Evidence for Environmental-Human Microbiota Transfer at a Manufacturing Facility with Novel Work-related Respiratory Disease

Abstract

Rationale: Workers' exposure to metalworking fluid (MWF) has been associated with respiratory disease.Objectives: As part of a public health investigation of a manufacturing facility, we performed a cross-sectional study using paired environmental and human sampling to evaluate the cross-pollination of microbes between the environment and the host and possible effects on lung pathology present among workers.Methods: Workplace environmental microbiota were evaluated in air and MWF samples. Human microbiota were evaluated in lung tissue samples from workers with respiratory symptoms found to have lymphocytic bronchiolitis and alveolar ductitis with B-cell follicles and emphysema, in lung tissue samples from control subjects, and in skin, nasal, and oral samples from 302 workers from different areas of the facility. In vitro effects of MWF exposure on murine B cells were assessed.Measurements and Main Results: An increased similarity of microbial composition was found between MWF samples and lung tissue samples of case workers compared with control subjects. Among workers in different locations within the facility, those that worked in the machine shop area had skin, nasal, and oral microbiota more closely related to the microbiota present in the MWF samples. Lung samples from four index cases and skin and nasal samples from workers in the machine shop area were enriched with Pseudomonas, the dominant taxa in MWF. Exposure to used MWF stimulated murine B-cell proliferation in vitro, a hallmark cell subtype found in the pathology of index cases.Conclusions: Evaluation of a manufacturing facility with a cluster of workers with respiratory disease supports cross-pollination of microbes from MWF to humans and suggests the potential for exposure to these microbes to be a health hazard.

Keywords: Pseudomonas pseudoalcaligenes; bioaerosols; environmental microbiota; microbiome; occupational disease.

Figure 1.

Compositional similarities between samples. (A) The graph shows a principal coordinate analysis based on the Bray-Curtis dissimilarity index for each sample type, coded by location. Overall, in-use metalworking fluid (MWF) and lung tissue clustered more closely than the other fluids obtained. The calculation between sample types and within MWF, skin, and nasal samples demonstrates significant compositional differences (see inset). (B–E) Degree of similarity between MWF and human samples. Microbiota similarities based on the Bray-Curtis dissimilarity index were explored for MWF (both preserved and nonpreserved) and lung samples (B), skin samples (C), nasal swab samples (D), and oral wash samples (E) (P value is based on Mann-Whitney test). Assem. = assembly; NP = nonpreserved; ns = not significant; P = preserved.

Figure 2.

A Pseudomonas operational taxonomic unit (OTU) found to be differentially enriched in human and environmental samples. (A) Violin plots display the adjusted P values for the comparison of the relative abundance of taxa between different groups, using DESeq2, within each sample type (cases vs. control for lung tissue; administration vs. assembly vs. machine shop for skin, nasal, oral, and air samples; and preserved vs. nonpreserved for metalworking fluid). Dot sizes are proportional to relative abundance for each OTU. (B) An OTU annotated to Pseudomonas (Pseudomonas_813945) was identified based on DESeq2 and found to be higher in case lung samples and consistently enriched in skin and nasal samples from employees working in the machine shop area. Lastly, this Pseudomonas OTU was enriched in nonpreserved metalworking fluid. *P value < 0.05 and ***P value < 0.001. Admin = administration; Assemb = assembly; Mach Shop = machine shop; MWF = metalworking fluid; NP = nonpreserved; P = preserved.

Figure 3.

In vitro evaluation of murine B-cell proliferation after exposure to metalworking fluid (MWF). Murine B cells were exposed to different MWFs because this cell subtype is found as a hallmark cell for the histological abnormalities identified among subjects from this facility who underwent lung biopsy because of respiratory symptoms and were found to have of lymphocytic bronchiolitis, alveolar ductitis, and emphysema with B-cell primary lymphoid follicles involving both respiratory bronchioles and alveolar ducts. Top: Representative bright-field microcopy images of murine B cells after 4-day in vitro culture with media supplemented with either BAFF (B-cell activating factor) or LPS as positive controls or with one of the four MWF samples (neat preserved, in-use preserved, neat nonpreserved, and in-use nonpreserved). In the absence of additional stimuli, signaling via BAFF in naive cells promotes survival but not proliferation. LPS (positive control) induces the proliferation of B cells via TLR4 signaling. Larger blasting cells are evident in wells where the proliferation of B cells is induced, whereas only small dying cells are found in the wells with MWF neat conditions. Scale bars, 100 μm. Bottom panels (gated on live single cells) show representative fluorescence-activated cell-sorting analysis of cell staining with intracellular proliferation dye (cell trace violet). The dye stains free amines inside the cells and is diluted as cells proliferate. The percentage of the population that underwent proliferation is indicated. Cells exposed to BAFF are used as a reference population and are shown in gray.