Bordetella pertussis

Bordetella pertussis is a Gram-negative, aerobic, pathogenic, encapsulated coccobacillus bacterium of the genus Bordetella, and the causative agent of pertussis or whooping cough. Its virulence factors include pertussis toxin, adenylate cyclase toxin, filamentous hæmagglutinin, pertactin, fimbria, and tracheal cytotoxin.

The bacteria are spread by airborne droplets and the disease's incubation period is 7–10 days on average (range 6–20 days). Humans are the only known reservoir for B. pertussis. The complete B. pertussis genome of 4,086,186 base pairs was published in 2003. Compared to its closest relative B. bronchiseptica, the genome size is greatly reduced. This is mainly due to the adaptation to one host species (human) and the loss of capability of survival outside a host body.

Like B. bronchiseptica, B. pertussis can express a flagellum-like structure, even though it has been historically categorized as a nonmotile bacterium.

Taxonomy
The genus Bordetella contains nine species: B. pertussis, B. parapertussis, B. bronchiseptica, B. avium, B. hinzii, B. holmesii, B. trematum, B. ansorpii, and B. petrii.

B. pertussis, B. parapertussis and B. bronchiseptica form a closely related phylogenetical group. B. parapertussis causes a disease similar to whooping cough in humans, and B. bronchiseptica infects a range of mammal hosts, including humans, and causes a spectrum of respiratory disorders.

Evolution
The disease pertussis was first described by French physician Guillaume de Baillou after the epidemic of 1578. The disease may have been described earlier in a Korean medical textbook. The causative agent of pertussis was identified and isolated by Jules Bordet and Octave Gengou in 1906. It is believed that the genus Bordetella may have evolved from ancestors that could survive in the soil according to 16S rRNA gene sequencing data. 16S rRNA is a component of all bacteria that allows for the comparison of phyla within a sample. The expansion of human development into the agricultural field caused there to be an influx of human to soil contact. This increase not only created more advantageous environments for the ancestors of Bordetella not only to thrive in, but to spread to humans as well. Over time, Bordetella, like B. pertussis, has adapted to specifically infect humans and they are still able to multiply and thrive in soil conditions.

It was initially determined that B. pertussis is a monomorphic pathogen in which majority of strains found had the same two types of alleles: ptxA1 or ptxA2. Modern developments in genome sequencing have allowed B. pertussis to be studied more allowing for the discovery of the ptxP region. Through studying the gene, there has been evidence of mutations within the gene that show missing genomes present on the DNA strand. A study by Bart et al., revealed that 25% of the genes on the Tohama I reference strain of B. pertussis sequence were missing in comparison to the ancestral strains. These mutations were noted to be caused by an increase in intragenomic recombination with loss of DNA. Genes controlled by the BvgAS system have transformed B. pertussis into a much more contagious pathogen. In particular, strains with the ptxP3 allele, that developed through mutations in the recent years, have an increased expression of toxins. Ultimately, this leads to higher acuteness of the disease when contracted. This has causes an upwards trend of most cases of B. pertussis being the ptxP3 strain, especially in developing countries. Since the 1990s, most cases in developed countries such as the United States have ptxP3 isolates rather than the ptxA1 causing it to become the more dominant strain.

Growth requirements
Bordetella pertussis prefers aerobic conditions in pH range of 7.0-7.5, optimal to thrive in the human body. The max pH level for their growth was at a pH level of 8.0. The minimum pH range for minimal growth was at pH 6.0-6.5. The bacteria are not able to reproduce at pH levels lower than 5.0.

In addition, Bordetella pertussis favors a temperature range of 35 °C to 37 °C. It is a strict aerobe as mentioned previously and its nutritional requirements are meticulous in its requirement for nicotinamide supplement. It has been identified that growth of the bacteria is hindered in the presence of fatty acids, peroxide media, metal ions, and sulfides.

As a strict aerobe, the bacteria requires oxygen to grow and sustain. Such aerobes undergo cellular respiration to metabolize substances using oxygen. In such respiration, the terminal electron acceptor for the electron transport chain is oxygen. The organism is oxidase positive, but urease, nitrate reductase, and citrate negative.

Metabolism
B. pertussis presents unique challenges and opportunities for metabolic modeling, especially given its reemergence as a pathogen. Elevated glutamate levels were found to slow growth due to oxidative stress, revealing a complex relationship. This effect is compounded by observations suggesting that a small starting population could amplify oxidative stress through quorum sensing, a phenomenon deserving further investigation.

When B. pertussis is in a balanced that medium of lactate and glutamate that does not accumulate ammonium, a partially faulty citric acid cycle in B. pertussis and its ability to synthesize and break down β-hydroxybutyrate is observed. Cultivating B. pertussis in this medium resulted in some production of polyhydroxybutyrate but no excretion of β-hydroxybutyrate, indicating a more efficient conversion of carbon into biomass compared to existing media formulations.

In biofilm conditions, B. pertussis cells exhibited increased toxin levels alongside reduced expression of certain proteins, indicating a metabolic shift towards utilizing the full tricarboxylic acid (TCA) cycle over the glyoxylate shunt. These changes correlated with heightened polyhydroxybutyrate accumulation and superoxide dismutase activity, potentially contributing to prolonged survival in biofilms. The interplay between protein expression and metabolic responses highlights the intricate mechanisms influencing B. pertussis growth and adaptation. Despite a less negative energy profile compared to host tissues like the human respiratory system, B. pertussis efficiently couples biosynthesis with catabolism, sustaining robust growth even after extended incubation periods.

Host species
Humans are the only host species of B. pertussis. Outbreaks of whooping cough have been observed among chimpanzees in a zoo, and among wild gorillas; in both cases it is considered likely that the infection was acquired as a result of close contact with humans. Several zoos have a long-standing custom of vaccinating their primates against whooping cough.

Research shows that some primate species are highly sensitive to B. pertussis, and developed a clinical whooping cough in high incidence when exposed to low inoculation doses. Whether the bacteria spread naturally in wild animal populations has not been confirmed satisfactorily by laboratory diagnosis. In research settings, baboons have been used as a model of the infection although it is not known whether the pathology in baboons is the same as in humans.

Pertussis
Pertussis is an infection of the respiratory system characterized by a "whooping" sound when the person breathes in. B. pertussis infects its host by colonizing lung epithelial cells. The bacterium contains a surface protein, filamentous haemagglutinin adhesin, which binds to the sulfatides found on cilia of epithelial cells. Other adhesins are fimbriae and petractin. Once anchored, the bacterium produces tracheal cytotoxin, which stops the cilia from beating. This prevents the cilia from clearing debris from the lungs, so the body responds by sending the host into a coughing fit. B. pertussis has the ability to inhibit the function of the host's immune system. The toxin, known as pertussis toxin, inhibits G protein coupling that regulates an adenylate cyclase-mediated conversion of ATP to cyclic adenosine monophosphate. The result is that phagocytes convert too much adenosine triphosphate to cyclic adenosine monophosphate, causing disturbances in cellular signaling mechanisms, and preventing phagocytes from correctly responding to the infection. Pertussis toxin, formerly known as lymphocytosis-promoting factor, causes a decrease in the entry of lymphocytes into lymph nodes, which can lead to a condition known as lymphocytosis, with a complete lymphocyte count of over 4000/μl in adults or over 8000/μl in children. Beside targeting lymphocytes, it limits neutrophil migration to the lungs. It also decreases the function of tissue-resident macrophages, which are responsible for some bacterial clearance.

The infection of B. pertussis occurs mostly in children under the age of one since this is when they are unimmunized, or children with faded immunity, normally around the ages 11 through 18. The signs and symptoms are similar to a common cold: runny nose, sneezing, mild cough, and low-grade fever. The patient becomes most contagious during the catarrhal stage of infection, normally two weeks after the coughing begins. It may become airborne when the person coughs, sneezes, or laughs. The paroxysmal cough precedes a crowing inspiratory sound characteristic of pertussis. After a spell, the patient might make a "whooping" sound when breathing in, or may vomit. Transmission rates are expected to rise as the host experiences their most contagious stage when the total viable count of B. pertussis is at its highest. After the host coughs, the bacteria in their respiratory airways will be exposed into the air by way of aerosolized droplets, threatening nearby humans.

A human host can exhibit a range of physical reactions as a result of the B. pertussis pathogen, depending on how well their body is equipped to fight infection. Adults have milder symptoms, such as prolonged coughing without the "whoop". Infants less than six months also may not have the typical whoop. A coughing spell may last a minute or more, producing cyanosis, apnea, and seizures.

Transmission and infection
B. pertussis is a highly contagious infection of the respiratory tract. However, for B. pertussis to persist in a population the bacterium needs an uninterrupted chain of transmission as there are no animal reservoirs and the bacteria do not survive in the environment. B. pertussis primarily spreads through respiratory droplets, requiring direct contact between individuals due to its short survival time outside the body.

It was noted that between 1991 and 2008, there were 258 deaths for infants 8 months old and younger.

Progression of disease
Pertussis manifests in three distinct stages. The dynamic progression of pertussis, characterized by its distinct phases from incubation to paroxysmal coughing, underscores the complexity of the disease's clinical manifestations and highlights the potential significance of toxin release in driving symptoms.

Following exposure, an incubation period of 5–7 days ensues before symptoms appear.

The catarrhal phase follows, characterized by cold-like symptoms lasting about a week, with a high isolation rate of the organism. This phase transitions into the paroxysmal phase, where the dry cough evolves into a severe, paroxysmal cough with mucous secretion and vomiting.

The coughing fits, characterized by efforts to expel respiratory secretions, may result in a distinctive whooping sound. Recovery of the organism diminishes significantly during this phase. Although the organism is seldom detected in the blood, it is theorized that the clinical symptoms primarily stem from toxin release. The paroxysmal phase typically persists for a minimum of 2 weeks.

Diagnosis
A nasopharyngeal swab or aspirate can be sent to the bacteriology laboratory for Gram stain (Gram-negative, coccobacilli, diplococci arrangement), with growth on Bordet–Gengou agar or buffered charcoal yeast extract agar with added cephalosporin to select for the organism, which shows mercury drop-like colonies. Endotracheal tube aspirates or bronchoalveolar lavage fluids are preferred for laboratory diagnostics due to their direct contact with the ciliated epithelial cells and higher isolation rates of the pathogen.

Laboratory diagnostic methods used to identify B. pertussis:


 * 1) Serology
 * 2) Identification of specific agglutinating antibodies in the patient's blood serum with a high sensitivity and specificity rate.
 * 3) Able to detect the level of virulence and measure the immune response to the pathogen.
 * 4) Recommend those corresponding to the catarrhal phase of the illness. Not used in infants due to delay of positive results, often indicating the disease has progressed.
 * 5) Sparked the development of ELISA kits.
 * 6) Microbiological culture
 * 7) Known for high specificity, the ability to subtype the colonies presented and limited sensitivity. Ideal for antimicrobial resistant monitoring. Specificity results can be affected by age, immunization status, duration of symptoms, and even specimen handling.
 * 8) It is very difficult to cultivate separate pathogens and only high bacterial loads can lead to a positive culture. The ideal stage for isolation is the catarrhal stage or the beginning of the paroxysmal stage. Vaccinated persons also have a lower rate of isolation.
 * 9) Plates are incubated at 36 °C under high humidity for 7–10 days before obtaining results.
 * 10) Classical PCR assay
 * 11) Being the test of choice, this procedure is known for its quick and high sensitivity, however; often inaccurate when identifying between Bordetella species.
 * 12) The primers used for PCR usually target the transposable elements IS481 and IS1001.
 * 13) Recommend to be performed on infants and those corresponding to the catarrhal phase of the illness. It can detect the pathogens in atypical manifestations and vaccinated patients for longer periods, compared to the culture.
 * 14) Target genes within B. pertussis are IS481, IS1002, ptxS1, Ptx-Pr, and BP3385, however, B. bronchispeticaand B. holmesii contain similar gene expression, leaving it difficult to differentiate between the bacterium in the laboratory. The most effective technique to differentiate between the two bacteria is by human and animal isolates. Singleplex PCR identifies the target gene ptxS1.
 * 15) Direct Fluorescent Antibody Testing (DFA)
 * 16) Inexpensive and direct results of Bordetella detection with poor sensitivity and specificity. This test stains the nasopharyngeal secretions with a fluorescent modified antibody that binds directly to the B. pertussis or B. parapertussis bacteria. If positive, the binding antibody would glow under the microscope. Because of the low specificity, it is common to receive false positives with polyclonal antibodies occurring.

Several diagnostic tests are available, particularly the enzyme-linked immunosorbent assay ELISA kits. These are designed to detect filamentous hemagglutinin (FHA) and/or anti-pertussis-toxin antibodies of IgG, IgA, or IgM. Some kits use a combination of antigens which leads to a higher sensitivity, but might also make the interpretation of the results harder since one cannot know which antibody has been detected.

Misdiagnosis is common due to diagnostic techniques, misidentification between species in laboratories, and clinician error. The misdiagnoses between Bordetella species further increase the likelihood of antibiotic resistance. These factors highlight the need for a procedure to target all species through specific and fast methods.

Treatment
Whooping cough is treated by macrolides, for example erythromycin. The therapy is most effective when started during the incubation period or the catarrhal period. It is ideal for treatment should be within 1–2 weeks from onset of symptoms. When applied during the paroxysmal cough phase, the time of reconvalescence is not affected, only further transmission is reduced to 5–10 days after infection.

Prevention
Pertussis vaccine has been widely used since the second half of the 20th century. The first vaccines were whole-cell vaccines (wP), composed of chemically inactivated bacteria and given intramuscularly. When give, the inactive bacteria and antigens trigger the immune response and mimics natural infection.

Due to the frequent reports of reactions at the injection site, scientists started to replaced whole cell vaccines with acellular pertussis (aP) vaccines which have, recently, shown a decreased time of immunity and level of protection against colonization. These acellular vaccines are also intramuscular and are composed of purified surface antigens, mainly fimbriae, filamentous hemagglutinin, pertactin and pertussis toxin. Both vaccines are still used today, with the aP vaccine predominantly used in developed countries.

The aP vaccine is also a part of the diphtheria, tetanus, and acellular pertussis (DTaP) immunization. Those being administered these vaccines are recommended to receive boosters as they are only afford protection for about 4–12 years; while natural infection offers 7–20 years. Cases in infants are common and often have serious impacts as they are more susceptible to Bordetella pertussis than adolescents and healthy adults. Therefore, to decrease likelihood of contracting and spreading this disease, parents are recommended to receive the preventative vaccine. 

With the resurgence of pertussis cases, there are concerns regarding the level of protection provided by the current vaccine. This vaccine does not offer protection against other species of Bordetella such as B. holmesii and B. bronchiseptica and further highlights the need for a revamped vaccine. Research is currently developing a novel vaccine such as the BPZE1, which is a live attenuated vaccine against B. pertussis and challenges the other pathogens in the 'Classical Bordetellae'. This new vaccine inactivates the gene encoding 3 major toxins with only a single intranasal dose. It is currently being studied for safety in immunocompromised patients and pregnant women. There are other promising vaccines that are under study and in trial periods for accuracy, efficacy, and safety.