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 Table of Contents  
ORIGINAL ARTICLE
Year : 2014  |  Volume : 27  |  Issue : 3  |  Page : 602-605

Prevalence of extended-spectrum β-lactamase-producing Enterobacteriaceae in Menoufia University Hospitals


1 Department of Clinical Pathology, Faculty of Medicine, Menoufia University, Menoufia, Egypt
2 Department of Tropical Medicine, Faculty of Medicine, Menoufia University, Menoufia, Egypt

Date of Submission01-Sep-2013
Date of Acceptance30-Nov-2013
Date of Web Publication26-Nov-2014

Correspondence Address:
Sheriene M Moussa
Department of Clinical Pathology, Faculty of Medicine, Menoufia University, Yassin Abdel-Ghaffar Street, 4, Shebin El-Koom, 32511 Menoufia
Egypt
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/1110-2098.145525

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  Abstract 

Objective
The aim of the study was to evaluate the prevalence of extended-spectrum β-lactamase (ESBLs) in Enterobacteriaceae among patients conducted at Menoufia University Hospitals.
Background
Resistance to β-lactam antibiotics is an increasing problem, and β-lactamase production is the most common mechanism of drug resistance, especially in Enterobacteriaceae. The Clinical and Laboratory Standard Institute (CLSI) interpretive guidelines stated that Enterobacteriaceae that produce ESBLs are resistant to therapy with penicillin, cephalosporins, and aztreonam, despite apparent in-vitro susceptibility to some of these agents. Therefore, detection of ESBLs in Enterobacteriaceae is crucial for optimal treatment of patients and to control the spread of resistance.
Patients and methods
The isolated strains of Enterobacteriaceae are subjected to phenotype detection by screening for ESBLs by disk diffusion test then to confirmatory tests for ESBLs by double disk test.
Results
A total of 97 of 160 (60.6%) isolates were found to be ESBL producers by the CLSI confirmatory method. Of 97 ESBL producers, 45 (46.4%) were Escherichia coli, 28 (28.9%) were Enterobacter spp., and 22 (22.7%) were Klebsiella spp. Of the various clinical samples, the most frequent ESBLs isolates were from urine samples (27.8%), followed by sputum samples (18.6%).
Conclusion
High prevalence of ESBL producers in our hospital (60.6%) calls for strict policies regarding antibiotic usage and their screening methods, and hospital-based clinical laboratories should screen isolates following hospitalization in patients in need for antibiotics to formulate effective antibiotic strategy and plan a proper hospital infection control strategy to prevent the spread of these ESBL strains.

Keywords: drug resistance, Enterobacteriaceae, extended-spectrum β-lactamase, prevalence


How to cite this article:
El-Hendi AA, El-Lehleh AM, El-Shalakany AH, Moussa SM. Prevalence of extended-spectrum β-lactamase-producing Enterobacteriaceae in Menoufia University Hospitals. Menoufia Med J 2014;27:602-5

How to cite this URL:
El-Hendi AA, El-Lehleh AM, El-Shalakany AH, Moussa SM. Prevalence of extended-spectrum β-lactamase-producing Enterobacteriaceae in Menoufia University Hospitals. Menoufia Med J [serial online] 2014 [cited 2020 May 26];27:602-5. Available from: http://www.mmj.eg.net/text.asp?2014/27/3/602/145525


  Introduction Top


Resistance to β-lactam antibiotics is an increasing problem, and β-lactamase production is the most common mechanism of drug resistance, especially in Enterobacteriaceae [1] .

Extended-spectrum β-lactamase (ESBL)-producing strains are a major problem in many hospitals leading to increased morbidity, mortality, and healthcare costs. Cephalosporins are first-line drugs in treatment of infections caused by Enterobacteriaceae family; however, extensive use of third-generation cephalosporin has resulted in increased prevalence of ESBL among organisms [2] .

The most important mechanism of resistance to β-lactam antibiotics is the production of β-lactamase enzymes. The β-lactamases confer significant antibiotic resistance to their bacterial hosts by hydrolysis of the amide bond of the β-lactam ring. Classes A, C, and D include enzymes that hydrolyze their substrates by forming an acyl (penicilloic or cephalosporoic) enzyme through an active-site serine, whereas class B β-lactamases are metalloenzymes that utilize at least one active-site zinc ion. These enzymes are especially important in Gram-negative bacteria, as they constitute the major defense mechanism against β-lactam-based drugs [3] .

β-Lactamases are remarkably diversified due to their continuous mutation [1] .

The Clinical and Laboratory Standard Institute (CLSI) interpretive guidelines stated that Enterobacteriaceae that produce ESBLs are resistant to therapy with penicillin, cephalosporins, and aztreonam, despite apparent in-vitro susceptibility to some of these agents [4] . Third-generation cephalosporin resistance is often mediated by TEM-type and SHV-type l-lactamases in Enterobacteriaceae [5].

The aim of the study was to evaluate the prevalence of ESBLs in Enterobacteriaceae among patients conducted at Menoufia University Hospitals.


  Patients and methods Top


Collection of samples

A total of 160 Enterobacteriaceae isolates from urine, sputum, stool, blood, pus, drains, and other samples were collected from patients who referred to Menoufia University Hospitals, a referral center offering tertiary care to about one-third of population inhabiting the Nile Delta at North of Egypt.

Isolation and identification

Colony isolates were obtained after culture of urine specimens on cysteine lactose electrolyte deficient, whereas pus and sputum specimens were cultured on blood agar and MacConkey's agar; thereafter, all specimens were incubated aerobically at 37°C for 24 h. Blood samples were inoculated into blood culture bottles, then subcultures were performed also on blood and MacConkey's agar. A single isolated colony was considered for further studies and identification was made using standard conventional, morphological, cultural, and biochemical tests [6] .

Detection of extended-spectrum β-lactamase

Screening for extended-spectrum 0β-lactamase producers

It was performed according to CLSI 2011 recommendations to identify potential ESBL-producing isolates using standard disk diffusion test.

Ceftazidime (30 μg), cefotaxime (30 μg), and ceftriaxone (30 μg) are used. If a zone diameter of less than 22 mm for ceftazidime, less than 27 mm for cefotaxime, and less than 25 mm for ceftriaxone was recorded, the strain was considered to be 'suspicious for ESBL production'. The use of more than one antimicrobial agent for screening improves the sensitivity of detection.

Phenotypic confirmatory test for extended-spectrum β-lactamase production by cephalosporin/clavulanate combination disks

All strains that were screened out for ESBL production were also subjected to confirmation using the PCDDT, as recommended by the CLSI 2011. The ceftazidime (30 μg) and cefotaxime (30 μg) disks alone and in combination with clavulanic acid (ceftazidime+clavulanic acid, 30/10 μg disks, cefotaxime+clavulanic acid, 30/10 μg) were applied onto a plate of Mueller-Hinton agar, which was inoculated with the test strain.

An increase of at least 5 mm in zone diameter for either antimicrobial agent tested in combination with clavulanic acid versus its zone when tested alone denoted ESBL-producing strains.

Reference strains used in this study were Escherichia coli ATCC 25922 as negative control and Klebsiella pneumoniae ATCC 700603 as positive control.


  Results Top


The age of the studied patients ranged from 10 to 75 years with a mean ± SD of 55.72 ± 8.94 years and a median of 45 years.

A total of 160 clinical samples were cultured from various sample sites from patients admitted at Menoufia University Hospitals. The distribution of isolates was 46.8% E. coli, 30.0% Enterobacter spp., 21.8% Klebsiella spp., and 1.3% Serretia spp.

Most patients presented with fever (73%) as the chief complaint; others presented with persistent dysuria (15%), persistent wheezy chest (10%), and wound infection (2%).

Clinically, eight patients had ascites, 10 had jaundice, eight had shrunken cirrhotic liver, eight had splenomegaly, four had encephalopathy, 41 had pallor, 15 had abdominal pain, 20 had wheezy chest, three had clinically infected wounds and CVP lines, 18 had suprapubic pain, and nine had recurrent loin pain.

Overall, 97 (60.6%) isolates were detected as ESBL producers by phenotypic confirmatory test for ESBL production (cephalosporin/clavulanate combination disks) and included 45 (46.4%) E. coli, 28 (28.9%) Enterobacter spp., 22 (22.7%) Klebsiella spp., and two (2.1%) Serretia spp.

Among 60.6% ESBL isolates, there were 45.6% ESBL isolates from patients with previous antimicrobial intake, which were more than isolates from patients without previous antimicrobial intake 15.0%; ESBLs isolates are higher in male individuals (35.6%) than in female individuals (25.0%).

The most frequent ESBLs isolates were from urine samples (27.8%), followed by sputum samples (18.6%), stool (12.4%), and pus (9.3%), whereas the least frequent ESBLs isolates were from nasal swabs and CVP line samples.


  Discussion Top


There is a problem that occurred among ESBL-producing Enterobacteriaceae, which have widely caused the spread of infections worldwide [7],[8],[9] . Although hundred variants of ESBLs have been described today [10],[11] , the most common of them are derivatives of TEM or SHV enzymes. In addition, in recent years non-TEM and non-SHV ESBLs have been reported, mainly the CTX-M enzymes [8, 12, 13].

ESBL-producing strains are a major problem in many hospitals, leading to increased morbidity, mortality, and healthcare costs. Therefore, detection of ESBLs in Enterobacteriaceae is crucial for optimal treatment of patients and to control the spread of resistance [4] .

The uncontrolled use of antibiotics at hospital could be a leading contributory factor to the high ESBL prevalence because third-generation cephalosporins are usually first line against many severely infectious diseases. Justifiable use will be an effective means of controlling and decreasing spread of ESBLs strains. This study showed that the highest rate of ESBL-producing Enterobacteriaceae was found in patients with history of antibiotic administration (45.6%) than in other patients without antibiotic administration (15.0%). Similar results were reported by Graffunder et al. [14] in the USA who found a correlation between the selective pressures of antimicrobial agents identified as risk factors for ESBL-producing organisms and the presence of related resistance genes residing on the plasmid.

Suggested approaches to the management of patients with serious infections due to ESBL-producing Enterobacteriaceae are as follows:

(1) First-line therapy:

(a) Carbapenems.

(b) Piperacillin-tazobactam (low inoculum).

(c) Fosfomycin (oral formulation for simple urinary tract infections).

(2) Second-line therapy:

(a) Tigecycline (not in urinary tract infections).

(b) Fluoroquinolone.

(c) Colistin 15.

In the present study, the prevalence of ESBL-producing Enterobacteriaceae isolates was 60.6%. Nearly same results were obtained by Olowe et al. [16] from Nigeria, who showed that the prevalence of ESBL-producing Enterobacteriaceae isolates was about 51.3%, and also by Dallal et al. [17] from Iran who found that 57.5% of isolates were ESBL-producing Enterobacteriaceae.

In several western studies, prevalence of ESBLs was less than that found in our study. The uncontrolled use of third-generation cephalosporins at our hospital could be a leading contributory factor to the high ESBL prevalence observed in this study.

In the present study, the maximum ESBL production was seen among the isolates of E. coli (46.4%), followed by Enterobacter spp. (28.9%), Klebsiella spp. (22.7%), and Serretia spp. (2.1%). Nearly similar results were observed by Balan [2] from India who reported that ESBLs were detected among the following isolates: 24 (52.2%) E. coli, 15 (32.6%) K. pneumoniae, three (6.5%) Proteus mirabilis, three (6.5%) Enterobacter spp., and one (2.2%) Citrobacter spp. Another study in Chennai reported that ESBL production was 47% among E. coli and 37% among K. pneumoniae in a tertiary care center [18] .

However, these results disagree with that of the study of Kaur and Aggarwal [19] , which showed that the maximum ESBL production was found among the isolates of K. pneumoniae (52.27%), followed by those of E. coli (46.43%) ([Figure 1] and [Table 1], [Table 2] and [Table 3]).
Figure 1: Confirmatory test for detection of ESBL: >5 mm increase in zone diameter for cefotaxime and ceftazidime when tested in combination with clavulanic acid versus its zone when tested alone. C, clavulanic acid; CAZ, ceftazidime; CTX, cefotaxime; ESBL, extended-spectrum b-lactamase.

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Table 1: Distribution of Enterobacteriaceae isolates according to extended-spectrum b -lactamases production


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Table 2: Distribution of extended-spectrum b -lactamases-producing Enterobacteriaceae isolates according to the type of sample


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Table 3: Comparison of Enterobacteriaceae isolates according to extended-spectrum b -lactamase production and history of antimicrobial intake


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  Conclusion Top


There is high prevalence of ESBL producers among Enterobacteriaceae isolates in our hospital (60.6%). This calls for strict policies regarding antibiotic usage and their screening methods.

It is important for the clinical microbiology laboratory to implement one or more methods to detect ESBLs, and hospital-based clinical laboratories should screen isolates from the community-based patients before hospitalization to prevent the spread of ESBL-mediated resistance.

The use of cephalosporins should be discouraged for empirical therapy of infections and should only be used according to results of culture and sensitivity.


  Acknowledgements Top


Conflicts of interest

None declared.

 
  References Top

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2. Balan K. Detection of extended spectrum β-lactamase among Gram negative clinical isolates from a tertiary care hospital in South India. Int J Res Med Sci 2013; 1 :28-30.  Back to cited text no. 2
    
3. Wilke MS, Lovering AL, Strynadka NC. β-lactam antibiotic resistance: a current structural perspective. Curr Opin Microbiol 2005; 8 :525-533.  Back to cited text no. 3
    
4. Song W, Bae IK, Lee YN, Lee CH, Lee SH, Jeong SH. Detection of extended-spectrum β-lactamases by using boronic acid as an AmpC β-lactamase inhibitor in clinical isolates of Klebsiella spp. and Escherichia coli. J Clin Microbiol 2007; 45 :1180-1184.  Back to cited text no. 4
    
5. Colom K, Perez J, Alonso R, Aranguiz AF, Larino E, Cisterna R. Simple and reliable multiplex PCR assay for detection of blaTEM, blaSHV and blaOXA-1 genes in Enterobacteriaceae. FEMS Microbiol Lett 2003; 223 :147-151.  Back to cited text no. 5
    
6. Chessbrough M. District laboratory practice in tropical countries. 2nd ed. New York, USA: Cambridge University; 2006. 50-70.  Back to cited text no. 6
    
7. Quinteros M, Radice M, Gardella N, Rodriguez M, Costa N, Korbenfeld D, Couto E. Extended-spectrum β-lactamases in Enterobacteriaceae in Buenos Aires, Argentina, Public Hospitals. Antimicrob Agents Chemother 2003; 47 :2864-2867.  Back to cited text no. 7
    
8. Al-Agamy MH, Shible AM, Tawfik AF. Prevalence and molecular characterization of extended spectrum β-lactamase-producing Klebsiella pneumoniae in Riyadh, Saudi Arabia. Ann Saudi Med 2009; 29 :253-257.  Back to cited text no. 8
    
9. Apisarnthanarak A, Kiratisin P, Mundy LM. Predictors of mortality from community-onset blood stream infections due to extended-spectrum β-lactamase producing Escherichia coli and Klebsiella pneumoniae. Infec Control Hosp Epidemiol 2008; 29 :671-674.  Back to cited text no. 9
    
10.Jacoby GA, Munoz-Price LS Mechanisms of disease, the new β-lactamases. N Engl J Med 2005; 352 :380-391.  Back to cited text no. 10
    
11.Rawat D, Nair D. Extended-spectrum β-lactamase in Gram negative bacteria. J Glob Infect Dis 2010; 2 :263-274.  Back to cited text no. 11
    
12.Mohamudha PR, Srinivas AN, Rahul D, Harish BN, Parija SC. Molecular epidemiology of multidrug resistant extended-spectrum β-lactamase producing Klebsiella pneumoniae outbreak in a neonatal intensive care unit. IJCRIMPH 2010; 2 :226-238.  Back to cited text no. 12
    
13.Munier GK, Johnson CL, Snyder JW, Moland ES, Hanson D, Thomson KS. Positive extended-spectrum β-lactamase (ESBL) screening results may be due to AmpC β-lactamases more often than to ESBLs. J Clin Microbiol 2010; 48 :673-674.  Back to cited text no. 13
    
14.Graffunder EM, Preston KE, Evans AM, Venezia RA. Risk factors associated with extended-spectrum β-lactamase producing organisms at a tertiary care hospital. J Antimicrob Chemother 2005; 56 :139-145.  Back to cited text no. 14
    
15.Kanj SS, Kanafani ZA. Current concepts in antimicrobial therapy against resistant gram-negative organisms: extended-spectrum β-lactamase producing Enterobacteriaceae, carbapenem-resistant Enterobacteriaceae, and multidrug-resistant Pseudomonas aeruginosa. Mayo Clin Proc 2011; 86 :250-259.  Back to cited text no. 15
    
16.Olowe OA, Oladipo GO, Makanjuola OA, Olaitan JO. Prevalence of extended spectrum beta-lactamases (ESBLs) carrying genes in Klebsiella spp. from clinical samples at Ile-Ife, South Western Nigeria. Int J Pharm Med Biosci 2012; 1 :129-138.  Back to cited text no. 16
    
17.Dallal MM, Sabaghi A, Mirzaiee HM, Mehrabadi JF, Lari AR, Eshraghian MR, et al. Prevalence of SHV β-lactamases in Escherichia coli. Afr J Microbiol Res 2012; 6 :5518-5522.  Back to cited text no. 17
    
18.Gururajan G, Kathireshan A, Kaliyaperumal, Balagurunathan R. Prevalence of extended spectrum beta lactamases in uropathogenic Escherichia coli and Klebsiella species in a Chennai suburban tertiary care hospital and its antibiogram pattern. Res J Microbiol 2011; 6 :796-804.  Back to cited text no. 18
    
19.Kaur M, Aggarwal A. Occurrence of the CTX-M, SHV and the TEM genes among the extended spectrum β-lactamase-producing isolates of Enterobacteriaceae in a tertiary care hospital of North India. J Clin Diagn Res 2013; 7 :642-645.  Back to cited text no. 19
    


    Figures

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    Tables

  [Table 1], [Table 2], [Table 3]



 

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Abstract
Introduction
Patients and methods
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