease, and overall generalization of results. Combining animal studies with other methodologies, such as molecular epidemiological studies, computational and mathematical models, controlled human infection models (CHIM), and intervention studies, can enhance the robustness and applicability of research findings. Colonization and transmission studies using “rewilded mice” (laboratory mice exposed to a semi-controlled outside environment) that more closely mimic a complex environment that pathogens encounter could potentially yield critical insights into the transmission process (202). Furthermore, high-throughput mutagenesis and sequencing approaches, in combination with Collaborative Cross (CC) and Diversity Outbred (DO) mouse panels that provide powerful mouse genetic tools, have the potential to identify genetic loci that protect against pathogen acquisition and transmission to another host (203). It is understood that the microbiome and microbial products can provide colonization resistance against incoming pathogens. Experimental fecal microbiota transplantation (FMT) to reduce the burden of MDR pathogens in patients has also resulted in decreased fomite-mediated transmission (204). Utilizing animal models of transmission could offer deeper insights into this process and help identify specific bacteria and their products that can decrease pathogen burden and lower the occurrence of transmission events. In addition, how maternal microbiota affects their offspring’s microbiota through vertical transmission, and the role of diet and host genetics in the acquisition of bacterial pathogens, remains to be further explored. Vaccines have reduced the severity and frequency of disease, in part by reducing transmission. How vaccines modulate the host immune response and the molecular pathways that block the acquisition of pathogens can be tested using tractable animal models.

Collectively, these areas of research may provide valuable new insights for disease prevention. As pathogens constantly adapt and evolve new ways to subvert therapeutics, a deeper understanding of the transmission mechanisms may facilitate improved interventions and vaccines, resulting in reduced incidence of infections and the associated loss of life and financial burden of treatment.