Saturday, May 27, 2006
The pneumococcus, a type of bacteria in the Streptococcus family (Streptococcus pneumoniae), is responsible for several types of diseases in adults and in children. In developing countries, pneumococcus is linked to high infant and child mortality rates. Even in more industrialized nations, pneumococcal diseases are common in adults and children. These diseases can lead to severe illness and even death, despite being preventable in many cases. The April 12, 2006, issue of JAMA includes an article about pneumococcal diseases and the beneficial effect of pneumococcal vaccination in infants and children.
TYPES OF PNEUMOCOCCAL DISEASE
Meningitis (infection of the covering membranes of the brain and spinal cord)
Pneumonia (infection of the lungs)
Otitis media (middle ear infection)
Sinusitis (infection of the sinus cavities behind the face)
Bacteremia (bacteria in the bloodstream)
Sepsis (a serious bloodstream infection that can be fatal)
Meningitis can lead to permanent neurological damage or death. In adults and older children, it presents with fever, a stiff neck, fatigue, and headache. In infants, these signs may be absent, and the infant may only display fussiness, lethargy, and inability to feed. Individuals with pneumonia usually have shortness of breath, high fever, a cough, fatigue, and chest pains. Meningitis and sepsis are the most life-threatening pneumococcal diseases. Individuals with meningitis or sepsis must receive rapid diagnosis and treatment to prevent severe multiple organ failure and death.
Early diagnosis of pneumococcal infection is crucial to receiving proper antibiotic treatment. Some strains of pneumococcus have developed resistance to commonly used antibiotics. Laboratory testing is needed to determine proper therapy. Supportive therapy such as oxygen, intravenous fluids, surgical drainage, or even life support in an intensive care unit may be required in severe cases of pneumococcal infection.
Young children (aged 2 months to 24 months) should receive a special type of vaccine called the pneumococcal conjugate vaccine against pneumococcus. It is specifically formulated for effectiveness in infants and younger children.
Any person who has had a splenectomy (removal of the spleen) should have the pneumococcal vaccine.
The polysaccharide pneumococcal vaccine should be considered for individuals older than 65 years and for those older than 2 years who have chronic diseases.
Frequent handwashing is important for preventing the spread of pneumococcal diseases, as well as other bacterial and viral infections.
FOR MORE INFORMATION
Additional information available:
Patient Page Index on JAMA's Web site .
Many are available in English and Spanish. A Patient Page on meningitis in children was published in the April 28, 1999, issue; one on diagnosing and treating pneumonia was published in the February 9, 2000, issue; and one on fever in infants was published in the March 10, 2004, issue.
Sources: National Institute of Allergy and Infectious Diseases; Centers for Disease Control and Prevention; American Academy of Family Physicians; American Academy of Pediatrics
The JAMA Patient Page is a public service of JAMA. The information and recommendations appearing on this page are appropriate in most instances, but they are not a substitute for medical diagnosis. For specific information concerning your personal medical condition, JAMA suggests that you consult your physician.
This page may be photocopied noncommercially by physicians and other health care professionals to share with patients. To purchase bulk reprints, call 203/259-8724
JAMA Patient Page - April 12, 2006
Saturday, May 20, 2006
Bacterial Skin Infections
Three forms of impetigo are recognized on the basis of clinical, bacteriologic, and histologic findings. The lesions of common or superficial impetigo may contain group A b-hemolytic streptococci, S aureus, or both, and controversy exists about which of these organisms is the primary pathogen. The lesions have a thick, adherent, recurrent, dirty yellow crust with an erythematous margin. This form of impetigo is the most common skin infection in children. Impetigo in infants is highly contagious and requires prompt treatment.
The lesions in bullous (staphylococcal) impetigo, which are always caused by S aureus, are superficial, thin-walled, and bullous. When a lesion ruptures, a thin, transparent, varnish-like crust appears which can be distinguished from the stuck-on crust of common impetigo. This distinctive appearance of bullous impetigo results from the local action of the epidermolytic toxin (exfoliation). The lesions most often are found in groups in a single reglon.
Ecthyma is a deeper form of impetigo. Lesions usually occur on the legs and other areas of the body that are generally covered, and they often occur as a complication of debility and infestation. The ulcers have a punched-out appearance when the crust or purulent materials are removed. The lesions heal slowly and leave scars.
Cellulitis and Erysipelas
Streptococcus pyogenes is the most common agent of cellulitis, a diffuse inflammation of loose connective tissue, particularly subcutaneous tissue. The pathogen generally invades through a breach in the skin surface, and infection is fostered by the presence of tissue edema. Cellulitis may arise in normal skin. However, the lesion of cellulitis is erythematous, edematous, brawny, and tender, with borders that are poorly defined.
No absolute distinction can be made between streptococcal cellulitis and erysipelas. Clinically, erysipelas is more superficial, with a sharp margin as opposed to the undefined border of cellulitis. Lesions usually occur on the cheeks.
Staphylococcal Scalded Skin Syndrome
Staphylococcal scalded skin syndrome (SSSS), also called Lyell's disease or toxic epidermal necrolysis, starts as a localized lesion, followed by widespread erythema and exfoliation of the skin. This disorder is caused by phage group II staphylococci which elaborate an epidermolytic toxin. The disease is more common in infants than in adults.
Folliculitis can be divided into two major categories on the basis of histologic location: superficial and deep.
The most superficial form of skin infection is staphylococcal folliculitis, manifested by minute erythematous follicular pustules without involvement of the surrounding skin. The scalp and extremities are favorite sites. Gram-negative folliculitis occurs mainly as a superinfection in acne vulgaris patients receiving long-term, systemic antibiotic therapy. These pustules are often clustered around the nose. The agent is found in the nostril and the pustules. Propionibacterium acnes folliculitis has been misdiagnosed as staphylococcal folliculitis. The primary lesion is a white to yellow follicular pustule, flat or domed. Gram stain of pus reveals numerous intracellular and extracellular Gram-positive pleomorphic rods. The lesions are more common in men than in women. The process may start at the age when acne usually appears, yet most cases occur years later.
In deep folliculitis, infection extends deeply into the follicle, and the resulting perifolliculitis causes a more marked inflammatory response than that seen in superficial folliculitis. In sycosis barbae (barber's itch), the primary lesion is a follicular pustule pierced by a hair. Bearded men may be more prone to this infection than shaven men.
A furuncle (boil) is a staphylococcal infection of a follicle with involvement of subcutaneous tissue. The preferred sites of furuncles are the hairy parts or areas that are exposed to friction and macerations. A carbuncle is a confluence of boils, a large indurated painful lesion with multiple draining sites.
Erysipeloid, a benign infection that occurs most often in fishermen and meat handlers, is characterized by redness of the skin (usually on a finger or the back of a hand), which persists for several days. The infection is caused by Erysipelothrix rhusiopathiae.
Pitted keratolysis is a superficial infection of the plantar surface, producing a punched-out appearance. The pits may coalesce into irregularly shaped areas of superficial erosion. The pits are produced by a lytic process that spreads peripherally. The areas most often infected are the heels, the ball of the foot, the volar pads, and the toes. Humidity and high temperature are frequent aggravating factors. Gram-positive coryneform bacteria have been isolated from the lesions.
Erythrasma is a chronic, superficial infection of the pubis, toe web, groin, axilla, and inframammary folds. Most lesions are asymptomatic, but some are mildly symptomatic with burning and itching. The patches are irregular, dry and scaly; initially pink and later turning brown. The widespread, generalized form is more common in warmer climates. Corynebacterium minutissimum is the agent. Because of its small size, the organism is difficult to observe in KOH preparations of infected scales; however, it is readily demonstrable by Gram staining of the stratum corneum. Coral red fluorescence of the infected scales under Wood's light is diagnostic.
Trichomycosis involves the hair in the axillary and pubic regions and is characterized by development of nodules of varying consistency and color. The condition is generally asymptomatic and not contagious. Underlying skin is normal. Infected hairs obtained for microscopic examination are placed on a slide in a drop of 10 percent KOH under a coverslip. The nodules on the hairs are composed of short bacillary forms.
Three types of coryneforms are associated with trichomycosis; one resembles C minutissimum, one is lipolytic, and the third is C tenuis.
Intertrigo is most commonly seen in chubby infants or obese adults. In the skin fold, heat, moisture, and rubbing produce erythema, maceration, or even erosions. Overgrowth of resident or transient flora may produce this problem.
Acute Infectious Eczematoid Dermatitis
Acute infectious eczematoid dermatitis arises from a primary lesion such as a boil or a draining ear or nose, which are sources of infectious exudate. A hallmark of this disease is a streak of dermatitis along the path of flow of the discharge material. Coagulase-positive staphylococci are the organisms most frequently isolated.
Pseudofolliculitis of the Beard
Pseudofolliculitis of the beard, a common disorder, occurs most often in the beard area of black people who shave. The characteristic lesions are usually erythematous papules or, less commonly, pustules containing buried hairs. This occurs when a strongly curved hair emerging from curved hair follicles reenters the skin to produce an ingrown hair. Gram-positive microorganisms that belong to the resident flora are associated with this disordera clear illustration of the opportunism of nonpathogenic bacteria when the host defense is impaired.
Toe Web Infection
The disease commonly referred to as athlete's foot has traditionally been regarded as strictly a fungal infection. This assumption has been revised, however, because fungi often cannot be recovered from the lesions throughout the disease course. Researchers now believe that the dermatophytes, the first invaders, cause skin damage that allows bacterial overgrowth, which promotes maceration and hyperkeratosis. The fungi, through the production of antibiotics, then create an environment that favors the growth of certain coryneform bacteria and Brevibacterium. Proteolytic enzymes, which are produced by some of these bacteria, may aggravate the condition. If the feet become superhydrated, resident Gram-negative rods become the predominant flora, and the toe webs incur further damage. The fungi are then eliminated either by the action of antifungal substances of bacterial origin or by their own inability to compete for nutrients with the vigorously growing bacteria.
Other Bacterial Skin Diseases
Skin Tuberculosis (Localized Form)
Localized skin tuberculosis may follow inoculation of Mycobacterium tuberculosis into a wound in individuals with no previous immunologic experience with the disease. The course starts as an inflammatory nodule (chancre) and is accompanied by regional lymphangitis and lymphadenitis. The course of the disease depends on the patient's resistance and the effectiveness of treatment. In an immune or partially immune host, two major groups of skin lesions are distinguished: tuberculosis verrucosa and lupus vulgaris.
Mycobacterium marinum Skin Disease
Many cases of M marinum skin disease occur in children and adolescents who have a history of using swimming pools or cleaning fish tanks. Often, there is a history of trauma, but even in the absence of trauma the lesions appear frequently on the sites most exposed to injury. The usually solitary lesions are tuberculoid granulomata that rarely show acid-fast organisms. The skin tuberculin test is positive.
Mycobacterium ulcerans Skin Disease
Lesions in M ulcerans skin disease occur most often on the arms or legs and occasionally elsewhere, but not on the palms or soles. Most patients have a single, painless cutaneous ulcer with characteristic undermined edges. Geographic association of the disease with swamps and watercourses has been reported. In some tropical areas, chronic ulcers caused by this organism are common.
In scrofuloderma, tuberculosis of lymph nodes or bones is extended into the skin, resulting in the development of ulcers.
A disseminated form of the disease occurs when bacteria are spread through the bloodstream in patients who have fulminating tuberculosis of the skin. When hypersensitivity to tubercle bacilli is present, hematogenously disseminated antigen produces uninfected tuberculous skin lesions such as lichen scrofilloslls.
There are several agents of actinomycetoma. About half of the cases are due to actinomycetes (actinomycetoma); the rest are due to fungi (eumycetoma). The most common causes of mycetoma in the United States are Pseudallescheria (Petriel lidium) boydii (a fungus) and Actinomyces israelii (a bacterium). Regardless of the organism involved, the clinical picture is the same. Causative organisms are introduced into the skin by trauma. The disease is characterized by cutaneous swelling that slowly enlarges and becomes softer. Tunnel-like sinus tracts form in the deeper tissues, producing swelling and distortion, usually of the foot. The draining material contains granules of various sizes and colors, depending on the agent.
Actinomyces israelii usually is the agent of human actinomycosis; Arachnia propionica (Actinomyces propinicus) is the second most common cause. The characteristic appearance of the lesion is a hard, red, slowly developing swelling. The hard masses soften and eventually drain, forming chronic sinus tracts with little tendency to heal. The sinus tracts discharge purulent material containing "sulfur" granules. In about 50 percent of cases, the initial lesion is cervicofacial, involving the tissues of the face, neck, tongue, and mandible. About 20 percent of cases show thoracic actinomycosis, which may result from direct extension of the disease from the neck or from the abdomen or as a primary infection from oral aspiration of the organism. In abdominal actinomycosis, the primary lesion is in the cecum, the appendix, or the pelvic organs.
Saturday, May 13, 2006
Proof That Viruses Cooperate with Bacteria in Producing Disease
In many experiments using animal models, bacterial infections have been superimposed at various times after inoculation of viruses. Enhancement of bacterial infection was demonstrated by comparisons of the bacterial contents of appropriate tissues with those of animals receiving bacteria alone. Exacerbation of disease was shown by more-severe lesions or higher death rates than for animals receiving either the bacteria or viruses alone.
The classical investigation using an animal model was that of Shope in 1931 (94) using influenza virus and H. influenzae in pigs. However, mice have been the experimental animals used for most investigations. Examples are as follows. Influenza virus enhanced respiratory infections with pneumococci (36,59,97), staphylococci (43), Listeria monocytogenes (28), group B streptococci (49), and Bacillus thuringiensis (39). Sendai virus enhanced respiratory infections with Mycoplasma pulmonis (41). Reovirus enhanced staphylococcal infection (53), and cytomegalovirus enhanced P. aeruginosa infection (33). Observations for other animal models included increased infection with H. influenzae in RSV-infected cotton rats (71), the effect of influenza virus on colonization of the nasopharynx of chinchillas by different phenotypes of S. pneumoniae (102), and enhanced streptococcal infection following influenza virus infection of ferrets (10, 29).
Neonatal ferrets infected with influenza virus provided an animal model for SIDS. An intranasally administered virulent strain (clone 7a) killed the neonates, and the pathology of some was akin to that seen in SIDS. In contrast to the virulent strain, killing of the neonates by a less virulent strain (PR8) could be prevented by antibiotic treatment, indicating that it was due to virus exacerbation of naturally acquired bacterial infection (42).
For obvious reasons, humans have not been deliberately infected with respiratory viruses followed by bacterial pathogens to study the progress of subsequent bacterial infection as described above for animal models. However, the effect of experimental influenza A virus infection of volunteers on selection of pathogens from the natural nasopharyngeal flora has been studied; S. pneumoniae was not isolated from any subject prior to virus challenge but was isolated in substantial numbers from 15% of the subjects on the sixth day following challenge (105).
Also, cells and fluids have been harvested, either from patients with natural infections or volunteers experimentally infected with viruses, and interacted with pathogenic bacteria in vitro in biological tests related to infection. For example, S. aureus, H. influenzae, and S. pneumoniae showed enhanced adherence to pharyngeal cells obtained from volunteers infected with influenza virus compared with cells from uninfected controls (23). Other examples are given below.
More information about this book is available from the ASM Press.
Saturday, May 06, 2006
A Novel Bacterium Associated with Lymphadenitis in a Patient with Chronic Granulomatous Disease
PLoS Pathog. 2006 April; 2(4): e28.
Published online 2006 April 14. doi: 10.1371/journal.ppat.0020028.
Copyright This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
David E Greenberg,1 Li Ding,1 Adrian M Zelazny,2 Frida Stock,2 Alexandra Wong,2 Victoria L Anderson,1 Georgina Miller,3 David E Kleiner,4 Allan R Tenorio,5 Lauren Brinster,3 David W Dorward,6 Patrick R Murray,2 and Steven M Holland1*
1 Laboratory of Clinical Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, United States of America
2 Microbiology Service, Department of Laboratory Medicine, W. G. Magnuson Clinical Center, National Institutes of Health, Bethesda, Maryland, United States of America
3 Diagnostic and Research Services Branch, National Institutes of Health, Bethesda, Maryland, United States of America
4 Laboratory of Pathology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, United States of America
5 Section of Infectious Diseases, Department of Medicine, Rush Medical College, Chicago, Illinois, United States of America
6 Microscopy Branch, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, Montana, United States of America
Pascale Cossart, Editor
Institut Pasteur, France
* To whom correspondence should be addressed. E-mail: firstname.lastname@example.org
Received April 14, 2005; Accepted March 2, 2006.
AbstractChronic granulomatous disease (CGD) is a rare inherited disease of the phagocyte NADPH oxidase system causing defective production of toxic oxygen metabolites, impaired bacterial and fungal killing, and recurrent life-threatening infections. We identified a novel gram-negative rod in excised lymph nodes from a patient with CGD. Gram-negative rods grew on charcoal-yeast extract, but conventional tests could not identify it. The best 50 matches of the 16S rRNA (using BLAST) were all members of the family Acetobacteraceae, with the closest match being Gluconobacter sacchari. Patient serum showed specific band recognition in whole lysate immunoblot. We used mouse models of CGD to determine whether this organism was a genuine CGD pathogen. Intraperitoneal injection of gp91phox −/− (X-linked) and p47 phox −/− (autosomal recessive) mice with this bacterium led to larger burdens of organism recovered from knockout compared with wild-type mice. Knockout mouse lymph nodes had histopathology that was similar to that seen in our patient. We recovered organisms with 16S rRNA sequence identical to the patient's original isolate from the infected mice. We identified a novel gram-negative rod from a patient with CGD. To confirm its pathogenicity, we demonstrated specific immune reaction by high titer antibody, showed that it was able to cause similar disease when introduced into CGD, but not wild-type mice, and we recovered the same organism from pathologic lesions in these mice. Therefore, we have fulfilled Koch's postulates for a new pathogen. This is the first reported case of invasive human disease caused by any of the Acetobacteraceae. Polyphasic taxonomic analysis shows this organism to be a new genus and species for which we propose the name Granulobacter bethesdensis.
As new bacteria continue to be discovered every year, it is inevitable that some of them will be found to cause human disease. The authors describe the isolation and characterization of a new bacterium, grown from a patient with chronic granulomatous disease (CGD). In this genetic disease, one of the main lines of defense against infection, the neutrophil, has a discrete defect in the generation of superoxide, leading to recurrent infections with a narrow spectrum of bacteria and fungi. This new organism was cultured from lymph nodes that had been inflamed for several months. To prove that this new bacterium was indeed a pathogen, Greenberg and colleagues measured specific antibody response in the patient: they inoculated CGD mice with this organism and reproduced the appearance of the human infection; they recovered the organism in pure growth from infected mouse spleens.
This new bacterium belongs to the family Acetobacteraceae, bacteria that are found widely in the environment. They have a variety of industrial uses, such as the production of vinegar, but have never been reported to cause invasive human disease. Disease-causing organisms remain to be discovered. The researchers outline some of the steps that can be taken to verify the pathogenicity of novel organisms.Introduction
Chronic granulomatous disease (CGD) is a rare inherited disease of the phagocyte NADPH oxidase system that leads to defective production of toxic oxygen metabolites and impaired killing of certain microbes . Clinically, patients develop recurrent life-threatening infections with catalase-producing organisms as well as tissue granuloma formation . The bacteria and fungi that commonly cause infection in CGD include: Staphylococcus aureus, Serratia marcescens, Burkholderia cepacia, Nocardia and Aspergillus species [3–7]. Rare infections with organisms only encountered in CGD, such as Paecilomyces sp. and Trichosporon inkin, suggest that patients with CGD have a unique susceptibility pattern . However, when a specific microbiologic diagnosis cannot be made, patients are frequently treated with broad-spectrum antimicrobials that cover the most likely pathogens.
In the course of evaluation of fever and lymphadenitis in a patient with CGD, we identified a novel gram-negative rod. This organism belongs to a family of bacteria that has not been reported to cause invasive human disease. To prove that this organism was the cause of this patient's clinical syndrome, we utilized classical methods first described by Robert Koch in the late 19th century , supplemented with modern immunologic and genetic approaches. When faced with new potential pathogens, there remain situations in which Koch's postulates are as necessary to understand the etiology of infections as they were over a hundred years ago.Case Resport
The patient was a 39-year-old man with X-linked chronic granulomatous disease (CGD) complicated by multiple episodes of S. aureus pneumonia and Aspergillus pneumonia, and a history of S. aureus liver abscesses that required drainage. He had remained healthy on trimethoprim-sulfamethoxazole alone for many years. In April 2003 he was hospitalized for fever and chills after returning from a trip to the Bahamas. Blood and urine cultures, cytomegalovirus serologies, cryptococcal antigen, and a transthoracic echocardiogram were not diagnostic. He had evidence of remote Epstein-Barr virus infection. A computed tomography scan of his chest showed new necrotic subcarinal and right hilar lymph nodes and two calcified granulomata in the right lower lobe. Inpatient intravenous vancomycin, ceftazidime, and voriconazole followed by outpatient oral voriconazole and trimethoprim-sulfamethoxazole were not curative. One month later, while on his outpatient regimen, repeat blood, sputum, and urine cultures were again negative. Cerebrospinal fluid examination was unremarkable. Gallium and Indium scans showed increased uptake in the right cervical and anterior mediastinal lymph nodes. Mediastinoscopy with biopsy of carinal lymph nodes showed caseating granulomatous inflammation. Calcofluor white, AFB (acid-fast bacillus), and gram stains were negative as were fungal and bacterial cultures. In addition, AFB cultures were negative after eight weeks. Because of three months of fever, chills, and 10-lb weight loss despite empiric therapy, the patient was referred to the National Institutes of Health.
MRI of the chest showed mediastinal lymphadenopathy in the precarinal, subcarinal, and hilar regions with extensive necrosis (in Figure 1, see Figure 1A). Fevers, chills, fatigue, and night sweats persisted despite addition of azithromycin and moxifloxacin. Five months after symptom onset, he developed new painful left supraclavicular lymphadenopathy. Given the lack of response to empiric antimicrobials and the lack of microbiologic diagnosis, four involved left cervical and supraclavicular lymph nodes were removed. Pathology showed extensive necrotizing pyogranulomatous lymphadenitis with microabscess formation (Figure 1B and 1D). Fungal, gram, and AFB stains on sectioned lymph nodes were negative. A Warthin-Starry stain showed scant coccobacillary forms (Figure 1C). A novel gram-negative rod grew from three lymph node cultures on buffered charcoal yeast extract agar plates after four days of incubation.
The patient was initially treated in the fall of 2003 with doxycycline, meropenem, and a steroid taper. His remaining lymph nodes decreased in size over subsequent months. However, fever and enlarging lymphadenopathy returned in the fall of 2004. Since that time, he has required cervical lymph node excisions on four separate occasions. In May, June, and November of 2005, this new organism was again cultured from excised lymph nodes.Results
Gram-negative rods were isolated from three of the four cervical lymph node cultures, on buffered charcoal yeast extract plates (Remel, Lenexa, Kansas, United States) after four days incubation at 35 °C. The organism was a non-motile coccobacillus that was catalase-positive and oxidase-negative. Lysine and ornithine decarboxylases and arginine dihydrolase all were negative. Urease was positive; there was weak fermentation of glucose, and there was no acid production from lactose, mannitol, sucrose, maltose, or xylose. These results were confirmed at the Centers for Disease Control and Prevention (L. Helsel, personal communication). The API 20 NE (BioMerieux, Durham, North Carolina, United States) and RapID NH (Remel) commercial kits generated codes 0200000 and 1501, respectively, neither of which yielded an identification.
In view of the failure of conventional tests to identify the organism and its fastidious growth characteristics, we sequenced the 16S rRNA gene and searched for related sequences in GenBank. The best 50 matches by BLAST search were all members of the family Acetobacteraceae, with the closest match being Gluconacetobacter sacchari (95.7% similarity) and Gluconacetobacter liquefaciens (95.5% similarity). The isolate from May of 2005 also had 16S sequencing performed and was identical to the original isolate. The 16S rDNA sequence similarity between the patient isolate and other commonly encountered pathogenic gram-negative rods was very low (~80%) (Figure 2).
Initial attempts to perform antibiotic susceptibility testing by microdilution and by a disk susceptibility test failed due to poor growth in broth and on Mueller-Hinton agar, respectively. Therefore, susceptibility testing was done on tryptic soy agar (TSA) sheep blood agar plates by the E-test. Minimum inhibitory concentrations (MICs) read after 48 h and 96 h of incubation at 35 °C showed the following results: ciprofloxacin (MIC > 32), azithromycin (MIC > 256), cefepime (MIC > 256), ceftriaxone (MIC = 16), meropenem (MIC > 32), and chloramphenicol (MIC > 256); amikacin showed an MIC = 32. At 48 h incubation, doxycycline showed MIC = 16 and trimethoprim-sulfamethoxazole showed MIC = 0.38; however, at 96 h of incubation, resistance to both drugs was observed.
The isolation of a previously unknown gram-negative rod from a patient with a rare immune disorder does not immediately prove causality. Contamination can occur during surgery or in the laboratory. To confirm the biological relevance of the isolated organism to our patient, we sought to determine whether this organism was immunologically and immunopathologically linked to his disease.
To prove that this organism stimulated an immune response in our patient, whole bacterial lysates were exposed to unfractionated patient plasma in an immunoblot. Four distinct bands were seen at approximately 38 kDa, 45 kDa, 47 kDa, and 62 kDa. To confirm unique band recognition, neither related organisms (Acetobacter estunensis, Gluconacetobacter xylinus) nor unrelated organisms (E. coli, Proteus mirabilis) showed similar band patterns when incubated with the patient's serum (Figure 3A). In addition, sera stored from the patient years prior to the onset of his symptoms showed no reactivity on immunoblot (courtesy of Dr. Mary Dinauer, Figure 3B). To further characterize the recognized bands, two-dimensional gels of bacterial protein extracts were performed. Proteins detected with patient plasma by immunoblot were subjected to peptide mass fingerprinting (unpublished data). To further identify antigens being recognized on the two-dimensional gel immunoblots, protein extracts were run over a column coated with purified patient IgG. These two methods identified at least one of the proteins as a methanol dehydrogenase (69 kDa; EC 188.8.131.52). The recent completion of the genome sequence of this novel organism allowed us to specifically match this recognized peptide to this organism. The amino acid sequence of the recognized methanol dehydrogenase is shown in Figure 4. Studies to identify the other immunoreactive peptides are under way. Unique immunoblot protein recognition continues to be evident at dilutions of patient plasma of 1:100,000. Of interest, immunoblot patterns similar to those of this patient against this novel organism have been found in 3/19 patients with CGD and in 1/20 healthy individuals tested, respectively.
In addition to immunoblots, scanning immunoelectron microscopy and immunotransmission electron microscopy were performed. Immunotransmission electron microscopy images were taken after incubating colonies with either patient or normal plasma followed by gold-labeled anti-human IgG. Whereas bacteria incubated with plasma from normal humans failed to label with colloidal gold, those probed with plasma from the patient were clearly decorated with gold particles (Figure 5).
Having confirmed that the patient had an immune response to the cultured organism, we sought to determine whether it showed pathogenicity in CGD. We used mouse models of CGD to determine whether this organism caused infection in the setting of CGD, and, if so, was it pathologically similar to that seen in our patient. gp91phox−/− (X-linked) and p47phox−/− (autosomal recessive) mice were injected with various amounts of organisms (102 to 108 CFU) i.p. and then organs and lymph nodes were harvested for culture and pathology at different time points. gp91phox−/− and p47phox−/− mice yielded histopathology similar to that seen in our patient's lymph nodes (Figure 6). In contrast, wild-type mice did not develop pyogranulomatous abscesses in the lung or lymph nodes, although a few demonstrated lymphoid hyperplasia. Splenomegaly was noted in both wild-type and CGD mice injected with the organism, but to a greater degree in the knockout mice. At all time points examined, organism recovery from spleen homogenates was greater in the CGD mice compared with wild-type mice. When 107 CFU of bacteria were injected i.p. into ten wild-type and ten gp91phox−/− mice, nine days post-inoculation the mean CFU/spleen in the wild-type mice was 11,550 compared with 41,720 in the gp91phox−/− mice (Figure 7) (p < .001; two-tailed t-test). Gram stains, growth characteristics, and specific antibody recognition of the bacteria isolated from organ homogenates were similar to those of the patient's original culture. To prove that the pathologic organism recovered from the animals was the same as the one isolated from the patient, the 16S sequence was determined and was identical to the original isolate.
To determine if the pathology observed in CGD mice was unique to this new organism, or whether other members of the Acetobacteraceae could cause a similar disease, we injected six CGD and four wild-type mice with this new organism (107 CFU) and another six CGD and five wild-type mice with another member of the Acetobacteraceae family, G. oxydans (ATCC 621H). Mice were sacrificed at nine days. All the mice, both wild-type and CGD, injected with the new bacterium showed pathologic changes in the lymph nodes ranging from moderate hyperplasia to the presence of plasmacytosis, atypical macrophages and lymphadenitis, although the changes in the wild-type mice were comparatively mild. In contrast, only 2/6 CGD mice and 2/5 wild-type mice injected with G. oxydans showed any pathologic changes, and these were limited to mild lymphoid hyperplasia and plasmacytosis. When taken together, the new bacterium caused more severe pathology when compared with another member of the Acetobacteraceae.Discussion
When we had identified a novel gram-negative rod from a CGD patient with fever and lymphadenitis, our first concern was to determine whether this organism was truly associated with his clinical syndrome. This question is critically important, insofar as this is a previously unrecognized organism without a body of literature to inform us as to its frequency or behavior.
Given that we have an animal model for CGD, we were able to utilize rules that were developed by Koch over one hundred years ago as he was trying to prove the etiology of tuberculosis. Although Koch's postulates cannot be applied in every situation, they are invaluable in the proof of causality. The isolation of this organism from our patient's diseased lymph nodes, its ability to cause similar disease when introduced into mouse models of CGD, the ability to isolate the same organism from these mice, and redemonstration of its identity prove that this novel bacterium in fact caused disease in our patient.
In addition to using an animal model to confirm pathogenicity of this organism in CGD, further evidence of its having infected this patient was demonstrated by his seroconversion from negative to high titer antibody. Screening of plasmas from other patients with CGD and people without it suggests that the bands at 38 kDa and 62 kDa may be characteristic of the human response to this organism. The recently completed genome sequence of this organism will prove valuable in determining the immunoreactive bands that are specific for this bacterium. When infected, both CGD and wild-type mice develop antibody. However, only CGD mice recapitulate the human immunopathology, confirming that this is a genuine pathogen in CGD.
This novel bacterium caused fever, lymphadenitis, and weight loss over almost six months in a patient with X-linked CGD, but was not fatal. Even at inocula as high as 108, there were no deaths among the mice used in our challenge studies. We followed animals up to four months after infection; all mice cleared the infection spontaneously over time, as indicated by decreasing colony counts from organ homogenates. Interestingly, although pathologic changes were seen in the infected CGD mice, a few of the normal mice had low-level positive cultures from spleen homogenates for up to 76 days. Both CGD and normal mice produced antibody, as did our patient. The repeated isolations of this organism from our patient suggest that antibody production alone is not protective. It remains unclear whether the repeated isolation of this bacterium from our patient's diseased lymph nodes represents relapse or reinfection from as yet unidentified environmental reservoir(s). In either case, given its apparent low virulence, further investigation of this organism may shed light on the mechanisms of microbial killing in CGD. Comparing the genome of this organism to other available genomes of CGD pathogens may provide insight into common virulence properties.
Since the initial acceptance of this manuscript, we have isolated Granulobacter bethesdensis from two more individuals with CGD who presented with similar clinical syndromes. G. bethesdensis was confirmed with similar biochemical and molecular techniques, and the isolates were shown to be molecularly distinct.
These are the first reported cases of invasive human disease caused by any of the Acetobacteraceae. The family Acetobacteraceae comprises several genera including the genus Gluconacetobacter and Acetobacter. The closest 16S rRNA gene sequence match was to G. sacchari at 95.7%. However, strains that have a 16S rDNA sequence similarity below 97% are not considered the same species . The sequence similarity of two species of Gluconacetobacter and two species of Acetobacter are shown in Figure 2. A polyphasic taxonomic approach shows that this organism belongs to a new genus and species in this family (unpublished data). Members of the genera Gluconacetobacter, Acetobacter, Gluconobacter, and Acidomonas  have been called the acetic acid bacteria, because they derive energy from the aerobic oxidation of ethanol to acetic acid. They have been cultured from fruits, fermented foods, plants, soil, and water [11–14], are utilized industrially in the production of vinegar, and are encountered during the fermentation of wine [15–18]. Given that this novel pathogen was isolated from a patient with CGD at the National Institutes of Health, we propose the name Granulobacter bethesdensis.
Patients with CGD are susceptible to infections caused by catalase-producing organisms. It is thought that organisms that produce catalase scavenge their own hydrogen peroxide and thus deprive the CGD phagocyte of the opportunity to use oxidative metabolites against it . However, many organisms are catalase-positive and do not cause disease in CGD. In fact, the list of catalase-positive organisms that cause disease in CGD is quite narrow. This suggests that there are mechanisms beyond catalase that confer pathogenicity upon organisms that infect patients with CGD. It is hoped that the addition of a new organism to the CGD-specific infection list will allow further characterization of these virulence pathways. Many questions remain regarding the clinical epidemiology of this newly identified organism, such as its environmental reservoir and its seroprevalence in other disease states. Given that the Acetobacteraceae are ubiquitous environmental organisms, it is striking that they have never been reported to cause invasive disease, although a case of peritonitis associated with Asaia bogorensis in a patient with a peritoneal dialysis catheter has been recently reported . The difficulty in culturing this organism from pathologic specimens, and the apparent seropositivity even among healthy people, suggest that there have been other encounters with this organism that have not been found previously. The clinical manifestations of this pathogen in other patients with CGD, and potentially in patients without CGD, are important subjects for future investigation.
Material and Methods
The growth of the isolate was assessed on various media under different incubation conditions. The organism grew best at 35°C on Acetobacter Medium  with the following modifications: glucose (50.0 g/L), CaCO3 (12.5 g/L), autolyzed yeast (5.0 g/L), agar (15.0 g/L). Colonies growing on this modified Acetobacter Medium developed a yellow color after 5 days. Good growth after 4–5 days of incubation was also obtained on buffered charcoal yeast extract agar and on TSA with sheep blood (Remel). Growth was not improved in a CO2 atmosphere.
Commercial biochemical kits, supplementary phenotypic tests, and sequencing were used to identify the isolate. Commercial kits used included the API 20 NE (BioMerieux) and the RapID NH (Remel). Additional phenotypic tests included oxidase, catalase, oxidative-fermentative medium with different sugars (OF medium King, Remel), lysine and ornithine decarboxylases, arginine dihydrolase, urease test (Rapid Urea Slant, Hardy Diagnostics, Santa Maria, California, United States), and motility. Susceptibility testing was performed on TSA with sheep blood by the E-test (Amersham Biosciences Biodisk, Piscataway, New Jersey, United States).
Sequencing and phylogenetic analysis.
The full 16S rRNA gene was sequenced using the MicroSeq Full Gene 16S rRNA Bacterial Isolation Sequencing Kit (Applied Biosystems, Foster City, California, United States), according to the manufacturer's protocol, and sequences were analyzed using the 3100 Genetic Analyzer (Applied Biosystems). The Lasergene program (version 5.51; DNASTAR, Madison, Wisconsin, United States) was used for sequence assembly and alignment. The assembled 16S rDNA sequence from the patient isolate was compared with 16S rDNA sequences available in GenBank databases using the standard nucleotide–nucleotide basic local alignment search tool (BLASTn) program (National Center for Biotechnology Information, Bethesda, Maryland, United States). Multiple sequence alignments of the 16S rRNA gene sequences from the isolate and the closest GenBank matches were done by the CLUSTAL W method.
Phylogenetic analyses were performed with the PHYLIP version 3.5c package . Distance matrices based on Kimura's two-parameter model were produced with DNADIST program, and a neighbor-joining tree constructed with the NEIGHBOR program. The resulting unrooted tree was depicted using the TreeView version 1.4 package .Immunology studies: Preparation of protein extracts, SDS gel electrophoresis, and immunoblot analysis.
Bacterial cells were pelleted by centrifugation and extracted with the Pierce (Rockford, Illinois, United States) bacterial protein extraction reagent. Protein concentrations were determined using the Pierce protein assay. 2μg of total protein per lane were electrophoresed on 10% SDS–PAGE, and the separated proteins transferred to PVDF membrane (Invitrogen Life Technologies, Carlsbad, California, United States). The membrane was blocked in TBS buffer containing 5% nonfat dried milk and 0.05% Tween-20 before incubation with human or mouse serum (1:100 to 1:100,000 dilution). After three washes, the membrane was incubated with horseradish peroxidase conjugated goat anti-human or goat anti-mouse IgG (1:10000 to 1:20000; Amersham Biosciences, Little Chalfont, United Kingdom). The blots were developed using the enhanced chemiluminescence kit (ECL Plus, Amersham Biosciences) as described by the manufacturer.
Challenge studies: Mice.
C57BL/6 wild-type (Jackson Lab, Bar Harbor, Maine, United States) and gp91phox−/− (Jackson Lab) and p47phox−/− (Taconic, Hudson, New York, United States) mice extensively backcrossed to the C57BL/6 background [23,24] were maintained at the National Institute of Allergy and Infectious Diseases animal facility under specific pathogen-free conditions. All experiments were approved by the Institute's Animal Care and Use Committee. Mice were 4–8 mo old and sex-matched for each set of experiments.Mouse infection and sample collection.
The organism propagated from the patient's cervical lymph node cultures served as the source of the bacteria used in the mouse challenge studies. For these challenge studies, bacterial colonies growing on TSA with sheep blood were used to inoculate trypticase soy broth which was incubated for 10–14 days at 37 °C with 5% CO2. Bacteria were harvested by centrifugation and the bacterial pellet was washed twice in Hanks' balanced salt solution. Bacterial density was determined spectrophotometrically at 650 nm. Concentrations of bacteria from 2 × 108 to 7 × 108 CFU were serially diluted in Hanks' balanced salt solution and used to infect mice, and simultaneously plated on trypticase soy agar + 5% sheep blood plates (Remel) to quantify the inoculum. Mice were injected i.p. with 0.1 ml diluted bacteria. At 2, 9, 20, 76, and 126 days after challenge, blood was collected either retro-orbitally or from the tail into sterile tubes for culture and serum separation. Mice were then sacrificed by CO2 asphyxiation. Spleens and selected lungs were homogenized in 0.5 ml PBS. In one of the mouse experiments, spleens were homogenized in an amount of PBS proportional to the spleen's weight, so CFU/spleen could be determined. Samples of blood, spleen, or lung homogenates were serially diluted and inoculated in duplicate onto TSA with 5% sheep blood plates and incubated at 37 °C with 5% CO2 for 96–120 h, and colonies were enumerated. For histopathologic analysis, 3 ml of 10% phosphate-buffered formalin was injected into each lung; the inflated lungs and specified lymph nodes (cervical, axillary, and inguinal) were fixed in 10% phosphate-buffered formalin before section and staining.
Accession NumbersGenBank (http://www.ncbi.nlm.nih.gov/Genbank) accession numbers are: 16S rDNA sequence from the novel gram-negative rod G. bethesdensis (AY7889500); G. liquefaciens (X75617); and G. sacchari (AF127412).
Drs. Mary Dinauer and John Curnutte for referral of the patient to the National Institutes of Health.
CFU - colony-forming units
CGD - chronic granulomatous disease
MICs - minimum inhibitory concentrations
PBS - phosphate-buffered saline
TSA - tryptic soy agar
Author contributions. DEG, AMZ, and SMH conceived and designed the experiments. DEG, LD, AMZ, FS, and AW performed the experiments. DEG, LD, AMZ, GM, DEK, LB, DWD, PRM, and SMH analyzed the data. LD, AMZ, FS, and AW contributed reagents/materials/analysis tools. DEG, VLA, ART, and SMH cared for patients. GM, DEK, and LB reviewed pathology. DEG, AMZ, and SMH wrote the paper.
Funding. This research was supported by the Intramural Research Program of the NIH, NIAID.
Competing interests. The authors have declared that no competing interests exist.
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