Emergence of TEM,SHV,and CTX-M-Extended Spectrum β-Lactamases and Class 1 Integron Among Enterobacter cloacae Isolates Collected from Hospitals of Tehran and Qazvin,Iran

Abstract

Extended-spectrum β-lactamase (ESBL) production is an important resistance mechanism among clinical isolates of Enterobacter cloacae. TEM, SHV, and CTX-M are the most common ESBL genotypes among Enterobacter spp. The main aims of this study were to determine the antimicrobial susceptibility pattern and to detect ESBL-encoding genes as well as intI1 genes. One hundred twenty isolates of E. cloacae were collected from hospitals of Tehran and Qazvin, Iran. The isolates were identified by standard laboratory methods and API 20E strips. ESBL screening was performed by the combined disk method. PCR and sequencing were conducted for detection of ESBL-encoding genes as well as intI1 genes. Clonal relatedness of ESBL-producing isolates was assessed by the enterobacterial repetitive intergenic consensus (ERIC)-PCR method. Of 120 isolates, 57 (47.5%) were characterized as multidrug resistant among those 48 (84.2%) isolates carried class 1 integron. Fifty-three (44.2%) isolates were found to be ESBL producers, in which bla_CTX-M-15 (60.4%) was the most common gene followed by bla_TEM-1 (32.1%), bla_TEM-169 (13.2%), and bla_SHV-12 (7.5%) either alone or in combination. Forty-four of the 53 (83.01%) ESBL-producing isolates were genetically unrelated. For the first time, this study describes the emergence of TEM-169, SHV-12, and CTX-M-15 ESBL genotypes in E. cloacae isolates in Iran.

Introduction

Enterobacter cloacae is one of the most important opportunistic pathogens in nosocomial infections, causing several clinical infections, including the urinary tract, lower respiratory tract, skin and soft tissue, and central nervous system infections.³⁶ Extensive and inappropriate use of broad-spectrum antibiotics has increased the appearance of multidrug-resistant E. cloacae (MDREC) isolates, which complicates the process of therapy and limits treatment options.^10,11,34 MDR is defined as resistance to at least three classes of antibiotics, including β-lactams, aminoglycosides, and fluoroquinolones.²⁰ Extended-spectrum β-lactamases (ESBLs) are one of the main causes of the β-lactam resistance in Enterobacteriaceae.^27,30 ESBL-producing isolates are also frequently resistant to other antibiotics such as quinolones, trimethoprim-sulfamethoxazole, and aminoglycosides.²⁹

Although ESBLs are most often found in Klebsiella pneumoniae and Escherichia coli strains, yet there are several reports regarding the frequency of ESBL production in other organisms, including Enterobacter, Serratia, and Citrobacter.^23,24,42 Nosocomial infections caused by ESBL-producing organisms significantly increased within the last decade and have been known to cause high mortality and morbidity.³¹ Most commonly, resistance of Enterobacter spp. to third-generation cephalosporins is caused by overproduction of chromosomal AmpC cephalosporinase.²⁷ ESBLs are usually plasmid-mediated enzymes that are capable of hydrolyzing a wide range of β-lactam antibiotics, including broad-spectrum cephalosporins, penicillins, and aztreonam, but they cannot hydrolyze cephamycins and are inhibited by clavulanic acid. Carbapenems are considered as the drugs of choice for treatment of severe infections caused by ESBL-producing Enterobacteriaceae.³

The first plasmid-mediated β-lactamase in gram-negative bacteria was TEM-1, described in the early 1960s in Greece.⁶ SHV-1 was the next plasmid-mediated β-lactamase found in K. pneumoniae in 1987.⁴³ Most ESBLs are derived from the TEM-1, TEM-2, and SHV-1 enzymes by a limited number of mutations in their sequences and one or more amino acid substitution.³ Recently, several studies revealed that CTX-M-type enzymes are becoming the most prevalent ESBL types detected in Enterobacteriaceae. CTX-M-1 was first reported in the E. coli strain in 1989 in Germany and since then has been observed worldwide. These enzymes are typically classified into five phylogenetic groups, including CTX-M-1, CTX-M-2, CTX-M-8, CTX-M-9, and CTX-M-25.³⁵

Several studies have shown that integrons play a significant role in spreading antibiotic resistance genes among Enterobacteriaceae isolates, as these resistant gene cassettes can be frequently located on integrons. To date, several classes of integrons have been identified, in which class 1 integron is the most common and widely disseminated among Enterobacteriaceae.⁴⁴ The prevalence of ESBLs and class 1 integron in the clinical isolates of E. cloacae from Iran is unknown. The aims of this study were (i) to identify the antimicrobial susceptibility pattern of E. cloacae isolated from selected educational hospitals in Qazvin and Tehran, (ii) to determine the prevalence of bla_TEM, bla_SHV, bla_VEB-1, bla_PER-1, and bla_CTX-M ESBL-encoding genes as well as the intI1 gene and their clonal relatedness, and (iii) to investigate the association between the presence of class 1 integron and MDR patterns among E. cloacae isolates.

Materials and Methods

Study design and identification

This was a cross-sectional study conducted over the period from July 2011 to December 2012. A total of 120 nonduplicate isolates of E. cloacae (1 isolate per patient) were collected from the patients admitted to university teaching hospitals in two cities of Tehran and Qazvin. The bacterial isolates were recovered from different clinical specimens, including urine (42 isolates; 35%), wound (30 isolates; 25%), sputum (6 isolates; 5%), bronchoalveolar lavage (4 isolates; 3.3%), trachea (19 isolates; 15.8%), blood (17 isolates; 14.2%), and cerebrospinal fluid (2 isolates; 1.7%). Isolates were obtained from patients admitted to intensive care units (55–45.8%), internal medicine (29–24.2%), infectious diseases (13–10.8%), neurology (6–5%), surgery (8–6.7%), and orthopedic (9–7.5%) wards. Seventy-four patients (61.7%) were males and 46 (38.3%) females aged between 17 and 85 years with a median of 49 years.

Written informed consent was obtained from all subjects enrolled in this study. The isolates were identified by conventional laboratory techniques²¹ and confirmed by the API 20 E (bioMérieux). The isolates were stored at −70°C in a trypticase soy broth containing 20% glycerol and subcultured twice before testing.

Antimicrobial susceptibility testing

Antimicrobial susceptibility testing was performed by the Kirby-Bauer disk diffusion method according to the Clinical and Laboratory Standards Institute (CLSI) guideline.⁴ The following antibiotic discs were used: gentamicin (10 μg), amikacin (30 μg), chloramphenicol (30 μg), Co-trimoxazole (25 μg), ceftazidime (30 μg), cefotaxime (30 μg), ceftriaxone (30 μg), cefpodoxime (10 μg), aztreonam (30 μg), cefepime (30 μg), imipenem (10 μg), meropenem (10 μg), norfloxacin (10 μg), nitrofurantoin (300 μg), piperacillin (100 μg), piperacillin-tazobactam (100/10 μg), tetracycline (30 μg), gatifloxacin (5 μg), and ciprofloxacin (5 μg). Antibiotic discs were purchased from Mast (Mast Diagnostics Group Ltd). E. coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853 were used as the quality control strains in antimicrobial susceptibility testing.

ESBL screening and confirmation by phenotypic methods

All isolates were initially screened for ESBL production by the standard disk diffusion method according to the CLSI guidelines using ceftazidime (30 μg), cefotaxime (30 μg), ceftriaxone (30 μg), cefpodoxime (30 μg), and aztreonam (30 μg) discs. The isolates with reduced susceptibility to selected antibiotics were then confirmed for ESBL production by the phenotypic combined disk method as recommended by the CLSI. The test was considered positive if an increase ≥5 mm in the zone diameter for either cefotaxime or ceftazidime in combination with clavulanate was observed compared with the zone diameter when tested alone.⁴ In the confirmatory method, K. pneumoniae ATCC 700603 was used as ESBL-positive control and E. coli ATCC 25922 as ESBL-negative control.

PCR and sequencing of ESBL-encoding genes and intI1 genes

Extraction of genomic DNA from E. cloacae isolates was performed by the boiling method. In brief, four or five colonies of overnight culture of each isolates were suspended in 300 μl of TE buffer (10 mM Tris, 1 mM EDTA, pH 8.0). The suspensions were heated for 5 min at 95°C and then cooled on ice. The cellular debris was removed by centrifugation at 12,000 g for 10 min. One microliter of supernatant was used as template DNA for the PCR method. PCR assay was performed for the detection of bla_SHV, bla_TEM, bla_PER-1, bla_VEB-1, bla_CTX-M-1, bla_CTX-M-2, bla_CTX-M-8, bla_CTX-M-9 groups, as well as bla_CTX-M-25, and the class 1 integrase conserved region intI1 gene using specific primers (Table 1). PCR amplifications were performed in a thermocycler (Applied Biosystems) as follows: 95°C for 5 min and 35 cycles of 1 min at 95°C, 1 min at specific annealing temperature for each primer (Table 2), and 1 min at 72°C. A final extension step of 10 min at 72°C was performed. Amplification reactions were prepared in a total volume of 25 μl (24 μl of PCR master mix plus 1 μl of template DNA), including 5 ng of genomic DNA, 2.0 U of Taq DNA polymerase, 10 mM dNTP mix at a final concentration of 0.2 mM, 50 mM MgCl₂ at a final concentration of 1.5 mM, 1 μM of each primer, and 1×PCR buffer (final concentration).

Table 1.

Sequences of Primers Used for Detection of ESBLs and Class 1 Integron Genes

PCR target	Primer sequence (5′-3′)	Annealing temperatures (°C)	References
TEM	F: ATAAAATTCTTGAAGACGAAA	50	²
	R: GAC AGTTACCAATGCTTAATC
SHV	F: TGGTTATGCGTTATATTCGCC	55	²
	R: GGTTAGCGTTGCCAGTGCT
CTX-M-1 group	F: ATGGTTAAAAAATCACTGCGTC	55	¹
	R: TTGGTGACGATTTTAGCCGC
CTX-M-2 group	F: ATGATGACTCAGAGCATTCG	55	¹
	R: GGGTTACGATTTTCGCCGC
CTX-M-8 group	F: ACTTCAGCCACACGGATTCA	55	¹
	R: CGAGTACGTCACGACGACTT
CTX-M-9 group	F: ATGGTGACAAAGAGAGTGCA	55	¹
	R: CCCTTCGGCGATGATTCTC
CTX-M-25	F: CACACGAATTGAATGTTCAG	50	³⁸
	R: TCACTCCACATGGTGAGT
PER-1	F: AATTTGGGCTTAGGGCAGAA	50	¹⁵
	R: ATGAATGTCATTATAAAAGC
VEB-1	F: CGACTTCCATTTCCCGATGC	55	¹⁵
	R: GGACTCTGCAACAAATACGC
Class 1 integron	F: ATCATCGTCGTAGAGACGTCGG	55	³³
	R: GTCAAGGTTCTGGACCAGTTGC
ERIC-PCR	F: ATGTAAGCTCCTGGGGATTCAC	52	³⁹
	R: AAGTAAGTGACTGGGGTGAGCG

ESBL, extended-spectrum β-lactamase; ERIC, enterobacterial repetitive intergenic consensus.

Table 2.

Antimicrobial Susceptibility of Enterobacter cloacae Isolated from Hospitals of Qazvin and Tehran

Antibiotics	S n (%)	I n (%)	R n (%)
Meropenem	115 (95.8)	4 (3.3)	1 (0.8)
Imipenem	110 (91.7)	8 (6.7)	2 (1.7)
Gatifloxacin	105 (87.5)	8 (6.7)	7 (5.8)
Amikacin	101 (84.2)	5 (4.2)	14 (11.7)
Norfloxacin	98 (81.7)	7 (5.8)	15 (12.5)
Cefepime	94 (78.3)	6 (5)	20 (16.7)
Ciprofloxacin	82 (68.3)	17 (14.2)	21 (17.5)
Piperacillin-tazobactam	77 (64.2)	25 (20.8)	18 (15)
Gentamicin	74 (61.7)	2 (1.7)	44 (36.7)
Chloramphenicol	68 (56.7)	12 (10)	40 (33.3)
Co-trimoxazole	63 (52.5)	0	57 (47.5)
Tetracycline	61 (50.8)	10 (8.3)	49 (40.8)
Aztreonam	60 (50)	6 (5)	54 (45)
Ceftazidime	58 (48.3)	6 (5)	56 (46.7)
Ceftriaxone	57 (47.5)	4 (3.3)	59 (49.2)
Piperacillin	55 (45.8)	3 (2.5)	62 (51.7)
Cefpodoxime	47 (39.2)	13 (10.8)	60 (50)
Cefotaxime	39 (32.5)	16 (13.3)	65 (54.2)
Nitrofurantoin	28 (23.3)	13 (10.8)	79 (65.8)

S, susceptible; I, intermediate; R, resistant.

PCR products were electrophoresed on a 1% agarose gel at 100 V and then were stained with the ethidium bromide solution and finally visualized in a gel documentation system (UVtec). E. coli ATCC 25922 was used as a negative control and K. pneumoniae ATCC 700603, E. coli ATCC 35218, and clinical isolates of E. coli harboring class 1 integron and ESBL-encoding genes (kindly provided by Dr Shahcheraghi, Iran) were used as positive controls.

The purified PCR products were sequenced by the Macrogen Company and sequence alignment and analysis were performed online using the BLAST program of the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov).

ERIC-PCR analysis

The epidemiological relationships of 53 ESBL-producing E. cloacae isolates were analyzed by enterobacterial repetitive intergenic consensus (ERIC)-PCR as previously described. Cycling conditions were as follows: denaturation at 94°C for 1 sec, annealing at 52°C for 10 sec, and extension at 72°C for 35 sec for 30 cycles, followed by a final extension at 72°C for 4 min. The resulting products were analyzed on 1.5% agarose gels. Fingerprints were compared visually, and the patterns differing by two or more bands were classified as different.³⁹

Statistical analysis

Statistical analysis was performed for descriptive statistics, including frequencies, cross tabulation of microbiological, clinical, and demographic characteristics using the computer software program SPSS, version 16 (SPSS). The chi-squared test and Fisher's exact test were used to determine the association between the integron carriage and MDR pattern. A p-value of <0.05 was considered as significant, statistically.

Results

In total, resistance rates varied between 4.1% and 76.4% against the antimicrobial agents used. The isolates showed a high rate of resistance against nitrofurantoin (76.4%) and cefotaxime (67.5%), while 95.8% and 91.7% of isolates were susceptible to meropenem and imipenem, respectively (Table 2). Fifty-seven (47.5%) isolates were characterized as MDREC, showing intermediate susceptibility or resistance to at least three different classes of antimicrobial agents tested. Forty-eight (84.2%) cases of MDR isolates were found to have the class 1 integron, whereas only 9 isolates (15.8%) with class 1 integron were non-MDR. Analysis of data revealed a significant association between the MDR pattern and the presence of class 1 integron (p<0.001). The results of this study also showed that the integron-positive isolates were statistically more frequently resistant to aminoglycosides and β-lactams, but not to quinolone compounds.

Out of 120 E. cloacae isolates, 76 (63.3%) isolates were resistant to at least one of the broad-spectrum cephalosporins used in the ESBL screening test. Fifty-three isolates (44.2%) were identified as potential ESBL producers using the combined disk method. ESBL-producing isolates were mostly recovered from urine (30.2%), followed by wound (28.3%) samples. The patients affected by these organisms were mostly admitted to ICU (54.7%) and the internal medicine (45.1%) wards, respectively (Table 3). All ESBL-producing isolates were found to be multidrug resistant, in which the highest susceptibility rate was observed against meropenem (98.1%), imipenem (90.6%) and gatifloxacin (79.2%), respectively.

Table 3.

Frequency of ESBL-Producing E. cloacae Isolates Based on Hospital Wards and Source of Clinical Specimens

ESBL-positive isolates (n=53)
Wards	Cases (n)	Percent	Specimens	Cases (n)	Percent
ICU	29	54.7	Urine	16	30.2
Internal medicine	11	20.8	Wound	15	28.3
Orthopedic	4	7.5	Trachea	9	17
Neurology	4	7.5	Blood	8	15.1
Infectious disease	3	5.7	Sputum	3	5.7
Surgery	2	3.8	CSF	1	1.9
			BAL	1	1.9

CSF, cerebrospinal fluid; BAL, bronchoalveolar lavage.

Of the 53 E. cloacae isolates with ESBL phenotype, 40 isolates (75.4%) were positive for ESBL genotypes. Overall, bla_CTX-M-15 was the most common (60.4%) gene followed by bla_TEM-1 (32.1%), bla_TEM-169 (13.2%), and bla_SHV-12 (7.5%) either alone or in combination. As shown in Table 4, bla_CTX-M-15 was found to coexist with bla_TEM-1 in 15 isolates (28.3%), 1 (1.9%) also carried only bla_SHV-12, and 2 (3.8%) carried bla_TEM-1, bla_CTX-M-15, and bla_SHV-12 genes. Isolates were negative for bla_VEB-1, bla_PER-1, bla_CTX-M-2, bla_CTX-M-8, bla_CTX-M-9 group genes, as well as the bla_CTX-M-25 gene.

Table 4.

Distribution of bla_SHV, bla_TEM, and bla_CTX-_M-ESBL Genes in E. cloacae Isolates

Resistance determinants	Cases (n)	Percent
bla _TEM-169	7	13.2
bla _SHV-12	1	1.9
bla _CTX-M-15	14	26.4
bla_TEM-1+bla_SHV-12	0	0
bla_CTX-M-15+bla_TEM-1	15	28.3
bla_CTX-M-15+bla_SHV-12	1	1.9
bla_TEM-1+bla_SHV-12+bla_CTX-M-15	2	3.8
No bla_TEM-1, bla_SHV-12, bla_CTX-M-15	13	24.5
Total	53	100

ERIC-PCR fingerprints showed that 44 of the 53 (83.01%) ESBL-producing isolates showed different DNA banding patterns, which means that most of them were clonally unrelated.

Discussion

In recent years, E. cloacae has been increasingly recognized as a cause of serious nosocomial infections. ESBL production among these isolates is a major concern because of its remarkable ability in developing resistance to several classes of antimicrobial agents and also high potential for transmission of resistance to other bacterial species.³⁶ ESBL detection is not routinely tested in laboratories in Iran. To our knowledge, this is the first report of ESBL genes and class 1 integron among E. cloacae isolates in Iran. Based on the antimicrobial susceptibility results of the present study, the MDR pattern was found in 57 isolates (47.5%) with a significant rate of resistance against common antibiotics. This may be mainly due to the selective pressure imposed by inappropriate and extensive use of broad-spectrum antibiotics in our hospital settings.

In the present study, the highest rates of resistance among the antimicrobials tested were associated with nitrofurantoin (76.4%) and cefotaxime (67.5%). These findings are in agreement with the results of other studies carried out in India,¹⁴ Greece,¹⁶ and Korea²⁵ with high resistance rates against the most common antibiotics used.

According to our data, 48 isolates (84.2%) were found to have class 1 integron among the MDREC isolates, showing that class 1 integron is widely distributed in our hospitals. This is considerably higher than that found in other geographical regions, including Poland (55%),²² Germany (21.9%)³⁷ and Malaysia (52.3%).¹³ We previously reported that class 1 integron (92.5%) is highly prevalent in MDR-Acinetobacter baumannii isolates.²⁸ This study also showed that the integron-positive isolates were statistically more resistant to aminoglycosides and β-lactams, but not for quinolone compounds. This is not surprising, since the class 1 integron carries many antibiotic resistance gene cassettes that encode resistance to a wide range of antibiotics in Enterobacteriaceae.^22,33 Moreover, resistance to quinolone compounds is reported to result mostly from chromosomal point mutations and plasmid-mediated qnr determinants.⁴⁵ In this study, 15.8% of integron-negative isolates were also resistant to these drugs indicating that this resistance could be acquired by chromosomal-encoded enzymes and other mobile genetic elements.

Based on our findings, 53 (44.2%) isolates of E. cloacae were ESBL producers. Similarly, Pai et al. from Korea reported that 43% of Enterobacter spp. were ESBL producers tested by the CD method.²⁵ The prevalence rate found in our study is higher than those reported by Garza-González et al. from Mexico (30%),⁹ Iabadene et al. from Algeria (17.7%),¹² and Park et al. from Korea (28.5%),²⁶ but lower than that found by Jain et al. from India, where 73.4% of Enterobacter spp. were positive for ESBL production.¹⁴

It should be noted that in the current study, 30.3% of broad-spectrum cephalosporin-resistant E. cloacae isolates were ESBL negative, suggesting that other mechanisms might alternatively be contributed in the resistance, most importantly, the production of AmpC, impermeability or reduced expression of outer membrane proteins, and efflux pump expression.⁵

In our study, all ESBL-producing isolates showed the MDR pattern and all were resistant to most β-lactams and non-β-lactams. These isolates showed a low level of susceptibility to most antimicrobial agents tested except for carbapenems. This could be explained by the fact that the ESBL producers usually carry a multiresistant plasmid, the genes conferring resistance to β-lactam and non-β-lactam antibiotics.⁸ Meropenem and imipenem were the most effective antibiotics against E. cloacae infections in this study. However, we encountered a low rate of carbapenem resistance among our E. cloacae isolates, which would have more clinical impact if these strains become more prevalent in the future.

Our study, like previous studies,^8,16 indicated that most ESBL-producing E. cloacae isolates were frequently collected from the patients admitted to intensive care units. The ICU stay, exposure to third-generation cephalosporins, and the use of invasive procedures such as urinary catheterization appear to predispose these patients to infections with these resistant organisms.

Overall, 60.4%, 32.1%, 13.2%, and 7.5% of E. cloacae isolates of the present study carried bla_CTX-M-15, bla_TEM-1, bla_TEM-169, and bla_SHV-12 ESBL genes either alone or in combination, respectively. We believe that this is the first report of TEM-169, SHV-12, and CTX-M-15 ESBL genotypes in E. cloacae isolates from Iran. CTX-M-15 and SHV-12 have already been identified in several countries, mostly in Asian ones. In Thailand, Tansawai et al. reported the coexistence of bla_SHV-12 and bla_TEM-1 in clinical isolates of E. cloacae.⁴¹ In a study from Korea, Kim and Lim reported that 38.8% and 11.1% of ESBL-producing E. cloacae isolates harbored bla_CTX-M-3 and bla_SHV-12, respectively.¹⁷ In the other report from Korea, Lee et al. showed the presence of bla_SHV-12 and bla_TEM-1 among clinical isolates of Enterobacter species.¹⁸ In Algeria, Iabadene et al. reported that 44%, 36%, 16%, and 4% of ESBL-positive E. cloacae isolates contained bla_CTX-M-15, bla_CTX-M-3, bla_SHV-12, and bla_VEB-1, respectively.¹² Garza-González et al. mentioned that CTX-M-15 (38.7%) was the most prevalent genotype among E. cloacae isolates in Mexico.⁹ The major ESBL gene in this study, the bla_CTX-M-15, was found to coexist with bla_TEM-1 in 15 isolates (28.3%). The coexistence of ESBL-encoding genes in clinical isolates of E. cloacae was also observed in Italy and in Philadelphia, USA.^19,40 There have been only rare reports of TEM-169-type ESBLs among Enterobacteriaceae worldwide. Ranjbar et al. reported the emergence of bla_TEM-169 and bla_CTX-M-88 in nontyphoidal Salmonella strains in Iran.³² In a study from India, Dhara et al. reported that 3.94% of ESBL-producing E. coli and K. pneumonia isolates carried the bla_TEM-169 gene.⁷

Genetic analysis using ERIC-PCR showed that more than 80% of ESBL-producing isolates were clonally unrelated, suggesting that dissemination of ESBL-producing isolates was not due to a clonal outbreak. Importantly, in the present study, the isolates were collected from seven hospitals in Tehran and five hospitals in Qazvin, two distant places in Iran.

In conclusion, we found that MDREC isolates and class 1 integron have a widespread distribution in Iran. The high prevalence of ESBL-producing isolates represents a serious therapeutic and epidemiological problem, which can be circumvented only through early detection and stringent control of such multidrug-resistant pathogens. In our study, for first time, the presence of bla_CTX-M-15, bla_TEM-169, and bla_SHV-12 ESBL genes in our E. cloacae isolates was reported from Iran. However, the initial identification of ESBL-producing isolates, careful and continuous monitoring of these organisms, and the use of an appropriate infection control policy are necessary in preventing further emergence and the spread of infection by these agents in our hospitals.

Footnotes

Acknowledgments

This study was financially supported by the Cellular and Molecular Research Center and Research Deputy of Qazvin University of Medical Sciences (grant number 90/437), Qazvin, Iran.

Disclosure Statement

No competing financial interests exist.

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