Evaluating the Antibiotic Susceptibility of Chlamydia - New Approaches for in Vitro Assays.

Pigs are the natural hosts of Chlamydia suis, the only Chlamydia species known to spontaneously acquire homotypic resistance conferred by a class C tetracycline resistance gene. Various susceptibility assays have existed for several years, but there is no widely accepted, standardized assay to determine chlamydial antibiotic susceptibility. In this study, we developed new approaches to determine the in vitro susceptibility of Chlamydia to different antibiotics in view of existing protocols. Specifically, the minimal inhibitory concentration (MIC) is based on a consensus of both inclusion number reduction and alteration of inclusion size and morphology upon antibiotic exposure. In addition to these, we employed a recovery assay, allowing observation of the chlamydial response to drug removal and subsequent recovery, as compared to both continued exposure and to the unexposed control. We propose a simple and fast screening method to detect tetracycline resistant C. suis strains within 2 to 3 days with minimal use of consumables. For proof of principle, we evaluated the susceptibility of three C. suis field strains and the reference strain S45/6 to tetracycline, sulfamethoxazole, and penicillin, antibiotics commonly used to prevent respiratory and gastrointestinal diseases on fattening pig farms. We found that tetracycline sensitive strains can easily be distinguished from resistant strains using the evaluation parameters proposed in this study. Moreover, we report that S45/6 is sensitive to sulfamethoxazole while all evaluated C. suis field strains showed some degree of sulfamethoxazole resistance. Finally, we confirm that Penicillin G induces the chlamydial stress response in all evaluated C. suis strains.

Role of host cell integrins in the microsporidium Encephalitozoon intestinalis adherence and infection in vitro.

Microsporidia are obligate intracellular, spore-forming, fungal-related pathogens that employ a unique organelle, the polar tube, to transfer infectious spore contents into host cells to initiate infection. Spore adherence to host cells may provide the proximity required for polar tube/host cell interaction during in vivo infection. In previous in vitro studies, host sulfated glycosaminoglycans (GAGs) or recombinant microsporidia endospore protein (EnP1) was implicated in the pathogen adherence and infection process; however, complete ablation of spore adherence and infection could not be achieved, suggesting that additional or alternative spore and host cell determinants of adherence and infection may exist. Analysis of the Encephalitozoon intestinalis genome revealed about 100 predicted proteins containing the canonical integrin-binding motif arginine-glycine-aspartic acid (RGD); and, many pathogens have been shown to engage integrin molecules on cell surfaces. We hypothesized that host cell integrins play a role in microsporidia adherence and infection. In this study, we demonstrated that addition of exogenous integrin ligands or recombinant alpha 3 beta 1 integrin or alpha 5 beta 1 integrin to assays of E. intestinalis adherence and infection significantly reduced spore adherence and infection of host cells, supporting our hypothesis and implicating these specific integrins as putative host cell receptors for E. intestinalis spores.

Productive and Penicillin-Stressed Chlamydia pecorum Infection Induces Nuclear Factor Kappa B Activation and Interleukin-6 Secretion In Vitro.

Nuclear factor kappa B (NFκB) is an inflammatory transcription factor that plays an important role in the host immune response to infection. The potential for chlamydiae to activate NFκB has been an area of interest, however most work has focused on chlamydiae impacting human health. Given that inflammation characteristic of chlamydial infection may be associated with severe disease outcomes or contribute to poor overall fitness in farmed animals, we evaluated the ability of porcine chlamydiae to induce NFκB activation in vitro. C. pecorum infection induced both NFκB nuclear translocation and activation at 2 hours post infection (hpi), an effect strongly enhanced by suppression of host de novo protein synthesis. C. suis and C. trachomatis showed less capacity for NFκB activation compared to C. pecorum, suggesting a species-specific variation in NFκB activation. At 24 hpi, C. pecorum induced significant NFκB activation, an effect not abolished by penicillin (beta lactam)-induced chlamydial stress. C. pecorum-dependent secretion of interleukin 6 was also detected in the culture supernatant of infected cells at 24 hpi, and this effect, too, was unchanged by penicillin-induced chlamydial stress. Taken together, these results suggest that NFκB participates in the early inflammatory response to C. pecorum and that stressed chlamydiae can promote inflammation.

Random Mutagenesis of the Aspergillus oryzae Genome Results in Fungal Antibacterial Activity.

Multidrug-resistant bacteria cause severe infections in hospitals and communities. Development of new drugs to combat resistant microorganisms is needed. Natural products of microbial origin are the source of most currently available antibiotics. We hypothesized that random mutagenesis of Aspergillus oryzae would result in secretion of antibacterial compounds. To address this hypothesis, we developed a screen to identify individual A. oryzae mutants that inhibit the growth of Methicillin-resistant Staphylococcus aureus (MRSA) in vitro. To randomly generate A. oryzae mutant strains, spores were treated with ethyl methanesulfonate (EMS). Over 3000 EMS-treated A. oryzae cultures were tested in the screen, and one isolate, CAL220, exhibited altered morphology and antibacterial activity. Culture supernatant from this isolate showed antibacterial activity against Methicillin-sensitive Staphylococcus aureus, MRSA, and Pseudomonas aeruginosa, but not Klebsiella pneumonia or Proteus vulgaris. The results of this study support our hypothesis and suggest that the screen used is sufficient and appropriate to detect secreted antibacterial fungal compounds resulting from mutagenesis of A. oryzae. Because the genome of A. oryzae has been sequenced and systems are available for genetic transformation of this organism, targeted as well as random mutations may be introduced to facilitate the discovery of novel antibacterial compounds using this system.

Expression and Localization of an Hsp70 Protein in the Microsporidian Encephalitozoon cuniculi.

Microsporidia spore surface proteins are an important, under investigated aspect of spore/host cell attachment and infection. For comparison analysis of surface proteins, we required an antibody control specific for an intracellular protein. An endoplasmic reticulum-associated heat shock protein 70 family member (Hsp70; ECU02_0100; "C1") was chosen for further analysis. DNA encoding the C1 hsp70 was amplified, cloned and used to heterologously express the C1 Hsp70 protein, and specific antiserum was generated. Two-dimensional Western blotting analysis showed that the purified antibodies were monospecific. Immunoelectron microscopy of developing and mature E. cuniculi spores revealed that the protein localized to internal structures and not to the spore surface. In spore adherence inhibition assays, the anti-C1 antibodies did not inhibit spore adherence to host cell surfaces, whereas antibodies to a known surface adhesin (EnP1) did so. In future studies, the antibodies to the 'C1' Hsp70 will be used to delineate spore surface protein expression.