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International Biodeterioration & Biodegradation 65 (2011) 460e464

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International Biodeterioration & Biodegradation

journal homepage: www.elsevier .com/locate/ ibiod

Quantitative real-time PCR detection of Pseudomonas oleovorans subsp. lubricantisusing TaqMan-MGB assay in contaminated metalworking fluids

Ratul Saha a,b,*, Robert S. Donofrio b, Susan T. Bagley a

aDepartment of Biological Sciences, Michigan Technological University, Dow 740, 1400 Townsend Drive, Houghton, MI 49931, USAbNSF International, Microbiology Department, 789 Dixboro Road, Ann Arbor, MI 48105, USA

a r t i c l e i n f o

Article history:Received 26 July 2010Received in revised form24 October 2010Accepted 26 October 2010Available online 21 February 2011

Keywords:Metalworking fluidsPseudomonasReal-time PCRgyrBTaqMan-MGB probe

Abbreviations: ATCC, American Type Culture CoColony Forming Units; Ct, Cycle Threshold; HPC, HeterMiddlebrook 7H11 Agar; MGB, Minor Groove Binding;PIA, Pseudomonas Isolation Agar; RGM, Rapidly GrowinTemperatures; TSA, Tryptic Soy Agar.* Corresponding author. Present address: NSF Inte

sion, 789 Dixboro Road, Ann Arbor, MI 48105, USA.fax: þ1 734 827 7190.

E-mail address: rsaha@mtu.edu (R. Saha).

0964-8305/$ e see front matter � 2011 Elsevier Ltd.doi:10.1016/j.ibiod.2010.10.013

a b s t r a c t

Metalworking fluids (MWFs) are highly prone to microbial contamination, which leads to their degra-dation and biofouling. Pseudomonas oleovorans subsp. lubricantis, a newly described subspecies, wasfound to be important to MWF fouling. However, the actual distribution of P. oleovorans subsp. lubricantisin MWF is difficult to study using standard culturing techniques. To overcome this, a study was conductedto design a specific quantitative real-time PCR (qPCR) assay using TaqMan�MGB (minor groove binding)probe for its identification and estimated quantification in contaminated MWFs. The gyrB housekeepinggene sequence was selected for designing a TaqMan� MGB primereprobe pair using the Allele ID� 5.0probe design software for the assay. Whole-cell qPCR was performed with MWF spiked directly withP. oleovorans subsp. lubricantis (eliminating DNA extractions using commercial kit); the primereprobepair’s sensitivity was 101 colony forming units (CFU) ml�1. The assay provided no amplificationwith otherclosely related Pseudomonas species found in MWFs indicating its specificity. It was successful in iden-tifying and enumerating P. oleovorans subsp. lubricantis from several used MWFs having between 104 and106 CFUml�1. The designed TaqMan� MGB probe thus can be successfully used for the subspecies-specificidentification of P. oleovorans subsp. lubricantis and facilitates the study of its impact on MWFs.

� 2011 Elsevier Ltd. All rights reserved.

1. Introduction

Microbial contamination causes degradation and biofouling ofmetalworking fluids (MWFs) (Rossmoore, 1995; van der Gast et al.,2001). There are multiple sources that can contribute to thecontamination of MWFs, such as water used for the dilution of freshfluid, the surrounding environment, and the sumps in which thefluid is stored when not in use (van der Gast et al., 2001). Previousstudies have documented that used MWFs are colonized by obli-gate and facultatively aerobic bacteria as the fluids are aeratedduring the machining processes. It was also observed that thedominant group of bacteria recovered from MWFs belongs to thegenus Pseudomonas (Foxall-VanAken et al., 1986; Virji et al., 2000;

llection; bp, Base Pair; CFU,otrophic Plate Count; M7H11,MWFs, Metalworking Fluids;g Mycobacteria; Tm, Melting

rnational, Microbiology Divi-Tel.: þ1 734 769 8010x2472;

All rights reserved.

Gilbert et al., 2010). The Pseudomonas sp. are capable of growingand surviving in the MWFs as they possess genes involved inhydrocarbon and alkanes utilization, indicating that they arepartially responsible for MWF degradation (Gilbert et al., 2010). It isalso reported that the growth of pseudomonads inMWFs favors thegrowth of other bacteria by reducing the pH and also by neutral-izing the effect of biocides (NIOSH, 1998; Gilbert et al., 2010). Themicrobial diversity of MWFs has commonly been studied usingtraditional microbiological techniques that usually do not recoverthe total microbial population present in the contaminated fluids(van der Gast et al., 2003; Gilbert et al., 2010), thus underestimatingthe actual microbial load or diversity at any given time.

During the investigation of ultraviolet irradiation as a means ofdisinfection for used MWFs, R2A agar, Pseudomonas isolation agar(PIA), and Middlebrook 7H11 (M7H11) agar were used for therecovery and identification of indicator bacteria, such as species ofPseudomonas and rapidly growing mycobacteria (RGM) (Saha et al.,2010b). Based on colony morphology, bacteria recovered onM7H11 from MWFs were initially suspected to be RGM. However,these colonieswere found to be gram-negative rods belonging to thegenus Pseudomonas. Further characterization using polyphasictaxonomy confirmed that these bacteria represent a new subspeciesof Pseudomonas oleovorans (belonging to themendocina sublineage).

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The type strain was named P. oleovorans subsp. lubricantis, as it wasrecovered from and capable of growing inMWFs (Saha et al., 2010b).However, P. oleovorans subsp. lubricantis would go undetected inMWFs as it failed to grow on PIA due to its sensitivity to triclosan; itwas also not recovered on theR2Aagar used for a heterotrophic platecount (HPC) due to its poor growth in this medium. So, a methodbased on molecular technique was developed for more accuratedetection and enumeration of P. oleovorans subsp. lubricantis inMWFs.

The present studywas conducted to design a specific qPCR assayusing a TaqMan-MGB (minor groove binding) probe for the iden-tification and estimation of P. oleovorans subsp. lubricantis incontaminated MWF samples. Based on a previous study (Saha et al.,2010a), a conventional TaqMan probe could not be designed forP. oleovorans subsp. lubricantis as it is closely related to the othersubspecies of P. oleovorans (Saha et al., 2010b), and a more specificprobe was needed for its discrimination and detection. Therefore,a TaqMan-MGB probe was developed because the presence of theMGB moiety increases the specificity and sensitivity of the assay. Itrequires a shorter oligonucleotide probe than the conventionalTaqMan probe and stabilizes the binding of the probe to the tar-geted single-stranded DNA (Ott et al., 2004; Zhao et al., 2005). Thenewly described P. oleovorans subsp. lubricantis belongs not only tothe genus Pseudomonas but also to themendocina sublineage groupof bacteria, such as P. oleovorans subsp. oleovorans and P. oleovoranssubsp. pseudoalcaligenes, which are commonly found in usedMWFsand act as potential deteriorgens (Lee and Chandler, 1941; van derGast et al., 2003; Gilbert et al., 2010). Therefore, it is important todetect and study the distribution and abundance of P. oleovoranssubsp. lubricantis using real-time technology to further investigateits impact on MWFs.

2. Materials and methods

2.1. Bacteria and culturing

P. oleovorans subsp. lubricantis (ATCC BAA1494), P. oleovoranssubsp. pseudoalcaligenes (ATCC 17440), P. oleovorans subsp. oleo-vorans (ATCC 8062), Pseudomonas mendocina (ATCC 2541), Pseu-domonas alcaliphila (ATCC BAA571), Pseudomonas aeruginosa (ATCC15442), and Pseudomonas fluorescens were used for the study. Allstrains were obtained from the American Type Culture Collection(ATCC; Manassas, VA) except for P. fluorescens (recovered fromcontaminated MWFs). All the strains were grown and maintainedon tryptic soy broth and agar (TSA) (BD, Franklin Lakes, NJ) at 37 �C(P. fluorescens was incubated at 30 �C) for 24 h, according to therequirement of the study.

Middlebrook 7H11 agar (Remel Laboratories, Lenexa, KS) wasused for the recovery of P. oleovorans subsp. lubricantis from usedMWFs collected from different locations. The samples were platedon M7H11 and incubated at 30 �C for 48 h. Biochemical andphenotypic characterization such as colony morphology, gramstain, oxidase and catalase, growth on MacConkey agar, PIA, Sim-mon’s citrate, nitrate reduction, arginine dihydrolase, starchhydrolysis, 5% NaCl test, urease test on Christensen agar, and sugarutilization tests were performed for the presumptive identificationof P. oleovorans subsp. lubricantis (Saha et al., 2010b).

2.2. Metalworking fluid samples

Two unused samples obtained from two different MWFmanufacturing companies and five used samples (synthetic, semi-synthetic, and water-soluble oils) from three separate machiningunits from different companies in Michigan, USA, were used.Unused MWFs were diluted (5% with sterile distilled water) before

utilization in assays for growth, specificity, and sensitivity of theprobe, and construction of standard graphs. Used samples wereapplied directly for the analysis without further dilution.

2.3. Growth in metalworking fluid

Erlenmeyer flasks (25 ml) were used with 5 ml of diluted MWFand inoculated with 100 ml (107 CFUml�1) of 48-h-grown culture ofP. oleovorans subsp. lubricantis. The flasks were incubated fordifferent time intervals (48, 72, and 96 h) at room temperature.After each incubation period serial dilutions were prepared in0.85% NaCl; spread plating was done on TSA. Viable counts weredetermined after 48 h at 30 �C. Sample pH was also measured.

2.4. DNA isolation from bacterial cultures

The UltraClean� Microbial DNA isolation kit (Mo Bio Labora-tories, Carlsbad, CA) was used for the isolation and purification ofgenomic DNA from different bacterial cultures used in the study.The purity and the yield of DNAwere estimated using a NanoDrop�

1000 spectrometer.

2.5. Preparation of whole cells for PCR

Five MWF samples collected from different machining opera-tions and freshly prepared samples spiked with P. oleovorans subsp.lubricantis were diluted 10-fold before being used for the whole-cell preparation. Whole-cell sample preparation was performed byplacing 1 ml of sample in a microcentrifuge tube, heating it at 94 �Cfor 10 min, and immediately chilling in ice. The samples werestored in ice or at 4 �C until the assay was performed. Prior towhole-cell preparations, the samples were diluted 10-fold toprevent inhibition of the PCR reaction.

2.6. Design of real-time TaqMan-MGB primereprobe pair

The sequences of the three housekeeping genes gyrB, rpoD, and16S rRNA obtained from P. oleovorans subsp. lubricantis (Saha et al.,2010b) were analyzed using BLAST, CLUSTAL X (Thompson et al.,1997), and Allele ID (v5.0; Premier Biosoft, Palo Alto, CA).Multiple alignments of the sequences were carried out usingCLUSTAL X to determine the sequence similarity and the phyloge-netic relationship between the closely related pseudomonads. TheAllele ID software was used to further analyze the sequences todesign the specific TaqMan-MGB primer-probe pair for P. oleovor-ans subsp. lubricantis. The probe was dual labeled with the fluo-rescent reporter (6-FAM) dye on the 50 end and the quencher (BHQ)on the 30 end of the sequence.

Real-time PCR assays were performed according to the protocoldescribed in Saha et al. (2010a). The only modificationwas in step 2of the PCR program, where the annealing time of the probe at 60 �Cwas for 30 s due to the presence of the MGB moiety.

3. Results

3.1. Enumeration, identification, and growth of P. oleovoranssubsp. lubricantis in MWFs

The viable counts of P. oleovorans subsp. lubricantis in differentused MWF samples recovered on M7H11 agar ranged from unde-tectable to nearly 104 CFU ml�1 (Table 1). The recovered colonieswere identified as presumptive P. oleovorans subsp. lubricantis byperforming different biochemical and phenotypic characterization.It was observed that P. oleovorans subsp. lubricantis was capable ofgrowing in MWF (Fig. 1). The MWF pH dropped from 9.2 to 8.0 in

Table 1Quantitative TaqMan-MGB real-time (qPCR) analysis of P. oleovorans subsp. lubricantis (PL) from used MWF samples. Values of qPCR count and Ct represent mean and standarddeviation (n ¼ 2).

Locationa Age of sample (years) Viable countb qPCR quantification of PL % Cultivatable

HPCc Presumptive PL qPCR count Ctd

A1 2.5 3.2 � 104 8.0 � 103 6.7 (�0.05) � 106 22.4 � 0.01 0.12B1 2 2.6 � 103 1.0 � 102 5.0 (�0.6) � 106 22.9 � 0.2 0.001C1 0.67 <1 � 101 <1 � 101 NDe ND NDC2 2 4.2 � 105 <1 � 101 3.5 (�1.14) � 104 36.7 � 0.6 <0.03C3 0.33 <1 � 101 <1 � 101 ND ND ND

a Used MWF samples collected from different industries located in Michigan, USA.b All viable counts of culturable bacteria are in colony forming units (CFU) per milliliter.c Middlebrook 7H11 (M7H11) was used for the culturable heterotrophic plate count (HPC) of bacteria.d Mean of cycle threshold value.e ND ¼ Not detected.

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96 h, indicating that it might act as a potential deteriorgen inMWFs, as lowering of pH alters fluid chemistry, which leads todegradation of the fluid and also causes corrosion and leaks in theMWF systems (NIOSH,1998). The lowering of the pH also favors thegrowth of other bacteria (Saha et al., 2010a).

3.2. Design of real-time TaqMan-MGB primereprobe pair

The analysis of the three housekeeping gene sequences (gyrB,rpoD, and 16S rRNA) with closely related species of Pseudomonasrevealed that the gyrB sequence of P. oleovorans subsp. lubricantishad more nucleotide differences compared to rpoD and 16S rRNA(Saha et al., 2010b). Therefore, it was selected to design the specificTaqMan-MGB primereprobe pair for the development of the qPCRassay. The unique sequences of the primereprobe pair designed forthe identification of P. oleovorans subsp. lubricantis were:

Sense Primer: 50-GTAGGTGTGTCGGTGGTCAAC-30;Anti-sense Primer: 50-GTTCCCAGATCTTACCGCTACG-30; andAnti-sense TaqMan-MGB Probe: 50-CAGTACCAGTTCCTT-30. The

properties of the unique probe were as follows: Tm ¼ 69.0 �C (afteradding MGB moiety); % GC ¼ 46.7; self dimer (Maximum DeltaG) ¼ �25.29 kcal/mol; length ¼ 15 bp, and the position of bindingof the probe is at 78 bp on the sequence.

3.3. Determination of the specificity and sensitivityof the unique TaqMan-MGB probe

The specificity of the assay was tested against both genomicDNA and whole cells of different Pseudomonas species. Whole-cell

Fig. 1. Growth and impact of P. oleovorans subsp. lubricantis on unused MWF incubatedat room temperature.

preparation and genomic DNA of P. oleovorans subsp. lubricantis,P. oleovorans subsp. oleovorans, P. oleovorans subsp. pseudoalcali-genes, P. alcaliphila, P. mendocina, P. aeruginosa, and P. fluorescenswere used for the qPCR assay. Of these cultures, amplificationsignals were observed only for P. oleovorans subsp. lubricantis forboth genomic DNA (data not shown) and whole cells (Fig. 2),indicating the specificity of the assay.

The sensitivity of the whole cell assay was determined by usingdifferent concentrations (100e104 CFU ml�1) of P. oleovorans subsp.lubricantis spiked into two different freshly diluted MWF samples(Fig. 3I). The assay was efficiently able to detect as low a level as101 CFU ml�1 of P. oleovorans subsp. lubricantis present in MWFs,indicating the sensitivity and the absence of any PCR inhibitoryfactors in theseMWFs. The standard graph obtainedwith thewholecell of P. oleovorans subsp. lubricantis indicated both the linearityand the sensitivity of the assay as evident from the value ofcorrelation coefficient (R2 ¼ 0.998) and the slope �3.69 (Fig. 3II).

3.4. Detection and enumeration of P. oleovorans subsp. lubricantisfrom used MWF samples

A standard graph was constructed with different concentrations(101e104 CFUml�1) of whole cells of P. oleovorans subsp. lubricantisafter the sensitivity of the assay was determined. A plot of cyclethreshold (Ct) against log of CFU ml�1 generated by the standardgraph was used to determine the concentrations of cells present indifferent used MWFs (Fig. 4).

The unique primereprobe pair was successful in detecting andestimating P. oleovorans subsp. lubricantis in different MWF

Fig. 2. Specificity assay of the TaqMan-MGB probe for P. oleovorans subsp. lubricantisusing whole cells targeting a specific region of the gyrB gene. Amplification signal wasobserved for (AeB) P. oleovorans subsp. lubricantis. No amplification signal wasdetected for (CeG) P. oleovorans subsp. oleovorans, P. oleovorans subsp. pseudoalcali-genes, P. alcaliphila, P. mendocina, or P. aeruginosa.

Fig. 3. Standard graph of P. oleovorans subsp. lubricantis constructed using whole-cellqPCR. (I) Logarithmic curve of P. oleovorans subsp. lubricantis using different concen-trations of whole cells (AeE: 104 to 1 CFU ml�1, respectively); F: negative control. (II)Linear representation of standard graph constructed using different concentrations ofwhole cells of P. oleovorans subsp. lubricantis. The TaqMan-MGB probe targeteda region in the gyrB gene. Each dilution was tested in duplicate.

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samples collected from different machining units. The results of theassays are represented in Table 1. The qPCR counts for the quanti-fication of P. oleovorans subsp. lubricantis were 99% higher than theviable counts determined using theM7H11 agar. The qPCR productsof the assays were also run on 2% agarose gel to confirm thepresence of P. oleovorans subsp. lubricantis in the samples. Thedifferences in the band intensity indicated the concentration ofP. oleovorans subsp. lubricantis in different samples (data notshown).

4. Discussion

Assays using qPCR have been developed for the study of RGMand Pseudomonas species in MWFs (Khan and Yadav, 2004; Rhodeset al., 2008; Gilbert et al., 2010), although genomic DNA samples

Fig. 4. Detection and enumeration of P. oleovorans subsp. lubricantis from used MWFsamples. Linear curve of real-time detection and enumeration of P. oleovorans subsp.lubricantis species from used MWFs (A, B, C, and D: 104 to 101 CFU ml�1 whole cells ofP. oleovorans subsp. lubricantis used for the construction of the standard curve; E and F:used MWF samples). Each sample was tested in duplicate.

were used for these assays (Suzuki et al., 2004; Rhodes et al., 2008;Gilbert et al., 2010). Only one study (Saha et al., 2010b) used qPCRwith whole cells for detecting certain Pseudomonas species in usedMWFs. Using whole-cell PCR eliminates the time-consuming DNAextraction step. This study is the first to also use the TaqMan-MGBtechnology for the identification of a specific type of Pseudomonasspecies, i.e., P. oleovorans subsp. lubricantis, in MWFs. In contrast toculture-based methods, where P. oleovorans subsp. lubricantis takes48 h to give well-isolated colonies in different growth media, thewhole-cell qPCR assay can detect P. oleovorans subsp. lubricantiswithin 2 h from used MWFs.

Based on the phylogenetic analysis of the three housekeepinggenes, a specific region of the gyrB gene bearing the signaturesequence for P. oleovorans subsp. lubricantis was targeted to designthe primereprobe pair. However, due to the high percentages ofsimilarity between the closely related bacteria, the regular TaqManprobe for species-specific real-time assay could not be used for theassay. Therefore, TaqMan-MGB technology was utilized to designthe probe for detecting P. oleovorans subsp. lubricantis. TaqMan-MGB probes usually have 10e15 bp and are shorter than conven-tional TaqMan probes, which minimally have 18 bp (Parashar et al.,2006). In an MGB probe the minor groove moiety is attached to the30 end of the sequence along with non-fluorescent quencher (Zhaoet al., 2005; Parashar et al., 2006; Yao et al., 2006). The MGB probewithout the MGB moiety has a lower melting temperature (Tm)than the regular TaqMan probe due to its shorter length, but theattachment of the MGB moiety on the 30 end helps to keep the Tmhigh and also stabilizes the binding of the probe as themoiety bindsto the minor groove of the target sequence (Ott et al., 2004; Traniet al., 2006). So, the advantage of using an MGB moiety is notonly having a high Tm but also having a more specific probe, asevident from this study. Previous studies have shown that the MGBprobe was successfully used for the purpose of investigating allelicdiscrimination (Cubero and Graham, 2005) and also to study singlenucleotide polymorphisms (SNPs) and other mutations (Yao et al.,2006; Morita et al., 2007). Similar to this present study, Ulrichet al. (2006) also used a TaqMan-MGB probe for the identificationof Burkholderia mallei, which is closely related and difficult todifferentiate from Burkholderia pseudomallei. This further empha-sizes the importance of the TaqMan-MGB probe to distinguishbetween closely related bacteria.

In this study the TaqMan-MGB probe was successful in the iden-tification and estimation of P. oleovorans subsp. lubricantis fromdifferentMWFsampleswith a lowerannealing timeof 30 s comparedto the regular TaqMan assay because theMGB probemakes the assayhighly specific for P. oleovorans subsp. lubricantis. The qPCR assaywasnot only successful in identification and estimation of P. oleovoranssubsp. lubricantis from different MWF samples but was also able todistinguish fromclosely related subspecies of P. oleovorans, indicatingthe high specificity of the TaqMan-MGB probe compared to theconventional TaqMan probe (Saha et al., 2010a). Additionally, theassay results indicated that recovery of bacteria using classicalculturing techniques may considerably underestimate the bacterialload present inmany samples (van der Gast et al., 2003; Gilbert et al.,2010). In the present study only 0.001e0.12% P. oleovorans subsp.lubricantis could be recovered by culturingmethods (Table 1). Similarresults were obtained in other studies, such as the 37.7% and 99.9%higher detection rates obtained in MWFs by qPCR in the case ofMycobacterium immunogenum by Rhodes et al. (2008) and Gilbertet al. (2010), respectively.

This investigation confirms that the qPCR assay using wholecells provides a more precise quantitative account of microbialload in environmental samples and can successfully be used forthe detection of specific bacteria such as P. oleovorans subsp.lubricantis from complex environment such as MWFs. Unlike its

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close relatives (P. oleovorans subsp. oleovorans and P. oleovoranssubsp. pseudoalcaligenes), P. oleovorans subsp. lubricantis growspoorly on R2A agar (commonly used for HPC), which adds to theprobability of its not being recovered from MWF samples. Theaccurate estimation of its abundance and distribution will help tofurther investigate its role and impact on MWFs.

Acknowledgments

The authors acknowledge the Biological Sciences Department,Michigan Technological University, MI, USA, and NSF International,MI, USA, for facilities and funding.

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