Pseudomonas aeruginosa is the most common Pseudomonas species affecting humans, causing several acute and chronic infections. It is regarded as an opportunistic, nosocomial pathogen known for hepatobiliary tract infections, keratitis, otitis, pneumonia, wound infections, urinary tract infections (UTI), and being the leading cause of death in cystic fibrosis patients. Pseudomonas spp. are most often isolated from water and soil. Its versatile characteristics allow it to colonize plants and animals. Contamination of tap water and agricultural soils are traced to be top environmental sources responsible for its transmission. The bacterium is regarded as a transient microbe that is not classified as normal flora (Chengappa, Kennedy et al. 2013). Ingestion, inhalation, and direct contact are the primary modes of transmission. Most P. aeruginosa infections remain localized with a 50 percent mortality rate (Wagner and Iglewski 2008). In non-human primates (NHPs), spontaneous Pseudomonas spp. infections causing notable morbidity or mortality are less common, but NHPs have been used as animal models for Pseudomonas antimicrobial and biofilm studies (Dohar, Hebda et al. 2005, Krause, Whu et al. 2013). However, Pseudomonas spp. have been cultured in common marmoset investigations relating to gastrointestinal diseases (Shigeno, Toyama et al. 2018, Mineshige, Inoue et al. 2020, Powers, Castell et al. 2023).

Marmosets are a New World NHP whose popularity as a research model has greatly expanded over the last three decades. These arboreal-natured NHP’s are characterized by their small body size (300–450 g) and short lifespan (15–16 years), making them cost efficient in biomedical research settings (Han, Powers et al. 2022). Cage enclosure requirements for marmosets are comparatively minimal compared to other NHP species (Pines, Kaplan et al. 2007, Duarte, Goulart et al. 2012). Superior breeding performance in marmosets is demonstrated by their ability to produce nearly three litters every other year with two-to-four offspring per litter for up to seven years of life (Wolfe-Coote 2005).

Within captive settings, marmosets develop spontaneous diseases which present in a broad range of symptoms and diagnoses (David, Dick et al. 2009, Shigeno, Toyama et al. 2018, Fox, Marini et al. 2019, Mineshige, Inoue et al. 2020, Han, Powers et al. 2022, Powers, Castell et al. 2023). Clinical presentations for typical bacterial infections include diarrhea, vomiting, poor appetite, and weight loss. Acute bacterial infections are localized with mild to moderate symptoms associated with the affected organ system, while chronic cases can involve multi-organ lesions and sepsis (Bleyer, Kunze et al. 2017). Herein, we describe the spectrum of spontaneous Pseudomonas spp. infections in 26 common marmosets with gross and histopathological findings and a brief note on environmental sources of Pseudomonas spp. infection in a marmoset colony.

The common marmoset has become an attractive animal model in biomedical research due to complete sequencing of their genome and physiological likeness to humans. Growth, metabolic, reproductive and immunity pathways have been positively observed as a result of the sequencing, enabling comparative analyses with Old World primates and humans (Worley, Warren et al. 2014). Additionally, the short lifespan of marmosets offers researchers the ability to observe complete aging-related and neurological degenerative disease progression. Biocontainment infectious diseases research on marmosets has been proven to appropriately mimic human disease progression and pathophysiology for several infectious agents (Artim, Sheh et al. 2019, Nelson, Salguero et al. 2021). However, only mild responses were displayed in some experimental infections, such as COVID-19 (Han, Powers et al. 2022). Marmosets have shown greatest homology with immunoglobulins and T-cell receptors of humans, making them relevant in immune toxicological research (Nelson and Loveday 2014).

Routinely reported bacterial species isolated from marmosets include Campylobacter spp., Clostridium spp., Escherichia coli, Francisella tularensis, Helicobacter spp., Klebsiella spp., Pseudomonas spp., Shigella spp., and Staphylococcus spp. Pseudomonas spp. are gram-negative bacilli that are environmentally ubiquitous. They cause acute and chronic persistent infections by quickly adapting to environmental changes (Lorenz, Pawar et al. 2016).

Pseudomonas spp. cause successful infections due to a variety of virulence factors contributing to host cell attachment, immune response inhibition, and antibiotic resistance (Qin, Xiao et al. 2022). Its polar flagella enable the pathogen to swim to epithelial surfaces where their flagella will transition into an irreversible attachment mechanism (Kazmierczak, Schniederberend et al. 2015). Exotoxin secretion systems, along with a highly impermeable lipopolysaccharide (LPS) outer membrane and biofilm shield, are key features for Pseudomonas’ intrinsic resistance against multiple antibiotics (Lorenz, Pawar et al. 2016).

The NHP colony at SNPRC is primarily composed of baboons, rhesus macaques, cynomolgus macaques, marmosets, and few chimpanzees. Over the 27-year period of analysis of case reports for Pseudomonas infections, other than marmosets only one baboon was reported to have Pseudomonas spp. infection. This could be indicative of higher susceptibility of marmosets to Pseudomonas infection or maybe the differences in housing (indoor, smaller cages, etc.) may influence the survival of Pseudomonas in the environment and result in greater number of cases. Since only animals with clinical symptoms or gross evidence of bacterial infection were cultured during necropsy procedures, the actual incidence of Pseudomonas spp. infections at SNPRC is likely higher and this study is not indicative of true morbidity and mortality rates caused by Pseudomonas spp. in captive marmoset populations. Clinical presentations of bacterial infections included lethargy, gastrointestinal issues (diarrhea, bloating, vomiting, weight loss, poor appetite), increased respiratory effort, and difficulty urinating. Gross lesions were observed in one or more organs during necropsy. Affected organs (larynx, lungs, liver, gallbladder, intestines, colon, uterus, and urinary bladder) appeared discolored, distended, enlarged, and/or with necrotic material. Microscopic findings varied in severity and confirmed the presence of inflammatory infiltrates, edema, necrotic material, hemorrhage, and pseudomembranous lesions. Upon examination, tubular digestive organs (cecum, intestines, and colon) displayed mild to severe regions of inflammatory infiltrates, edema, and necrosis. The mucosal layers showed greatest degrees of necrosis, ulceration, and fibrin deposits. Lymphocytes, plasma cells, and neutrophils were most often observed in the lamina propria. Submucosal and muscularis layers were marked with edema and inflammatory infiltrates. Some chronic cases presented with transmural edema, necrosis, fibrin, and infiltrates. Livers and gallbladders were often diagnosed with hepatitis, cholecystitis and cholangiohepatitis, indicating an ascending infection occurred. Portal areas of the liver were frequently expanded by edema and low numbers of inflammatory cells. However, variable numbers of neutrophils were contained in the hepatic lobes and admixed with fibrin deposits. Gallbladder infections developed with or without liver infections. Gallbladder lumens were filled with suppurative inflammation or necrotic material, and lymphocyte follicular formations were observed throughout the wall. Epithelial hyperplasia and ulceration were frequently present. Transmural inflammation from epithelium to the submucosal layers affirmed lesion chronicity in severe cases. The lesions in the tubular organs are similar to other bacterial infections including Francisella tularensis, Yersinia spp., and Helicobacter spp. Shigella spp. that cause necrotizing, hemorrhagic, and ulcerating typhlitis. Colonic mucosal surfaces and crypts were observed to undergo structural changes including hyperplasia, sloughing, edema, ulceration, and necrosis when infected with Klebsiella spp., Clostridium spp., Shigella spp., Escherichia coli, and Helicobacter spp. Hepatic lesions, including hepatic necrosis and inflammation, are regularly observed in Clostridium spp., Yersinia spp., and Francisella tularensis infections (Fox, Marini et al. 2019).

Literature on Pseudomonas spp. infections in NHPs is limited; few reports involving marmosets were identified. Marmosets are known for presenting gastrointestinal issues in captive settings, prompting investigations into factors influencing digestive disease outbreaks. Previously, five marmosets were reported with proliferative cholecystitis and severe, necrosuppurative cholangiohepatitis which was accompanied by positive Pseudomonas aeruginosa cultures (Powers, Castell et al. 2023). The accompanied histopathological findings included bile duct hyperplasia, periportal hepatitis, bile peritonitis, ulcerative gastroenteritis, and typhlitis (Powers, Castell et al. 2023), similar to the lesions reported in this study. Two other research institutes sought to identify causative agents for the gastrointestinal issues experienced by their marmosets. They concluded that Pseudomonas spp. may be a contributing factor for chronic diarrhea, bloating, and weight loss (Shigeno, Toyama et al. 2018, Mineshige, Inoue et al. 2020).

Pneumonia cases in the present study displayed alveolar spaces filled with abundant erythrocytes, proteinaceous fluid, and alveolar macrophages. Alveolar septa and bronchi were necrotic and occasionally covered by Pseudomonas spp. bacterial aggregates. Francisella tularensis, Bordetella spp., and Klebsiella spp. are also known to affect the respiratory system resulting in similar lesions of interstitial pneumonia and bronchopneumonia (Bleyer, Kunze et al. 2017, Fox, Marini et al. 2019). Both cases of metritis in the present study had transmural suppurative inflammation of the uterus with lumens occupied by fibrin, hemorrhage, necrotic debris, and neutrophils. Two other bacterial species reported to cause urogenital infections in NHPs are Staphylococcus spp. and Helicobacter spp. (Fox, Marini et al. 2019). Staphylococcus spp. has been cultured from the vagina and penis. Infections with Helicobacter spp. had reported pathological changes including glomerular and interstitial nephritis. Other urogenital diseases implicated to result from bacterial agents include glomerulosclerosis, tubular lesions, and renal failure (Fox, Marini et al. 2019). Only one marmoset from our dataset had Pseudomonas spp. cultured from a large external abdominal wound. Nonetheless, skin infections should be regarded as an atypical presentation in marmosets.

Several infections presented microscopically with bacterial colonies admixed with necrotic, fibrinous, or hemorrhagic material. Gram-stained slides demonstrated clusters of gram-negative bacilli bacteria consistent with Pseudomonas spp. (not shown). Not all cases had Gram stains performed, but dense bacilli bacterial colonies were observed in H&E slides. For cases with polymicrobial culture results (Table 1), the Pseudomonas spp. bacterial colonies were most abundant. This aligns with Lorenz et al.’s review on animal model responses to Pseudomonas aeruginosa during polymicrobial studies (Lorenz, 2016 #27).

Pseudomonas spp. contamination of tap water is a major source of infection. Tap water contamination may be a result of tap fixtures becoming colonized with the bacterium as it exists in protective biofilms (Whitacre 2009). In our case study, a recent spike in Pseudomonas spp. infections in the marmosets could be attributed to the drinking water source since some of the investigated drinking bottles tested positive for the bacteria. In addition to direct ingestion of the tap water, Pseudomonas spp. can be found on medical equipment (endotracheal tubes, laryngoscopes, bronchoscopes) if washed and rinsed with contaminated tap water. Moreover, contaminated water sources and dampened feces may become pathogenic aerosols through evaporation and humidity that can cause infection from inhalation. Chlorine, chloramines, ozone, and iodine have proven to be effective water treatments against Pseudomonas spp. (Whitacre 2009). Powers et al. (2023) reported a failed drinking water chlorination system to be the identified source of Pseudomonas spp. infections in five of seven diseased marmosets.