|Year : 2016 | Volume
| Issue : 4 | Page : 835-845
Phenotypic and molecular characterization of multidrug-resistant Enterobacteriaceae clinical isolates from intensive care units at Menoufia University hospitals
Ahmed B Mahmoud1, Naira A Eissa1, Nahed A El-Raghy1, Hatem M El-Sebaey2, Nahla A Melake1, Elham T Awad3, Sanaa S. M. Hamam MSc 1
1 Department of Microbiology, Faculty of Medicine, Menoufia University, Menoufia, Egypt
2 Department of Biochemistry, Faculty of Medicine, Menoufia University, Menoufia, Egypt
3 Department of Microbiology, Teaching Hospital, Menoufia, Shebin El-koum, Egypt
|Date of Submission||02-Aug-2015|
|Date of Acceptance||01-Oct-2015|
|Date of Web Publication||21-Mar-2017|
Sanaa S. M. Hamam
Department of Microbiology, Faculty of Medicine, Menoufia University, Textile Street, Shebin El Kom, Menoufia Governorate
Source of Support: None, Conflict of Interest: None
The objectives of the study were to determine the prevalence of multidrug-resistant Enterobacteriaceae spp. isolated from different ICUs at Menoufia University hospitals; to detect the presence of extended-spectrum β-lactamases (ESβLs), AmpC β-lactamases, and carbapenemases among the isolated pathogens by using phenotypic methods; and to investigate the presence of blaKPCand blaNDMresistance genes using real-time PCR.
ESβL producers are associated with increased morbidity and mortality, especially among patients in ICUs. Carbapenemases are diverse enzymes that vary in the ability to hydrolyze carbapenems and other β-lactams.
Materials and methods
This study involved 270 clinical specimens from 160 patients. ESβL production was detected by resistance to third-generation cephalosporins and aztreonam and confirmed by combined disk test. AmpC production was detected by cefoxitin disk test and confirmed by AmpC disk test. Carbapenemases were detected by resistance to imipenem, meropenem, and ertapenem, and then confirmed by modified Hodge test, phenylboronic acid combined disk (PBA-CD) test, and imipenem/EDTA combined disk (IPM/EDTA-CD) test. Carbapenemase gene (blaKPCand blaNDM) detection was performed by real-time PCR.
In all, 52.89% of Enterobacteriaceae isolates were extreme drug resistant, 26.45% were pandrug resistant, and 14.88% were multidrug resistant. Carbapenem resistance was 60.33% for imipenem, 65.29% for meropenem, and 85.95% for ertapenem. Risk factors associated with ICUs were ICU stay greater than and equal to 14 days, exposure to invasive procedures, and comorbid conditions. ESβL production occurred in 41.32% and AmpC occurred in 32.23% of Enterobacteriaceae spp. Carbapenemase production was detected in 69.86%; Klebsiella pneumoniae carbapenemase (KPC) was detected in 60.27%; and metallo-β-lactamase was detected in 75.34%. blaKPC gene was detected in 26.03% of imipenem-resistant Enterobacteriaceae spp., whereas blaNDMgene was not detected in all tested isolates.
Surveys of the prevalence, antibacterial susceptibility patterns, and identification of resistance patterns of bacterial isolates are important for determining appropriate empirical therapy for infections in critically ill patients.
Keywords: AmpC, blaKPC, blaNDM, carbapenemases, extended-spectrum β-lactamase, metallo-β-lactamases
|How to cite this article:|
Mahmoud AB, Eissa NA, El-Raghy NA, El-Sebaey HM, Melake NA, Awad ET, Hamam SS. Phenotypic and molecular characterization of multidrug-resistant Enterobacteriaceae clinical isolates from intensive care units at Menoufia University hospitals. Menoufia Med J 2016;29:835-45
|How to cite this URL:|
Mahmoud AB, Eissa NA, El-Raghy NA, El-Sebaey HM, Melake NA, Awad ET, Hamam SS. Phenotypic and molecular characterization of multidrug-resistant Enterobacteriaceae clinical isolates from intensive care units at Menoufia University hospitals. Menoufia Med J [serial online] 2016 [cited 2020 Feb 24];29:835-45. Available from: http://www.mmj.eg.net/text.asp?2016/29/4/835/202511
| Introduction|| |
Hospital-acquired infection (HAI) has been defined as an infection occurring during hospitalization that was not present, or incubating, at the time of admissionand appears within 48 h or more after admission . HAIs constitute a global health problem and contribute to significant morbidity and mortality, longer duration of hospitalization, as well as increased cost of treatment in both developed and resource-poor countries .
In a hospital, the most important problem is multidrug-resistant (MDR) organisms such as methicillin-resistant Staphylococcus aureus, vancomycin-resistant Enterococci spp., extended-spectrum β-lactamases (ESβLs), and carbapenem-resistant Enterobacteriaceae spp. . The major concern is the outbreaks caused by these organisms and the increased risk of endemic infections in ICU .
The genes encoding β-lactamases can be located on the bacterial chromosome, on plasmids, or on transposons. The genetic environment of the β-lactamase (bla) gene dictates whether the β-lactamases are produced in a constitutive or inducible manner. An increasing number of bla genes are being discovered on integrons .
ESβLs producing isolates are commonly resistant to the multiple antibiotics used for treating infections caused by Gram-negative bacteria . They are able to confer resistance to all extended-spectrum oxyimino cephalosporins (e.g., cefotaxime, ceftazidime, and cefepime) and monobactams (e.g., aztreonam) but do not hydrolyze 7-α-methoxy β-lactams, including cephamycins (e.g., cefoxitin and cefotetan) or oxacephems (e.g., moxalactam and flomoxef), and are inhibited by clavulanic acid .
AmpC enzymes confer resistance to penicillin and oxyimino cephalosporins (ceftazidime, cefotaxime, ceftriaxone, ceftizoxime, and cefuroxime), 7-α-methoxy cephalosporins (cefoxitin, cefotetan, cefmetazole, and moxalactam), and monobactams, and they are not inhibited by clavulanic acid, whereas the activity of cefepime and cefpirome is little or not affected .
It is mandatory to maintain the clinical efficacy of carbapenems (imipenem, ertapenem, meropenem, and doripenem), which are crucial for preventing and treating life-threatening nosocomial infections. However, the alarm has been raised regarding the spread of resistance to carbapenems among Enterobacteriaceae spp. .
Carbapenem resistance may be mediated by carbapenemases or noncarbapenemases. Resistance mediated by noncarbapenemases may be due to decreased membrane permeability to antibiotics because of a change in the number or activity of bacterial porins by mutation of porin proteins or loss of expression of porins, which may be associated with overexpression of a chromosomal AmpC gene or overexpression of AcrA , an efflux pump component, which was described to be responsible for imipenem resistance in Enterobacter aerogenes .
Carbapenemases are divided into molecular classes A, B, and D. Klebsiella pneumoniae carbapenemase (KPC) enzymes are currently the most clinically significant enzymes among the class A carbapenemases, as they confer high levels of resistance not only to carbapenems but also to most β-lactams, including broad-spectrum cephalosporins . The rapid spread and growing list of pathogens in which the blaKPC gene has been isolated is probably because of its carriage on plasmids. The gene is carried on a Tn3-based transposon, Tn4401 .
Class B β-lactamases exhibit a broad spectrum of hydrolytic activity including all penicillins, cephalosporins, and carbapenems, with the exception of monobactam and aztreonam. Their activity is not inhibited by commercially available β-lactamase inhibitors. Hydrolysis is dependent on the interaction of the β-lactam with Zn 2+ ion (s) in the active site, explaining the inhibition of their activity by EDTA .
New Delhi metallo-β-lactamase-1 (NDM-1) is the most recently discovered transferable molecular class B β-lactamase. It was first described in K. pneumoniae and Escherichia More Details coli isolated in Sweden in 2008 from an Indian patient transferred 1 day previously from a New Delhi hospital . NDM-1 gene is carried by diverse plasmids that also harbor multiple resistance genes associated with carbapenemase, cephalosporinase, macrolide, rifampin, and sulfamethoxazole resistance, a combination that makes these strains MDR, which leaves few treatment options .
By May 2011, over 100 isolates with NDM-1 had been identified in the UK, widely scattered among different hospitals but still mostly with epidemiological links to India or Pakistan. It was recorded in clinical Enterobacteriaceae spp. other than K. pneumoniae and E. coli; these include Klebsiella oxytoca, Proteus mirabilis, Enterobacter cloacae, Citrobacter freundii, and Providencia spp. NDM-1 has also been found in clinical Acinetobacter baumannii isolates from Germany, India, the UK, and China. When sewage samples and tap water in New Delhi were examined, NDM-1 was found in a wider diversity of Gram-negative species including Enterobacteriaceae spp., Pseudomonas spp., Stenotrophomonas spp., Aeromonas spp., and Vibrio cholerae. This distribution reflects the association of blaNDM-1 with promiscuous plasmids .
The aim of this study was to determine the prevalence of MDR Enterobacteriaceae spp. isolated from different ICUs at Menoufia University hospitals; to detect the presence of ESβLs, AmpC β-lactamases, and carbapenemases among the isolated pathogens using phenotypic methods; and to investigate the presence of blaKPC and blaNDM resistance genes using real-time PCR.
| Materials and Methods|| |
This study was conducted over the period from September 2013 to March 2015. The study protocol was approved by the local ethics committee of the Menoufia University. All patients gave written informed consent before inclusion in the study. It included 160 patients admitted to different ICUs of Menoufia University hospitals and suffering from HAIs. Personal and clinical history were taken as name, age, sex, occupation, socioeconomic status, residence, cause of admission to ICUs, duration of hospitalization, history of antibiotic intake, exposure to invasive procedure, and associated comorbidities.
Two hundred and seventy clinical samples were collected from patients, including urine, sputum, tracheal aspirate, blood, wound, and bed sores swabs. The specimens were processed according to standard microbiological methods. One hundred and twenty-one Enterobacteriaceae isolates were isolated and identified by conventional techniques  and bacterial isolates subjected to molecular diagnosis were identified using the API system (Microbact 12A; Oxoid, Ashley Road, Altrincham, Cheshire, England).
Antimicrobial susceptibility screening test
It was done for Enterobacteriaceae isolates by disk diffusion method against different antimicrobial agents (Oxoid). Procedures were performed and results were interpreted according to the Clinical and Laboratory Standard Institute (CLSI) guidelines .
Extended-spectrum β-lactamase detection methods
- Screening disk diffusion test: ESβLs were suspected by resistance to ceftazidime (30 µg) with a zone diameter less than and equal to 22 mm, by resistance to cefotaxime (30 µg) with a zone diameter less than and equal to 27, by resistance to ceftriaxone (30 µg) with a zone diameter less than and equal to 25 mm, and by resistance to aztreonam cefotaxime (30 µg) with a zone diameter less than and equal to 27 
- Combined disk diffusion confirmatory test: Ceftazidime (30 μg) and ceftazidime/clavulanic acid (30/10 μg), cefotaxime (30 μg) and cefotaxime/clavulanic acid (30/10 μg) were placed at a suitable distance on a Mueller–Hinton agar (MHA) plate. An organism was considered as an ESβL producer if there was a greater than and equal to 5-mm increase in diameter of ceftazidime/clavulanic and cefotaxime/clavulanic acid compared with that of ceftazidime and cefotaxime alone, respectively .
AmpC detection methods
- Cefoxitin disk test: Resistant or intermediate-resistant isolates to cefoxitin and to at least one of cefotaxime, ceftriaxone, or ceftazidime and/or monobactams according to CLSI criteria were considered as plasmid AmpC producers and were subjected to other confirmatory tests 
- AmpC disk test: The surface of an MHA plate was inoculated with cefoxitin-susceptible E. coli ATCC 25922 according to the standard disk diffusion method. A 30-μg cefoxitin disk was placed on the inoculated surface of the MHA. Sterile filter paper disks were inoculated with several colonies of the test organism and then placed almost touching the antibiotic disk with the inoculated disk face in contact with the agar surface. Indentation or a flattening of the zone of inhibition, indicating enzymatic inactivation of cefoxitin, was considered as a positive result .
Detection of classes A and B carbapenemase production
- Screening for susceptibility to carbapenems: This can be performed by using imipenem, meropenem, and ertapenem disk diffusion method (10 μg for each) according to the guidelines of CLSI, 2014 (for imipenem and meropenem; S≥23 and R≤19 and for ertapenem; S≥22 and R≤18)
- Confirmatory tests:
- Modified Hodge test (MHT): This test was done according to the CLSI guidelines (2014). The indicator organism (E. coli ATCC 25922) was inoculated on an MHAplate. One disk of imipenem (10 μg) was placed on the plate and three to five colonies of test and quality control organisms (K. pneumoniae ATCC BAA-1705, MHT positive and K. pneumoniae ATCC BAA-1706, MHT negative, Microbiologics, 200 Cooper Avenue North St. Cloud, Minnesota USA) were picked and inoculated in a straight line out from the edge of the disk. The streak was at least 20–25 mm in length. After incubation at 37°C for 18–24 h, the plate was examined for enhanced growth around the test or quality control organism streak at the intersection of the streak and the zone of inhibition (clover leaf-like indentation), which indicate positive for carbapenemase production 
- Inhibitor-based methods: PBA-CD test was used for detection of class A carbapenemases (KPCs), and IPM/EDTA-CD test was used for detection of class B carbapenemases [metallo-β-lactamase (MβLs)].
PBA-CD test: Two (10 μg) imipenem disks were placed on an MHA plate and 20 µl of a 20 mg/ml PBA solution (400 µg of PBA/disk) was added to one of the imipenem disks and incubated aerobically at 37°C for 18–24 h. A 5-mm increase in the inhibition zone diameter of imipenem/PBA disk compared with imipenem disk alone revealed KPC producer .
IPM/EDTA-CD test: Two (10 μg) imipenem disks were placed on the plate at a distance of 15 mm apart and 5 μl of sterile EDTA solution (930 μg EDTA) was added to one of the imipenem disks and incubated aerobically at 37°C for 18–24 h. If the increased inhibition zone with imipenem/EDTA disk was greater than and equal to 7 mm compared with the imipenem disk alone, it was considered as MβL positive .
A total of 44 and 55 Enterobacteriaceae clinical isolates demonstrating resistance to carbapenems by the screening method were examined for the presence of blaKPC and blaNDM genes, respectively, by real-time PCR using QuantiTect probe PCR kit (Qiagen, Hilden, Germany). Plasmid DNA extraction was performed using QIAprep Miniprep extraction kit (Qiagen). PCR reaction mix was prepared by mixing 12.5 μl of master mix, 1 μl of primer A, 1 μl of primer A, 0.5 μl of probe, 5 μl of template DNA, and 5 μl of RNase-free water in every PCR tube (primers used in the study shown in [Table 1]). The real-time cycler was programmed as follows: first, PCR initial activation step was set for 15 min at 95°C, to activate the hotstart Taq DNA polymerase. Second, the two-step cycling consisted of the denaturation step that was set for 15 S at 94°C and combined annealing/extension step set for 60 S at 60°C. The cycles were repeated 35–45 times. PCR tubes were placed in the real-time cycler (Applied Biosystems 7500 Cycler, USA, Frankfurter Straße, Darmstadt, Germany) and the cycling program was started according to the instructions of the manufacturer.
Computer SPSS program, version 17 (Chicago, Illinois), was used. The results were expressed by applying ranges, means ± SD, c2-test, and P values. P value less than 0.05 was considered significant.
| Results|| |
This study included 160 patients. The mean age of studied patients was 47.06 ± 5.94 years.
In the present study, 51.48% of samples yielded positive cultures. Urinary tract infection was the most prevalent HAI in ICUs (41.01%), followed by respiratory tract infection (36.69%), surgical wound infection (12.23%), bed-sore infection (7.91%), and bloodstream infection (2.16%). Among positive cultures, 121 Enterobacteriaceae spp. were isolated (80.13%). The most frequent species of Enterobacteriaceae spp. was K. pneumoniae, as it was represented by 44.8%, followed by E. coli (19.83%), Enterobacter spp. (14.88%), Citrobacter spp. and Serratia marcescens (6.61% for each), P. mirabilis (4.96%), and Morganella morganii (3.31%) [Figure 1].
Enterobacteriaceae isolates showed 100% resistance to levofloxacin, norfloxacin, piperacillin, piperacillin/tazobactam, and trimethoprim/sulfamethoxazole. They showed high resistance to most of the antibiotics used: they had 98.35% resistance to amoxicillin/clavulanic acid, ceftazidime, cefepime, cefotaxime, and ceftriaxone, and 96.69% resistance to tobramycin, ciprofloxacin, and cefoxitin. They had 95.04% resistance to gentamicin, 94.21% resistance to aztreonam, 93.39% resistance to chloramphenicol, and 92.56% resistance to amikacin. Susceptibility pattern to carbapenems was as follows: highest resistance was to ertapenem (85.95%), followed by meropenem (65.29%) and imipenem (60.33%). Resistance to the new therapeutic drug tigecycline was 58.68% ([Table 2]).
|Table 2 Antimicrobial susceptibility pattern of Enterobacteriaceae isolates by disk diffusion method|
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Frequency of MDR, extreme drug-resistant (XDR), and pandrug-resistant (PDR) Enterobacteriaceae isolates was demonstrated. In all, 52.89% of Enterobacteriaceae isolates were XDR, 26.45% were PDR, and 14.88% were MDR [Figure 2].
|Figure 2: Frequency of multidrug-resistant, extreme drug-resistant, and pandrug-resistant Enterobacteriaceae isolates. MDR, nonsusceptibility to at least one agent in three or more antimicrobial categories; XDR, nonsusceptibility to at least one agent in all but two or fewer antimicrobial; PDR, resistance to almost all commercially available antimicrobials or resistance to all antimicrobials routinely tested.|
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About 53% of patients with imipenem-resistant isolates were among the age group of 51–70 years compared with 41.67% in other patients (imipenem-sensitive), with no statistically significant difference between two groups (P > 0.05). Around 58.9% of patients with imipenem-resistant isolates were male and 41.1% were female compared with 52.08 and 47.92% in other patients, respectively, with no statistically significant difference between the two groups (P > 0.05). There was a statistically significant difference regarding duration of hospital stay more than 14 days (63.01%), patients with invasive procedures (100%), associated comorbid conditions (68.49%), and history of carbapenem intake (71.23%). No statistically significant difference was found between patients from rural areas and those coming from urban areas among the two studied groups (P > 0.05) ([Table 3]).
|Table 3 Relation between imipenem resistance of Enterobacteriaceae isolates and demographic and clinical data of the studied patients|
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Regarding results of phenotypic tests used for detection of ESβL-producing and AmpC-producing Enterobacteriaceae isolates, most of the isolated Enterobacteriaceae spp. (98.35%) were found to be potential ESβL producers by the screening method depending on the resistance to certain antibiotics with specific zone diameter for ESβL detection. Only 41.32% were confirmed to be ESβL producers by using combined disk method, with a high statistically significant difference between the two tests (P < 0.001); most of the isolated Enterobacteriaceae spp. (96.69%) were found to be potential AmpC producers by the screening method (cefoxitin disk test), whereas only 32.23% were confirmed to be AmpC producers by using the AmpC disk test, with a high statistically significant difference between the two tests (P < 0.001) ([Table 4] and [Figure 3].
|Table 4 Comparison between positive results of phenotypic tests used for detection of extended-spectrum β-lactamases-producing and AmpC-producing Enterobacteriaceae isolates|
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|Figure 3: AmpC disk test. A positive result indicated by indentation of inhibition zone around cefoxitin disk due to presence of AmpC positive tested strain present on filter paper adjacent to cefoxitin disk.|
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Among 73 imipenem-resistant Enterobacteriaceae isolates, 51 (69.86%) isolates were MHT positive, and 44 (60.27%) isolates were confirmed to be KPC-producing by being positive by combined imipenem/boronic acid synergy test, and 55 (75.34%) isolates were confirmed to be MβL producing by being positive by combined imipenem/EDTA synergy test [Figure 4] and [Figure 5].
|Figure 4: Modified Hodge test (MHT). Positive-control strain is Klebsiella pneumoniae (ATCC BAA-1705) and negative control strain is K. pneumoniae (ATCC BAA-1706). C, positive test strain. Positive MHT is detected by the extension of growth of the indicator strain toward an imipenem disk (an indentation).|
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|Figure 5: Detection of carbapenemases-producing isolates by modified Hodge test in comparison with inhibitory based methods among imipenem-resistant Enterobacteriaceae isolates.|
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The sensitivity and specificity of MHT in relation to real-time PCR results of blaKPC gene detection among tested Enterobacteriaceae isolates showed that MHT was positive in 12/19 (63.16%) blaKPC gene-positive isolates (sensitivity was 63% and specificity was 8%). However, combined imipenem/boronic acid synergy test detected 19/19 (100%) isolates of blaKPC-positive isolates (sensitivity was 100% and specificity was 80%) ([Table 5]).
|Table 5 Sensitivity and specificity of modified Hodge test and combined imipenem/boronic acid synergy test in relation to real-time polymerase chain reaction results of blaKPC gene detection among tested Enterobacteriaceae isolates|
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Most of the patients with blaKPC-positive Enterobacteriaceae strains (17/19) were above 30 years of age and had prolonged ICU stay greater than and equal to 14 days (14/19). All patients had a history of invasive procedures. BlaKPC gene was detected among 10/40, 5/12, 2/5, and 2/10 of imipenem-resistant K. pneumonia, Enterobacter spp., S. marcescens, and E. coli isolates, respectively. About 26% of imipenem-resistant Enterobacteriaceae isolates carry blaKPC. Ten out of 19 patients had a history of carbapenem intake, two had a history of tigecycline intake, and 13/19 had comorbid conditions, the most common of which is diabetes mellitus. Four out of 19 blaKPC gene-harboring strains were ESBL producers, 8/19 were AmpC producers, 19/19 were KPC producers, and 17/19 were MβL producers ([Table 6]).
|Table 6 Characteristics of intensive care unit patients with infections caused by blaKPC-positive Enterobacteriaceae spp.|
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| Discussion|| |
Infections caused by MDR bacteria constitute a serious problem for ICU patients throughout the world. The mortality rate associated with MDR bacteria in these patients is high in ICUs . The spread of MDR Enterobacteriaceae isolates in clinical samples of MUHs, including ESβL, AmpC β-lactamase, and carbapenemase producers, was studied.
The most common isolated species of Enterobacteriaceae was K. pneumonia followed by E. coli, Enterobacter spp., Citrobacter spp., S. marcescens, P. mirabilis, and M. morganii. The high rates of these microorganisms in the nosocomial infections might be because most of them are found in the intestinal tract and pharynx. They are also able to survive in wet places, in mechanical respiration equipments, and in pipes of difficult access that are to be washed and/or dried. These results are in agreement with those of Nageeb et al.  and Raghunathan et al. and disagreed with that of Kandasamy et al. , who found that Pseudomonas aeruginosa was the most frequently isolated bacteria.
Our investigations in this study demonstrated that resistance to carbapenems was high: 60.33% for imipenem, 85.95% for ertapenem, and 65.29% for meropenem. The high prevalence of carbapenem-resistant isolates in ICUs can be explained by the immunocompromised state of most ICUs patients, heavy pressure of antibiotic use especially carbapenems, and excessive exposure to invasive procedures that provide optimum chance to catch infections. These results were in accordance with those of Seibert et al.  and Zhou et al.  whofound that rates of carbapenem-resistant strains were higher against ertapenem than against imipenem or meropenem. They stated that it may be mediated by the presence of ESβLs or plasmid-borne AmpC in combination with impermeability due to porin loss and/or efflux pumps. The results were not in agreement with those of Daef and Elsherbiny  in Egypt who reported in their study that resistance of Enterobacteriaceae spp. to imipenem ranged between 14.7 and 27.0%. Interestingly, Cai et al.  reported 0.00% resistance to imipenem, whereas resistance to meropenem ranged between 0.00 and 11.76% among Enterobacteriaceae spp. isolated in their study and resistance of other Gram-negative bacteria to both drugs ranged between 14.29% in P. aeruginosa and 100% in Stenotrophomonas maltophilia.
In this study, there was a high statistically significant difference between patients harboring carbapenem-resistant strains and those having carbapenem-sensitive isolates regarding duration of ICU stay, comorbid conditions, and history of antibiotic use including carbapenems. These results are in accordance with Ghoneim et al. , who concluded that the most important risk factors significantly associated with HAIs caused in ICUs were central venous catheters, heavy use of antibiotics, and duration of ICU stay.
In the present study, 98.35% of Enterobacteriaceae isolates were potential ESβL producers by disk diffusion method and 41.32% were confirmed to be ESβL producers by combined disk test. This low rate of confirmed ESβL may be because of the effect of clavulanic acid, which may induce expression of high-level AmpC production and antagonize rather than protect the antibacterial activity of the partner β-lactam. Thus, the presence of an ESβL can be masked by the expression of an AmpC-type enzyme in the same strain. Current results are in agreement with the results of Hassanein et al. , who reported that 84.2% of isolates were potential ESβL producers and 63.2% were confirmed ESβL producers. On the other hand, Akujobi and Ewuru reported lower incidence of ESβL production (11.4%).
In our study, the most common ESβL-producing species was P. mirabilis, followed by E. coli and K. pneumoniae. These results were comparable to those ofPoupet et al. , who reported that the most frequent Enterobacteriaceae spp. ESβL producer was E. coli followed by K. pneumoniae, Citrobacter spp., Enterobacter spp., and P. mirabilis.
In the present study, 96.69% of isolates were potential AmpC producers by resistance to cefoxitin and only 32.23% were confirmed to be AmpC producers by AmpC disk test. This low rate of confirmed AmpC may be because of other mechanisms of cephamycin resistance such as porin deficiency or carbapenemase production . The most common AmpC-producing species were M. morganii, followed by Enterobacter spp., E. coli, S. marcescens, K. pneumoniae, and Citrobacter spp. The results of the current study are in agreement with those of Parveen et al. , who reported that potential AmpC-producing species were 57.02% of all isolates and only 26.81% were confirmed to be AmpC producers.
Carbapenemase production was screened by resistance to carbapenems and confirmed by MHT. The results revealed that 73 out of 121 (60.33%) Enterobacteriaceae isolates were imipenem-resistant and only 56 out of 121 Enterobacteriaceae isolates were imipenem-resistant by MHT (46.28%). MHT interpretation is often difficult and subjectiveand does not distinguish between carbapenemase types. KPC production was detected by combined imipenem/boronic acid synergy test, in which 44 out of 73 (60.27%) imipenem-resistant Enterobacteriaceae isolates were positive for the test [35 (79.5%) isolates were MHT positive and nine (20.5%) were MHT negative] and MβL was detected by combined imipenem/EDTA synergy test, in which 55 out of 73 imipenem-resistant Enterobacteriaceae isolates (75.34%) were positive for the test (45 (85.45%) isolates were MHT positive and 10 (14.55%) were MHT negative]. The most frequent Enterobacteriaceae spp. producing KPC detected by imipenem/boronic acid synergy test was K. pneumoniae, followed by Enterobacter spp., E. coli, S. marcescens, P. mirabilis, and M. morganii. The most frequent Enterobacteriaceae spp. producing MβL detected by imipenem/EDTA synergy test was K. pneumoniae, followed by Enterobacter spp., E. coli, S. marcescens, and P. mirabilis. The results are in agreement with the study by Kandasamy et al. , in which 40% of imipenem-resistant species were MHT positive, Alshara et al. , in which 77.8% of the isolates were MβL producers, andEndimiani et al. , in which theprevalence rates of KPC were more than 30%. In contrast to the current results, Doi et al.  reported that MHT was positive in all isolates that were positive for imipenem/boronic acid synergy test and was negative in all isolates that were negative for imipenem/boronic acid synergy test. Recently, in Egypt, Fattouh et al.  found that KPC production was detected mostly in K. pneumoniae, followed by E. coli and Enterobacter spp., in a study conducted in Sohag University.
In the current study, the prevalence rates of different β-lactamase production were 41.32% for ESBLs, 32.32% for AmpC, 60.27% for KPC, and 75.34% for MβLs, and 52.89% of isolates had more than one type of β-lactamase.
In the present study, 52.89% of Enterobacteriaceae isolates were XDR, 26.45% were PDR, and 14.88% were MDR. In accordance with our results, Alshara et al.  in Iraq reported that the incidence of MDR isolates in their study was 11.1% in Gram-negative isolates, 55.5% of isolates were XDR, and the incidence of PDR isolates was 33.3%.
The molecular study revealed that 19 out of 44 (43.18%) imipenem-resistant Enterobacteriaceae isolates were positive for blaKPC gene. It was detected mostly among K. pneumoniae (10 isolates), followed by Enterobacter spp. (five isolates) and E. coli and S. marcescens (two isolates for each). Urine was the most frequent specimen from which blaKPC gene-positive isolates were detected, followed by tracheal aspirate and sputum.
Among 35 MHT-positive isolates, only 12 isolates were PCR positive and among nine MHT-negative isolates seven were PCR positive. MHT was not proved as a useful method for detection of blaKPC gene production, as it had 63% sensitivity and 8% specificity by comparing it with results of real-time PCR. However, combined imipenem/boronic acid synergy test had 100% sensitivity, 80% specificity, and 89% accuracy for detection of blaKPC gene.
In agreement with this study, Kandasamy et al. reported that they found blaKPC gene in 20% of imipenem-resistant Enterobacteriaceae spp. Results of Hung et al.  are coincident with the current study, as they reported the presence of blaKPC gene mostly inK. pneumonia, C. freundii, E. coli, and S. marcescens, and with the Seibert et al.  study, who reported that blaKPC-positive organisms were frequently isolated from urine and tracheal aspirate following rectal swabs, which were not sampled in the present study.
Raghunathan et al. reported a strong correlation between the results of the MHT and PCR. They explained the presence of positive isolates for MHT and negative for PCR either by being positive for AmpC or because of the presence of inhibitory substances in the reaction or technical inexperience.
In 2009, the CLSI  guidelines recommended the MHT to definitively identify KPC producers in Enterobacteriaceae isolates, but in 2013  it explained that not all carbapenemase-producing isolates of Enterobacteriaceae spp. are MHT positive, and MHT-negative results may be encountered in isolates with carbapenem-resistance mechanisms other than carbapenemase production and some isolates showed a slight indentation but did not produce carbapenemases.
The enzyme NDM-1 is expressed by certain bacteria that carry the blaNDM-1 gene. Drug resistance genes can be transferred by plasmid; hence, these features of NDM-1 may cause global public health security .
Many reports have indicated the spread of NDM-1 producers in many different countries, and some originated from the Indian subcontinent and others from Balkan countries. Reports of emerging carbapenemases among Enterobacteriaceae spp. have been published worldwide, including Middle East countries such as Kuwait, Oman, United Arab Emirates, Iraq, Egypt, Lebanon, Tunisia, and Turkey . The first identification of a blaNDM gene in a clinical isolate (A. baumannii) originating from Egypt was reported by Kaase et al. , with no obvious link with the Indian subcontinent.
In the current study, molecular assessment of blaNDM among 55 MDR Enterobacteriaceae isolates positive for combined imipenem/EDTA synergy test gave negative results. Positive phenotypic methods might be because of the presence of MβLs other than blaNDM. Therefore, there are no documented data about its detection in Enterobacteriaceae spp. in Egypt until now.
| Conclusion|| |
Large percentages of MDR Enterobacteriaceae strains have been observed, making therapeutic options very limited. Surveys of the prevalence, antibacterial susceptibility patterns, and identification of resistance patterns of bacterial isolates are important for determining appropriate empirical therapy for infections in critically ill patients.
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Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6]