Jm520507 403.408

Journal of Medical Microbiology (2003), 52, 403–408 Expression stability of six housekeeping genes: aproposal for resistance gene quantification studiesof Pseudomonas aeruginosa by real-timequantitative RT-PCR Hakan Savli,1 Aynur Karadenizli,2 Fetiye Kolayli,2 Sibel Gundes,3Ugur Ozbek4 and Haluk Vahaboglu3 1–3Tibbi Biyoloji AD1, Mikrobiyoloji ve Klinik Mikrobiyoloji AD2 and Enfeksiyon Hastaliklari ve Klinik Mikrobiyoloji AD3, Kocaeli Universitesi, Tip Fakultesi, Sopali Ciftligi, 41900 Kocaeli, Turkey 4Genetik AD, DETAE, Istanbul Universitesi, Istanbul, Turkey Constantly expressed genes are used as internal controls in relative quantification studies. Suitableinternal controls for such studies have not yet been defined for Pseudomonas aeruginosa. In thisstudy, the genes ampC, fabD, proC, pbp-2, rpoD and rpoS of P. aeruginosa were compared in termsof expression stability by real-time quantitative RT-PCR. A total of 23 strains with diverse resistancephenotypes were studied. Stability of expression among the housekeeping genes was assessed onthe basis of correlation coefficients, with the best-correlated pair accepted as being the most stableone. Eventually, proC and rpoD formed the most stable pair (r ¼ 0·958; P , 0·001). Next, in fourciprofloxacin-selected nfxC-like mutants, levels of oprD, oprM and oprN mRNA were compared withthose of their wild-type counterparts. The comparison was made after correcting the raw values bythe geometric mean of the internal control genes proC and rpoD. The level of oprN mRNA wassignificantly up-regulated, while the oprD gene was down-regulated (although this difference wasstatistically insignificant), in the mutants. This expression pattern was consistent with that of the expected expression profile of nfxC-type mutants; this experiment therefore lends further support to the use of proC and rpoD genes simultaneously as internal controls for such studies.
Levels of expression of these genes have traditionally beenstudied by Western blotting with the aid of mAbs. Unfortu- Pseudomonas aeruginosa is an important nosocomial patho- nately, this method is unable to help in studying different gen, particularly in intensive care units 2000).
proteins simultaneously and so is weak in relative compari- Members of this species are inherently resistant to a range of sons. Moreover, mAbs are not commercially available. The antibiotics. In addition, they are capable of conferring quantification of mRNAs by real-time RT-PCR has been resistance to others by shifting the regulation levels of various used with great success in other fields , 2000; , 1999). We suggest that this highly sensitivemethod may also be useful in relative comparison of Porins (particularly porin D) and efflux pumps of the resistance-nodulation-division (RND) family are the cur- The reliability of a relative comparison depends largely on the rently recognized genetic systems that are associated sig- normalization of unwanted variations between samples.
nificantly with antibiotic resistance , 2001).
Constantly expressed genes, often selected from among The exact links between the expression patterns of these and housekeeping genes, are used as internal controls for normal- other resistance phenotypes have not been fully elucidated, ization of the results. The proportion of mRNA of constantly and inconsistent conclusions appear in the literature expressed genes in the total cellular RNA is assumed to be equal between different samples. Normalization of raw values by means of internal controls therefore serves to topic still attract considerable interest.
eliminate sample-to-sample variation of the RNA isolationand reverse transcription steps and, even more importantly,serves to eliminate variations in total transcriptional activity For this purpose, six housekeeping genes were compared in 39 8C (with an increase of 0·3 8C every cycle), 2 min at 72 8C and 1 min this study. These were pyrroline-5-carboxylate reductase at 94 8C and 30 cycles of 2 min at 44 8C, 3 min at 72 8C and 1 min at (proC), malonyl CoA : acyl carrier protein (ACP) transacyl- 94 8C. A final extension for 1 h at 72 8C completed the procedure.
ase (fabD), sigma factors RpoD (rpoD) and RpoS (rpoS), PCR products were separated on a 2 % agarose gel and visualized by penicillin-binding protein 2 (pbp-2) and chromosomal beta- ethidium bromide staining. For better resolution, they were also run on lactamase (ampC). The expression stability of these house- a 6 % acrylamide/bis-acrylamide gel (data not shown). The banding keeping genes was first investigated as proposed by patterns of both gels were analysed by the freely distributed gel analysis et al. (2002) and, later, the levels of oprM, software LabImage (version 2.62). Molecular sizes of the bands werecalculated by this software relative to the marker DNA.
oprN and oprD mRNA were compared in a set of P.
aeruginosa strains.
RNA isolation and reverse transcription. Total RNA was isolatedfrom 5 ml fresh overnight (approx. 18 h) broth culture (MH broth) byusing the NucleoSpin RNA II kit (Macherey-Nagel), as described by the manufacturer. Genomic DNA was eliminated by RNase-free DNase Itreatment during the isolation procedure. Finally, RNAs were eluted Strains, resistance tests and beta-lactamase assays. We selected from the silica membranes in a volume of 40 ìl diethyl pyrocarbonate- 17 P. aeruginosa strains from a set obtained from university hospitals in treated double-distilled water. The A260 of the resulting RNA solution four different geographical regions of Turkey. The bacteria had been was between 1 and 10. Reverse transcription was performed at 42 8C for identified in those universities by various identification systems. The 90 min by using random hexamer primers so as to obtain cDNA copies strains were, nevertheless, reidentified in our institute by classical of mRNAs (2 ìl) with 100 IU MMuLV reverse transcriptase (MBI methods and, if required, by the non-fermenter ID panel of the Becton Fermentas) in 20 ìl total volume. Concentrations of cDNAs were Dickinson system (Diagnostic Instrument Systems).
adjusted on a LightCycler (Roche Diagnostics). For every sample, 1 ìlcDNA and 9 ìl SYBR Green I (the same concentration as indicated by Of the 17 strains selected, 10 were fully susceptible to antibiotics of the the manufacturer for the PCR assay) were mixed in capillary tubes. After main classes, while the others were of different resistance phenotypes.
incubation at 95 8C for 5 min, fluorescence emissions were read at 55 8C We challenged the susceptible strains in Mueller–Hinton (MH) broth with the real-time fluorometry facility of the LightCycler. This enabled with ciprofloxacin (0·1 ìg mlÀ1) in order to obtain resistant variants.
us to compare the total cDNA concentrations of the samples with the Consequently, six isogenic mutants with various resistance patterns control transcript, which was approximately 1 mg mlÀ1 at the highest were selected. These mutants, as well as their counterparts, were dilution. Concentrations of cDNAs of the samples were adjusted to a level close to the second dilution (10À1) of the control cDNA. Thisadjustment was critical for performing successful calculations. The aim Resistance patterns were determined by the disk diffusion method on was to keep the cDNA concentrations of the samples between the MH agar plates. MICs were determined by either E-test strips on MH concentrations of the controls in order to avoid large variations during agar plates or by the agar dilution method as described by the NCCLS.
calculations by the LightCycler software.
Antibiotic disks and MH agar were obtained from Oxoid while E-teststrips were sourced from AB Biodisc. Powder forms of the antibiotics Real-time PCR. The sequences of the genes studied were obtained from were obtained as gifts from the respective companies.
GenBank and the primers were designed with the aid of the OLIGOsoftware (version 5.0; Molecular Biology Insights). The sequences of the Beta-lactamases were analysed as described elsewhere 1998). Extracts obtained by freezing and thawing of dense bacterialsuspensions were applied to polyacrylamide gels with ampholytes PCR was performed in the LightCycler in capillary glass tubes with the ranging from pH 3 to 10. Nitrocefin overlay and migration relative to LightCycler FastStart DNA Master SYBR Green I kit (Roche). Work was TEM-1 and SHV-1 standards enabled us to evaluate the pI values of always carried out on desktop coolers (pre-cooled to 4 8C). Master beta-lactamases. Precise identification of the beta-lactamases depended mixtures were prepared exactly as recommended by the manufacturer, on sequence analysis of PCR products as described previously except for the concentration of Mg2þ. The final concentrations of Mg2þ and primers were respectively 2·5 mM and 50 pmol per reaction.
The control cDNA was from P. aeruginosa ATCC 27853 and the primers Random amplified polymorphic DNA (RAPD) typing. Clonal of the control reactions were specific for pbp-2. An arbitrary concentra- variability was further ensured by RAPD typing of eight selected strains, tion value of 1·5 3 104 copies of the pbp-2 gene was assigned to the two from each region. DNA was isolated from fresh overnight agar control transcript. Tenfold dilutions of this down to 15 copies of the cultures. A loopful of bacteria was homogenized in 50 ìl TE buffer (10 pbp-2 gene were always included in the reactions.
mM Tris/HCl, 1 mM EDTA, pH 8) and lysis was accomplished with500 ìl guanidium thiocyanate (6 M) plus 0·5 % sodium lauroylsarco- PCR was accomplished after a 5 min activation and denaturation step at sine for 10 min at room temperature. DNA was extracted first by 95 8C, followed by 45 cycles of 15 s at 95 8C, 10 s at 60 8C and 15 s at phenol/chloroform and then by chloroform/isoamyl alcohol (24 : 1, v/v) and then precipitated with 0·1 vols sodium acetate (3 M, pH 5·4) Primer dimers and other artefacts were evaluated by melting curve plus an equal volume of 2-propanol at room temperature. Precipitates analysis and eventually only dimer- and artefact-free reactions were were collected by centrifugation (10 min at 12 000 g) and then the pellets considered valid. Results were read with the ‘second derivative maxi- were washed twice with 70 % ethanol, air-dried for 2 min and mum’ algorithm of the software provided. The LightCycler software resuspended in 30 ìl double-distilled water.
generated a standard curve by plotting ‘crossing cycle number’ versus RAPD PCR was performed as described elsewhere logarithms of the given concentrations for each control. Eventually, a regression line was drawn between these points. The software calculated GGATTCA-39) and ERIC-2 (59-AAGTAAGTGACTGGGGTGAGCG- the concentrations of the studied genes with the aid of this standard 39). Master mixtures were prepared as described in the above references.
However, the amplification program was modified as follows: one cycleof denaturation for 5 min at 95 8C followed by 25 cycles of 3 min at Statistical analysis. The stability of mRNA expression was assessed by Housekeeping genes of Pseudomonas aeruginosa Table 1. Primers used in quantification studies calculation of Spearman’s correlation coefficients of the raw concentra-tion data with the aid of the statistical package SPSS (version 9.0). The best-correlated pair was considered to be the most stable one.
Stability was further evaluated by a freely distributed MS Excelapplication (geNorm). Detailed information on this application can be obtained al. (2002). This approach assumesthat minimally regulated, stably expressed genes stay in a constant ratio to each other. In other words, in a given set of genes, it must be the pair ofmost stable genes that will be able to keep the ratio to each otherconstant in different samples. Importantly, co-regulated genes areexceptions to this assumption and they are not included.
The applet geNorm helps to calculate the gene expression stabilitymeasure (M), which is the mean pair-wise variation for a gene from all other tested control genes al., 2002). A higher value ofM means greater variation in expression. The stepwise exclusion ofgenes with the highest M values allows the ranking of the tested genesaccording to their expression stability. The proposed threshold foreliminating a gene as unstable was an expression stability measure of> 0·5.
Raw quantities were corrected by dividing a value by the geometricmean of proC and rpoD genes of the same sample. Relative comparisonswere done between corrected values with the ANOVA test for Fig. 1. (a) RAPD fingerprints (negative exposure). Lanes: M, mole- cular size marker (100 bp DNA ladder); 1–7, selected strains (strains2 and 5–7 are the parents of nfxC mutants). (b) Fingerprints A total of 23 strains were included in the study. The group according to the lengths (bp) of fragments calculated by gel analysis was composed of 17 wild-type strains and six ciprofloxacin- software. Lanes are the same as in (a).
selected, isogenic mutants. Clonal heterogeneity was inves-tigated by DNA typing of eight of these wild-type strains. Onesample did not provide a readable pattern, probably becauseof inhibitors carried over from the DNA isolation step.
nfxC mutants because of the concurrent down-regulation of However, the other seven were amplified successfully and the fingerprints were sufficiently polymorphic to confirm the phenotypes of the mutants were in agreement with this.
Interestingly, the MICs of ceftazidime were variable; one was Of the 17 wild-type strains, seven were already resistant to increased and one decreased while the other two were various antibiotics. It is noteworthy that three of these did unchanged. One study reported an unexplained decrease in not produce a beta-lactamase other than the chromosomal the ceftazidime MICs of nfxC mutants 2000).
one strains were probably resistant because However, another study showed that the functional subunit of activated porin and/or efflux systems. Of the six cipro- of the mexEF–oprN operon is not related to beta-lactam floxacin-selected mutants, two were resistant only to the hypersusceptibility 2000). These observations fluoroquinolones, while the other four expressed nfxC-like suggest the existence of other co-operating systems respon- multiple-resistance phenotypes In addition to sible for beta-lactam susceptibility or resistance in nfxC fluoroquinolones, the MIC of imipenem increases among mutants. This issue remains unresolved.
Table 2. Antibiotic-resistance phenotypes and extrinsic beta-lactamases in strains examined Seventeen strains were examined: strains R1–7, resistant wild-type; S1–10, susceptible wild-type. Strains were classed asresistant (R) or susceptible (S) according to NCCLS criteria.
*Other than chromosomal AmpC beta-lactamase.
Table 3. MICs of ciprofloxacin-selected (nfxC-like) mutants compared with wild-types MICs are given in ìg mlÀ1 for the parent strain/mutant.
The housekeeping metabolism of prokaryotes has been These data led us to assume that these genes might also be shown to be highly variable et al., 2001), so expressed in sufficient quantities in P. aeruginosa, a further genes expressed stably under one condition might not be so advantage in optimization of the PCR test.
under others. Stability in terms of mRNA expression inprokaryotic cells, therefore, should be tested under equiva- The raw quantities of mRNA of the six genes studied lent conditions with the investigated setting. In other words, internal controls intended for use in resistance gene quanti- coefficients indicated proC and rpoD as the most significant fication studies should be tested in strains with changing pair (r ¼ 0·958; P , 0·001). Similarly, the stepwise exclusion resistance phenotypes. The diversity in the resistance phe- of the genes with the highest M values by geNorm left proC notypes of this study group fulfils this requirement.
(M ¼ 0·36) and rpoD (M ¼ 0·36) as the most stable genes.
Next, the levels of oprD, oprM and oprN mRNA of the fournfxC-like isogenic mutants were compared with those of Six genes were compared in this study group. In order to their wild-type counterparts. In this experiment, the geo- avoid co-regulated genes, we carefully selected genes that are metric mean of the levels of proC and rpoD in a sample was its distantly related in metabolic function and chromosomal normalization factor. Comparison of the normalized quan- order. The selected genes participate in critical functions.
titative values of these genes is shown mean pbp-2 has a central role in peptidoglycan metabolism, while it level of oprN was 1·3 times higher in the mutants, while the has some relation to the rod-shape-determining protein.
mean level of oprD was 1·28 times lower. Values were comparable for all the mutants. Interestingly, the statistical The sigma factor-encoding rpoD is a critical housekeeping comparison was significant only for oprN concentrations.
gene , 1995). proC is involved in amino acidbiosynthesis, while fabD is involved in a different class of Data on expression of these resistance genes in the literature metabolic function , 1999). However, their have to date been obtained by immunoblotting. Evaluations metabolic importance was not the only reason for selecting were dependent on crude differences and lacked precise these genes. Equally important is that they all were shown in numerical values. Therefore, comparison of the results of this an Escherichia coli DNA array study to be expressed at levels study with the literature was not possible. Moreover, immunoblotting indicates differences in the level of mature Housekeeping genes of Pseudomonas aeruginosa Table 4. Raw concentrations of mRNA of six genes from 23 strains obtained by RT-PCR Concentrations are given as copies ìlÀ1 (310À3). S1–S10 are susceptible strains; S5 and S6 are theparents of M1 and M2 (ciprofloxacin-selected non-nfxC-type mutants) and S7–S10 are theparents of NFX1–NFX4 (ciprofloxacin-selected nfxC-type mutants). R1–R7 are wild-typeresistant isolates.
Table 5. Comparison of mRNA concentrations of nfxC-like are regulated individually to some extent. The levels of mRNA of these genes may, therefore, not be exactly in accordwith protein levels. However, the quantification results with RT-PCR in this study were in agreement with the expected expression profile of nfxC-type mutants.
Resistance due to the altered regulation of intrinsic genes in P. aeruginosa is not well understood. This type of resistance may depend on the regulation of a more composite co- regulated network of multiple operons as well as the ‘quorum-sensing systems’ of P. aeruginosa 2001ortunately, our understanding of this is extremely limited due to the limited power of the methods used at present. Therefore, further studies using new meth- ods deserve increased scientific interest. We believe that real- time quantification with the selection of suitable internal control genes will facilitate studies and provide new insights into the regulatory alterations of innate genes and themultiple antibiotic resistance problem of P. aeruginosa.
This study showed that proC and rpoD form the most stablepair in a set of clonally unrelated P. aeruginosa strains with protein. It is known that mRNA expression predicts mature diverse resistance phenotypes. Thus, this pair may be used as protein levels poorly and there may be up to 30-fold internal controls in relative comparison studies of resistance differences ). Transcription and translation Negative regulation of the Pseudomonas aeruginosa outer membraneporin OprD selective for imipenem and basic amino acids. Antimicrob We are grateful to Dr Serhat Unal (Hacettepe Universitesi), Dr Mehmet Ali Inal (Ege Universitesi) and Dr Hakan Leblebicioglu (Ondokuz MayısUniversitesi) for providing strains and to Jason John Nash and Victor L.
Okamoto, K., Gotoh, N. & Nishino, T. (2001). Pseudomonas aeruginosa Yu for English reading of this manuscript.
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