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US Army Medical Research Institute of Infectious Diseases, Fort Detrick, MD (NAT, CAW, ELS, HEHCDF, SG, NK, KES), and Microbial Diseases Laboratory, Division of Communicable Diseases, California Department of Health Services, Richmond, CA (SA, JMJ)
Abstract
Invasive Klebsiella pneumoniae is an emerging disease of humans characterized by abscesses in the liver or other sites involving bacteria with the unique hypermucoviscosity phenotype. Over several months, 7 African green monkeys in our research colony developed abscess formation in multiple locations and succumbed to disease. K. pneumoniae was identified by bacterial culture in 6 monkeys and immunohistochemistry in 1 additional monkey. All monkeys had been housed in, or had contact with monkeys housed in, 1 animal room in our facility. All affected monkeys had 1 or more abscesses, most notably in the abdomen, but also affecting the lungs, cerebellum, and skin. Abdominal abscesses and associated adhesions entrapped loops of bowel, forming palpable masses. Abdominal masses were located at the root of the mesentery, the ileocecocolic junction, or the pelvic inlet. In 1 case, culture, serotyping, and polymerase chain reaction (PCR) analysis of the bacterial isolate identified K. pneumoniae expressing the hypermucoviscosity phenotype and capsular serotype K2 and determined that the K. pneumonia was genetically rmpA+/magA–.
Key words: Abscess; African green monkeys; hypermucoviscosity; invasive; Klebsiella pneumoniae.
Klebsiella pneumoniae is a gram-negative, aerobic, nonmotile bacillus and is a common cause of a wide range of infections in humans and animals.8,12 In Old and New World monkeys, infection with K. pneumoniae causes pneumonia, meningitis, peritonitis, cystitis, and septicemia.7,14 K. pneumoniae also constitutes normal fecal and oral flora in many nonhuman primates. In the past 2 decades, a new type of invasive K. pneumoniae disease has emerged in humans in Taiwan and other Asian countries, and more recently from non-Asian countries, including the USA.2,10,11,13,17 Fatal human infections with invasive strains of K. pneumoniae involve pulmonary emboli or abscess, meningitis, endophthalmitis, osteomyelitis, or brain abscess.3,10 Recently, a highly invasive K. pneumoniae causing primary liver abscesses in humans has also been reported.2,10,17 These invasive, abscess-forming strains of K. pneumoniae are associated with the so-called hypermucoviscosity (HMV) phenotype, a bacterial colony trait identified by a positive string test.5,6,9 The HMV phenotype is seen in K. pneumoniae expressing either the capsular serotypes K1 or K2. K1 serotypes of K. pneumoniae have 2 potentially important genes, rmpA, a transcriptional activator of colanic acid biosynthesis,16 and magA, which encodes a 43-kD outer membrane protein.5 K2 serotypes of K. pneumoniae also have rmpA but do not have magA. Capsular serotypes K1 and K2 are reported to play an important role in the invasive ability of HMV K. pneumoniae.18 The role of rmpA and magA in the pathogenesis of invasive K. pneumonia, however, seems less certain.
To our knowledge, K. pneumoniae expressing the HMV phenotype has not been reported to cause natural disease in nonhuman primates, nor in other animal species. In this report, we describe the clinical, gross and histopathologic features, and the bacteriologic findings of a single African green monkey (Chlorocebus aethiops—Africa green monkey [AGM]) with invasive K. pneumoniae infection, in which the bacterial isolate expressed the HMV phenotype and 6 additional AGMs in which Klebsiella was identified by bacterial culture or by immunohistochemistry. All monkeys described in this report were maintained in a facility at the US Army Medical Research Institute of Infectious Diseases accredited by the American Association for Accreditation of Laboratory Animal Care. All research was conducted under approved animal protocols in adherence with the Guide for the Care and Use of Laboratory Animals (Committee on Care and Use of Laboratory Animals of the Institute of Laboratory Animal Resources, National Research Council, National Institutes of Health Publication No. 86–23, revised 1996).
During a routine physical examination, an adult male AGM, V513, presented with an 8- to 10-cm-diameter mass on the right dorsal flank. No other abnormal findings were observed at the time, and the animal had no history of illness or injury before being transferred to our facility in 2003. One day after the initial presentation, the fluid-filled mass ruptured and was draining a thick, mucoid discharge. The abscess was cultured, surgically debrided and lavaged, a Penrose drain was placed, and antibiotic treatment was initiated. An abdominal mass was palpated 24 hours following surgery and was described as a 3 cm x 1 cm irregularly shaped, firm mass in the cranial abdomen. The mass enlarged over several days, and exploratory laparotomy revealed mesenteric abscesses centered at the ileocecocolic junction and also involving the duodenum, ileum, cecum, colon, the right ureter, and the body wall inferior to the right kidney. Surgical removal was not possible, and the animal was euthanatized. A complete necropsy was performed, samples were taken for bacterial culture, and a full set of tissues was fixed in 10% neutral buffered formalin and processed routinely for histopathologic evaluation.
Bacteria in the abscess fluid were identified as K. pneumoniae by routine culture and biochemical methods and were further characterized as K. pneumoniae subsp. pneumoniae serotype K2 by capsular serotyping using a slide agglutination technique.1 Furthermore, bacterial colonies plated on a blood agar plate exhibited a hypermucoviscosity phenotype, as demonstrated by a positive string test (formation of a >5 mm mucoviscous string when a loop is passed through a colony; Fig. 1). Genetic analysis of the sequence of a 760-bp amplicon of the bacterial 16S rRNA from the isolate was submitted to a basic local alignment search tool search and found to be 99% identical to several K. pneumoniae entries. Additional analysis for 2 novel genes, magA (mucoviscosity-associated gene A) and rmpA gene (regulator of the mucoid phenotype) suggested as potential hypermucoviscosity-associated genes in human cases,5,6 was performed by PCR using previously described primers.4,19 The bacterial isolate from this case was positive for the rmpA gene and was negative for the magA gene.
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Histologically, the mesentery, mesenteric lymph nodes, and serosa of multiple organs contained multifocal abscesses composed of viable and degenerate neutrophils and cellular debris surrounded by many macrophages, fewer lymphocytes, plasma cells, and a fibrous connective tissue capsule. The macrophages contained abundant, often granular or vescicular cytoplasm (Fig. 6a). Chronic abscesses were also present in the mesentery and serosa of multiple abdominal organs. The mesenteric lymph nodes at the ileocecocolic junction contained multiple areas of necrosis, hemorrhage, and degenerate neutrophils that were surrounded by many macrophages with abundant foamy cytoplasm (Fig. 5). Fibrosis was present in the capsule, subcapsular sinus, and surrounded and replaced necrotic follicles. Some mesenteric lymph nodes were nearly replaced by fibrosis, and only remnants of their architecture were identifiable. The subcutis of the right flank contained histiocytic panniculitis with hemorrhage, necrosis, collagenolysis, and neutrophilic inflammation. Immunohistochemistry was performed on select tissue sections by the immunoperoxidase method (Envision System, DAKO Inc., Carpinteria, CA) using a rabbit antiserum to K. pneumoniae proteins (United States Biological, Swampscott, MA) diluted 1 : 4000 following heat-induced antigen retrieval with Tris base/Ethylenediamine Tetraacetic Acid (Tris/EDTA) buffer. Macrophages in sections of mesentery, serosa, and the mesenteric lymph nodes contained many immunolabeled bacteria (Figs. 5b, 6b).
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The rmpA+/magA– genotype demonstrated in the bacterial isolate from 1 monkey is consistent with human HMV isolates of K. pneumoniae, in which the rmpA gene is present in both K1 and K2 capsular serotypes, as well as nearly 67% of non–K1/K2 serotypes,17 but the magA gene appears restricted to isolates of the K1 serotype.15 Furthermore, the magA gene appears to correlate particularly with the development of primary liver abscesses, or liver abscesses combined with metastatic abscesses, in humans.2,10,16 Of note in that regard, liver abscesses were not evident in any of the monkeys at our facility, all of which were presumably magA–.
Our demonstration of the K2 capsular serotype in the one monkey isolate is of particular significance, as capsular serotype appears to play a key role in the virulence of invasive K. pneumoniae. Serotype K1 and K2 isolates of K. pneumoniae have been reported to be significantly more resistant to phagocytosis and more virulent in the mouse model than non-K1/K2 isolates,17 even non-K1/K2 isolates that were rmpA+. However, among the non-K1/K2 strains, those that possessed the rmpA gene were more resistant to phagocytosis than the rmpA– strains, so rmpA may still contribute to the pathogenesis of K. pneumoniae in vivo. The magA gene may also contribute to virulence, but like rmpA, it does not appear to be as significant a contributor to virulence as the K1 or K2 phenotype.17
The means by which the causative K. pneumonia may have spread or caused disease in individual monkeys in our colony is unknown. The only significant epidemiologic factor we identified was that affected monkeys were maintained in the same room or had contact with a monkey housed in that room. Of note, no clinical or pathological cases have been identified in the 14 months between the last identified case and the preparation of this manuscript. Also, bacteriological testing of fecal and oral swabs of 20 African green monkeys in our colony did not yield any isolates of K. pneumoniae (data not shown). However, as we were completing this manuscript in June 2007, a routine oral swab from a recently obtained AGM in quarantine at our institute yielded K. pneumoniae showing the HMV phenotype and rmpA+/magA– genotype. This AGM exhibited no abnormal clinical signs during quarantine. The animal was euthanatized for precautionary reasons and a full necropsy was performed which revealed no gross abnormalities. The epidemiological significance of this case remains uncertain.
Mice are susceptible to experimental infection with HMV strains of K. pneumoniae and appear to provide a useful model for pathogenesis studies of invasive K. pneumoniae. To our knowledge, however, this report represents the first time that invasive K. pneumoniae with the HMV phenotype associated with abscess formation has been identified to occur spontaneously in animals. The fact that 7 AGMs over a few months' time demonstrated abscessation in multiple tissues shows that this species is quite susceptible to invasive K. pneumoniae infection. Therefore, veterinarians, laboratory workers and research pathologists should be aware of this pathogen as a cause of abdominal masses and multisystemic abscessation in the AGM. In addition, the AGM may provide another useful animal model to understand the pathogenesis of this emerging human pathogen.
Acknowledgements
We are grateful to Stephen Libby, Department of Laboratory Medicine, University of Washington for providing us with the magA+ K. pneumoniae strains used as positive controls and to Polly Kaufmann, Health protection Agency, Centre for Infections, London, UK, for K1 and K2 antisera. We also thank Jeff Brubaker, Neil Davis, Gale Krietz, John Kondig Chris Mech, and Larry Ostby (USAMRIID) for assistance in their respective fields of expertise. The views, opinions, and/or findings contained herein are those of the authors and should not be construed as official government position, policy, or decision unless so designated by other documentation.
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