**3.7. CCCP test**

**3.6. D-test**

148 Trends in Infectious Diseases

*3.6.1. Procedure*

Step 4: Incubate at 37o

**Figure 12.** Preparation of a D-test.

*3.6.2. Interpretation*

shown in Figure 14.

The D-test is used for the detection of inducible AmpC beta-lactamases [39]. An antibiotic is used as an inducer for AmpC production (imipenem or cefoxitin) whereas others are used as

Step 3: Place substrate discs (for example ceftazidime and piperacillin/tazobactam) near the

The test is positive when a D-shaped inhibition zone is observed for one of the substrate discs (Figure 13) because of the imipenem-mediated induction of the AmpC production and the subsequent inactivation of the substrate antibiotic by the beta-lactamase. An important advantage of the test is that it can be easily incorporated within any routine antibiogram as

substrates (ceftazidime, cefotaxime, piperacillin/tazobactam).

Step 2: Inoculate with a sterile cotton swab and place an imipenem disc.

Step 1: Make a 0.5 McFarland bacterial suspension.

C for 18-24h.

imipenem disc as shown in Figure 12.

CCCP (Carbonyl cyanide m-chlorophenyl hydrazone) is an efflux pump inhibitor that can be added in Mueller-Hinton agar during its preparation. The test is used to detect efflux pump overexpression that contributes to or determines carbapenem resistance in the study isolate [40].

**Figure 13.** Interpretation of the D-test.

**Figure 14.** Incorporation of the D-test in a common antibiogram.

#### *3.7.1. Procedure*

Step 1: Prepare agar plates containing CCCP at a concentration of 12.5 μM.

Step 2: Make a 0.5 McFarland bacterial suspension.

Step 3: Inoculate with a sterile cotton swab on a CCCP-supplemented plate and in parallel on a CCCP-free plate. For economy reasons, two isolates may be inoculated on the same plate as shown in Figure 15.

Step 4: Place a meropenem disc on both plates for each inoculation.

Step 5: Incubate at 37o C for 18-24h.

**Gene Primers (5'- 3') Product size Reference aac(6')-Ia** ATGAATTATCAAATTGTG 558 bp [41]

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**aac(6')-Ic** CTACGATTACGTCAACGGCTGC 130 bp [42]

**aac(3)-Ia** ACCTACTCCCAACATCAGCC 169 bp [43]

**aac(3)-Ic** GATGATCTCTACTCAAACC 472 bp [44]

**aac(3)-IV** GTTACACCGGACCTTGGA 675 bp [45]

**aphA-3** GGGACCACCTATGATGTGGAACG 595 bp [46]

**aph(3')-Via** ATACAGAGACCACATACAGT 235 bp [47]

**aad(2'')-Ia** ATGTTACGCAGCAGGGCAGTCG 188 bp [48]

**aph(3')-IIIa** GGCTAAAATGAGAATATCACCGG 523 bp [49,50]

**ant(4')-Ia** CAAACTGCTAAATCGGTAGAAGCC 294 bp [49,50]

**strA-strB** TATCTGCGATTGGACCCTCTG 519 bp [51]

**armA** AGGTTGTTTCCATTTCTGAG 590 bp [52]

**rmtA** CTAGCGTCCATCCTTTCCTC 635 bp [53]

**rmtB** ATGAACATCAACGATGCCCT 769 bp [54]

**gyrA (***E. coli***)** ACGTACTAGGCAATGACTGG 190 bp [56]

**qnrA** TCAGCAAGAGGATTTCTCA 627 bp [57]

**Qnr** CCGTATGGATATTATTGATAAAG 661 bp [58]

**Table 2.** Primers used for the detection of aminoglycoside and quinolone resistance determinants.

AAATCTGCCCGTGTCGTTGGT 343 bp [55]

TTACTCTTTGATTAAACT

TTGCTTCGCCCACTCCTGCACC

ATATAGATCTCACTACGCGC

TTAGGCAGCAGGTTGAGG

AACGGCATTGAGCGTCAG

CAGGCTTGATCCCCAGTAAGTC

GGACAATCAATAATAGCAAT

CGTCAGATCAATATCATCGTGC

CTTTAAAAAATCATACAGCTCGCG

GGAAAGTTGACCAGACATTACGAACT

CATTGCTCATCATTTGATCGGCT

TCTCTTCCATTCCCTTCTCC

TTTGCTTCCATGCCCTTGCC

CCTTCTGATTGGCTTATCCA

GCCATACCTACGGCGATACC

AGAAGTCGCCGTCGATAGAAC

GGCAGCACTATTACTCCCA

CTAATCCGGCAGCACTATTA

**gyrA** (*A. baumannii*)

**Figure 15.** Preparation of the CCCP test.

#### *3.7.2. Interpretation*

The test is considered positive when synergy between meropenem and CCCP is observed on the CCCP-supplemented plate (Figure 16).

**Figure 16.** Interpretation of the CCCP test. Isolate 1 is inoculated on the upper side and isolate 2 is inoculated on the lower side of the plates. (+): Positive; (-): Negative.

## **4. Molecular methods**

Genetic methods for the detection of resistance genes are based on nucleic acid hybridization and amplification. Therefore, the knowledge of specific primers (amplification nucleotides) and probes (labeled single-stranded ologonucleotides) is necessary in order to detect the genetic target of interest. The technique used depends on the type of resistance that is sus‐ pected. A simple polymerase chain reaction (PCR) may be applied searching for a gene that confers a certain level of resistance when it is expressed. This is the case for example, of genes encoding for antibiotic-inactivating enzymes. In Tables 2 and 3, primers for the detection of aminoglycoside or fluorquinolone resistance-conferring enzymes and beta-lactamases are shown, respectively.


**Figure 15.** Preparation of the CCCP test.

the CCCP-supplemented plate (Figure 16).

lower side of the plates. (+): Positive; (-): Negative.

**4. Molecular methods**

shown, respectively.

The test is considered positive when synergy between meropenem and CCCP is observed on

**Figure 16.** Interpretation of the CCCP test. Isolate 1 is inoculated on the upper side and isolate 2 is inoculated on the

Genetic methods for the detection of resistance genes are based on nucleic acid hybridization and amplification. Therefore, the knowledge of specific primers (amplification nucleotides) and probes (labeled single-stranded ologonucleotides) is necessary in order to detect the genetic target of interest. The technique used depends on the type of resistance that is sus‐ pected. A simple polymerase chain reaction (PCR) may be applied searching for a gene that confers a certain level of resistance when it is expressed. This is the case for example, of genes encoding for antibiotic-inactivating enzymes. In Tables 2 and 3, primers for the detection of aminoglycoside or fluorquinolone resistance-conferring enzymes and beta-lactamases are

*3.7.2. Interpretation*

150 Trends in Infectious Diseases

**Table 2.** Primers used for the detection of aminoglycoside and quinolone resistance determinants.


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

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**blaVIM** TCTACATGACCGCGTCTGTC 748 bp [74]

**blaVIM-1** GTTAAAAGTTATTAGTAGTTTATTG 799 bp [73]

**blaVIM-2** ATGTTCAAACTTTTGAGTAAG 801 bp [73]

**blaSPM-1** CCTACAATCTAACGGCGACC 649 bp [75]

**blaNDM-1** GGTTTGGCGATCTGGTTTTC 621 bp [76]

**blaOXA-1** CCAAAGACGTGGATG 540 bp [77]

**blaOXA-10** CGTGCTTTGTAAAAGTAGCAG 652 bp [78]

**blaOXA-23** CCTCAGGTGTGCTGGTTATTC 513 bp [79]

**blaOXA-24** TTCCCCTAACATGAATTTGT 1020 bp [80]

for such resistance determinants in *P. aeruginosa* are shown in Table 4).

**Gene Primers (5'- 3') Reference ampC** CGCCGTACAACCGGTGAT [81]

**OprD** CTACGGCTACGGCGAGGAT [81]

**mexA** AACCCGAACAACGAGCTG [81]

**Table 3.** Primers used for the detection of beta-lactamases frequently encountered among Gram negative pathogens.

In cases in which resistance depends upon the expression level (overexpression or downre‐ gulation) of the gene, real time Reverse Transcriptase-PCR (rt RT-PCR) is used to detect not only the presence, but also the mRNA expression of the gene. The results are consequently confronted with the expression level of the same gene in a control strain. This technique is useful for the study of the expression of specific porins and efflux pumps (primers and probes

TGTGCTTTGACAACGTTCGC

CTACTCGGCGACTGAGC

CTACTCAACGACTGAGCG

TCGCCGTGTCCAGGTATAAC

CGGAATGGCTCATCACGATC

GTTAAATTCGACCCCAAGTT

CATGATTTTGGTGGGAATGG

CCCAACCAGTCTTTCCAAAA

GTACTAATCAAAGTTGTGAA

CGGCCGTCCTCTTTCGA

**probe** [DFAM]TCAGCCTGAAAGGAGAACCGCATTACTTC[DTAM]

GACCGGACTGGACCACGTACT **probe** [DFAM]CACCACGAAACCAACCTCGAAGCC[DTAM]


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

**blaSHV** GGTTATGCGTTATATTCGCC 867 bp [59]

**blaTEM** ATGAGTATTCAACATTTCCG 867 bp [59]

**blaCTX-M** CGCTTTGCGATGTGCAG 550 bp [60]

**blaCTX-M-2** ATGATGACTCAGAGCATTCG 884 bp [61]

**blaCTX-M-9** GTGACAAAGAGAGTGCAACGG 857 bp [62]

**blaCTX-M-10** GCTGATGAGCGCTTTGCG 684 bp [63]

**blaGES/IBC** GTTTTGCAATGTGCTCAACG 371 bp [64]

**blaPER-1** ATGAATGTCATTATAAAAGC 926 bp [65]

**blaPER-2** CGCTTCTGCTCTGCTGAT 469 bp [66]

**blaPSE** ACCGTATTGAGCCTGATTTA 321 bp [67]

**blaTLA-1** TCTCAGCGCAAATCCGCG 974 bp [68]

**blaVEB-1** CGACTTCCATTTCCCGATGC 643 bp [69]

**blaKPC** TGTCACTGTATCGCCGTC 331 bp [70]

**blaSME-1** AACGGCTTCATTTTTGTTTAG 830 bp [71]

**blaIMP** CTACCGCAGCAGAGTCTTTG 587 bp [72]

**blaIMP-1** ATGAGCAAGTTATCTGTATTC 741 bp [73]

**blaIMP-2** ATGAAGAAATTATTTGTTTTATG 741 bp [73]

TTAGCGTTGCCAGTGCTC

152 Trends in Infectious Diseases

CTGACAGTTACCAATGCTTA

TTATTGCATCAGAAACCGTG

ATGATTCTCGCCGCTGAAGCC

TTACAAACCGTTGGTGACG

TGCCATAGCAATAGGCGTAG

AATTTGGGCTTAGGGCAAGAAA

GGCAGCTTCTTTAACGCC

ATTGAAGCCTGTGTTTGAGC

CTATTTCCCATCCTTAACTAG

GGACTCTGCAACAAATACGC

TATTTTTCCGAGATGGGTGAC

GCTTCCGCAATAGTTTTATCA

AACCAGTTTTGCCTTACCAT

TTAGTTGCTTGGTTTTGATGG

TTAGTTACTTGGCTGTGATG

ACCGCGATATCGTTGGT

**Table 3.** Primers used for the detection of beta-lactamases frequently encountered among Gram negative pathogens.

In cases in which resistance depends upon the expression level (overexpression or downre‐ gulation) of the gene, real time Reverse Transcriptase-PCR (rt RT-PCR) is used to detect not only the presence, but also the mRNA expression of the gene. The results are consequently confronted with the expression level of the same gene in a control strain. This technique is useful for the study of the expression of specific porins and efflux pumps (primers and probes for such resistance determinants in *P. aeruginosa* are shown in Table 4).



Consequently, the combined and rational use of the available methodologies seems to be the optimal solution for the cost-effective detection of resistance mechanisms in Gram negative

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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.

and Sianou Effrosyni3

1 Research Assistant, Aristotle University of Thessaloniki, School of Medicine, Thessaloniki,

2 Clinical Microbiologist, Department of Clinical Microbiology, St. Paul's General Hospital,

3 Clinical Microbiologist, Department of Clinical Microbiology, Hippokration General Hos‐

[1] Meletis G. Editorial: war against multi-drug-resistant pathogens: what is new in the

[2] Hanson ND. Editorial: Resistance in gram-negative pathogens: a threat to global

[3] Savard P, Perl TM. A call for action: managing the emergence of multidrug-resistant Enterobacteriaceae in the acute care settings. Curr Opin Infect Dis 2012;25. 371-377.

[4] Grundmann H, Klugman KP, Walsh T, et al. A framework for global surveillance of

[5] Campbell S. The need for a global response to antimicrobial resistance. Nurs Stand

armory? Recent Pat Antiinfect Drug Discov 2012;7. 173-174.

antibiotic resistance. Drug Resist Updat 2011;14. 79-87.

health. Curr Pharm Des 2013;19. 163.

pathogens by the Clinical Microbiology laboratory.

**Acknowledgements**

**Author details**

Thessaloniki, Greece

**References**

pital, Thessaloniki, Greece

2007;21. 35-40.

Greece

Meletis Georgios1,2\*, Tzampaz Egki3

\*Address all correspondence to: meletisg@hotmail.com

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

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.
