**Acknowledgements**

**Gene Primers (5'- 3') Reference**

**mexC** GGAAGAGCGACAGGAGGC [81]

**mexE** TACTGGTCCTGAGCGCCT [81]

**mexX** GGCTTGGTGGAAGACGTG [81]

**Table 4.** Primers and probes used in real-time RT PCR for the determination of the expression levels for specific

Finally, sequencing [82-84] of the PCR product allows its confrontation with the already known gene sequences that are available in genetic databases. This can lead to the detection of mutations or to the characterization and classification of the gene within a genetic family.

There are several benefits and limitations using either phenotypic or molecular methods for the detection of resistance mechanisms in Gram negative pathogens. Phenotypic tests require bacteria in pure culture from a clinical sample thus needing 24-48h to obtain a final result. Molecular techniques on the other hand, can be performed directly with clinical speciments

The detection of low-level resistance is by definition problematic using phenotypic tests thus interpretation problems may appear. In such cases, molecular techniques are an option for

Moreover, genetic detection gives a definite answer for the presence or not of specific resistance determinants within a study isolate (a specific beta-lactamase for example) whereas this is not possible with the phenotypic tests which provide only general information about the resistance

Genetic assays however, present also some major limitations: (i) It is possible to screen exclusively for known mechanisms and for one gene at the time (unless a multiplex PCR assay [85-88] can be applied) and; (ii) their cost is high and becomes higher when screening for

clarifying the possible involvement of any known resistance mechanism.

ATGGCCTTCTGCTTGACG **probe** [DFAM]CATGTTCGTTCACGCGCAGTTG[DTAM]

CTGCACCGTCAGGCCCTC **probe** [DFAM]CCGAAATGGTGTTGCCGGTG[DTAM]

TCAGCGGTTGTTCGATGA **probe** [DFAM]CGGAAACCACCCAAGGCATG[DTAM]

GGCTGATGATCCAGTCGC **probe** [DFAM]CCGACACCCTGCAGGGCC[DTAM]

resistance mechanisms in *P. aeruginosa.*

reducing significantly the procedure time.

**5. Conclusion**

154 Trends in Infectious Diseases

mechanisms involved.

multiple resistance determinants.

We are grateful to our teacher, Dr. Danai Sofianou for the knowledge that she has horizontally transferred to us during our collaboration at Hippokration General Hospital of Thessaloniki.
