**7. Conclusions**

*Growing and Handling of Bacterial Cultures*

laboratory stuff.

**6. Therapeutic options**

The test was called carbapenem inactivation method (CIM), and it was based on degradation of meropenem by carbapenemase. The water suspension of carbapenemresistant microorganism is incubated with a 10-ug meropenem disk for 2 h. After incubation, the disk is removed from the suspension and placed onto a Mueller-Hinton agar plate previously inoculated with the carbapenem-sensitive microorganism (usually *Escherichia coli*). If the carbapenem-resistant microorganism produces carbapenemase, consequently, meropenem will be hydrolyzed and the indicator (carbapenem susceptible) microorganism will grow close to the disk [90]. CIM method has high sensitivity, comparable to that of Carba NP test, as reported recently [91]. Modified CIM test includes preparation of the bacterial suspension in tryptic soy broth and extending the time of incubation to 4 hours improving the detection of some carbapenemases in *Enterobacteriaceae* [92]. Recently, the use of mass spectrometry (matrix-assisted laser desorption/ionization-time of flight—MALDI-TOFF) based on analysis of degradation of carbapenem-molecule-enabled rapid detection of KPC carbapenemase (in 45 min) or MBL (150 min) [93]. Finally, simplex or multiplex PCR, real-time PCR, or hybridization tests could significantly improve detection of carbapenemase genes in clinical laboratory bypassing the sensitivity and specificity problems with phenotypic tests [89]. However, molecular methods require expensive equipment and trained

Colistin is usually the last resort antibiotic for the treatment of infections associated with carbapenemase-producing Gram-negative bacteria. However, nephrotoxicity and neurotoxicity are of clinical concern [94]. Colistin-resistant isolates of *A. baumannii, P. aeruginosa*, and *K. pneumoniae* have emerged recently leading to pan-drug resistant phenotypes with modified outer membrane lipopolysaccharide [95–97]. The optimal treatment approach for infection caused by carbapenemaseproducing Gram-negative bacilli remains a controversy. Still, a combination therapy is strongly recommended and includes two or more antibiotics active in vitro such as colistin, tigecycline, amikacin, aztreonam, or carbapenem in different combination schemes [98, 99]. Colistin is not recommended as monotherapy because of the development of heteroresistance. The combinations of colistin with rifampicin, vancomycin, and meropenem were shown to be synergistic in *A. baumannii* in vitro by chequerboard, 2-well and time-kill metod, but the results of the randomized, controlled clinical trials are controversial [99–101]. If the pathogen is suspected to be MBL producer, aztreonam may present the core drug (if the isolate does not possess other extended-spectrum or ampC β-lactamase), but if the patient is critically ill, the combination therapy with three drugs is preferred. If aminoglycosides or fosfomycin retain in vitro activity, for any serious infection, then they should not be using monotherapy. However, oral fosfomycin should not be used for the management of any infection outside the urinary tract, and in critically ill patients, combination therapy should be considered. Tigecycline is recommended for skin and soft-tissue infection with carbapenemase-producing *A. baumannii* and *Enterobacteriacea*e. If MICs to meropenem are low or moderately high (up to 16 μg/ml), high-dose meropenem can be administered by prolonged infusion [99, 100]. Dual carbapenem-based regimen for carbapenem-resistant *Enterobacteriaceae* usually includes high-dose meropenem or high-dose doripenem with ertapenem, and it is also most effective if the third drug is added [99]. In bloodstream and respiratory tract infections colistin could be added, while in gastrointestinal and biliary tract infections colistin and tigecycline should be included. Newer β-lactamβ-lactamase inhibitor combinations such as ceftolozan/tazobactam seem promising

**70**

In the last decade, an epidemic spread of carbapenamases among *Enterobacteriaceae* and Gram-negative nonfermentative bacilli was observed worldwide. The type of the most prevalent carbapenemase in a geographic region differs and might be associated with historical or cultural connections and exchange of people between countries where certain types of carbapenemase are endemic including the transfer of patients or staff across the borders, medical tourism, and migration of refugees. Particularly alarming is the fact that carbapenemases are not restricted only to hospital isolates any more. They have been reported and are continuously circulating between hospitals, long-term care facilities, community, and the environment. In conclusion, facing the global dissemination of carbapenemase-producing Gram-negative bacteria, and awaiting for the new antimicrobials to appear, a reasonable approach including hand hygiene, contact precautions, rational antibiotic usage, and active surveillance should be part of an intensive strategy aimed to reduce the incidence of colonization and infections with carbapenem-resistant microorganisms and to stop the spread of these bacteria into the community or the environment and vice versa.
