Rapid detection of rRNA group I pseudomonads in contaminated metalworking fluids and biofilm formation by fluorescent in situ hybridization

Abstract

Metalworking fluids (MWFs), used in different machining operations, are highly prone to microbial degradation. Microbial communities present in MWFs lead to biofilm formation in the MWF systems, which act as a continuous source of contamination. Species of rRNA group I Pseudomonas dominate in contaminated MWFs. However, their actual distribution is typically underestimated when using standard culturing techniques as most fail to grow on the commonly used Pseudomonas Isolation Agar. To overcome this, fluorescent in situ hybridization (FISH) was used to study their abundance along with biofilm formation by two species recovered from MWFs, Pseudomonas fluorescens MWF-1 and the newly described Pseudomonas oleovorans subsp. lubricantis. Based on 16S rRNA sequences, a unique fluorescent molecular probe (Pseudo120) was designed targeting a conserved signature sequence common to all rRNA group I Pseudomonas. The specificity of the probe was evaluated using hybridization experiments with whole cells of different Pseudomonas species. The probe's sensitivity was determined to be 10(3) cells/ml. It successfully detected and enumerated the abundance and distribution of Pseudomonas indicating levels between 3.2 (± 1.1) × 10(6) and 5.0 (± 2.3) × 10(6) cells/ml in four different industrial MWF samples collected from three different locations. Biofilm formation was visualized under stagnant conditions using high and low concentrations of cells for both P. fluorescens MWF-1 and P. oleovorans subsp. lubricantis stained with methylene blue and Pseudo120. On the basis of these observations, this molecular probe can be successfully be used in the management of MWF systems to monitor the levels and biofilm formation of rRNA group I pseudomonads.

Potential biological control ofErwinia tracheiphilaby internal alimentary canal interactions inAcalymma vittatumwithPseudomonas fluorescens

“…Pseudomonas fluorescens was visualized using probe Pseudo120 labelled with a 6‐FAM fluorophore (Saha et al . ). To visualize E. tracheiphila , we designed a species‐specific probe.…”

The microbiology of metalworking fluids

“…Due to the continuous release of microbial biomass from biofilm sources, there exists a steady concentration of microorganisms (10 7 CFU/ml) capable of growth (within 12 h after cleaning; Veillette et al 2004), which makes control application critical in MWF systems with biofilms (Skerlos et al 2001). A recent study by Saha et al (2011b) demonstrated that, under a low concentration of cells, biofilm formation (Fig. 3) by some of the predominant species of Pseudomonas could be delayed by 24 h, indicating that implementation of suitable disinfection methods along with a proper fluid management system could prolong the life of in-use MWFs.…”

“…The BactiFluor method was performed successfully in all the 107 MWF samples used along with 30 other metal-processing fluids. Saha et al (2011b) also developed a FISH method for the rapid detection of rRNA group I pseudomonads in contaminated MWFs and to study the biofilm-forming capabilities of some of the predominant Pseudomonas species. The unique Pseudo120 probe successfully detected and enumerated the abundance and distribution of Pseudomonas, indicating levels between 3.2 (± 1.1)×10 6 and 5.0 (± 2.3)×10 6 cells/ml of used MWFs.…”

Microorganisms in metalworking fluids: Current issues in research and management

“…The number of the publications on biofilms growing in the MWFs environment is limited. In a recent work [41], the biofilms formed by Pseudomonas isolates were grown on microscope slides and FISH technique was used to evaluate which species of the bacteria was actually forming the biofilm. This technique allowed the detection of live bacteria in the biofilms, both grown in the laboratory, as well as these existing in the coolant samples collected from industrial plants.…”