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 Table of Contents  
ORIGINAL ARTICLE
Year : 2016  |  Volume : 29  |  Issue : 4  |  Page : 855-861

Multidrug-resistant Enterobacteriaceae nosocomial uropathogens at Menoufia University Hospitals: phenotypic characterization and detection of resistance genes using real-time PCR


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, Shebin Elkom, Egypt

Date of Submission02-Aug-2015
Date of Acceptance01-Oct-2015
Date of Web Publication21-Mar-2017

Correspondence Address:
Amira H El-Khayat
Department of Microbiology, Faulty of Medicine, Menoufia University, Tala City, Menoufia Governorate, 32611
Egypt
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/1110-2098.202515

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  Abstract 

Objectives
The aims of this study were as follows: to determine the incidence of multidrug-resistant Enterobacteriaceae isolated from the urine of patients admitted in different departments at Menoufia University Hospitals; to detect the presence of extended spectrum β-lactamases (ESβLs), AmpC β-lactamases, and carbapenemases among the isolated pathogens using phenotypic methods; and to investigate the presence of bla KPC and bla NDM genes using real-time PCR.
Background
Urinary tract infections are the most common type of nosocomial infections. ESβLs and AmpC β-lactamases are clinically significant because they may confer resistance to multiple classes of antibiotics. Carbapenemases are diverse enzymes that vary in the ability to hydrolyze carbapenems and other β-lactams.
Materials and methods
This study included 260 urine samples collected from 260 patients. ESβL production was detected on the basis of resistance to third-generation cephalosporins and aztreonam and then confirmed using the combined disk test. AmpC production was detected using the cefoxitin disk test and confirmed using the AmpC disk test. Carbapenemases were detected on the basis of resistance to imipenem, meropenem, and ertapenem, and then confirmed using the modified Hodge test, the phenylboronic acid combined disk test, and the imipenem/EDTA combined disk test. Carbapenemase gene (bla KPC and bla NDM) detection was performed using real-time PCR.
Results
Carbapenem resistance was 42.5% to ertapenem, 40.8% to meropenem, and 45% to imipenem. Risk factors associated with Hospital aquired infection were hospital stay of 14 days or more, exposure to invasive procedures, and comorbid conditions. ESβL production occurred in 55.8% and AmpC occurred in 30.8% of Enterobacteriaceae spp. Carbapenemase production was detected in 87%, Klebsiella pneumoniae carbapenemase (KPC) was detected in 37%, metallo-β-lactamase was detected in 54%, and bla KPCgene was detected in 24% of imipenem-resistant Enterobacteriaceae spp. However, bla NDM gene was not detected in all tested isolates.
Conclusion
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-lactamases, KPC, metallo-β-lactamases


How to cite this article:
Elraghy NA, Zahran WA, Makled AF, El-Sebaey HM, El-Hendawy GR, Melake NA, Awad E, El-Khayat AH. Multidrug-resistant Enterobacteriaceae nosocomial uropathogens at Menoufia University Hospitals: phenotypic characterization and detection of resistance genes using real-time PCR. Menoufia Med J 2016;29:855-61

How to cite this URL:
Elraghy NA, Zahran WA, Makled AF, El-Sebaey HM, El-Hendawy GR, Melake NA, Awad E, El-Khayat AH. Multidrug-resistant Enterobacteriaceae nosocomial uropathogens at Menoufia University Hospitals: phenotypic characterization and detection of resistance genes using real-time PCR. Menoufia Med J [serial online] 2016 [cited 2020 Feb 24];29:855-61. Available from: http://www.mmj.eg.net/text.asp?2016/29/4/855/202515


  Introduction Top


Urinary tract infection (UTI) is a serious health problem affecting millions of people each year. It has been estimated that about six million patients visit outpatient departments and about 300 000 are treated in the wards every year for UTI worldwide [1].

UTIs are the most common type of nosocomial infection, accounting for 40–50% of all hospital-acquired infections. Risk factors include catheterization, diabetes mellitus, long-time hospitalization, immunosuppression, and female sex [2].

The vast majority of UTIs are caused by Enterobacteriaceae originating from the gut before entering the urethra.  Escherichia More Details coli are the most common uropathogen, responsible for over 80% of community-acquired and ˜50% of hospital-acquired UTIs [3].

The war waged between microorganisms and antimicrobials continues to flare up unabated, with each partner developing new weaponry and seeking novel ways of combat. β-Lactam group of antibiotics are the workhorse antimicrobial agents. Favored by their comparatively high effectiveness, low toxicity, and low cost, β-lactams are prescribed more often compared with any other antibiotic. Heavy use of this antibiotic has resulted in the selection of drug-resistant bacteria caused by the production of β-lactamases, and is now an increasing problem, especially in Enterobacteriaceae [4].

A dramatic clinically significant increase in the antimicrobial resistance of uropathogens, especially hospital acquired, over the past 10 years calls for new concepts in the treatment of UTIs. Resistance of urinary pathogens to common antibiotics is currently a topic of concern [5].

Extended-spectrum β-lactamases (ESβL) are often plasmid mediated. Although they occur predominantly in E. coli and Klebsiella spp., they have also been described in other genera of the Enterobacteriaceae [6].

AmpC β-lactamases are clinically significant because they may confer resistance to penicillins, cephalosporins, oxyimino-cephalosporins (e.g., ceftriaxone, cefotaxime, and ceftazidime), cephamycins (e.g., cefoxitin and cefotetan), and monobactams. AmpC β-lactamase activity is not affected by the ESβL inhibitor clavulanic acid [7].

Carbapenemases are diverse enzymes that vary in the ability to hydrolyze carbapenems and other β-lactams [8]. Production of active carbapenemase is a major carbapenem resistance mechanism among clinical Gram-negative isolates [9].

The aims of this study were as follows: to determine the incidence of multidrug-resistant Enterobacteriaceae isolated from the urine of patients admitted in different departments 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 bla KPC and bla NDM resistance genes using real-time PCR.


  Materials and Methods Top


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 participants provided written informed consent before inclusion in the study. The study included 260 patients admitted in different departments of Menoufia University Hospitals and suffering from UTI. Personal and clinical history was taken, such as name, age, sex, occupation, socioeconomic status, residence, duration of hospitalization, history of antibiotic intake, exposure to invasive procedure, and associated comorbidities.

Bacterial strains

In all, 260 urine samples were collected from patients. The specimens were processed according to standard microbiological methods. A total of 120 Enterobacteriaceae isolates were isolated and identified using conventional techniques [10], and bacterial isolates subjected to molecular diagnosis were identified using API system (Microbact 12ATM, Oxoid, Thermo Fisher Scientific, Inc. Waltham, MA, USA).

Antimicrobial susceptibility test

It was carried out for Enterobacteriaceae isolates using the disk diffusion method against different antimicrobial agents (Oxoid). Procedures were performed and results were interpreted according to the Clinical and Laboratory Standard Institute guidelines [11].

Extended spectrum β-lactamase detection methods

Screening disk diffusion test

ESβLs were suspected on the basis of resistance to ceftazidime (30 µg) with zone diameter of 22 mm or less, cefotaxime (30 µg) with zone diameter of 27 or less, ceftriaxone (30 µg) with zone diameter of 25 mm or less, and aztreonam cefotaxime (30 µg) with zone diameter of 27 or less [11]. Combined disk confirmatory test: Ceftazidime (30 μg) and ceftazidime/clavulanic acid (30/10 μg) and cefotaxime (30 μg) and cefotaxime/clavulanic acid (30/10 μg) were placed at a suitable distance on Mueller Hinton agar (MHA) plate. Organism was considered as ESβL producer if there was at least 5 mm increase in the diameter of ceftazidime/clavulanic and cefotaxime/clavulanic acid than that of ceftazidime and cefotaxime alone, respectively [11].

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 [12].

The AmpC disk test

The surface of a 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 [13].

Detection of class A and class B carbapenemase production

Screening for susceptibility to carbapenems

Susceptibility to carbapenems was screened 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; for ertapenem, S ≥ 22 and R ≤ 18).

Confirmatory tests

Modified Hodge test (MHT): The MHT test was carried out according to the CLSI guidelines (2014). The indicator organism (E. coli ATCC 25922) was inoculated on MHA plate. One disk of imipenem (10 μg) was placed on the plate and 3–5 colonies of test and quality control organisms (QC: K. pneumoniae ATCC BAA-1705, MHT positive; K. pneumoniae ATCC BAA-1706, MHT negative; Microbiologics, 200 Cooper Ave N, St Cloud, MN 56303, 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 QC organism streak at the intersection of the streak and the zone of inhibition (cloverleaf-like indentation), which indicates positive for carbapenemase production [14].

Inhibitor-based methods: The phenylboronic acid combined disk (PBA-CD) test was used for the detection of class A carbapenemases (KPCs), and the imipenem/EDTA combined disk (IPM/EDTA-CD) test was used for the detection of class B carbapenemases metallo-β-lactamase (MβLs).

PBA-CD test: Two (10 μg) imipenem disks were placed on 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. An increase of 5 mm in the inhibition zone diameter of imipenem/PBA disk compared with imipenem disk alone revealed KPC producer [15].

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 disk and incubated aerobically at 37°C for 18–24 h. If the increased inhibition zone with imipenem/EDTA disk was at least 7 mm compared with imipenem disk alone, it was considered as MβL positive [16].

Molecular study: Fifty-four Enterobacteriaceae clinical isolates demonstrating resistance to carbapenems using the screening method were examined for the presence of bla KPC and bla NDM genes with real-time PCR using QuantiTect probe PCR kit (Qiagen).

Plasmid DNA extraction was performed using QIAprep Miniprep extraction kit (Qiagen). PCR reaction mix was prepared by mixing 12.5 μl master mix, 1 μl primer A, 1 μl primer A, 0.5 μl probe, 5 μl template DNA, and 5 μl 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, two-step cycling consisted of 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 for 35–45 times. PCR tubes were placed in the real-time cycler (Applied Biosystems 7500 cycler; Thermo Fisher Scientific, Inc., Waltham, MA, USA) and the cycling program was started according to the instructions of the manufacturer.
Table 1 Primers used in the study

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Statistical analysis

Computer SPSS (SPSS Inc. Released 2008. SPSS Statistics for Windows, Version 17.0. Chicago: USA, SPSS Inc.) program version 17 was used. The results were expressed by applying ranges, means ± SD, c2-test, and P values. A P value less than 0.05 was considered to be significant.


  Results Top


This study included 260 patients. The mean age of studied patients was 31.41 ± 14.52 years. There were 120 Enterobacteriaceae isolates. E. coli was the most common urinary isolate (48.4%), followed by K. pneumoniae (18.3%), Enterobacter spp. (13.3%), K. oxytoca (7.5%), Proteus spp. (5%), Citrobacter spp. (3.3%), Serratia spp. (2.5%), and Morganella morganii (1.7%), as shown in [Table 2].
Table 2 Distribution of the Enterobacteriaceae obtained during the study

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As regards antibiotic susceptibility of isolated Enterobacteriaceae, all isolates were resistant to piperacillin and amoxicillin/clavulanic acid. They were highly resistant to cotrimoxazole (95%), ciprofloxacin (94.2%), cefoxitin (90%), aztreonam (85%), cefotaxime (84.2%), ceftriaxone (83.3%), and ceftazidime (82.5%). Susceptibility to amikacin, levofloxacin, piperacillin/tazobactam, ertapenem, imipenem, meropenem, and tigecycline was observed in 51.7, 37.5, 49.5, 57.5, 55, 59.5, and 80.8% of isolates, respectively, as shown in [Table 3].
Table 3 Antimicrobial susceptibility pattern of Enterobacteriaceae isolates using the disk diffusion method

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About 85% of Enterobacteriaceae were detected as ESβL producers using the disk diffusion screening method, whereas the combined disk confirmatory method detected 67 (55.8%) Enterobacteriaceae as ESβL producers. Statistically, there was a high significant difference between them (P < 0.001), as shown in [Table 4].
Table 4 Results of the disk diffusion screening method and the combined disk confirmatory method for the detection of extended spectrum β-lactamase-producing Enterobacteriaceae isolates

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About 90% of Enterobacteriaceae were detected as AmpC producers using the cefoxitin disk test screening method, whereas the confirmatory AmpC disk test detected 37 (30.8%) Enterobacteriaceae as AmpC producers. Statistically, there was a high significant difference between them (P < 0.001), as shown in [Table 5].
Table 5 Comparison between the cefoxitin disk screening method and the AmpC disk confirmatory method for the detection of AmpC β-lactamase production among Enterobacteriaceae isolates

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About 47 isolates out of 54 imipenem-resistant Enterobacteriaceae (87%) were positive with MHT. However, 12/54 (22.2%) were positive using the imipenem/boronic acid synergy test, and 21/54 (39%) were positive using the imipenem/EDTA synergy test. However, eight isolates (14.8%) were positive using both the imipenem/boronic acid synergy test and imipenem/EDTA synergy test, as shown in [Table 6].
Table 6 Phenotypic profiles of Enterobacteriaceae isolates detected as imipenem-resistant using the disk diffusion screening method

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About 24.07% (13/54) of all imipenem-resistant Enterobacteriaceae isolates were positive for bla KPC and were distributed as follows: K. pneumonia e was the predominant KPC producer by 61.5%, followed by E. coli (15.4%) and K. oxytoca (15.4%), and finally Enterobacter spp., which represented 7.7% of KPC producers in this study according to the results of real-time PCR. However, none of the imipenem-resistant Enterobacteriaceae isolates were positive for bla NDM, as shown in [Table 7].
Table 7 Results of real-time PCR for the detection of blaKPC and blaNDM among imipenem-resistant Enterobacteriaceae isolates

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  Discussion Top


In the current study, Enterobacteriaceae comprised 78% of the isolates, of which E. coli (48.4%) and K. pneumoniae (18.3%) were the two most common species, followed by Enterobacter spp. (13.3%), K. oxytoca (7.5%), Proteus spp. (5%), Citrobacter spp. (3.3%), Serratia spp. (2.5%), and Morganella morganii (1.7%); this correlates with the findings of Shaaban et al. [17] and Abd El-Mongy and Reyad [18].

In this study, 55.8% of Enterobacteriaceae were found to be ESβL producers using the combined disk confirmatory method. This is in agreement with the findings of Thabit et al. [19], who reported that 53% of isolated Enterobacteriaceae in Assiut University Hospital were ESβL producers.

Of the 120 isolates of Enterobacteriaceae studied, 37 (30.8%) isolates were positive using the confirmatory AmpC disk test. This result is much higher than the result of Miró et al. [20] in Spain; they found a prevalence of 0.64% for pAmpC. However, our result is similar to the results obtained in other Egyptian studies, as that presented by El-Hady and Adel [7], Ain Shams University Hospital, who reported that 33.8% of isolated Enterobacteriaceae were AmpC producers.

In the present study, 120 isolates of Enterobacteriaceae were obtained from the urine of hospitalized patients. Among these isolates, 54 isolates (45%) were resistant to imipenem using the disk diffusion method, which is nearer to the result of El-Kazzaz and Abou El-khier [21], who found that 47% of clinical isolates of Gram-negative pathogens obtained from patients admitted in Mansoura University Hospital were imipenem resistant.

Carbapenemases production was screened on the basis of resistance to carbapenems and confirmed using the MHT, in which 87% were MHT positive. KPC production was confirmed using combined the imipenem/boronic acid synergy test, in which 34% were positive for the test, and MβL was confirmed using the combined imipenem/EDTA synergy test, in which 54% were positive for the test. Moreover, this study also demonstrated the coproduction of KPC and MβL in eight (14.8%) isolates using the disk enhancement test with both PBA and EDTA.

In a similar study presented by Fattouh et al. [22] in Sohag University Hospital, they found that, of the total 59 carbapenem-resistant isolates, MHT identified 52 (88.14%) isolates as carbapenemase producers. MβL activity was detected in 20 (33.9%) isolates using the imipenem/EDTA synergy test, KPC in 14 (23.73%) isolates using the PBA test, and coexistence of both KPC and MβLs in nine (15.25%) isolates of Gram-negative pathogens.

In the current study, 13 of 54 carbapenem-resistant isolates (24.07%) were positive for bla KPC using PCR and 41 of 54 isolates (75.93%) were negative; this is in agreement with a study by Girgis et al. [23], in Ain Shams University Hospitals; they reported that 21% of isolates were bla KPC gene positive using PCR. Fortunately, none of IPM-resistant Enterobacteriaceae isolates in our study was positive for bla NDM ([Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5].
Figure 1: Combined disk test for the detection of extended spectrum β-lactamase (ESβL) production.

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Figure 2: AmpC disk test.

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Figure 3: Modified Hodge test.

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Figure 4: Species identification using API system.

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Figure 5: Results of real-time PCR.

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  Conclusion Top


Successful implementation of infection control measures is a must to reduce the problem of bacterial resistance, especially in countries where patients can have access to antibiotics without a prescription.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

 
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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6], [Table 7]



 

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