Personal exposure of dairy workers to dust, endotoxin, muramic acid, ergosterol, and ammonia on large-scale dairies in the high plains Western United States.

Dairy workers experience a high degree of bioaerosol exposure, composed of an array of biological and chemical constituents, which have been tied to adverse health effects. A better understanding of the variation in the magnitude and composition of exposures by task is needed to inform worker protection strategies. To characterize the levels and types of exposures, 115 dairy workers grouped into three task categories on nine farms in the high plains Western United States underwent personal monitoring for inhalable dust, endotoxin, 3-hydroxy fatty acids (3-OHFA), muramic acid, ergosterol, and ammonia through one work shift. Eighty-nine percent of dairy workers were exposed to endotoxin at concentrations exceeding the recommended exposure guidelines (adjusted for a long work shift). The proportion of workers with exposures exceeding recommended guidelines was lower for inhalable dust (12%), and ammonia (1%). Ergosterol exposures were only measurable on 28% of samples, primarily among medical workers and feed handlers. Milking parlor workers were exposed to significantly higher inhalable dust, endotoxin, 3-OHFA, ammonia, and muramic acid concentrations compared to workers performing other tasks. Development of large modern dairies has successfully made progress in reducing worker exposures and lung disease prevalence. However, exposure to endotoxin, dust, and ammonia continues to present a significant risk to worker health on North American dairies, especially for workers in milking parlors. This study was among the first to concurrently evaluate occupational exposure to assayable endotoxin (lipid A), 3-hydroxy fatty acids or 3-OHFA (a chemical measure of cell bound and noncell-bound endotoxins), muramic acid, ergosterol, and ammonia among workers on Western U.S. dairies. There remains a need for cost-effective, culturally acceptable intervention strategies integrated in OHS Risk Management and production systems to further optimize worker health and farm productivity.

Spectinamide MBX-4888A exhibits favorable lesion and tissue distribution and promotes treatment shortening in advanced murine models of tuberculosis.

The spectinamides are novel, narrow-spectrum semisynthetic analogs of spectinomycin, modified to avoid intrinsic efflux by Mycobacterium tuberculosis . Spectinamides, including lead MBX-4888A (Lee-1810), exhibit promising therapeutic profiles in mice, as single drugs and as partner agents with other anti-tuberculosis antibiotics including rifampin and/or pyrazinamide. To demonstrate that this translates to more effective cure, we first confirmed the role of rifampin, with or without pyrazinamide, as essential to achieve effective bactericidal responses and sterilizing cure in the current standard of care regimen in chronically infected C3HeB/FeJ mice compared to BALB/c mice. Thus, demonstrating added value in testing clinically relevant regimens in murine models of increasing pathologic complexity. Next we show that MBX-4888A, given by injection with the front-line standard of care regimen, is treatment shortening in multiple murine tuberculosis infection models. The positive treatment responses to MBX-4888A combination therapy in multiple mouse models including mice exhibiting advanced pulmonary disease can be attributed to favorable distribution in tissues and lesions, retention in caseum, along with favorable effects with rifampin and pyrazinamide under conditions achieved in necrotic lesions. This study also provides an additional data point regarding the safety and tolerability of spectinamide MBX-4888A in long-term murine efficacy studies.

Comparative studies evaluating mouse models used for efficacy testing of experimental drugs against Mycobacterium tuberculosis.

Methodologies for preclinical animal model testing of drugs against Mycobacterium tuberculosis vary from laboratory to laboratory; however, it is unknown if these variations result in different outcomes. Thus, a series of head-to-head comparisons of drug regimens in three commonly used mouse models (intravenous, a low-dose aerosol, and a high-dose aerosol infection model) and in two strains of mice are reported here. Treatment with standard tuberculosis (TB) drugs resulted in similar efficacies in two mouse species after a low-dose aerosol infection. When comparing the three different infection models, the efficacies in mice of rifampin and pyrazinamide were similar when administered with either isoniazid or moxifloxacin. Relapse studies revealed that the standard drug regimen showed a significantly higher relapse rate than the moxifloxacin-containing regimen. In fact, 4 months of the moxifloxacin-containing combination regimen showed similar relapse rates as 6 months of the standard regimen. The intravenous model showed slower bactericidal killing kinetics with the combination regimens tested and a higher relapse of infection than either aerosol infection models. All three models showed similar outcomes for in vivo efficacy and relapse of infection for the drug combinations tested, regardless of the mouse infection model used. Efficacy data for the drug combinations used also showed similar results, regardless of the formulation used for rifampin or timing of the drugs administered in combination. In all three infection models, the dual combination of rifampin and pyrazinamide was less sterilizing than the standard three-drug regimen, and therefore the results do not support the previously reported antagonism between standard TB agents.

Synthesis, Structure-Activity Relationship, and Mechanistic Studies of Aminoquinazolinones Displaying Antimycobacterial Activity.

Phenotypic whole-cell screening against Mycobacterium tuberculosis (Mtb) in glycerol-alanine-salts supplemented with Tween 80 and iron (GASTE-Fe) media led to the identification of a 2-aminoquinazolinone hit compound, sulfone 1 which was optimized for solubility by replacing the sulfone moiety with a sulfoxide 2. The synthesis and structure-activity relationship (SAR) studies identified several compounds with potent antimycobacterial activity, which were metabolically stable and noncytotoxic. Compound 2 displayed favorable in vitro properties and was therefore selected for in vivo pharmacokinetic (PK) studies where it was found to be extensively metabolized to the sulfone 1. Both derivatives exhibited promising PK parameters; however, when 2 was evaluated for in vivo efficacy in an acute TB infection mouse model, it was found to be inactive. In order to understand the in vitro and in vivo discrepancy, compound 2 was subsequently retested in vitro using different Mtb strains cultured in different media. This revealed that activity was only observed in media containing glycerol and led to the hypothesis that glycerol was not used as a primary carbon source by Mtb in the mouse lungs, as has previously been observed. Support for this hypothesis was provided by spontaneous-resistant mutant generation and whole genome sequencing studies, which revealed mutations mapping to glycerol metabolizing genes indicating that the 2-aminoquinazolinones kill Mtb in vitro via a glycerol-dependent mechanism of action.

Metronidazole lacks antibacterial activity in guinea pigs infected with Mycobacterium tuberculosis.

Metronidazole, which is used for the treatment of infections caused by anaerobic organisms, was evaluated in Mycobacterium tuberculosis-infected guinea pigs. M. tuberculosis can adapt to hypoxia, which is present in the primary lesions of infected guinea pigs. Metronidazole treatment (for 6 weeks at 100 mg/kg of body weight) resulted in no reduction in the bacillary burden and significantly worsened lesion inflammation.

Bedaquiline and Pyrazinamide Treatment Responses Are Affected by Pulmonary Lesion Heterogeneity in Mycobacterium tuberculosis Infected C3HeB/FeJ Mice.

BALB/c and Swiss mice are routinely used to validate the effectiveness of tuberculosis drug regimens, although these mouse strains fail to develop human-like pulmonary granulomas exhibiting caseous necrosis. Microenvironmental conditions within human granulomas may negatively impact drug efficacy, and this may not be reflected in non-necrotizing lesions found within conventional mouse models. The C3HeB/FeJ mouse model has been increasingly utilized as it develops hypoxic, caseous necrotic granulomas which may more closely mimic the pathophysiological conditions found within human pulmonary granulomas. Here, we examined the treatment response of BALB/c and C3HeB/FeJ mice to bedaquiline (BDQ) and pyrazinamide (PZA) administered singly and in combination. BALB/c mice consistently displayed a highly uniform treatment response to both drugs, while C3HeB/FeJ mice displayed a bimodal response composed of responsive and less-responsive mice. Plasma pharmacokinetic analysis of dissected lesions from BALB/c and C3HeB/FeJ mice revealed that PZA penetrated lesion types from both mouse strains with similar efficiency. However, the pH of the necrotic caseum of C3HeB/FeJ granulomas was determined to be 7.5, which is in the range where PZA is essentially ineffective under standard laboratory in vitro growth conditions. BDQ preferentially accumulated within the highly cellular regions in the lungs of both mouse strains, although it was present at reduced but still biologically relevant concentrations within the central caseum when dosed at 25 mg/kg. The differential treatment response which resulted from the heterogeneous pulmonary pathology in the C3HeB/FeJ mouse model revealed several factors which may impact treatment efficacy, and could be further evaluated in clinical trials.