Case 1 introduction

Diagnosis of community-acquired pneumonia is made clinically based on symptoms of cough, especially with blood, and chest x-ray indicating infiltrates, cavitary lesions, or pleural effusions. Specific diagnosis of pneumonia is made by culture of expectorated sputum. Sputum samples must be of good quality (many white blood cells and rare squamous epithelial cells) represent the flora of the lower respiratory tract and not mouth flora. In a small percentage of cases of community-acquired pneumonia blood cultures will also be positive for the affecting organism.

Treatment of K. pneumoniae pneumonia would be based on the susceptibility of the isolate. Treatment can be complicated by the presence of multidrug-resistant strains. Most strains are susceptible to extended spectrum cephalosporins such as cefepime as well as fluoroquinolones such as gatifloxacin. In cases of strains that produce an extended spectrum -lactamase, the treatment of choice would be imipenem.

[12.1] The most common mechanism by which Klebsiella pneumoniae attains its antibiotic resistance is by plasmid acquisition. Which of the following best describes the direct transfer of a plasmid between two bacteria?

[12.2] A specimen of thick, bloody sputum from a hospitalized 80-year-old patient with diabetes mellitus and difficulty in breathing is sent for laboratory analyses. The tests yield heavy growth of a lactose-positive, nonmotile, gram-negative rod with a large capsule. Which of the following bacteria is most likely to be the cause of the pulmonary problems?

[12.3] A 65-year-old diabetic man presents to the emergency room with a severe productive cough producing thick bloody sputum resembling a "currant-jelly" like appearance. Culture using MacConkey agar reveals pink colonies, with large mucoid colonies on routine laboratory media. Which of the following organisms is most likely responsible for this patient's pneumonia?

[12.4] The O antigens that are used to help characterize members of the Enterobacteriaceae family are found on which of the following?

[12.1] B. The three important processes by which DNA is transferred between bacteria are via transformation, transduction, and conjugation. Transformation is defined as the uptake of soluble DNA by a recipient cell. Transduction refers to the transfer of DNA by a virus from one cell to another. Conjugation refers to the direct transfer of soluble DNA (plasmids) between cells. Examples of such plasmids are the sex factors and the resistance (R) factors.

[12.2] C. Whereas all of the above listed organisms are gram-negative rods, only Klebsiella pneumoniae fulfill all of the laboratory criteria listed in the question, such as the presence of a very large capsule, which gives a striking mucoid appearance to its colonies.

[12.3] C. Patients with K. pneumoniae infections usually have predisposing conditions such as alcoholism, advanced age, chronic respiratory disease, and diabetes. The "currant-jelly" sputum distinguishes K. pneumoniae from the other organisms. Infections can lead to necrosis and abscess formation. Please refer to the discussion for Question 12.2.

[12.4] B. There are three surface antigens associated with several members of the Enterobacteriaceae. The cell wall antigen (somatic or O antigen) is the outer polysaccharide portion of the lipopolysaccharide (LPS/endotoxin). The H antigen is on the flagellar proteins (Escherichia and Salmonella). The capsular or K polysaccharide antigen is particularly prominent in heavily encapsulated organisms such as Klebsiella.
CASE 13

INTRODUCTION

An 18-day-old female infant is brought to the pediatric emergency room by her panicked mother. The child has developed a fever and has been crying nonstop for the past 4 hours. She has fed only once today and vomited all of the ingested formula. The baby was born by vaginal delivery after an uncomplicated, full-term pregnancy to a healthy 22-year-old gravida₁ para₁ (one pregnancy, one delivery) woman. The mother has no history of any infectious diseases and tested negative for group B Streptococcus prior to delivery. The immediate postpartum course was routine. The baby had a routine check-up in the pediatrician's office 3 days ago, and no problems were identified. On exam, the child has a temperature of 38.3C (100.9F), pulse of 140 beats per minute, and respiratory rate of 32 breaths per minute. She is not crying at the moment. She has poor muscle tone, will not regard your face or respond to loud stimuli. Her anterior fontanelle is bulging. Her mucous membranes are moist, and her skin is without rash. Her heart is tachycardic but regular, and her lungs are clear. Her white blood count is elevated, a urinalysis is normal, and a chest x-ray is clear. A Gram stain of her cerebrospinal fluid (CSF) from a lumbar puncture shows gram-positive coccobacilli.

 What organism is responsible for this infection?

 How does this organism avoid antibody-mediated host defenses?

 Organism responsible for this infection: Listeria monocytogenes.

This cycle is then repeated in the new host cell, allowing Listeria to spread without being exposed to antibodies or other humoral immunity factors. For this reason, host cellular immunity factors protect against infection and those with impaired cellular immunity are vulnerable.

Clinical diagnosis is difficult based on the nonspecific signs and symptoms. Clinically Listeria meningitis in neonates resembles group B Streptococci, both are a significant cause of bacteria meningitis in that age group. Definitive diagnosis of Listeria is made by culture of cerebrospinal fluid (CSF) and/or blood. Gram stain of the CSF would demonstrate small gram-positive bacilli, appearing similar to corynebacteria or Streptococcus pneumoniae.

Culture of Listeria from food may require cold enrichment, which would enhance the growth of Listeria. Food samples would be sent to a public health laboratory where some of the food would be enriched in a selective broth media at room temperature or lower.

Treatment of Listeria septicemia or meningitis is with ampicillin plus or minus gentamicin. Of significance is the inherent resistance of Listeria to cephalosporins, which are commonly chosen as empiric therapy for meningitis in adults and would be appropriate for treatment of streptococcal meningitis in children. Prevention involves the avoidance of the consumption of undercooked foods, especially in high-risk patients.

[13.1] A 22-year-old medical student suffers diarrhea for more than a week since his return from a short vacation in Mexico. While in Mexico, he consumed a large quantity of raw cheeses almost every day. Which of the following is the most likely organism causing his diarrhea?

[13.2] A premature neonate suffers pneumonia and sepsis. Sputum culture on blood agar plate yields pinpointed beta-hemolytic colonies. Which of the following is a simple test to determine whether the organism is Streptococcus agalactiae or Listeria monocytogenes (these two organisms are important neonatal pathogens)?

[13.3] The most frequent source of infection with Listeria monocytogenes is through which of the following?

[13.1] C. Outbreaks of gastroenteritis, as a consequence of Listeria monocytogenes, are related to the ingestion of unpasteurized milk products, for example, cheese. Bacillus cereus, causing food poisoning, is associated with spore survival and germination when rice is held at warm temperatures. Escherichia coli is usually associated with the enterohemorrhagic Escherichia coli (EHEC) form (verotoxin) that causes a bloody diarrhea and is associated with improperly cooked hamburger. Salmonella causes a diarrhea associated with contaminated chicken consumption. Finally, Shigella is usually associated with enterocolitis outbreaks among children in mental institutions and daycare centers.

[13.2] B. Streptococcus agalactiae (group B streptococci) is the leading cause of neonatal sepsis and meningitis. All streptococci (including Streptococcus agalactiae) are catalase-negative, whereas Staphylococci are catalase-positive. Listeria monocytogenes is also catalase-positive.

[13.3] C. Unpasteurized milk is a common vector for transmission of Listeria. See answer to question [13.1].

A 50-year-old man, a recent immigrant from Vietnam, is brought to the emergency room with a cough productive of bloody sputum. He first noticed a cough about 2 months ago, but there was not much sputum. In the past several days his sputum production has increased and become mixed with blood. He reports having lost approximately 15 lb in this time frame as well. He also notes that he's had drenching night sweats 2 or 3 nights a week for the past month. He has a 50-pack-year smoking history but no other medical history. He came to the United States from Vietnam 7 months ago. On examination, he is a thin, frail-appearing male. His vital signs are normal. His head and neck exam is normal. He has no palpable adenopathy in his neck or axilla. His lung exam is notable only for decreased breath sounds diffusely. A chest x-ray shows a cavitary infiltrate of the left upper lobe.

 What type of organism is likely to be seen on Gram stain of a sputum sample?

 What technique of staining is most commonly used to identify this organism?

 What is the histologic characteristic of Langhans' cells?

ANSWERS TO CASE 14: Mycobacteria

 Likely Gram stain findings of sputum sample: M. tuberculosis appear as colorless ("ghost") cells.

 Most commonly used staining technique for M. tuberculosis: acid-fast staining.

 Histologic characteristics of Langhans' cells: multinucleated giant cells of fused macrophages.

It is estimated that M. tuberculosis infects approximately one-third of the world's population. M. tuberculosis is spread from person to person via aerosolized respiratory droplets that travel to the terminal airways. The bacteria are phagocytized by alveolar macrophages but inhibit destruction by the phago-some and proceed to replicate. Circulating macrophages are attracted to the site of infection and create multinucleated giant cells, composed of fused macrophages called Langhans cells. Extrapulmonary sites are infected through the spread of infected macrophages via lymphatic or hematogenous dissemination. Because of the intracellular nature of M. tuberculosis infections, antibody mediated defenses are relatively ineffective. Persons with conditions of reduced cellular immunity, such as AIDS, alcoholism, or drug abuse, or persons living in crowded, close quarters, such as prisoners, are at increased risk for infection. Organisms can remain dormant in granulomas for many years and reactivate following immunosuppression at a later date. At that time the organisms usually infect extrapulmonary sites.

 Langhans cells: Multinucleated giant cells composed of fused macrophages.

In general, Mycobacterium species are slow-growing organisms, with a generation time of 1520 hours, compared to about 1 hour for most bacteria. Mycobacterium can be divided into groups as characterized by Runyon, based on their growth characteristics, particularly pigment production. The photo-chromogens, which are pigmented only in the presence of light, include M. kansasii, as well as other saprophytic Mycobacteria. The scotochromogens, which are pigmented even without the presence of light, include M. szulgai, as well as the nonpathogenic M. gordonae, which has an orange pigment. The nonchromogens are not pigmented in the light or dark including M. avium-intracellulare, as well as M. haemophilum. The fourth runyon group is composed of the rapidly growing Mycobacteria such as M. fortuitum, M. chelonae, and M. abscessus. The M. tuberculosis complex includes M. tuberculosis, M. africanum, M. ulcerans, and M. bovis, as well as other rarely identified Mycobacterium. These colonies appear buff or tan color and are dry when growing on Lowenstein-Jensen agar. M. leprae is not considered in that classification because it cannot be cultured in the laboratory.

One of the virulence factors of M. tuberculosis is cord factor. This can be visualized microscopically as organisms grown in broth culture will demonstrate a ropelike pattern indicating cording. The rapid growing Mycobacterium include M. fortuitum complex, M. chelonae complex, and M. abscessus, as well as other uncommonly isolated nonpathogenic Mycobacteria. These organisms by definition will grow within seven days of subculture onto routine microbiological media such as a blood agar plate

Diagnosis

Diagnosis of tuberculosis is initially made based on a history (exposure to patient with tuberculosis, immigration, a stay in a jail or homeless shelter) and physical exam in patients with a productive cough, night sweats, and fever. A positive PPD test would indicate exposure to M. tuberculosis and warrant further testing with chest x-ray. Patients with the characteristic upper lobe cavitary lesion would have sputum collected and cultured for Mycobacterium.

A fluorescent, direct smear, of the respiratory specimen after decontamination to remove bacterial flora is reported within 24 hours of receipt of the respiratory specimen in the laboratory. Several first early morning deep cough specimens should be collected. Growth of M. tuberculosis on Lowenstein-Jensen (LJ) agar can take 38 weeks because of the slow dividing time of the organism; however, with the use of broth medium growth time has been decreased to as short as 1 week. Newly designed automated broth systems read bottles for growth based on CO2 production of the organisms on a daily basis for up to eight weeks. Bottles which are determined to be positive are stained by Kinyoun stain to visualize the presence of Mycobacteria. Positive bottles can be tested directly for M. tuberculosis, M. kansasii, M. avium-intracellulare, or M. gordonae using DNA probes. Other Mycobacteria species are identified either by routine biochemical tests, which require several weeks, or by high-pressure liquid chromatography, which can speciate Mycobacterium based on mycolic acids extracted from their cell surface. Optimal growth temperature for Mycobacterium species is 35C (95F); however, the Mycobacterium that infect the skin such as M. haemophilum grow best at lower temperatures, and organisms such as M. szulgai prefer 42C (107.6F). Growth on solid media is also enhanced in the presence of 510% CO².