Evaluation of Antibiotic Resistance Pattern and Efficacy of Modified Hodge Test for Detection of Carbapenem-Resistant Klebsiella pneumoniae Strains Isolated From Clinical Samples

© 2018 The Author(s); Published by Alborz University of Medical Sciences. This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial 4.0 International License (http://creativecommons.org/licenses/by-nc/4.0/) which permits copy and redistribute the material just in noncommercial usages, provided the original work is properly cited. Background Klebsiella pneumoniae is a gram-negative opportunistic pathogen that can cause infections such as pneumonia, septicemia, urinary tract infection (UTI), and soft tissue infection.1,2 Carbapenems are a class of effective therapeutic agents for the treatment of these infections, however, in recent years resistance to carbapenems has been increased.3,4 The mechanisms of resistance to carbapenems may be related to a series of causes including weakness in bacterial outer membrane permeability, increasing production of extended-spectrum betalactamases (ESBLs), AmpC beta-lactamases and expression of betalactamases like carbapenemases.5,6 The production of carbapenemases especially K. pneumoniae carbapenemase (KPC) is the most important mechanism of enzymatic resistance. Carbapenemaseproducing k. pneumoniae are a group of emerging, highly drug-resistant bacilli, which cause infections associated with significant morbidity and mortality.7 A number of carbapenemases have been reported including KPC, GES, SME, NMC-A and IMI types (Amber class A), IMP, VIM and NDM type (Amber class B), metallo-β-lactamases and OXA type (Amber class D), and oxacillinases.8,9 KPCproducing K. pneumoniae was reported to emerge in some countries such as the northeastern USA, Greece, Israel, Columbia and Puerto Rico. Moreover, France, Sweden, Norway, Scotland, China, Colombia, Brazil, Trinidad and Tobago, and Poland are the countries that have reported pathogen-harbouring KPCs.10, 11 Information on this issue is limited in our country.


Modified Hodge Test
The modified Hodge Test (MHT) was performed according to the CLSI recommendations.At first, the aliquot of E. coli ATCC 25922 in 5 mL saline was adjusted to 0.5 McFarland standard, and then the suspension was diluted 1:10.Next, the sterile cotton swab was dipped into the suspension and inoculated on Müeller-Hinton agar plate, then a 10 μg meropenem disk was placed in the center of the plate.In a straight line, by a sterile swab, suspected bacteria (resistant or semi-susceptible isolates to one or more antibiotics of the carbapenem family and third generation cephalosporins) were streaked from the edge of the meropenem disc (MEM) to the plate edge.The plate was incubated overnight at 35 ± 2°C in ambient air for 16-24 hours.In negative isolates, the clear zones around the disk remain homogeneous, while carbapenemase-producing isolates cause cloverleaf like indentation.

Results
The samples tested comprised 100 clinical samples of K. pneumoniae that were identified by biochemical tests and also were confirmed by determination of the ureD gene.Analysis for presence of ureD gene demonstrated that all isolates were positive for ureD gene which confirmed their identity as K. pneumoniae (Figure 1).
Among 100 clinical isolates of K. pneumonia, 62 were females and 38 were males (P = 0.01).The highest prevalence was related to the urine specimens with 46 (46%) isolates, while blood and cerebrospinal fluidderived samples each with 2 (2%) were the rare ones.The clinical profile of K. pneumoniae isolates are demonstrated in Table 1.The ICU ward with 53 (53%) and the infant ward with 7 (7%) samples were the most and the least frequent cases, respectively.
Results of antibiotic susceptibility test are shown in Table 2.The highest and the lowest rates of resistance were observed for piperacillin (84%) and ertapenem (50%), respectively.
The MHT was performed for suspected carbapenemaseproducing isolates (Figure 2).
The MHT was positive for 68 (68%) isolates.Urine samples (64.7%) accounted for the majority of cases, while abdominal and cerebrospinal fluids (0%) were the lowest frequent groups.
Based on the results (Table 3), the ICU wards with 47 (69.1%) and the emergency wards with 4 (5.9%)samples were the most and the least frequent cases in MHT positive group, respectively.

Discussion
Klebsiella pneumoniae is a gram-negative opportunistic pathogen of nosocomial infections that can remain on environmental surfaces and on human skin and respiratory tract. 12,13The increasing appearance of resistance to various antibiotics in K. pneumoniae isolates is worrisome.This study was designed to evaluate the prevalence of carbapenemase-producing K. pneumoniae strains among various clinical specimens obtained from different wards of Isfahan hospitals, Iran.In our study, 68% of isolates were positive as carbapenemaseproducing.The urinary specimens from the ICU wards were the most frequent cases and the highest resistance was to piperacillin.In our findings, there was a raise in the resistant of K. pneumoniae isolates, especially carbapenem resistant strains in our city; this means that the frequency of carbapenemase is higher in Isfahan and needs further investigation and consideration.Although tests other than MHT, such as the aminophenylboronic acid and dipicolinic acid tests, may also be suitable options for the phenotypic screening of carbapenemases, the facilities necessary for these tests are not routinely available in the majority of laboratories.In this study, MHT was a suitable method for approving carbapenemase production.In Italy, 84% (32 of of clinical isolates showed the production of carbapenemase. 14In several other studies like that of Krishnappa et al and Shanmugam et al, the frequency of carbapenemase-producing cases were 82% (38/46) and 95%, respectively. 15,16In another study in Brazil, 36 of the 44 carbapenem non-susceptible K. pneumoniae isolates were phenotypic carbapenemase producers as determined by the MHT. 17In the studies conducted in Iran like that of Roudbari et al and Shokri et al, the prevalence of carbapenem-resistant K. pneumoniae were   87% and 7.1%, respectively. 18,19These differences show that hospitalization for a long time and the widespread use of broad-spectrum cephalosporins and carbapenems can increase carbapenemase-producing K. pneumoniae infections.The study of Agha-Seyed Hosseini et al in Kashan, Iran, indicated that among 181 K. pneumoniae isolates, 26.5% of cases were imipenem-resistant and the isolates showed high resistance to ampicillin, cefalotin, and cefotaxime, while the low resistance was found to ertapenem and doripenem.The urinary and respiratory samples from the ICU wards accounted for the most frequent infections. 20Bina et al reported that the highest and the lowest resistance were to piperacillin and imipenem. 21In our study, the results for the most frequent clinical samples and the highest resistance were similar to the results of studies of Agha-Seyed Hosseini et al and Bina et al, respectively.In comparison to the studies of Bina et al 21 and Roudbari et al, 18 in our study there was a raise in carbapenem-resistant strains, meaning that in our regions, there is a need for further guidance/information on infection prevention and for control team.Moreover, if phenotypic methods are used, we suggest the laboratories to employ PCR for all carbapenemase-encoding genes.We hope the results of this study be useful in the application of an effective control approach on infectious diseases to avoid and decrease the prevalence of carbapenemresistant K. pneumoniae.

Table 1 .
Prevalence of Klebsiella Pneumoniae Isolates by Type of Clinical Specimens

Table 2 .
Antimicrobial Resistance Profile of Klebsiella Pneumoniae Isolates

Table 4 .
Antimicrobial Resistance Profile of Klebsiella Pneumoniae MHT Positive

Table 3 .
Prevalence of Klebsiella pneumoniae MHT Among the Clinical Locations