Bacterial isolates
During the study period, 122 nonduplicated isolates of K. pneumoniae, A. baumannii, and E. coli, which showed resistance to at least one of the tested carbapenems, were collected. The isolates were obtained from urine specimens (43, 35.2%), followed by tracheal aspirate/fluid (35, 28.7%), wound and soft tissue specimens (34, 27.9%), blood (6, 4.9%), sputum (3, 2.5%), bronchoalveolar lavage (1, 0.8%). The source of isolates was as follows: intensive care units (46, 37.7%), internal medicine (25, 20.5%), surgery (23, 18.9%), outpatient clinics (11, 9.0%), emergency (10, 8.2%), cardiac care unit (3, 2.5%), orthopedic (3, 2.5%), and pediatric intensive care unit (1, 0.8%). Patient's age ranged from 1 to 93 years old (61.4 ± 20.43), which 73 (59.8%) were from female patients, while 49 (40.1%) from males.
Antimicrobial susceptibility testing
The results of the antimicrobial susceptibility testing indicated that majority of K. pneumoniae and A. baumannii isolates were resistant to ceftriaxone, cefotaxime, cefepime, tazocin, amoxicillin clavulanic acid, ceftazidime and gentamicin, which indicates that these isolates were extensively drug-resistant (XDR). Antimicrobial susceptibility profiles of the isolates were shown in Table 1.
Phenotypic detection of carbapenemase production
To evaluate phenotypical tests for carbapenemase production, three methods the MHT, Carba NP test and mCIM were selected. All the positive controls were positive in all the three methods. There were no false positive results with all three methods. Discriminatory power of the methods in comparison to PCR was statistically significant (p < 0.0001). The positivity of MHT, Carba NP and mCIM tests according to the bacteria are shown in tables 2.
MHT test results
All 122 isolates of carbapenem-resistant bacteria were tested, and among them 70 (57.3%) isolates showed positive results by MHT. There were observed that 24 isolates had false negative results by MHT. All the 24 isolates of positive carbapenemase gene, which were negative by the MHT test, included 13, 10 and one isolates of A. baumannii, K. pneumoniae and E. coli respectively. The false negative results missed by MHT test were linked to blaVIM (11/24), blaKPC (6/24), blaIMP (4/24) and blaOXA-48 (3/24) carbapenemases. The sensitivity of the MHT test was determined as 67.33%, and a specificity of 100% in the isolates studied (Table 3).
CarbaNP test results
Among the studied carbapenem-resistant bacteria, 84 (68.8%) isolates were positive by CarbaNP. The positivity of the CarbaNP test according to bacteria is given in Table 2. There were false negative results in 16 isolates in Carba NP test. The false negative results missed by Carba NP test were linked to blaVIM (8/16), blaIMP (5/16) and blaKPC (3/16) carbapenemases. The test was found negative in all those isolates displaying no reduced sensitivity against any of the carbapenems. The sensitivity of the CarbaNP test was determined as 80.8%, and a specificity of 100% in the isolates studied (Table 3).
mCIM test results
All 122 isolates of carbapenem-resistant bacteria were tested, and among them 87 (71.3%) isolates showed positive results by mCIM (Table 2). It was observed that 12 isolates tested by mCIM were determined as false negative. All the 12 isolates of positive carbapenemase gene, which were negative by the mCIM test, included 11 and one isolates of A. baumannii and K. pneumoniae, respectively. The false negative results missed by mCIM test were linked to blaVIM (6/12), blaIMP (3/12) blaKPC (2/12), and blaOXA-48 (1/12) carbapenemases.
All the isolates were detected to be positive by the end of 6 h. The negative isolates were incubated overnight, but the result did not change. All the isolates displaying no reduced sensitivity against any of the carbapenems were found to be negative by the mCIM test. The sensitivity and specificity of the mCIM test in the isolates studied was determined to be 90.4% and 100%, respectively. We observed that the mCIM test has a slightly higher sensitivity than the CarbaNP test (90.4%% vs. 80.8%, respectively; p = 0.018).
Detection of carbapenemase genes
All isolates were tested for five carbapenemase genes by PCR. Of all the carbapenem-resistant isolates, PCR results demonstrated that 119 (97.5%) isolates harbored at least a carbapenemase genes, with none of the five carbapenemase genes being detected in three isolates by the PCR method (Table 4). One isolate yielding carbapenemase production by mCIM test was negative for the evaluated genes. The number of isolates that carried these genes is shown in Table 5. The most frequently carbapenemase genes identified were blaKPC identified in 59 isolates (48.3%), blaVIM identified in 49 isolates (40.1%), followed by blaIMP (31, 25.4%) and blaOXA-48 (9, 7.3%), whereas blaNDM was not detected in this study. In addition, a high percentage 54 (70.1%) of K. pneumoniae isolates carried blaKPC, while only three and two isolates of A. baumannii and E. coli, respectively, contained this resistant genes. Co-production of ‘KPC and VIM’, ‘KPC and IMP’ and ‘KPC and OXA-48’ was detected in nine (K. pneumoniae), seven (five K. pneumoniae and two E. coli) and three (K. pneumoniae) isolates, respectively.