**5. Laboratory detection**

Appropriate and timely laboratory detection of carbapenemase-producing microorganisms is of crucial importance to implement adequate antimicrobial therapy. Screening for carbapenemase production is usually based on reduced inhibition zones around carbapenem disks in routine disk-diffusion testing or elevated MICs for carbapenems. However, the level of resistance depends on the type of carbapenemase, allelic variant, gene expression, and/or the presence of additional resistance mechanisms such as porin loss or upregulation of efflux pumps.

Recommendations for carbapenem susceptibility breakpoints have been established by both Clinical Laboratory Standards Institute (CLSI) and The European Committee on Antimicrobial Susceptibility Testing (EUCAST), but no global consensus exists with EUCAST breakpoints being generally lower [85, 86]. However, some strains with low-level resistance are still being missed. EUCAST has also established epidemiologic breakpoints to distiguish between wildtype isolates and those with carbapenem-resistant determinants [86].

Ideally, methods for identifying carbapenemase should have a short turnaround time to ensure timely implementation of control measures. This could be challenged by difficulties in detecting carbapenemase producers, since MICs to carbapenems could be elevated but within susceptible range or even low, as described in *Enterobacteriacea*e and *A. baumannii*. Particularly in OXA-48-producing organisms, the MICs of carbapenemas can be very low. The screening usually relies on the reduction of the inhibition zone around ertapenem disk [87, 88]. However, relevant methodology with specific laboratory test has not yet been standardized. Modified Hodge test is the only test recommended by CLSI for the phenotypic detection of carbapenemase producers but often lacking sensitivity and specificity. There are also several inhibitor-based tests using different inhibitors (EDTA and phenantroline as inhibitors of MBLs, phenylboronic acid as inhibitor of KPC) in combination with carbapenem (e.g., meropenem) or cephalosporin (e.g., ceftazidime) in different formats—disk-diffusion or broth dilution or E-test [88]. There is no specific inhibitor that could be used in detection of class D carbapenemases, but there are reports on using temocillin disk (or combined with avibactam) for this purpose [88]. Carba NP (derived from the names Nordmann-Poirel) test is a simple biochemical test based on hydrolysis of imipenem detectable by a change of color of the indicator due to decrease of pH. It is applicable in most microbiological laboratories, although the reference standard in detection of carbapenemase production is spectrophotometric measurement of carbapenem hydrolysis in the presence or absence of inhibitor, but it is still reserved for reference laboratories [89]. Recently, a new method for the detection of carbapenemases was described [90].

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 laboratory stuff.

### **6. Therapeutic options**

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

**71**

*Carbapenemases*

**7. Conclusions**

**Author details**

Croatia

*DOI: http://dx.doi.org/10.5772/intechopen.76282*

in treatment of carbapenem-resistant *Pseudomonas aeruginosa* infections [102]. However, it is not effective against MBL-producing organisms. Continued reports should be acknowledged in efforts of optimizing therapy for infections caused by carbapenem-resistant Gram-negative bacili. Furthermore, there is an urgent need to develop new antimicrobials. Although antibiotics still represent the mainstream direction for the treatment, the alternatives to them should be taken into account, including vaccines, bacteriocins, and probiotics. The revival of old antibiotics should be supported by expanding the knowledge of their pharmacokinetics and pharmacodynamics in order to avoid side effects and resistance developments.

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

1 Department of Microbiology, School of Medicine, University of Zagreb, Zagreb,

2 Clinical Department of Clinical and Molecular Microbiology, University Hospital

© 2018 The Author(s). Licensee IntechOpen. 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,

3 Clinical Department of Microbiology and Parasitology, University Hospital

\*Address all correspondence to: branka.bedenic@kbc-zagreb.hr

the community or the environment and vice versa.

Branka Bedenić1,2\* and Sanda Sardelić3

provided the original work is properly cited.

Centre Zagreb, Zagreb, Croatia

Centre Split, Split, Croatia

#### *Carbapenemases DOI: http://dx.doi.org/10.5772/intechopen.76282*

in treatment of carbapenem-resistant *Pseudomonas aeruginosa* infections [102]. However, it is not effective against MBL-producing organisms. Continued reports should be acknowledged in efforts of optimizing therapy for infections caused by carbapenem-resistant Gram-negative bacili. Furthermore, there is an urgent need to develop new antimicrobials. Although antibiotics still represent the mainstream direction for the treatment, the alternatives to them should be taken into account, including vaccines, bacteriocins, and probiotics. The revival of old antibiotics should be supported by expanding the knowledge of their pharmacokinetics and pharmacodynamics in order to avoid side effects and resistance developments.
