Microbiology: Mycoplasmas & Fastidious Gram-Negative Bacteria

Microbiology: Mycoplasmas & Fastidious Gram-negative Bacteria

Kimberly Watkinspg. 1

Slide 1

Today we are going to talk about the listed bacteria.

Slide 2

We will use the same formatas before in terms of talking about the organisms and their characteristics, the epidemiology of their diseases, how they produce diseases, and how we detect and identify them.

Slide 3

The first one we are going to talk about is Haemophilus, which means “blood-loving.” It is a gram-negative coccobacillus (seen on the gram stain). It is a facultative anaerobe and it is also non-hemolytic, which means that you have to provide it with things that it otherswise would get from the lysis of RBCs. We say that this organism isfastidious because most Haemophilus will not grow on un-supplemented blood agar since they are not hemolytic (they cannot get to the things that they need from RBCs). We use an enriched medium such as chocolate agar. Some of the strains are invasive and some are colonizing, primarily in the respiratory tract. In the upper respiratory tract (URT), the non-encapsulated strains of H. influenzae and other species are very commonly found. The encapsulated strains are the ones that tend to cause more severe disease.

Slide 4

These are organisms that primarily cause disease in the respiratory tract. They are transmitted through aerosols. In many causes, it is actually activation of endogenous disease. A typical example is someone with an underlying lung disease, such as chronic obstructive pulmonary disease (COPD), where you may be colonized with the organisms in the respiratory tract, even the non-encapsulated strains that are there. These are often times older people that have smoked that have impaired cilia activity, impaired cough reflex, maybe have chronic bronchitis, and have a lot of excessive secretions in the respiratory tract. These bacteria will then grow and proliferate (grow to very large numbers) and can cause symptoms such as fever and productive cough, so you have an exacerbation of chronic bronchitis that you see in that setting,

The major virulence factor for H. influenzae (prototype pathogen) is an anti-phagocytic capsule. There are several different capsule serotypes (at least 6) named in letters a-f forH. influenzae and it’s the serotype b that is the most significant pathogen. Like we have discussed with some of the other bacteria that have multiple serotypes that are immunologically distinct and in this case one of them tends to be more pathogenic than others. This was formerly an important cause of meningitis, epiglottis and bacteremia in young children who did not develop an antibody to protect them and this was the basis for the vaccine developed in the 1980s (all children get as part of their immunization). It is based on the antigen for the serotype b.

Since it gram-negative, it has endotoxin as part of the cell wall and is part of the pathogenesis. It damages the respiratory epithelium which can lead to bacteremic spread of the organism. Unlike some of the bacteria we have talked about before (staphylococci and streptococci), the Haemophilus do not produce any exotoxins of consequence; they mediate disease primarily through their endotoxin.

They do have an IgA protease and about 30% of the Haemophilus strains will produce a beta-lactamase enzymethat will break down penicillin. So non beta lactamase stable penicillin such as ampicillin will no longer work on those organisms.

Slide 5

If you are going to detect H. influenzae in the lab, you have to grow it on chocolate agar (RBCs have been heated so that they lyse and will release the things that the Haemophilus needs)> it will not grow on MacConkey agar. It is capnophillic (give it CO2). The Haemophilus species will vary on whether that will require the X and V factors. We put them on a nutrient agar plate (which the organism would otherwise not grow) and use filter paper strips that contain the X or V factor or both. In the lower right, you can see the haze of growth of the Haemophilus on the agar plate only around the X and the V and that is how you can tell that it isH. influenzae. Other species would require either X or V but not both. H. influenzae requires X (hemin) and V (NAD, an enzyme co-factor for catabolizing reactions). Something else that is seen sometimes in culture is if you put a S. aureus strain (strongly beta-hemolytic) on a blood agar plate it will hemolyze the RBCs and that will release the X and V factor and the Haemophilus will then grow around where the staph is growing. This is called satelliting (see satellite colonies in lower left).

Slide 6

H. influenzae is still an important cause of otitis media in children because this is usuallynot the type b encapsulated strains that cause this. The type b encapsulated strains have largely been eliminated as pathogens in children because of the H. influenzae vaccine but we still see this organism as a cause of disease in adults (in the non-encapsulated strains primarily) such as sinusitis, epiglottis and bacteremia. Epiglottis and bacteremia are not nearly as common as they used to be just because that was usually the invasive encapsulated type b strains and at least in children and in young adults we do not see these diseases very often. You can get laryngotracheobronchitis, meningitis (which is very rare because it is almost always the encapsulated type b strains that have been wiped out by the vaccine), and exacerbation of chronic bronchitis in COPD. It is also an important cause of community acquired pneumonia and also causes cellulitis and conjunctivitis.

Slide 7

Because of the vaccine, we hardly ever see the invasive disease in young children. The vaccine that is given now as part of the series to all infants is a polysaccharide based vaccine to the capsular serotype b. Polysaccharides are poor immunogens in young children so it is conjugated to a protein carrier to make it more immunogenic. Three to four doses are given in infancy. Since introduced over 90 % of invasive disease has been eliminated. However, this does not have any impact on the non-typeable (non-encapsulated) H. influenzae infections.

Slide 8

Some other Haemophilus species that are just for note. H. ducreyi is a cause of STD (not a common cause of STD in the US) and it produces a chancroid (painful genital ulcer) which can be mistaken for herpes virus infection. H. aegyptius (very similar to H. influenzae) is also a cause of bacterial conjunctivitis.

Slide 9

Bordetella pertussis is the cause of pertussis or whooping cough. It is an encapsulated gram-negative coccobacillus (seen in gram stain) that is slow-growing, fastidious and aerobic. It does not do a lot biochemically (does not utilize any sugars so it is not easily picked up in the lab). It generates metabolic energy by oxidizing amino acids.

Slide 10

Whooping cough is a very contagious disease and before the vaccine it was a very common disease encountered in pediatrics (could cause death especially in young infants). Generally there are 3 different stages after an incubation period of 5-21 days:

1. Catarrhal stage: cough and sneeze which gets worse and progresses to the second stage
2. Paroxysmal stage: you have a severe irritation of the airways in the URT because of the exotoxin produced and it causes necrosis of the airways and stimulates the coughing. You get very rapid coughing which makes it hard to breathe and then you take a very deep breath which sounds like a whooping sound. The child can literally turn blue because they are not oxygenating well due to the severe coughing.
3. Convalescent stage: after you get over it and your immunity develops can take several months to get back to normal. You can cough for a long time with this organism.

You will typically see lymphocytosis in the blood and if you ever have whooping cough you get a lifelong immunity (it simulates the immune system to make Abs against several of the toxins).

Slide 11

This is an old disease (known since the 16th century) and even though we vaccinate for it in the in US we do not have complete eradication. The vaccine that has been used in the past and the immunity wanes over time and then adults can get this disease as a result of their immunity waning, especially if you get exposed to non-vaccinated individuals. Worldwide there have been 285,000 cases (deaths) as of 2001. Also, in 2005 there were 25,000 cases in the US and it is increasing. There is no environmental or animal reservoir; it is person-to-person spread. Over half of the cases in the US are in adolescents and young adults (vaccinated as infants but their vaccine immunity has waned). If adults and adolescents are getting this disease, they can spread it to young children before they have been adequately immunized.

Slide 12

Data from Alabama over the past few years shows that it is a reportable disease that is increasing.

Slide 13

Why is it going up? Infants are being under-vaccinated (immigrants may not get their infants immunized and also no health insurance) and they can acquire the disease and spread it to other children as well as adults. The other reason is that physicians have been taught that this is a vaccine preventable illness and that everybody gets vaccinated and so you do not see this and they do not think about it when diagnosing patients. A person is very contagious until you get them out of circulation and on the proper antibiotics. In addition, with the older vaccine it sort of wears out in early adulthood (do not have enough Abs) and this is contributing to the reservoir.

Slide 14

How do you get the disease? For successful bacteria to cause disease, they have to have a way to attach to the host. Pertussis has a number of different structures that allows it to attach and affiliate with the epithelium of the URT. You have the filamentous hemagglutinin (FHA) and the pertussis toxin (PTx), which are part of the attachment moieties. The toxin not only helps attach but also damages the cells. Once the bacteria get in, they have to get the host immune system blocked in some way so it does not get rid of them. One of things that the pertussis does is it impairs chemotaxis of WBCs and then the exotoxins damage the respiratory epithelium and it can help it spread throughout the body. The main disease that you get is in the respiratory tract.

Slide 15

There are a lot of different toxins. Ido not know that you have to learn the details of these toxins but the main things to remember are 1) there are several of them 2) this is an exotoxin-mediated disease 3) toxins not only facilitate the attachment but also the local damage to the cell over the respiratory tract and stimulate the host immune system and 4) are the basis for the vaccine. These toxins actually inhibit the leukocyte migration and phagocytosis, cause necrosis of the respiratory tract epithelium, stimulate the release of interleukins (why you get the fever: IL-1 is a mediator of fever) and also the endotoxin (LPS). So the bug hits you with two different things: all the exotoxins and (since it is gram-negative) the endotoxin. The endotoxin activates the alternate complement pathway and stimulates cytokine release, which accounts for some of the systemic effects that can spread beyond the respiratory tract.

Slide 16

If you encounter someone that you suspect has pertussis, the diagnostic test of choice is first to collect a nasopharyngeal swab (all the way in the posterior nasopharynx because that is where it is replicating). You have to have a special media, such as the Bordet-Gengou or Regan-Lowe that is enriched with horse blood and charcoal. You incubate it in a moist environment and then identify by immunoflourescence or agglutination. This will not be done in a community hospital lab because it is not seen often (would be sent to reference lab). You can measure antibody titers but is retrospective. PCR is recommended to augment the sensitivity of culture (enhance the diagnosis). A direct fluorescent antigen test (DFA) is done on NP secretions but like so many DNA based tests you have to have a very high organism load to detect (since it is uncommon this test is not very good).

Slide 17

The old vaccine contained whole heat-killed organisms that presumably had all the antigens for the toxins that you would make Abs against. People had a lot of side effects to the vaccine because you were injecting whole killed bacteria and your body was reacting to a lot of things that were not really the way the organisms were mediating disease. In 1996, a new acellular vaccine was developed called the DaPT (D: diphtheria, aP: acellular pertussis, T: tetanus) that is given in 5 doses (beginning at 2 months up to the time of school age). The acellular vaccine was developed to contain primarily just the toxins that cause disease (help prevent the attachment of the organism and the introduction of disease, the cell damage). Because of the concern of the resurgence of pertussis and the waning immunity, there is a new vaccine for adults and adolescents (ages 11-64) as a single dose. This is separate from the tetanus toxoid vaccine. The Td protects against tetanus and diphtheria but not against pertussis and is recommended every ten years.

Slide 18

Legionella is a very fastidious (why it was not identified until the 70s), catalase-negative and facultative intracellular organism. It does not gram stain very well (very pale because it does not take up the saffarin well). It is non-fermentative (so you will not see sugar fermentations). There are over 30 different species and multiple serogroups.

Slide 19

Came to attention with the convention in 1976 of WWII veterans; they knew that there was something else causing the pneumonia; developed a medium to grow the organism in vitro. The organism was infected through air conditioning. It lives in water systems, cooling systems, and A/C. It is a somewhat opportunistic organism; tends to cause severe disease in people with underlying lung disease. We normally think of pneumonia as being contagious from person to person. Legionellais usually not spread from person to person; it is spread from an environmental source. The organism is inhaled, complement deposits on the bacteria and then the bacteria bind macrophages. Since it is facultative intracellular it lives in the macrophage and it is not killed because it prevents the phagosome and the lysosome from fusing. It lives and multiplies in the phagosome and produces enzymes. The cell dies and the bacteria are released. When the cells die in the respiratory tract, it stimulates inflammation and also coughing. You can get a purulent pneumonia with abscesses because of the intense stimulation of the host immune response and bacteria growing in the cells. Fairly high mortality if it is not treated.

Slide 20

To culture Legionella we can use a medium with buffered charcoal yeast extract and cysteine (why it looks black). It may take several days for them to grow. A tracheal aspirate or a lung biopsy tends to be more reliable than sputum to get the organism out. It is grown very rarely in the hospital lab. If it grows on the agar, it is sent off to be identified by agglutination or immunofluorescence.

Slide 21

There are other ways to detect Legionella. You can stain lung specimens with silver or also a direct florescent antibody (it is not a good test unless you have lots of organisms). A test that is commonly used is to test for Legionella antigen that has spilled over into the urine. The infection can become systemic and after several days you will start excreting the antigen in the urine. Culture is the only method that will pick up all the species. Antibody paired serum can be measured for Legionella pneumophila but will not pick up some of the other species. PCR is also being looked at as a way to pick up Legionella.

Slide 22

If you take all the community acquired pneumonias (CAP), maybe 5-10% is due to Legionella. The unique things about Legionella as a cause of CAP is point source outbreaks. You may have a lot of people that come down with this at one time if they have been exposed to the same source but it is not generally contagious from person to person. It is used associated with water. In addition, you can get vague non-specific complaints such as diarrhea and kidney problems because this organism doe spread throughout the body and can cause damage. The main people that tend to get this are older men with obstructive lung disease and transplant recipients that are immunosuppressed (not really seen in young children or otherwise healthy individuals). Because of the unique epidemiology, it is more common in the summer.

Slide 23

There is not a vaccine. Cell mediated immunity seems to be more important than Ab mediated immunity and is why transplant recipients that have had a depressed cellular immunity because of suppression tend to get it. The way to prevent it is to monitor the sources of contamination and you clean them or eliminate them.