Abstract
Introduction:
Klebsiella pneumoniae is one of the main opportunistic pathogens which can cause different types of infections. Production of beta-lactamases like AmpC and ESBL mostly lead to beta-lactam resistance in these Gram-Negative bacteria. The aim of this study was the detection of AmpC-producing K. pneumoniae in clinical isolates.
Materials and Methods:
Three hundred and three isolates of K. pneumoniae were identified. Double disc method including cefoxitin with cefepime and using boronic acid with cloxacillin were performed as two phenotypic methods for detection of AmpC. Amplification of AmpC gene was performed by PCR.
Results:
Eight and three isolates showed positive results in double disc method and by using boronic acid with cloxacillin, respectively. Five isolates had specific band for AmpC gene after electrophoresis.
Conclusion:
Our results were indicated the low prevalence of AmpC-producer-K. pnemoniae in Iran. On the other hand these two tested phenotypic methods showed low sensitivity for detection of AmpC.
Keywords: Beta-lactamases, Boronic acid, Cloxacillin, Double disc method, Enterobactriaceae, Iran
Introduction
K. pneumoniae is the third common gram negative bacteria of nosocomial infections in burn patients after P. aeruginnosa and A. baumannii [1-3]. This microorganism can cause diverse type of infection like pneumonia, urinary tract infection, and septicemia etc [3,4]. These infections cause increasing morbidity and mortality among patients [3,4]. K. pneumoniae can persist on hospital environmental and can colonize human skin [4]. Therefore, it can easily transmit among patients via hands of health care personnel [4]. On the other hand over use of antibiotic and/or inappropriate antibiotic therapy are the major causes for the emergence of antibiotic resistant K. pneumoniae [3,5,6]. Resistance to antibiotics can contribute by different mechanism like efflux-pump, production of different enzymes, etc [4]. One of the main mechanism for resistance to beta-lactam antibiotics is the production of beta-lactamases including AmpC betalactamases (AmpC) and extended-spectrum beta-lactamases (ESBLs) [7,8]. AmpC beta-lactamases are group I cephalosporinases and can cause resistance to wide variety of beta-lactam antibiotics including third generation of cephalosporins and aztreonam [6,9]. Even AmpC may also mediate resistance to carbapenems in combination with porin loss [6]. One of the main characterizations of this enzyme is related to poor inhibition by beta-lactamases inhibitor like clavulanic acid [10]. AmpC is originally mediated chromosomally in Enterobactriaceae; however, plasmid-mediated AmpC beta-lactamases has arisen through the transfer from chromosome to plasmid and can lead to spread among different bacteria population including Klebsiella spp. which typically do not have chromosomal AmpC [6,10]. The increasing frequency of plasmid mediated AmpC isolates posing therapeutic challenges for health care worldwide by causing resistance to a variety type of beta-lactam antibiotics [6]. The aim of this study was to determine the AmpC-producer -K. pneumoniae isolated from different clinical isolates.
Materials and Methods
Bacterial Isolations
This cross-sectional study was conducted from August to December 2013. Three hundred and three K. pneumoniae were collected from different clinical sources in different wards of hospital including, burn wound, pneumonia, septicemia and urinary tract infections, inpatients 17% (52) and 83% (251) outpatients, from three teaching hospital in Tehran. The plate of bacteria sent to Antimicrobial Resistance Research Center, Iran University of Medical Sciences for future examines.
These isolates were identified with conventional biochemical and microbiological tests. Like, TSI, urea, SIM and citrate etc.
Antibiotic Susceptibility Testing
Antibiotic susceptibility testing was done by disc diffusion agar method according to CLSI guide line [11]. Standard antibiotic discs were prepared from MAST Company (Mast Diagnostics, UK) and include:
Cefoxitin (30μg), ceftriaxone (30μg), cefotaxime (30μg), cefepime (30μg), imipenem (10μg), meropenem (10μg), amoxicillin-clavulanic acid (20/10μg), aztreonam (30μg), tobramycin (10μg), gentamicin (10μg), amikacin (30μg), trimethoprim-sulfamethoxazole (1.25/23.75 μg), chloramphenicol (30μg) and tetracycline (30μg), ceftazidime (30 μg), cefoxitin, ceftriaxone), piperacillin (100μg).
Phenotypic detection of AmpC
Two phenotypic tests were used for identification of AmpC:
1. The increasing inhibition zone at least 5mm around meropenem (10μg) plus 600 μg/disc boronic acid and meropenem (10μg) plus 750 μg/disc cloxacillin simultaneously in comparison to meropenem (10μg) alone. The stock of boronic acid was prepared 30mg/ml and the stock of clocacillin was prepared 37.5 mg/ml [12].
2. Double disc methods with cefoxitine (30μg) and cefepime (30μg). The isolates which susceptible to cefepime and resistant to cefoxitine were consider as an AmpC-producer isolate [13].
Genotypic detection of AmpC
PCR was used as a molecular test for detection of AmpC plasmid mediate (cmy) gene. The forward primer was 5-ATTCCGGGTATG GCCGT-3 and the reverse was 3-5-GGGTTTACCTCAACGGC [14].
The polymerase chain reaction was performed in following condition:
The first denaturation at 94°C for 5 min and 30 cycles of 94°C for 60 s, annealing at 58°C for 60 sec, extension at 72°C for 60 sec and at last the final extension at 72°C for 5 min.
Isolates which showed specific band after PCR and electrophoresis have been sent for sequencing to Genfanavaran, Macrogen, Seoul, Korea.
Results
Three hundred and three isolates of K. pneumoniae were confirmed according to the results of biochemical and microbiological tests. The percentage of antibiotic resistance was showed in [Table/Fig-1].
[Table/Fig-1]:
(Number) Percentage of resistance to tested antibiotics.
| Antibiotics | CRO | FOX | CTX | CAZ | CEF | IMI | MEM | AT | TO | GM | AK | SXT | PRL | C | T |
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Percentage of resistance | 20.8 | 50 | 55 | 50 | 43 | 23 | 23 | 54 | 100 | 37 | 79 | 91 | 65 | 97 | 91 |
| (63) | (151) | (167) | (163) | (130) | (69) | (69) | (164) | (303) | (112) | (239) | (276) | (197) | (294) | (276) |
CRO: ceftriaxone, FOX: cefoxitin, CTX: cefotaxime, CAZ: ceftazidime, CEF: cefepime, IMI : imipenem, AT, aztreonam, MEM: meropenem, TO: tobramycin, GM: gentamicin, AK: amikacin, SXT: trimethoprim sulfamethoxazole, PRL: piperacillin-Tazobactam, , C: chloramphenicol, T: tetracycline
Eight isolates had less than 14mm inhibition zone around cefepime and more than 18 mm around cefoxitin according to CLSI table 2013. On the other hand, only three isolates showed at least 5 mm in diameter of inhibition zone around meropenem plus boronic acid and meropenem plus cloxacillin simultaneously in comparison to meropenem alone. None of the isolates showed positive results in both of detection phenotypic tests. Three positive PCR isolates were positive in at least one of phenotypic methods that were used in this study (two out of eight double disc method positive and one out of three when use of boronic cid and cloxcillin combined with meropenem). Two isolates were negative in both of phenotypic methods with positive results in PCR [Table/Fig-2].
[Table/Fig-2]:
Number of isolates showing positive AmpC by all three methods
| Phenotypic methods | No. of strains | PCR |
|---|---|---|
| Meropenem+boronic acid/ Meropenem+cloxacillin | 3 | 1 |
| Cefoxitine/ cefepime | 8 | 2 |
| Strains without positive results in both phenotypic methods | 292 | 2 |
| Total | 303 | 5 |
Number of confirmed AmpC positive strains with PCR according to source of isolated bacteria was showed in [Table/Fig-3].
[Table/Fig-3]:
Number of confirmed AmpC positive strains with PCR according to source of isolated bacteria
| Source of specimens | Burns infection | Ventilator association pneumonia |
|---|---|---|
| No. of confirmed AmpC positive strains | 4 | 1 |
Totally, in five strains AmpC specific band was observed after PCR [Table/Fig-4] and gel electrophoresis in agaros 1.5%. Direct sequencing of PCR amplified products was carried out using ABI 3730X capillary sequencer (Genfanavaran, Macrogen, Seoul, Korea).
[Table/Fig-4]:
![[Table/Fig-4]:](https://cdn.ncbi.nlm.nih.gov/pmc/blobs/cb40/4437065/0ef918f90cb2/jcdr-9-DC01-g001.jpg)
PCR amplification fragments for detection of AmpC agene among K.pneumoniae isolates M: 1kb DNA size marker; lane 1; Psitive Control of AmpC; lane 2-4; Positive strains; lane 5; Negative Control of AmpC gene
Discussion
K. pneumoniae is one of the important Gram - negative bacteria in nosocomial infections [1-3,15]. Antibiotic resistant isolates make therapeutic challenge for physicians [3]. K. pnemoniae can produce variety of beta – lactamase enzymes [3,5]. Recent study was conducted in Iran reported the prevalence of OXA-48-producer K. pneumoniae [15]. Also, this study indicated the AmpC – producer K. pneumoniae but in low prevalence with the rate of 1.6%. The results of study was conducted in Turkey in west-north of Iran were showed the prevalence of 8% AmpC-producer K. pneumoniae isolated from blood culture [16]. This prevalence is more than our results because K. pneumoniae were isolated from hospitalized patients in Turkey but in our study most of specimens were isolated from outpatients. Despite of the low prevalence of AmpC producer K. pneumoniae in our tested isolates, it is notable that the different number of isolates was isolated from different clinical specimens. Other studies in Iran have indicated the higher resistance in K. pneumoniae which isolated from burn patients [3]. This can be due to the expand using of beta-lactam antibiotics in patients whome were hospitalized in burn ward in Iran [3,5,6]. Since the AmpC is the cephalosporinase, this low prevalence of AmpC producer K. pneumoniae in our tested isolates can related to use of other broad spectrum antibiotics for empirical treatment except cephalosporins and pressure of the overuse of antibiotics for selection of this mechanism of antibiotic resistant is low. On the other hand, we can inhibit the increasing prevalence of AmpC producer K. pneumonia by prevention of broad spectrum cephalosporins misuse.
In Pakistan that is in east – south border of Iran the rate of AmpC – producer – K. pneuminiae was 12% [7]. The results of study in China were showed 12% AmpC – producer – K. pneuminiae [4]. In a study that was conducted in India six out of 44 Klebsiella spp. (13.6%) were positive for AmpC [17].These isolates were collected from different clinical specimens [17]. In other study that was done in India five out of 120 K. pneumonia (94.1%) were positive for detection of AmpC [18]. This difference between two studies that were conducted in the same country (India) can be related to different centers that were selected for study and also, source of specimens. The influence of antibiotic treatment and their pressure for resistant bacteria selection can be different in different health care settings. In this regard, six of 22 K. pneumonia (27.2%) were positive for AmpC [19]. These results indicated the relation with the source of isolated bacteria, because these isolates were collected from blood specimens. Generally, broad spectrum antibiotics are using for treatment of septicemia and the pressure of antibiotic over use can lead to select more resistance bacteria. These results are equal to our finding from burn patients in current study. On the other hand, this subject should be considered that AmpC gene is plasmid mediated and can transfer to other bacteria and make complication in treatment of patients [10].
Conclusion
According to finding of this study it seems that both phenotypic tests have low sensitivity. Thus the phenotypic method can be useful for primary screening. On the other hand, phenotypic methods are more economical than molecular methods such as PCR and also, phenotypic methods do not require expertise and professionals. But it is notable that PCR is more reliable test for detection of AmpCproducing strains and can use for critical and important cases.
Acknowledgment
This study was supported by a grant (M/T 91-04-134-20187) from Iran University of Medical Sciences, Tehran, Iran.
Financial or Other Competing Interests
As declared above.
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