Use of MALDI-TOF Mass Spectrometry and a Custom Database to Characterize Bacteria Indigenous to a Unique Cave Environment (Kartchner Caverns, AZ, USA)

MALDI-TOF mass spectrometry has been shown to be a rapid and reliable tool for identification of bacteria at the genus and species, and in some cases, ...

Arizona State University

Forty percent of hazardous waste sites in the United States are co-contaminated with organic and metal pollutants. Data from both aerobic and anaerobic systems demonstrate that biodegradation of the organic component can be reduced by metal toxicity. Metal bioavailability, determined primarily by medium composition/soil type and pH, governs the extent to which metals affect biodegradation. Failure to consider bioavailability rather than total metal likely accounts for much of the enormous variability among reports of inhibitory concentrations of metals. Metals appear to affect organic biodegradation through impacting both the physiology and ecology of organic degrading microorganisms. Recent approaches to increasing organic biodegradation in the presence of metals involve reduction of metal bioavailability and include the use of metal-resistant bacteria, treatment additives, and clay minerals. The addition of divalent cations and adjustment of pH are additional strategies currently under investigation.

Impact of metals on the biodegradation of organic pollutants.

Metals have been reported to inhibit organic pollutant biodegradation; however, widely varying degrees and patterns of inhibition have been reported. To investigate the roles of medium composition and metal bioavailability on these different degrees and patterns of inhibition, we assessed the impact of cadmium on naphthalene biodegradation by a newly isolated strain of Comamonas testosteroni in three chemically-defined minimal salts media (MSM): Tris-buffered MSM, PIPES-buffered MSM, and Bushnell-Haas medium. Cadmium (total concentrations of 100 and 500 microM) inhibited biodegradation in each medium. Degrees of inhibition were different in each medium. Cadmium was most inhibitory in PIPES-buffered MSM and least inhibitory in Bushnell-Haas. For example, in Bushnell-Haas medium, 100 microM cadmium reduced the cell yield more than 4-fold compared to controls not containing cadmium. The same concentration of cadmium completely inhibited growth in PIPES-buffered MSM. No difference in inhibition was observed in any medium when cadmium was added 24 h before inoculation rather than when added within one minute of inoculation. Two patterns of inhibition were observed. Inhibition occurred in a dose dependent pattern in Tris- and PIPES-buffered MSM and in a non-dose dependent pattern in Bushnell-Haas. Specifically, in Bushnell-Haas, 100 microM total cadmium extended the lag phase by 23+/-8.66 h, whereas 500 microM did not extend the lag phase. Soluble, ionic cadmium (Cd2+) concentrations were measured and modeled in each medium to assess cadmium bioavailability. In media containing 500 microM total cadmium, bioavailability was highest in Tris- and PIPES-buffered MSM and lowest in Bushnell-Haas. In Bushnell-Haas, cadmium bioavailability was initially higher in the 500 microM treatments (196+/-21.2 microM) than in the 100 microM treatments (78.2+/-2.04 microM); however, after 12 h, bioavailability was higher in the 100 microM treatments (56.4+/-24.8 micro) than the 500 microM treatments (13.3+/-1.2 microM). These data suggest that the type of medium determines the degrees and patterns by which metals inhibit biodegradation and emphasize the importance of coupling metal toxicity and bioavailability data.

Medium composition affects the degree and pattern of cadmium inhibition of naphthalene biodegradation.

Rush Lake (WI, USA), the largest prairie-pothole lake east of the Mississippi River, has been contaminated with lead pollution as a result of over 140 years of waterfowl hunting. We examined: (1) the extent of lead pollution in Rush Lake sediments and (2) whether lead pollution in Rush Lake is affecting the abundance and community structure of indigenous sediment bacteria. Sediment lead concentrations did not exceed 59 mg Pb kg(-1) dry sediment. No relationship was observed between sediment lead concentration and the abundance of aerobic (P=0.498) or anaerobic (P=0.416) heterotrophic bacteria. Similarly, lead did not appear to affect bacterial community structure when considering both culturable and nonculturable community members. In contrast, the culturable fraction of sediment bacteria in samples containing 59 mg Pb kg(-1) exhibited a unique community structure. While factors other than lead content likely play roles in determining bacterial community structure in the sediments of Rush Lake, these data suggest that the culturable fraction of sediment bacterial communities is affected by elevated lead levels.

Lead pollution in a large, prairie-pothole lake (Rush Lake, WI, USA): effects on abundance and community structure of indigenous sediment bacteria.

A rapid and reliable bacterial source tracking (BST) method is essential to counter risks to human health posed by fecal contamination of surface waters. Genetic fingerprinting methods, such as repetitive sequence based-PCR (rep-PCR), have shown promise as BST tools but are time-consuming and labor-intensive. In this work, we investigate the ability of MALDI-TOF-MS to characterize and discriminate between closely related environmental strains of Escherichia coli and to classify them according to their respective sources. We compared the performance of a rapid MALDI-TOF-MS-based method to a commonly used rep-PCR-based method that employs the BOX-A1R primer. Among the criteria evaluated were repeatability and the ability of each method to group E. coli isolates according to their respective sources. Our data suggest that the MALDI-TOF-MS-based approach has a lower repeatability level compared to rep-PCR but offers an improved ability to correctly assign E. coli isolates to specific source groups. In addition, we have identified five biomarkers that appear conserved among avian species. We conclude that MALDI-TOF-MS may represent a promising, novel and rapid approach to addressing the problem of fecal contamination of surface waters and warrants further investigation.

Discrimination and characterization of environmental strains of Escherichia coli by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS).

Currently available bacterial source-tracking tools are often technically demanding, time consuming, and have limited accuracy in grouping isolates according to their respective sources. There is a need for the development of bacterial source-tracking tools that would allow for more rapid and accurate grouping of isolates by source. We examined the use of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) for the characterization of environmental isolates of Enterococcus. Our main objectives were to develop sample preparation protocols for obtaining reproducible MALDI-TOF mass spectra from Enterococcus isolates and to evaluate methods of data analysis to maximize repeatability of the method and its ability to group isolates according to their respective sources. Our data showed that treatment of 21 Enterococcus isolates from seven unique sources with lysozyme for 20 h, followed by calculation of similarity coefficients using the Pearson product-moment correlation coefficient, facilitated a repeatability level of 91% as well as grouping by source for isolates obtained from several sources including human waste. Our data suggest that MALDI-TOF-MS-based fingerprinting of environmental isolates of Enterococcus has potential as a rapid and accurate bacterial source tracking (BST) tool, but requires further development, specifically regarding the time requirements needed for pre-treatment of isolates with lysozyme.

Characterization of environmental isolates of Enterococcus spp. by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

It has been widely reported that pH mediates cadmium toxicity to bacteria. We used a tripartite approach to investigate mechanisms by which pH affects cadmium toxicity that included analyses of: (1) growth kinetics, (2) global gene expression, and (3) cadmium speciation. Cadmium extended the lag phase at pH 7, but not at pH 5. DNA microarray analysis revealed that stress response genes including hdeA, otsA, and yjbJ were more highly expressed at pH 5 than at pH 7 after only 5 min of exposure to cadmium, suggesting that acidic pH more rapidly induced genes that confer cadmium resistance. In addition, genes involved in transport and many hypothetical genes were more highly expressed at pH 5 than at pH 7 in the presence of cadmium. Concentrations of two cadmium species, including one previously implicated in the mechanism by which pH mediates cadmium toxicity (CdOH+), increased with pH. Our data demonstrate that transcriptional responses of Escherichia coli to cadmium are substantially affected by pH and suggest that several stress response, transport, and hypothetical genes play roles in the mechanism by which pH mediates cadmium toxicity.

Environmental pH affects transcriptional responses to cadmium toxicity in Escherichia coli K-12 (MG1655).

Hazardous waste sites are commonly contaminated with both organic and metal pollutants. Many metal pollutants have been shown to inhibit organic pollutant biodegradation. We investigated the ability of a modified, polydentate cyclodextrin (carboxymethyl-beta-cyclodextrin, CMCD) to reduce the toxicity of 33.4 microM cadmium, cobalt or copper during naphthalene degradation by a Burkholderia sp. in 120 h aerobic, batch studies. The highest investigated concentration of CMCD, 3340 microM, reduced cadmium, cobalt, and copper toxicity. With each metal, the length of the lag phase was reduced (by as much as 108 h with cobalt or copper), the cell yield was increased (by as much as a factor of 16 with cobalt), and the growth rate was increased (by as much as a factor of 31 with cobalt). The degrader was unable to use CMCD as the sole source of carbon and energy. Our data suggest that the ability of CMCD to complex metals plays an important role in its ability to mitigate metal toxicity and that CMCD has the potential to enhance biodegradation in organic and metal co-contaminated environments.

Carboxymethyl-beta-cyclodextrin mitigates toxicity of cadmium, cobalt, and copper during naphthalene biodegradation.

High concentrations of Escherichia coli in mats of Cladophora in the Great Lakes have raised concern over the continued use of this bacterium as an indicator of microbial water quality. Determining the impacts of these environmentally abundant E. coli, however, necessitates a better understanding of their ecology. In this study, the population structure of 4285 Cladophora-borne E. coli isolates, obtained over multiple three day periods from Lake Michigan Cladophora mats in 2007-2009, was examined by using DNA fingerprint analyses. In contrast to previous studies that have been done using isolates from attached Cladophora obtained over large time scales and distances, the extensive sampling done here on free-floating mats over successive days at multiple sites provided a large dataset that allowed for a detailed examination of changes in population structure over a wide range of spatial and temporal scales. While Cladophora-borne E. coli populations were highly diverse and consisted of many unique isolates, multiple clonal groups were also present and accounted for approximately 33% of all isolates examined. Patterns in population structure were also evident. At the broadest scales, E. coli populations showed some temporal clustering when examined by year, but did not show good spatial distinction among sites. E. coli population structure also showed significant patterns at much finer temporal scales. Populations were distinct on an individual mat basis at a given site, and on individual days within a single mat. Results of these studies indicate that Cladophora-borne E. coli populations consist of a mixture of stable, and possibly naturalized, strains that persist during the life of the mat, and more unique, transient strains that can change over rapid time scales. It is clear that further study of microbial processes at fine spatial and temporal scales is needed, and that caution must be taken when interpolating short term microbial dynamics from results obtained from weekly or monthly samples.

Multi-scale temporal and spatial variation in genotypic composition of Cladophora-borne Escherichia coli populations in Lake Michigan.

The use of matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometry (TOF MS) has emerged as a promising tool to rapidly profile bacteria at the genus and species level and, more recently, at the sub-species (strain) level. Recently, it has been proposed that the approach can be enhanced with regard to reproducibility and throughput by automating spectrum acquisition; however, effects of automating spectrum acquisition on spectrum quality and reproducibility have not been investigated. Using an intact cell-based sample preparation method, we directly compared the quality and reproducibility of spectra acquired in a fully automated fashion to those acquired manually by two operators with different levels of experience. While automation tended to increase base peak resolution, other measures of spectrum quality, including signal-to-noise (S:N) ratio, data richness, and reproducibility were reduced. Negative effects of automation on the performance of this approach to bacterial profiling may be particularly important during profiling of closely related strains of bacteria that yield very similar spectra.

Automating data acquisition affects mass spectrum quality and reproducibility during bacterial profiling using an intact cell sample preparation method with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

Since the advent of the use of matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometry (TOF MS) as a tool for microbial characterization, efforts to increase the taxonomic resolution of the approach have been made. The rapidity and efficacy of the approach have suggested applications in counter-bioterrorism, prevention of food contamination, and monitoring the spread of antibiotic-resistant bacteria. Strain-level resolution has been reported with diverse bacteria, using library-based and bioinformatics-enabled approaches. Three types of characterization at the strain level have been reported: strain categorization, strain differentiation, and strain identification. Efforts to enhance the library-based approach have involved sample pre-treatment and data reduction strategies. Bioinformatics approaches have leveraged the ever-increasing amount of publicly available genomic and proteomic data to attain strain-level characterization. Bioinformatics-enabled strategies have facilitated strain characterization via intact biomarker identification, bottom-up, and top-down approaches. Rigorous quantitative and advanced statistical analyses have fostered success at the strain level with both approaches. Library-based approaches can be limited by effects of sample preparation and culture conditions on reproducibility, whereas bioinformatics-enabled approaches are typically limited to bacteria, for which genetic and/or proteomic data are available. Biological molecules other than proteins produced in strain-specific manners, including lipids and lipopeptides, might represent other avenues by which strain-level resolution might be attained. Immunological and lectin-based chemistries have shown promise to enhance sensitivity and specificity. Whereas the limits of the taxonomic resolution of MALDI TOF MS profiling of bacteria appears bacterium-specific, recent data suggest that these limits might not yet have been reached.

MALDI TOF MS profiling of bacteria at the strain level: a review.

MALDI-TOF MS has been shown capable of rapidly and accurately characterizing bacteria. Highly reproducible spectra are required to ensure reliable characterization. Prior work has shown that spectra acquired manually can have higher reproducibility than those acquired automatically. For this reason, the objective of this study was to optimize automated data acquisition to yield spectra with reproducibility comparable to those acquired manually. Fractional factorial design was used to design experiments for robust optimization of settings, in which values of five parameters (peak selection mass range, signal to noise ratio (S:N), base peak intensity, minimum resolution and number of shots summed) commonly used to facilitate automated data acquisition were varied. Pseudomonas aeruginosa was used as a model bacterium in the designed experiments, and spectra were acquired using an intact cell sample preparation method. Optimum automated data acquisition settings (i.e., those settings yielding the highest reproducibility of replicate mass spectra) were obtained based on statistical analysis of spectra of P. aeruginosa. Finally, spectrum quality and reproducibility obtained from non-optimized and optimized automated data acquisition settings were compared for P. aeruginosa, as well as for two other bacteria, Klebsiella pneumoniae and Serratia marcescens. Results indicated that reproducibility increased from 90% to 97% (p-value[Formula: see text]0.002) for P. aeruginosa when more shots were summed and, interestingly, decreased from 95% to 92% (p-value [Formula: see text] 0.013) with increased threshold minimum resolution. With regard to spectrum quality, highly reproducible spectra were more likely to have high spectrum quality as measured by several quality metrics, except for base peak resolution. Interaction plots suggest that, in cases of low threshold minimum resolution, high reproducibility can be achieved with fewer shots. Optimization yielded more reproducible spectra than non-optimized settings for all three bacteria.

Todd R. Sandrin

School of Mathematical and Natural Sciences

Arizona State University

Use of MALDI-TOF Mass Spectrometry and a Custom Database to Characterize Bacteria Indigenous to a Unique Cave Environment (Kartchner Caverns, AZ, USA)

Todd R. Sandrin

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Arizona State University

Use of MALDI-TOF Mass Spectrometry and a Custom Database to Characterize Bacteria Indigenous to a Unique Cave Environment (Kartchner Caverns, AZ, USA)

School of Mathematical and Natural Sciences