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 Table of Contents  
ORIGINAL ARTICLE
Year : 2018  |  Volume : 8  |  Issue : 3  |  Page : 175-179

Molecular characteristics of extended-spectrum-beta-lactamase-producing Klebsiella pneumoniae isolates in the West of Iran


1 Department of Microbiology, School of Medicine, Kermanshah University of Medical Sciences, Kermanshah, Iran
2 Students Research Committee, Kermanshah University of Medical Sciences, Kermanshah, Iran

Date of Web Publication24-Sep-2018

Correspondence Address:
Parviz Mohajeri
Department of Microbiology, School of Medicine, Kermanshah University of Medical Sciences, Shirudi Shahid Boulevard, Daneshgah Street, Kermanshah 67148-69914
Iran
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/AIHB.AIHB_20_18

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  Abstract 


Background: Klebsiella pneumoniae causes community and nosocomial infections. Drug treatment of extended-spectrum beta-lactamases (ESBLs) by K. pneumoniae isolates is costly, long, and sometimes problematic. Objectives: The aim of this study was to determine the antibiotic resistance patterns and the occurrence of bla CTX-M, bla TEM and bla SHV genes in the third-generation cephalosporin-resistant K. pneumoniae isolated from urinary tract infection in Kermanshah region, Iran. Materials and Methods:Fifteen K. pneumoniae isolates were collected and confirmed by the analytical profile index-20E system. Phenotypic confirmatory test was performed using combination disk method. The genes of bla CTX-M, bla TEM and bla SHV were investigated by polymerase chain reaction. SPSS version 20 (IBM, Chicago, IL, USA) was used for sample analysis. Results: Resistant isolates to ampicillin and imipenem represented 96% and 4%, respectively, which were the highest and lowest resistance. Seventeen (34%) out of the 50 isolates were ESBL producers by the synergy test. The prevalence of bla CTX-M, bla TEM and bla SHV among these isolates was 88% (n = 15), 70% (n = 12) and 58% (n = 10), respectively. Conclusions: Our research showed a possibility of the spread of multidrug-resistant ESBL-producing isolates. Monitoring control of risk factors and drug-resistant patterns with the use of phenotypic and/or genotypic analyses are very important to prevent the occurrence and dissemination of resistant strains due to ESBL-producing K. pneumoniae in hospital settings.

Keywords: Bla CTX-M, bla SHV, bla TEM, extended-spectrum beta-lactamases, Klebsiella pneumoniae


How to cite this article:
Mohajeri P, Kavosi S, Esmailzadeh T, Farahani A, Dastranj M. Molecular characteristics of extended-spectrum-beta-lactamase-producing Klebsiella pneumoniae isolates in the West of Iran. Adv Hum Biol 2018;8:175-9

How to cite this URL:
Mohajeri P, Kavosi S, Esmailzadeh T, Farahani A, Dastranj M. Molecular characteristics of extended-spectrum-beta-lactamase-producing Klebsiella pneumoniae isolates in the West of Iran. Adv Hum Biol [serial online] 2018 [cited 2019 Jul 19];8:175-9. Available from: http://www.aihbonline.com/text.asp?2018/8/3/175/241924




  Introduction Top


Klebsiella pneumoniae, a Gram-negative rod, is classified in the Enterobacteriaceae family and causes community and nosocomial infections.[1],[2]Klebsiella involved in 6%–17% of urinary tract infections and among Gram-negative bacteria is the second leading cause of urinary tract infections.[3]K. pneumoniae strains have the capability to cause severe morbidity and mortality, especially in Intensive Care Units, paediatric and surgical wards in hospitals.[2] Extended-spectrum beta-lactamase (ESBL)-producing K. pneumoniae was first detected during 1983 and 1989 in Europe and the USA, respectively,[4] and since then, many outbreaks caused by this pathogen have been reported worldwide.[2] ESBL-producing isolates are not susceptible to penicillins and narrow- and extended-spectrum cephalosporins [1] but susceptible to cephamycins and carbapenems.[4],[5] Several studies have revealed failure of therapy, poor clinical results and increased mortality for patients infected with bacteria-encoding ESBLs.[6],[7],[8] The most prevalent ESBLs belong to three groups: TEM, SHV, and CTX-M. The TEM enzyme reflects the name of a patient from which it was derived Temoneira. The SHV enzymes are named after the thiol variable active site and are often associated with K. pneumonia.[5] It is interesting to note that SHV-1 and TEM-1 have a relatively narrow spectrum of activity, and the first ESBLs were mutants derived from them.[9] A CTX-M enzyme cleaves cefuroxime, cefotaxime and cefepime better than ceftazidime. However, the types of CTX-M with increased hydrolysing activity against ceftazidime have emerged.[6],[7] This group of ESBLs has spread quickly and is now one of the main types of ESBLs in many countries.[5] The genes coding for ESBLs are usually carried by plasmid, which facilitates their spread among Gram-negative bacteria. ESBL-producing isolates are multidrug resistant (MDR) since the relevant plasmids may also carry other genes responsible for resistance.[10] Therefore, development of resistance to multiple antimicrobial agents in pathogenic bacteria has become an important public health concern since there are fewer or even sometimes no effective antimicrobial agents for infections caused by these bacteria. The problem of increasing antimicrobial resistance is even more threatening when considering the very limited number of new antimicrobial agents that are in development. The aim of this study was to determine the antibiotic resistance patterns and the occurrence of bla CTX-M, bla TEM and bla SHV genes in the third-generation cephalosporin-resistant K. pneumoniae isolated from urinary tract infection in Kermanshah region, Iran.


  Materials and Methods Top


Bacterial isolates and identification

This cross-sectional study was conducted from January 2015 to June 2016. A total of 50 K. pneumoniae were isolated from the urine of outpatients with urinary tract infections from Kermanshah (Central Laboratory), West of Iran.

Urinary tract infection was defined as the presence of ≥105 colony-forming units per millilitre of urine.[11] It isolates were identified using conventional tests,[12] and then, isolates were confirmed by the analytical profile index-20E system (bioMerieux, France).  Escherichia More Details coli ATCC 25922 was used for quality control.

Antibiotic susceptibility test

Antimicrobial susceptibility testing was carried out by disk diffusion method on Mueller-Hinton agar (Merck, Germany) as recommended by the Clinical and Laboratory Standard Institute (CLSI).[13] The antibiotics tested were ampicillin (20 μg), gentamicin (10 μg), ceftazidime (30 μg), imipenem (10 μg), ciprofloxacin (5 μg), ofloxacin (5 μg), cefepime (30 μg), nalidixic acid (30 μg), tetracycline (30 μg), nitrofurantoin (30 μg) and azithromycin (30 μg) (Mast, England). E. coli ATCC 25922 was used as a control. MDR isolates were defined as resistance to at least one agent of three or more antimicrobial families, except for ampicillin (because of intrinsic resistance).[13]

Extended-spectrum beta-lactamase confirmatory test

Phenotypic confirmatory test was performed using combination disk method according to the recommendations of the CLSI.[13] Discs contained ceftazidime (30 μg) and cefotaxime (30 μg) in combination with and without clavulanate (10 μg) were used to detect ESBL producers. The test was considered positive when the difference of zone diameters between the beta-lactam disk and disk containing antibiotic plus clavulanic acid was equal to or more than 5 mm.[13] E. coli ATCC 25922 and K. pneumoniae ATCC 700603 were used as a negative and a positive control, respectively.

Polymerase chain reaction amplification

DNA templates of isolates were prepared by boiling method. DNA of ESBL-producing isolates was targeted for the bla CTX-M, bla TEM and bla SHV genes using the primers [14],[15] (SinaClon, Iran) as listed in [Table 1].
Table 1: Primers of target genes used in this study

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Polymerase chain reaction (PCR) was carried out in 25 μl volume reaction mixtures containing 10 pmol of each primer, 200 μM dNTP, 1.5 mM MgCl2, 1.5 μl of crude template DNA and 1 U Taq polymerase in the reaction buffer provided by the manufacturer (CinnaGen, Tehran, Iran). PCR amplification conditions were 4 min denaturation at 94°C followed by 35 cycles of 1 min at 94°C, 1 min at the annealing temperature (55°C for bla SHV, bla CTX-M and bla TEM) and 1 min at 72°C with a final extension period of 5 min at 72°C. After electrophoresis of PCR products on 1% agarose gel (Merck Co, Germany) and staining with ethidium bromide, the DNA bands were visualised by GelDoc apparatus (Bio-Rad, USA).

Statistical analysis

All statistical analyses were performed using SPSS version 20 (IBM, Chicago, IL, USA). Chi-square and Fisher's exact tests were used to determine the relationship between antibiotic-resistant pattern and ESBL-producing patterns and the positive genotype of bla CTX-M, bla TEM and bla SHV genes. Statistical significance was defined as the P < 0.05.


  Results Top


During the 6-month period, a total of 50 K. pneumoniae isolates were obtained from urinary tract infection in the Central Laboratory in Kermanshah, Iran. They were collected from 34 (68%) females and 16 (32%) males, with an average age of 43.4 years. The results of antimicrobial susceptibility testing are shown in [Table 2]. The highest rate of resistance was ampicillin (96%) and gentamicin (8%) and the lowest imipenem (4%). Seventy per cent of isolates were MDR and 17 (34%) of isolates were ESBL producers [Figure 1]. In addition, 13 (76.5%) of the ESBL producers isolates were MDR.
Table 2: Antibiotic susceptibility of 50 urinary isolates of Klebsiella pneumoniae

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Figure 1: Result of combination disk method. The extended-spectrum beta-lactamase producing (b) and not producing (a) organisms were differentiated by increasing of growth inhibition zone only around the cefotaxime/clavulanate (or ceftazidime/clavulanate) for at least 5 mm in extended-spectrum beta-lactamase-producing ones.

Click here to view


By PCR experiments, the following genes were detected: Bla CTX-M (15 isolates, 88%), bla TEM (12 isolates, 70%) and bla SHV (10, 58%) [Figure 2]. Interestingly, the following bla gene's combinations were observed: bla SHV + bla CTX-M + bla TEM (47%), bla SHV + bla TEM (47%) and bla CTX-M + bla TEM (58%).
Figure 2: Polymerase chain reaction amplification of β-lactamase genes in extended-spectrum beta-lactamase-producing Klebsiella pneumoniae isolates M, 100 bp DNA ladder, lanes 2, 4, 6, negative controls, lane 1 bla TEM, (431 bp), lane 3 bla CTX-M, (550 bp), lane 5, bla SHV (868 bp) amplification products.

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There was coexistence of genes bla SHV, bla CTX-M and bla TEM in 47%; bla SHV and bla TEM in 47% and bla CTX-M and bla TEM in 58% of isolates. Resistance to most antibiotics, except ampicillin, was significantly higher in ESBL-producing isolates (P = 0.001) [Table 2].


  Discussion Top


The rapid expansion of ESBLs among Gram-negative bacteria has been the serious concern to control infections since their discovery in 1980.[16] The increasing reports of these enzymes in K. pneumoniae have been considered as a serious issue discussed by a clinical microbiologist in the 21st century.[17] In Asia, the prevalence of ESBL-producing K. pneumoniae varies in different areas. The studies in the Middle East revealed a higher prevalence of ESBL than other's parts of the world.[18] The frequency of ESBL-producing K. pneumoniae isolates in other countries is as follows: 66.7% in India, 54.7% and 61% in Turkey and 41% in the United Arab Emirates.[18],[19] The prevalence of ESBL-producing K. pneumoniae in Tehran has been reported to be 50%; high frequencies were also reported in another part of Iran: 56.1% in Mashhad, 39.4% in Ilam, and 41.3% in Kerman.[19],[20],[21],[22],[23] Similarly, our study showed a high rate (34%) of ESBL-producing K. pneumoniae recovered from urinary infections in Kermanshah. Comparison of rate of ESBL-producing isolates in different countries and also in different cities of Iran indicates the prevalence of ESBL varies in different locations and even in different times in a location.[22],[23],[24],[25] The high prevalence of ESBL among isolates in Kermanshah is probably the result of excessive use of third-generation cephalosporins and dissemination of resistant plasmids among bacterial isolates.[19] The high resistance to ampicillin is consistent with the inherent resistance of K. pneumoniae to penicillins [26] and has been reported by previous studies.[21],[27],[28] However, there was low resistance to imipenem, and it is still used as antibiotics effective at K. pneumoniae, as reported in other studies in Iran.[21],[29]

The high frequency of MDR isolates could reflect the widespread use of antibiotics, lack of the control of risk factors for the acquisition of these strains and dissemination of resistant genes.

According to Podschun and Ullmann,[30] integrons play an important role in the spread of genes encoding antibiotic resistance and so to the emergence of MDR strains. Therefore, it is plausible that the MDR observed in our ESBL producers isolates would have linked to the occurrence of integrons. Resistances to ciprofloxacin, even to aminoglycoside and carbapenem in ESBL-producing isolates, can seriously restrict the therapeutic choice of antibiotics in the future.

CTX-M is more prevalent among isolates in Kermanshah and indicates the simultaneous resistance to beta-lactam antibiotics by the CTX-M enzyme globally and in Iran. In most studies of genes, SHV and TEM had the lowest and the highest prevalence among isolates of K. pneumoniae.[31],[32] The result of this study is not much because SHV genes had the lowest prevalence in comparison with CTX-M. Several genes were responsible for producing ESBL phenotype based on other studies.[20],[30] Resistance of three isolates to imipenem is an alarm for treatment of infections in Kermanshah therapeutic centres and suggests that other genes may be responsible for resistance in these isolates.


  Conclusions Top


The results of this study showed high resistance to cephalosporins and ampicillin and other antibiotics. On the other hand, the results of this study indicated the possibility of the future spread of ESBL-producing isolates in Kermanshah. Due to increasing complexity of resistance to beta-lactams and fluoroquinolone, the key to effective surveillance is the use of both phenotypic and genotypic analyses in concert and investigation of other genes is not studied in Kermanshah. The control of risk factors and the drug resistance patterns with use of genotypic analyses are very important to prevent the occurrence and dissemination of resistant strains due to ESBL-producing K. pneumoniae in hospital settings.

Financial support and sponsorship

We gratefully acknowledge Vice-Chancellor for Research and Technology, Kermanshah University of Medical Sciences, for financial support of this study resulting from MSc Medical Microbiology thesis of Sahar Kavosi, Kermanshah University of Medical Sciences, Iran (Grant No. 97178).

Conflicts of interest

There are no conflicts of interest.



 
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