UNIVERSITY OF COLORADO SCHOOL OF MEDICINE DEPARTMENT OF PATHOLOGY RESIDENCY TRAINING PROGRAM MICROBIOLOGY VIII: Miscell

1. UNIVERSITY OF COLORADO SCHOOL OF MEDICINE DEPARTMENT OF PATHOLOGY RESIDENCY TRAINING PROGRAM MICROBIOLOGY VIII: Miscellaneous Gram Negative Bacilli--1 CASE STUDY #1: Recurrent Fever

2. CASE STUDY I: Recurrent Fever The case study presented here occurred in one of our local hospitals, serving as a teaching exercise for future reference. A 40-year old man developed intermittent low-grade fever and a general feeling of tiredness and weakness that occurred about 2 weeks following a family gathering in his home town in rural Mexico. Blood cultures were drawn during one of the fever spikes. A positive culture did not “alert” until after 5 days of incubation. Although considered by the clinical staff to be a contaminant, the specimen was subcultured to blood agar (see next screen). The one relevant note of clinical history was the ingestion of home-made cheese prepared with raw, unpastuerized milk.

3. CASE STUDY I: Recurrent Fever Blood Culture Isolate The colonies growing on the surface of the blood agar plate illustrated in the photograph were observed after 48 hours incubation. They are relatively small, measuring 0.5 - 1.0mm in diameter. They are entire, convex, and glistening, with no evidence of hemolysis.

4. CASE STUDY I: Recurrent Fever Gram Stain Features The bacterial cells observed in a gram stain prepared from an isolated colony were tiny, averaging 0.5 - 0.7 x 0.6 - 1.5um in diameter, appearing as Gram-negative coccobacilli. (see photograph). The appearance of the bacterial cells as observed in direct gram stains of clinical materials has been described as “grains of sand”.

5. CASE STUDY I: Recurrent Fever Recommendation It is the recommendation of Public Health laboratories that all slowly growing isolates with the appearance of tiny gram-negative cocco-bacilli, particularly if poorly staining, be referred immediately to them for work up, bypassing attempts to make an in-house definitive identification. In this case, the clinical history should have provided an additional clue. The slow-growing isolate was considered to be a contaminant. Several attempts were made to make the laboratory identification of the blood culture isolate; however, it did not key out on any of the commercial identification systems. It was ultimately sent out for definitive identification. Two days later the report of Brucella species was received. As indicated in the algorithm on the following page, a rapid urease reaction is key to making the identification. This isolate was a very weakly urease positive on Christensen’s agar, with the reaction so weak and delayed that a false negative reaction occurred in the commercial system used.

6. ABBREVIATED IDENTIFICATION OF Brucella species Slow growth of tiny gray-white, shiny colonies on BA Tiny pale-staining cocco-bacilli on gram stain Positive cytochrome oxidase reaction: Spot test Rapid hydrolysis of urea: reaction in 1- 4 hours on Christians agar History of close contact with animals or ingestion of raw animal products History of recurrent fevers Suspect Brucella species Consult public health laboratory immediately

7. CASE STUDY I: Recurrent Fever Follow Up Since this isolate had been worked up on the open bench in the laboratory, and because of the known ease of inter-human communicability, each of the laboratory microbiologists were put on a 5-day course of antibiotics. Again to reiterate, the recovery of a slowly growing, poorly staining gram-negative cocco-bacillus is considered to be suspicious for an agent of bioterrorism and should be referred to a public health laboratory shortly after recovery. Brucella species has been recognized as one of the more common bacterial agents involved in laboratory-acquired infections.

8. Brucellosis: Review Brucellosis is a zoonotic disease caused by any of four Brucella species: B. abortus, B. melitensis, B. suis, and B. canis. Brucellosis is endemic in many animal species. Animals acquire disease either sexually or by ingesting contaminated milk or other animal products. Humans most commonly develop disease following ingestion of contaminated milk or milk products (goat cheese, for example). Cutaneous infections occur following direct contact with abraded animal skin. Human infection is less commonly acquired through the conjunctiva or by inhalation. Person to person transmission occurs only rarely, if at all. In the United States, brucellosis is an occupational disease, most commonly in abittoir workers, butchers, farmers, and veterinarians.

9. Brucellosis: Clinical Presentations Incubation period for the onset of until clinical signs of acute brucellosis is between 7 and 21 days after exposure. The infective dose = 10 - 100 organisms. The onset of disease may be insidious--low grade fever, malaise, weakness, fatigue, headache, backache and myalgies. There may be a paucity of physical findings--splenomegaly (10 - 20% of patients), lymphadenopathy (15% of patients), and hepatomegaly (<10%). Because of the ability of the organisms to persist intra-cellularly, the acute disease usually extends into chronic illness with periods of remission interspersed with recurrent episides of fever, chills, malaise, and other symptoms mentioned above ("undulant fever").

10. Brucellosis: Pathology Localized disease may involve almost any organ system--osteomyelitis, splenic abscess, genitourinary tract, pulmonary disease, and endocarditis are among the most common infections. Osteomyelitis usually involves the vertebrae--disc space abscesses are also common. Splenic abscesses, with the formation of circumscribed granulomas that may undergo secondary calcification, often are found. Endocarditis is the most common cause of mortality among patients with chronic disease. Skin lesions--animal handlers, particularly veterinarians, are prone to develop cutaneous lesions.

11. Brucellosis: Pictorial Retrospective The skin lesions evolve as an erythematous macular, papular, or pustular lesion on the hands and arms at the sites of direct exposure to infected animal material. The lesions illustrated in the photograph are from an archival slide collection depicting cutaneous brucellosis on the arm of a farmer who hand-milked cows.

12. Brucellosis: Pictorial Retrospective Localized disease may involve almost any organ system, including the spleen, in which multiple granulomas may be seen. The granulomas in the photograph range from 1 - 3 cm in diameter. Initially, multiple splenic abscesses may be observed, that later develop into circumscribed granulomas that characteristically undergo secondary fibrosis and calcification.

13. Brucellosis: Pictorial Retrospective The reaction in lymph nodes, spleen, liver, and other organs is granulomatous. Although caseous necrosis per se is rarely observed, focal areas of stellate necrosis may be observed, with festooning of macrophages at the periphery , the so-called Splendore-Hoeple effect (blue arrow).

14. Brucellosis: Pictorial Retrospective The granulomatous reaction often also is characterized by the presence of numerous multi-nucleated giant cells (blue arrows). This reaction occurs because of the ability of organisms to remain viable intra-cellularly Organisms are difficult to demonstrate with tissue gram stains. Culture is necessary to establish a diagnosis in most instances. Pathologists should include chronic brucellosis in the differential diagnosis of all granulomatous lesions with histology as seen in the photograph.

15. UNIVERSITY OF COLORADO SCHOOL OF MEDICINE DEPARTMENT OF PATHOLOGY RESIDENCY TRAINING PROGRAM MICROBIOLOGY VIII: Miscellaneous Gram Negative Bacilli--I CASE STUDY #2: Pancreatic Abscess Original Presentation: CACMLE Teleconference Jan 22, 1992 Elmer W. Koneman, M.D., Paul C. Schreckenberger, Ph.D. William Janda, Ph.D. Microbiology Laboratory University of Illinois/Chicago

16. Pancreatic Abscess CASE HISTORY The case presented here is from a report by Bullock and Devitt (J Infection 3:32-85, 1981) of a 29 year-old alcoholic male who developed sudden onset of septicemia related to a necrotic, purulent pancreatic abscess. The authors indicate in the paper that septic complications of acute pancreatitis are not uncommon. Their review of the literature indicates that 90% of pancreatic abscesses are culture positive, with E. coli, Klebsiella, Bacteroides, Pseudomonas, Proteus, Staphylococcus and Streptococcus species being the bacterial agents most commonly recovered. Blood cultures were drawn and became positive in 72 hours with the organism presented here.

17. Pancreatic Abscess Illustrated in this photograph is the appearance of the colonies on the surface of selective haemophilus recovery culture media, supplemented with X & V factors and bacitracin. The colonies are off-white yellow, entire, flat to slightly convex, smooth, and measuring 0.5 mm to 2.0 mm in diameter after 48 hours incubation at 35o C. Hemolysis is not observed.

18. Pancreatic Abscess Illustration of enhanced growth on the surface of a chocolate agar plate. Classic chocolate agar, in which erythrocytes are lysed by the heat treatment during preparation, release Factor V and Factor X in abundance, suitable for the recovery of Haemophilus species from clinical specimens. Commercial chocolate agar, prepared by synthetically adding derived Factors X and V, also support the growth of Haemophilus species. This isolate was identified as Haemophilus segnis. The colonies here appear off-yellow, entire, slightly convex, smooth, and measure between 1.0 and 2.0 mm in diameter after 48 hours incubation at 35o C in CO2.

19. Pancreatic Abscess Staphylococcus aureus and other bacterial species provide a rich source for Factor V. Note in the photograph the tiny satelliting colonies of Factor-V dependent Haemophilus species growing adjacent to the Factor-V producing staphylococcal colonies.

20. Pancreatic Abscess A hemolytic strain of Staphylococcus aureus is commonly used as a source for Factor V. The “staph streak” procedure takes advantage of this property in the recovery of Factor-V dependent Haemophilus species on routine sheep blood agar. Note in the photograph of the surface of a sheep blood agar plate the growth of tiny colonies of Haemophilus species adjacent to the staph streak on the right side. This is known as the “satellite phenomenon”.

21. Pancreatic Abscess Illustrated in this photo-micrograph is a gram stain prepared from one of the isolated colonies illustrated in the previous screen. The bacterial cells are usually described as short, cocco-bacillary, and poorly staining, often admixed with pleomorphic and filamentous forms. These features are well captured in this photograph.

22. Pancreatic Abscess The assessment of X and V factor requirements is performed in many laboratories using reagent strips selectively impregnated with these substrates. Note in the photograph that this Haemophilus species is X-Factor dependent, but not V factor as it grows only around the strips containing Factor X, provided either by the “X” strip or the “XV” strip. Currently commercial tri- and quad-plates are available, supplemented with X, V, and XV quadrants, providing for identification based on differential growth factor requirements.

23. Pancreatic Abscess Illustrated in this photograph is the porphyrin test. This test is based on the ability of an organism to synthesize porphyrin from the base substrate, delta amino levulinic acid, which in this case is contained in the culture medium. Strains that are not X-Factor dependent can synthesize porphyrin and will produce a red fluorescence when observed under ultraviolet light (see photograph). This test is valuable to exclude extraction of extraneous hemin when obtaining inocula from the original blood agar isolation plate.

24. Pancreatic Abscess Disk test for performing the porphyrin synthesis test. These filter paper strips are impregnated with delta amino-levulinic acid. Each has been inoculated with a bacterial isolate. The fluorescence observed on the left disk indicates porphyrin synthesis and non-dependence on Factor X; the absence of fluorescence in the disk to the right indicates Factor X dependence.

25. Haemophilus: Review IDENTIFICATION OF HAEMOPHILUS SEGNIS No growth on BA Growth on chocolate agar and Haemophilus isolation media or as satellite colonies Gram-negative coccobacilli and filaments Factor V required ALA and X-V disk Tests + Acid from sucrose and fructose No acid from ribose and xylose. Composite photograph illustrating cultural characteristics for the identification of Haemophilus species.

26. Haemophilus: Laboratory Features By definition members of the genus Haemophilus have absolute growth requirements for nicotinamide adenine dinucleotide (NAD, coenzyme I, Factor V) and/or iron-containing pigments (hemin, hematin, X Factor). Sheep erythrocytes, upon lysis, release NADases that render sheep blood deficient in V Factor, precluding growth of any Haemophilus species requiring this factor (notably H. influenzae and in this case, H. segnis). This phenomenon does not occur with rabbit or horse blood. Selective culture media is set up in most laboratories for the recovery of Haemophilus factor-dependent species from select specimens, particularly from sputum and nasopharyngeal secretions obtained from children under 2-years old, and from cerebrospinal fluid. Physicians should let laboratory personnel know if H. influenzae infections are clinically suspected so that selective recovery media can be set up and appropriate follow-up tests be performed.

27. Haemophilus segnis: Recapitulation Haemophilus segnis is an uncommon isolate in clinical laboratories, and a rare cause of infection. The case presented here, however, serves as a reminder that such isolates on occasion may be encountered, and the means for making a definitive identification must be pursued. Haemophilus segnis is part of the human upper respiratory flora, and may be recovered from dental plaques, and on occasion from the pharynx as a secondary commensal in cases of acute pharyngitis of other causes. The cause of a pancreatic abscess infection as reviewed in this case presentation is an unusual circumstance. It is a V-dependent but can be distinguished from Haemophilus influenzae by not requiring extraneous X-factor, giving a positive ALA test result, and producing acid from fructose and sucrose. Haemophilus segnis shares many features with Haemophilus parainfluenzae. The majority of the latter biotypes are urease and/or ornithine positive while H. segnis is negative for these reactions.