Loci Encoding Compounds Potentially Active against Drug-Resistant Pathogens amidst a Decreasing Pool of Novel Antibiotics.

Since the discovery of penicillin, microbes have been a source of antibiotics that inhibit the growth of pathogens. However, with the evolution of multidrug-resistant (MDR) strains, it remains unclear if there is an abundant or limited supply of natural products to be discovered that are effective against MDR isolates. To identify strains that are antagonistic to pathogens, we examined a set of 471 globally derived environmental Pseudomonas strains (env-Ps) for activity against a panel of 65 pathogens including Achromobacter spp., Burkholderia spp., Pseudomonas aeruginosa, and Stenotrophomonas spp. isolated from the lungs of cystic fibrosis (CF) patients. From more than 30,000 competitive interactions, 1,530 individual inhibitory events were observed. While strains from water habitats were not proportionate in antagonistic activity, MDR CF-derived pathogens (CF-Ps) were less susceptible to inhibition by env-Ps, suggesting that fewer natural products are effective against MDR strains. These results advocate for a directed strategy to identify unique drugs. To facilitate discovery of antibiotics against the most resistant pathogens, we developed a workflow in which phylogenetic and antagonistic data were merged to identify strains that inhibit MDR CF-Ps and subjected those env-Ps to transposon mutagenesis. Six different biosynthetic gene clusters (BGCs) were identified from four strains whose products inhibited pathogens including carbapenem-resistant P. aeruginosa BGCs were rare in databases, suggesting the production of novel antibiotics. This strategy can be utilized to facilitate the discovery of needed antibiotics that are potentially active against the most drug-resistant pathogens.IMPORTANCE Carbapenem-resistant P. aeruginosa is difficult to treat and has been deemed by the World Health Organization as a priority one pathogen for which antibiotics are most urgently needed. Although metagenomics and bioinformatic studies suggest that natural bacteria remain a source of novel compounds, the identification of genes and their products specific to activity against MDR pathogens remains problematic. Here, we examine water-derived pseudomonads and identify gene clusters whose compounds inhibit CF-derived MDR pathogens, including carbapenem-resistant P. aeruginosa.

Environmental Pseudomonads Inhibit Cystic Fibrosis Patient-Derived Pseudomonas aeruginosa.

Pseudomonas aeruginosa is an opportunistic pathogen which is evolving resistance to many currently used antibiotics. While much research has been devoted to the roles of pathogenic P. aeruginosa in cystic fibrosis (CF) patients, less is known of its ecological properties. P. aeruginosa dominates the lungs during chronic infection in CF patients, yet its abundance in some environments is less than that of other diverse groups of pseudomonads. Here, we sought to determine if clinical isolates of P. aeruginosa are vulnerable to environmental pseudomonads that dominate soil and water habitats in one-to-one competitions which may provide a source of inhibitory factors. We isolated a total of 330 pseudomonads from diverse habitats of soil and freshwater ecosystems and competed these strains against one another to determine their capacity for antagonistic activity. Over 900 individual inhibitory events were observed. Extending the analysis to P. aeruginosa isolates revealed that clinical isolates, including ones with increased alginate production, were susceptible to competition by multiple environmental strains. We performed transposon mutagenesis on one isolate and identified an ∼14.8-kb locus involved in antagonistic activity. Only two other environmental isolates were observed to carry the locus, suggesting the presence of additional unique compounds or interactions among other isolates involved in outcompeting P. aeruginosa This collection of strains represents a source of compounds that are active against multiple pathogenic strains. With the evolution of resistance of P. aeruginosa to currently used antibiotics, these environmental strains provide opportunities for novel compound discovery against drug-resistant clinical strains. IMPORTANCE: We demonstrate that clinical CF-derived isolates of P. aeruginosa are susceptible to competition in the presence of environmental pseudomonads. We observed that many diverse environmental strains exhibited varied antagonistic profiles against a panel of clinical P. aeruginosa isolates, suggesting the presence of distinct mechanisms of inhibition among these ecological strains. Understanding the properties of these antagonistic events offers the potential for discoveries of antimicrobial compounds or metabolic pathways important to the development of novel treatments for P. aeruginosa infections.

Tradeoffs Between Evolved Phage Resistance and Antibiotic Susceptibility in a Highly Drug-Resistant Cystic Fibrosis-Derived Pseudomonas aeruginosa Strain.

Background: Multi-drug resistant pathogens pose significant challenges towards the effective resolution of bacterial infections. A promising alternative strategy is phage therapy in which limited applications has afforded lifesaving resolution from drug resistant pathogens. However, adoption of this strategy is hampered by narrow bacteriophage host ranges, and as with antibiotics, bacteria can acquire resistance to phage. Methods: To address these issues, we isolated 25 broad-host range phages against multiple cystic fibrosis (CF)-derived P. aeruginosa clinical strains thus promoting their application against conspecific pathogens. To investigate evolved resistance to phage in relation to antibiotic resistance, one CF-derived P. aeruginosa strain was exposed to a lytic phage over a short time scale. Results: Trade-offs were observed in which evolved phage resistant P. aeruginosa strains showed decreased resistance to antibiotics. These traits that likely reflect single nucleotide polymorphisms. Conclusion: Results suggest phage and antibiotics may be a combined approach to treat bacterial infections.

The effect of aquaponics on tomato (Solanum lycopersicum) sensory, quality, and safety outcomes.

Resource-efficient food production practices are needed to support a sustainable food system. Aquaponics, a system where fish and produce are grown symbiotically in the same water circulating system, minimizes water usage, fertilizer input, and waste production. However, the impact of aquaponics on produce quality is underexplored. We utilize objective testing, descriptive analysis, and consumer acceptance to characterize the impact of aquaponics on tomato quality. Two tomato varieties were grown in an aquaponics system and compared with soil-grown controls across 3 years. Safety was assessed by analyzing coliforms and confirming the absence of Escherichia coli. Weight, texture, color, moisture, titratable acidity, brix, and phenolic and antioxidant measurements were assessed. A semitrained descriptive sensory panel assessed 13 tomato attributes and acceptance was determined using untrained participants. Aquaponic tomatoes were frequently lighter and yellower in color and lower in brix. Descriptive analysis indicated significant differences in several sensory attributes, though these findings were inconsistent between years and varieties. Nutrient deficiencies may explain quality differences, as iron supplementation improved outcomes. Notably, the objective and descriptive differences minimally impacted consumer acceptance, as we found no significant differences in taste, texture, or appearance liking between production method in either variety. Despite variation in produce quality across years, aquaponics tomatoes pose minimal E. coli risk and are liked as much as soil-grown tomatoes. These findings demonstrate that aquaponics can produce products that are as acceptable as their soil-grown counterparts. PRACTICAL APPLICATION: Aquaponic tomatoes are as safe as soil-grown tomatoes. Furthermore, aquaponics tomatoes are liked as much as soil-grown tomatoes. Careful monitoring of nutrients in an aquaponic system may optimize quality. Overall, aquaponics has a minimal impact on tomato quality and thus is a sustainable food production method that can compete with conventional products on quality.

Metapopulation structure of Vibrionaceae among coastal marine invertebrates.

Although animal-associated microbial communities (microbiomes) are increasingly recognized to influence health, the extent to which animals represent highly selective habitats for microbes leading to predominance of high host specificity remains poorly understood. Here, we show that vibrios, which are well-known commensals and opportunistic pathogens of marine animals, overall display little host preference, likely because of efficient dispersal-colonization dynamics mediated by food items. We isolated 1753 strains from water and animal samples, which are linked in a food chain and display different degrees of similarity (respiratory and digestive tract of mussels and crabs, live and dead zooplankton, and whole water samples). Multilocus sequence data served as input for modelling and statistical analysis of spatiotemporal population structure. These data showed that the majority of populations occurred broadly within and among hosts, with the dominant population being a near perfect generalist with regard to seasons, host taxa and body regions. Zooplankton harboured the fewest and most specific populations, while crabs and mussels contained the highest diversity with little evidence for host preferences. Most mussel- and crab-associated populations were detected in water samples at similar frequencies, particularly in filter-feeding mussels where populations were also evenly distributed across host individuals. The higher variation among individuals observed in crabs and zooplankton is consistent with stochastic clonal expansions. These patterns suggest that evolution of a high degree of host specificity is surprisingly rare even though these animals represent long-lived habitats, and vibrios are consistent members of their microbiome. Instead, many of the populations show stronger association with planktonic (micro)habitats while the microbiome may be a fairly open system for vibrios in which high rates of immigration can outpace selection for specialization.

O-antigen diversity and lateral transfer of the wbe region among Vibrio splendidus isolates.

The O-antigen is a highly diverse structure expressed on the outer surface of Gram-negative bacteria. The products responsible for O-antigen synthesis are encoded in the wbe region, which exhibits extensive genetic diversity. While heterogeneous O-antigens are observed within Vibrio species, characterization of these structures has been devoted almost exclusively to pathogens. Here, we investigate O-antigen diversity among coastal marine Vibrio splendidus-like isolates. The wbe region was first identified and characterized using the sequenced genomes of strains LGP32, 12B01 and Med222. These regions were genetically diverse, reflective of their expressed O-antigen. Additional isolates from physically distinct habitats in Plum Island Estuary (MA, USA), including within animal hosts and on suspended particles, were further characterized based on multilocus sequence analysis (MLSA) and O-antigen profiles. Results showed serotype diversity within an ecological setting. Among 48 isolates which were identical in three MLSA genes, 41 showed gpm genetic diversity, a gene closely linked to the wbe locus, and at least 12 expressed different O-antigen profiles further suggesting wbe genetic diversity. Our results demonstrate O-antigen hyper-variability among these environmental strains and suggest that frequent lateral gene transfer generates wbe extensive diversity among V. splendidus and its close relatives.

Expanding Tiny Earth to genomics: a bioinformatics approach for an undergraduate class to characterize antagonistic strains.

The evolution of multidrug resistant pathogens and the diminishing supply of effective antibiotics are global crisis. Tiny Earth (TE) is undergraduate curriculum that encourage students to pursue science careers by engagement in authentic drug discovery research. Through the TE program, students identify environmental strains that inhibit other bacteria. Although these isolates may produce antibiotics based on the antagonistic phenotype, understanding the activity in regard to genome content remains elusive. Previously, we developed a transposon mutagenesis module for use with TE to identify genes involved in antibiotic production. Here, we extend this approach to a second semester undergraduate course to understand the origin of antagonism and genome diversity. Using a bioinformatics strategy, we identified gene clusters involved in activity, and with annotated genomes in hand, students were able to characterize strain diversity. Genomes were analyzed using different computational tools, including average nucleotide identity for species identification and whole genome comparisons. Because the focus of TE involves the evolution of drug resistance, predicted products in strains were identified and verified using a drug susceptibility assay. An application of this curriculum by TE members would assist in efforts with antibiotic discovery.

Exploring Rain as Source of Biological Control Agents for Fire Blight on Apple.

Poor survival on plants can limit the efficacy of Biological Control Agents (BCAs) in the field. Yet bacteria survive in the atmosphere, despite their exposure to high solar radiation and extreme temperatures. If conditions in the atmosphere are similar to, or more extreme than, the environmental conditions on the plant surface, then precipitation may serve as a reservoir of robust BCAs. To test this hypothesis, two hundred and fifty-four rain-borne isolates were screened for in vitro inhibition of Erwinia amylovora, the causal agent of fire blight, as well as of other plant pathogenic bacteria, fungi and oomycetes. Two isolates showed strong activity against E. amylovora and other plant pathogenic bacteria, while other isolates showed activity against fungal and oomycete pathogens. Survival assays suggested that the two isolates that inhibited E. amylovora were able to survive on apple blossoms and branches similarly to E. amylovora. Pathogen population size and associated fire blight symptoms were significantly reduced when detached apple blossoms were treated with the two isolates before pathogen inoculation, however, disease reduction on attached blossoms within an orchard was inconsistent. Using whole genome sequencing, the isolates were identified as Pantoea agglomerans and P. ananatis, respectively. A UV-mutagenesis screen pointed to a phenazine antibiotic D-alanylgriseoluteic acid synthesis gene cluster as being at the base of the antimicrobial activity of the P. agglomerans isolate. Our work reveals the potential of precipitation as an under-explored source of BCAs, whole genome sequencing as an effective approach to precisely identify BCAs, and UV-mutagenesis as a technically simple screen to investigate the genetic basis of BCAs. More field trials are needed to determine the efficacy of the identified BCAs in fire blight control.

Nutrient Capture from Aqueous Waste and Photocontrolled Fertilizer Delivery to Tomato Plants Using Fe(III)-Polysaccharide Hydrogels.

Inexpensive and sustainable methods are needed to reclaim nutrients from agricultural waste solutions for use as a fertilizer while decreasing nutrient runoff. Fe(III)-polysaccharide hydrogels are able to flocculate solids and absorb nutrients in liquid animal waste from Confined Animal Feeding Operations (CAFOs). Fe(III)-alginate beads absorbed 0.05 mg g-1 NH4 + and NO3 - from 100 ppm solutions at pH = 7, with > 80% phosphate uptake and ∼30% uptake of ammonium and nitrate. Ammonium uptake from a raw manure solution (1420 ppm NH4 +) showed a significant 0.7 mg g-1 uptake. Tomato plant trials carried out with Fe(III)-alginate hydrogel beads in greenhouse conditions showed controlled nutrient delivery for the plants compared to fertilizer solution with the same nutrient content. Plants showed an uptake of Fe from the gel beads, and Fe(III)-alginate hydrogel beads promoted root growth of the plants. The plants treated with nutrient-loaded Fe(III)-alginate hydrogels yielded comparable tomato harvest to plants treated with the conventional fertilizer solution.

Genetic interactions of smc, ftsK, and parB genes in Streptomyces coelicolor and their developmental genome segregation phenotypes.

The mechanisms by which chromosomes condense and segregate during developmentally regulated cell division are of interest for Streptomyces coelicolor, a sporulating, filamentous bacterium with a large, linear genome. These processes coordinately occur as many septa synchronously form in syncytial aerial hyphae such that prespore compartments accurately receive chromosome copies. Our genetic approach analyzed mutants for ftsK, smc, and parB. DNA motor protein FtsK/SpoIIIE coordinates chromosome segregation with septum closure in rod-shaped bacteria. SMC (structural maintenance of chromosomes) participates in condensation and organization of the nucleoid. ParB/Spo0J partitions the origin of replication using a nucleoprotein complex, assembled at a centromere-like sequence. Consistent with previous work, we show that an ftsK-null mutant produces anucleate spores at the same frequency as the wild-type strain (0.8%). We report that the smc and ftsK deletion-insertion mutants (ftsK' truncation allele) have developmental segregation defects (7% and 15% anucleate spores, respectively). By use of these latter mutants, viable double and triple mutants were isolated in all combinations with a previously described parB-null mutant (12% anucleate spores). parB and smc were in separate segregation pathways; the loss of both exacerbates the segregation defect (24% anucleate spores). For a triple mutant, deletion of the region encoding the FtsK motor domain and one transmembrane segment partially alleviates the segregation defect of the smc parB mutant (10% anucleate spores). Considerable redundancy must exist in this filamentous organism because segregation of some genomic material occurs 90% of the time during development in the absence of three functions with only a fourfold loss of spore viability. Furthermore, we report that scpA and scpAB mutants (encoding SMC-associated proteins) have spore nucleoid organization defects. Finally, FtsK-enhanced green fluorescent protein (EGFP) localized as bands or foci between incipient nucleoids, while SMC-EGFP foci were not uniformly positioned along aerial hyphae, nor were they associated with every condensing nucleoid.

Aquatic Pseudomonads Inhibit Oomycete Plant Pathogens of Glycine max.

Seedling root rot of soybeans caused by the host-specific pathogen Phytophthora sojae, and a large number of Pythium species, is an economically important disease across the Midwest United States that negatively impacts soybean yields. Research on biocontrol strategies for crop pathogens has focused on compounds produced by microbes from soil, however, recent studies suggest that aquatic bacteria express distinct compounds that efficiently inhibit a wide range of pathogens. Based on these observations, we hypothesized that freshwater strains of pseudomonads might be producing novel antagonistic compounds that inhibit the growth of oomycetes. To test this prediction, we utilized a collection of 330 Pseudomonas strains isolated from soil and freshwater habitats, and determined their activity against a panel of five oomycetes: Phytophthora sojae, Pythium heterothalicum, Pythium irregulare, Pythium sylvaticum, and Pythium ultimum, all of which are pathogenic on soybeans. Among the bacterial strains, 118 exhibited antagonistic activity against at least one oomycete species, and 16 strains were inhibitory to all pathogens. Antagonistic activity toward oomycetes was significantly more common for aquatic isolates than for soil isolates. One water-derived strain, 06C 126, was predicted to express a siderophore and exhibited diverse antagonistic profiles when tested on nutrient rich and iron depleted media suggesting that more than one compound was produced that effectively inhibited oomycetes. These results support the concept that aquatic strains are an efficient source of compounds that inhibit pathogens. We outline a strategy to identify other strains that express unique compounds that may be useful biocontrol agents.

Environmental vibrios represent a source of antagonistic compounds that inhibit pathogenic Vibrio cholerae and Vibrio parahaemolyticus strains.

With the overuse of antibiotics, many pathogens including Vibrio cholerae and Vibrio parahaemolyticus have evolved multidrug resistance making treatment more difficult. While understanding the mechanisms that underlie pathogenesis is crucial, knowledge of bacterial interactions of V. cholerae and V. parahaemolyticus could provide insight to their susceptibility outside of the human host. Based on previous work showing competition among environmental strains, we predict that marine-derived bacteria should inhibit Vibrio pathogens and may be a source of unique antibiotic compounds. We tested a collection of 3,456 environmental Vibrio isolates from diverse habitats against a panel of V. cholerae and V. parahaemolyticus, and identified 102 strains that inhibited the growth of these pathogens. Phylogenetic analysis revealed that 40 pathogen-inhibiting strains were unique at the hsp60 gene sequence while 62 of the isolates were identical suggesting clonal groups. Genomic comparisons of ten strains revealed diversity even between clonal isolates and were identified as being closely related to known Vibrio crassostreae, Vibrio splendidus, and Vibrio tasmaniensis strains. Further analysis revealed multiple biosynthetic gene clusters within all sequenced genomes that encoded secondary metabolites with potential antagonistic activity. Thus, environmental vibrios represent a source of compounds that inhibit Vibrio pathogens.

Antibiotic discovery throughout the Small World Initiative: A molecular strategy to identify biosynthetic gene clusters involved in antagonistic activity.

The emergence of bacterial pathogens resistant to all known antibiotics is a global health crisis. Adding to this problem is that major pharmaceutical companies have shifted away from antibiotic discovery due to low profitability. As a result, the pipeline of new antibiotics is essentially dry and many bacteria now resist the effects of most commonly used drugs. To address this global health concern, citizen science through the Small World Initiative (SWI) was formed in 2012. As part of SWI, students isolate bacteria from their local environments, characterize the strains, and assay for antibiotic production. During the 2015 fall semester at Bowling Green State University, students isolated 77 soil-derived bacteria and genetically characterized strains using the 16S rRNA gene, identified strains exhibiting antagonistic activity, and performed an expanded SWI workflow using transposon mutagenesis to identify a biosynthetic gene cluster involved in toxigenic compound production. We identified one mutant with loss of antagonistic activity and through subsequent whole-genome sequencing and linker-mediated PCR identified a 24.9 kb biosynthetic gene locus likely involved in inhibitory activity in that mutant. Further assessment against human pathogens demonstrated the inhibition of Bacillus cereus, Listeria monocytogenes, and methicillin-resistant Staphylococcus aureus in the presence of this compound, thus supporting our molecular strategy as an effective research pipeline for SWI antibiotic discovery and genetic characterization.

Ecological populations of bacteria act as socially cohesive units of antibiotic production and resistance.

In animals and plants, social structure can reduce conflict within populations and bias aggression toward competing populations; however, for bacteria in the wild it remains unknown whether such population-level organization exists. Here, we show that environmental bacteria are organized into socially cohesive units in which antagonism occurs between rather than within ecologically defined populations. By screening approximately 35,000 possible mutual interactions among Vibrionaceae isolates from the ocean, we show that genotypic clusters known to have cohesive habitat association also act as units in terms of antibiotic production and resistance. Genetic analyses show that within populations, broad-range antibiotics are produced by few genotypes, whereas all others are resistant, suggesting cooperation between conspecifics. Natural antibiotics may thus mediate competition between populations rather than solely increase the success of individuals.

Differential Salmonella survival against communities of intestinal amoebae.

Predation from intestinal amoebae may provide selective pressure for the maintenance of high genetic diversity at the Salmonella enterica rfb locus, whereby serovars better escape predators in particular environments depending on the O-antigens they express. Here, the hypothesis that amoebae from a particular intestinal environment collectively prefer one serovar over another is tested. Collections of Acanthamoeba, Tetramitus, Naegleria and Hartmannella were isolated from the intestinal tracts of several vertebrate hosts, including bullfrog tadpoles, goldfish, turtles and bearded dragons, and their feeding preferences were determined. Congeneric amoebae from the same environment had significantly similar feeding preferences. Strikingly, even unrelated amoebae - such as Naegleria and Tetramitus from goldfish - also had significantly similar feeding preferences. Yet amoebae isolated from different environments showed no similarity in prey choice. Thus, feeding preferences of amoebae appear to reflect their environment, not their taxonomic relationships. A mechanism mediating this phenotypic convergence is discussed.

Genetic variation at the mitochondrial large-subunit rRNA locus of Pneumocystis isolates from simian immunodeficiency virus-infected rhesus macaques.

The nucleotide sequences of a segment of the Pneumocystis mitochondrial large-subunit (mt LSU) rRNA gene from rhesus macaques coinfected with simian immunodeficiency virus (SIV) and Pneumocystis carinii were examined. Of 12 isolates examined, 3 were found to be identical and the others showed substantial sequence variation, with up to 13% divergence among variants. We identified two general sequence types that differed at several sites, including a conserved 26-nucleotide insertion. Four monkeys had evidence of two Pneumocystis variants present simultaneously, indicative of a mixed infection. There was a high degree of variance between the rhesus macaque-derived Pneumocystis mt LSU rRNA gene sequence and the cognate sequences in Pneumocystis organisms derived from other hosts. Analysis of the mt LSU rRNA genes of Pneumocystis organisms derived from rhesus macaques and several other mammalian hosts supports the observation that rhesus macaque-derived Pneumocystis is most closely related to human-derived PNEUMOCYSTIS: In addition, the data identify the mt LSU rRNA gene as an informative locus for transmission and epidemiological studies of the SIV-rhesus macaque model of Pneumocystis infection.

Protozoan predation, diversifying selection, and the evolution of antigenic diversity in Salmonella.

Extensive population-level genetic variability at the Salmonella rfb locus, which encodes enzymes responsible for synthesis of the O-antigen polysaccharide, is thought to have arisen through frequency-dependent selection (FDS) by means of exposure of this pathogen to host immune systems. The FDS hypothesis works well for pathogens such as Haemophilus influenzae and Neisseria meningitis, which alter the composition of their O-antigens during the course of bloodborne infections. In contrast, Salmonella remains resident in epithelial cells or macrophages during infection and does not have phase variability in its O-antigen. More importantly, Salmonella shows host-serovar specificity, whereby strains bearing certain O-antigens cause disease primarily in specific hosts; this behavior is inconsistent with FDS providing selection for the origin or maintenance of extensive polymorphism at the rfb locus. Alternatively, selective pressure may originate from the host intestinal environment itself, wherein diversifying selection mediated by protozoan predation allows for the continued existence of Salmonella able to avoid consumption by host-specific protozoa. This selective pressure would result in high population-level diversity at the Salmonella rfb locus without phase variation. We show here that intestinal protozoa recognize antigenically diverse Salmonella with different efficiencies and demonstrate that differences solely in the O-antigen are sufficient to allow for prey discrimination. Combined with observations of the differential distributions of both serotypes of bacterial species and their protozoan predators among environments, our data provides a framework for the evolution of high genetic diversity at the rfb locus and host-specific pathogenicity in Salmonella.

Phylogenetic relationships among Pneumocystis from Asian macaques inferred from mitochondrial rRNA sequences.

The presence of Pneumocystis organisms was detected by nested-PCR at mitochondrial large subunit (mtLSU) rRNA gene in 23 respiratory samples from Asian macaques representing two species: Macaca mulatta and M. fascicularis. A very high level of sequence heterogeneity was detected with 18 original sequence types. Two genetic groups of Pneumocystis could be distinguished from the samples. Within each group, the extent of genetic divergence was low (2.5+/-1.4% in group 1 and 2.3+/-1.7% in group 2). Genetic divergences were systematically higher when macaque-derived sequence types were compared with Pneumocystis mtLSU sequences from other primate species (from 5.3+/-2.7% to 19.3+/-3.0%). The two macaque-derived groups may be considered as distinct Pneumocystis species. Surprisingly, these Pneumocystis species were recovered from both M. mulatta and M. fascicularis suggesting that host-species restriction may not systematically occur in the genus Pneumocystis. Alternatively, these observations question about the species concept in macaques.