**2. Antibiotic resistance mechanisms among Gram negative nosocomial pathogens**

Antibiotic resistance may be intrinsic (the microorganism is by definition resistant against a certain antibiotic) or acquired. Acquired refers to resistance that is a consequence of mutational events or gene acquisition via horizontal gene transfer.

© 2014 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Four general mechanisms leading to acquired antibiotic resistance have been described: (1) decreased entrance of the antibiotic into the bacterial cell; (2) increased extrusion of the antibiotic by bacterial efflux systems; (3) mutational modification of the antibiotic's target and; (4) production of antibiotic-inactivating enzymes. Characteristic examples for each mechanism are presented in Table 1.

**3.1. Double Disc Synergy Test (DDST)**

**Figure 1.** Double Disc Synergy Test preparation.

Step 2: Make a 0.5 McFarland bacterial suspension.

center of the plate (20 μg amoxicillin+10 μg clavulanic acid).

around the central amoxicillin/clavulanic acid disc (Figure 1).

C for 18-24h.

results negative (Figure 2-upper plates).

testing non-SPICE bacteria.

Step 5: Incubate at 37o

*3.1.2. Interpretation*

*3.1.1. Procedure*

preparation in order to prevent any AmpC interference [27].

The DDST is used for the detection of beta-lactamases that are inhibited by beta-lactamase inhibitors such as clavulanic acid (Ambler class A beta-lactamases and especially ESBLs). For SPICE organisms, cloxacillin should be incorporated in Mueller-Hinton agar during its

Phenotypic and Molecular Methods for the Detection of Antibiotic Resistance Mechanisms in…

http://dx.doi.org/10.5772/57582

141

Step 1: Prepare agar plates containing 200μg/ml cloxacillin (by adding 1ml solution containing 80 mg cloxacillin in 399 ml Mueller-Hinton agar at the liquid phase). Omit this step when

Step 3: Inoculate with a sterile cotton swab and place an amoxicillin/clavulanic acid disc at the

Step 4: Place ceftazidime, imipenem, ceftriaxone, cefotaxime, aztreonam and cefepime discs

The DDST is considered positive when the inhibition zone of any of the antibiotics is larger towards the clavulanic acid disc (Figure 2-lower left plate) or a ghost inhibition zone appears between the central disc and any of the other antibiotics (Figure 2-lower right plate). This is happening because of the ESBL's inhibition by the clavulanic acid. In proximity to the central disc the enzyme's activity is blocked. Thus, the growth inhibition zone appears only towards the clavulanic acid disc. If resistance to cephalosporins is not due to ESBL production, the test


**Table 1.** Examples of antibiotic resistance mechanisms.

Among the aforementioned mechanisms, the production of beta-lactamases is considered of major importance because these enzymes are commonly transferable and inactivate multiple beta-lactam antibiotics. Within this large enzymatic family, carbapenemases (class B metallobeta-lactamases (MBLs) [20] that contain zinc in their active center and class A KPC [21]) hydrolyze in vitro all or almost all beta-lactams, including carbapenems [22]. Class A extended spectrum beta-lactamases (ESBLs) hydrolyze penicillins, monobactams and cephalosporins whereas are inhibited by the beta-lactamase inhibitors [23,24]. Class C cephalosporinases (AmpC) present various spectrums of cephalosporin hydrolysis but are not inhibited by the beta-lactamase inhibitors [25]. Additionaly, AmpC enzymes may be inducible in *Serratia* spp, *Pseudomonas* spp, Indole-positive *Proteus*, *Citrobacter* spp and *Enterobacter* spp (SPICE group of bacteria) complicating the treatment of infections caused by these pathogens. Finally, molecular class D beta-lactamases (OXA) comprise numerous enzymes with variable spec‐ trums of beta-lactam hydrolysis [26].
