Spectrum of antibiotic resistance of the Spain14-5 Streptococcus pneumoniae clone over a 22 year period

Emilio Pérez-Trallero1,2,*, José María Marimón1, Alberto Gonzalez1, Diego Vicente1 and José María García-Arenzana on behalf of The Spanish Pneumococcal Infection Study Network§

1 Microbiology Service, Hospital Donostia, Paseo Dr. Beguiristain s/n, 20014 San Sebastián (Gipuzkoa); 2 Department of Preventive Medicine and Public Health, Faculty of Medicine, University of the Basque Country, San Sebastián, Spain

Received 4 November 2003; returned 23 November 2003; revised 10 December 2003; accepted 11 December 2003


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Objective: To study the characteristics and the evolution through time of a single Streptococcus pneumoniae multidrug-resistant international clone.

Methods: From 1981 to 2002, the presence of the multidrug-resistant Spain14-5 clone was studied among the 4201 S. pneumoniae isolated in Gipuzkoa (northern Spain).

Results: Overall, 93 isolates belonging to the Spain14-5 clone were identified. The first isolate of this clone was detected in 1981 and was already resistant to ß-lactams, erythromycin, clindamycin and chloramphenicol. The reference strain from the international collection for this clone was susceptible to macrolides and lincosamides whereas most of the isolates studied, including the first isolate detected in 1981, were resistant to macrolides and had the erm(B) gene encoding macrolide resistance.

Conclusions: The clone was genetically stable through time, was multiresistant since its inception and has recently become highly resistant to fluoroquinolones. The characteristic antibiotic resistance pattern of this clone should include erythromycin resistance.

Keywords: multiresistance, PFGE, MLST, fluoroquinolone resistance, macrolide resistance


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Since the emergence and spread of penicillin resistance among clinical isolates of Streptococcus pneumoniae, some serotypes have shown an association between penicillin resistance and resistance to other antibiotics.14 These isolates, resistant to ß-lactams and two or more other classes of antibiotics, are usually known as multidrug-resistant S. pneumoniae.5 In different parts of the world, multidrug-resistant isolates were observed to belong to a restricted number of serotypes, a finding that led to the description and characterization of the major multidrug-resistant international clones.6

Resistant pneumococci probably emerged in Spain earlier than in other parts of the world, with penicillin-resistant strains appearing in the late 1970s and early 1980s.7,8 Multidrug-resistant pneumococci appeared in Gipuzkoa at about the same time.9

Reports of multidrug-resistant pneumococci have increased in the last few years.1013 Although much is known about the prevalence of antibiotic resistance, the mechanisms and determinants of these resistances, and the international spread of the major antibiotic-resistant clones, few studies have analysed the evolution of these clones over long periods. In this study we describe the evolution of one of the major multidrug-resistant international clones—the Spain14-5 clone—over more than 20 years in a region of the north of Spain. At the same time, the phenotypic and genotypic characterization of this international clone initially described by Coffey et al.14 in 1996 is completed.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Bacterial isolates

From January 1981 to December 2002, all clinical isolates of S. pneumoniae in Gipuzkoa, Basque Country, northern Spain, were collected and stored at –80°C. All were identified to the species level by their colony morphology, optochin sensitivity and/or bile solubility tests. Isolates were also serogrouped or serotyped by the Quellung reaction (Quellung antisera, Staten Seruminstitut, Copenhagen, Denmark).

Susceptibility testing

Susceptibility to antibiotics was initially undertaken using the disc diffusion method on Mueller–Hinton agar or Iso-Sensitest agar supplemented with 5% horse or sheep blood following the recommendations of the NCCLS in force at the time.15 The antibiotic MICs for all invasive isolates (blood, CSF, pleural fluid, etc.) and all penicillin non-susceptible isolates (1 µg oxacillin disc inhibition zone < 20 mm) isolated between 1981 and 1997, as well as the antibiotic MICs for all S. pneumoniae isolated between January 1998 and December 2002, were determined by the broth microdilution method using microtitre trays (Sensititre; Trek Diagnostics Systems, West Sussex, UK) and Mueller–Hinton broth supplemented with 3% lysed horse blood and interpreted according to the NCCLS criteria.16 S. pneumoniae ATCC 49619 was used as control for the disc diffusion and broth microdilution methods.

Molecular typing methods

For the molecular typing methods, the previously characterized6 isolate of the Spain14-5 international multiresistant clone, strain MS22 ATCC 700902, was used as a control.

PFGE, multilocus sequence typing (MLST) and BOX-PCR were carried out as previously described.17 The PFGE patterns were analysed with the Diversity Database fingerprinting software version 2 (Bio-Rad, Hercules, CA, USA) and a dendrogram was constructed by the unweighted pair group method with arithmetic averages, the Dice coefficient and a position tolerance of 1%. Isolates with a genetic relatedness of >85% were considered to represent the same PFGE type.

Materials and methods common for all protocols using PCR

Bacterial DNA was extracted after lysozyme and proteinase K incubations using Qiagen columns (Valencia, CA, USA), according to the manufacturer’s instructions. All the primers were synthesized by Amersham Pharmacia (Buckinghamshire, UK). PCR products were automatically sequenced with the ABI 300 Genetic Analyzer (Perkin-Elmer Applied Biosystems, Foster City, CA, USA).

Mechanisms of antibiotic resistance

The mechanisms of macrolide, rifampicin and fluoroquinolone resistance were investigated as previously described.18 The cat (chloramphenicol acetyltransferase) gene and the tet(M) and tet(O) genes were detected as previously described.1921 Resistance to trimethoprim was investigated in all susceptible isolates and in a sample of resistant isolates after sequencing of the dihydrofolate reductase (dhfr) gene.22

PCR-restriction fragment length polymorphism (RFLP) assay for differentiation of alleles 16 and 47 of the ddl gene

To differentiate between alleles 16 and 47 of the ddl (D-Ala-D-Ala ligase) gene, 10 µL of the unpurified amplicon of 513 bp obtained after PCR using the primers and conditions described by Enright & Spratt23 was digested for 1 h at 37°C with 10 U each of AccI and AluI. As expected, after analysing the sequences described in the GenBank, isolates displaying allele 16 (GenBank accession no. AJ232256) were not digested with AccI, and gave four fragments of 216, 161, 107 and 29 bp after digestion with AluI. In contrast, isolates displaying allele 47 (GenBank accession no. AJ387980) gave two fragments of 275 and 238 bp after digestion with AccI, and gave two fragments of 484 and 29 bp after digestion with AluI.

Analysis of the pbp2b gene

Genetic polymorphism of the penicillin-binding protein 2b (pbp2b) gene was studied by a PCR-RFLP analysis using the primers and PCR conditions previously described.24 Amplification products were digested with the restriction endonuclease HinfI and separated by electrophoresis in 2.5% agarose gels stained with ethidium bromide.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
From 1981 to 2002, 1327 of 4201 isolates of S. pneumoniae were penicillin non-susceptible (penicillin MIC >= 0.12 mg/L) and 1091 isolates had a penicillin MIC >= 0.5 mg/L, an MIC two-fold lower than the penicillin MIC (2 mg/L) for the reference isolate of the Spain14-5 clone.6 Among them, 294 isolates were serotype 14, the most frequent serotype among S. pneumoniae clinical isolates with a penicillin MIC >= 0.5 mg/L in the 22 year period of the study. Other serogroups frequently found were serogroup 23 (210 isolates), serogroup 6 (208 isolates), serogroup 19 (172 isolates) and serogroup 9 (113 isolates). The remaining 94 isolates with a penicillin MIC >= 0.5 mg/L belonged to other serogroups or were non-typeable.

All serotype 14 isolates were studied using PFGE and 93/294 (31.6%) showed a pattern with a similarity >85% with the PFGE pattern of the S. pneumoniae reference strain of the international multiresistant Spain14-5 clone (strain MS22, ATCC 700902). Eighty-nine of these 93 isolates showed exactly the same PFGE pattern (100% similarity) and only four strains showed a pattern with less than three bands difference with the reference strain (similarity >85%). These four isolates were further characterized by MLST and gave the same ST17 as most of the isolates of this clone (see below).

These 93 isolates were from 72 patients (11 patients had multiple isolates within an interval that ranged from 8 days to more than 1.5 years). They were isolated mainly from specimens of the respiratory tract (63 isolates) and from blood (17 isolates). The remainder were isolated from ear secretions (10 isolates), eye secretions (two isolates) and CSF (one isolate).

The first multidrug-resistant S. pneumoniae belonging to the Spain14-5 clone was isolated in Gipuzkoa in April 1981 from the throat of a 1-year-old child with meningococcal meningitis. This first isolate had 100% homology with the PFGE and BOX-PCR patterns of the reference strain of this clone and a unique PCR-RFLP profile of the pbp2b gene (Figure 1, lane 1) while MLST revealed it to be ST17, the same type as other isolates of this clone. The isolate was resistant to penicillin, chloramphenicol and erythromycin but was susceptible to trimethoprim–sulfamethoxazole. Until 1987, no other isolate of the Spain14-5 clone was detected. From 1989 to 1998, the number of isolates of this clone ranged from 1 to 6 per year, but in 1999, 34 isolates of this clone were obtained from 23 patients (all except two patients were aged more than 65 years old). Only one of the isolates was from blood culture, the remaining 33 being from sputum. In the last years of the study, the number of isolates of this clone decreased to 12 in 2000, four in 2001 and eight in 2002.



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Figure 1. PCR-RFLP patterns of the pbp2b gene of S. pneumoniae. Lanes 1–4: patterns ‘a’, ‘b’, ‘c’ and ‘d’, respectively. Lane 5: pattern ‘b’ of the reference strain (ATCC 700902). Lane 6: molecular weight control (100 bp ladder).

 
Typing techniques

With BOX-PCR, all the 93 isolates with PFGE profiles similar to the Spain14-5 clone showed two patterns, arbitrarily named pattern I and pattern II (Table 1) that differed in only two bands. Pattern I, the same as that of the reference strain of the Spain14-5 clone was observed in 42 isolates whereas pattern II was found in 51 isolates. Of 34 isolates examined by MLST, eight belonged to ST18 (allelic profile: 1-5-4-11-9-3-16), the ST of the reference strain of the Spain14-5 clone, whereas 26 belonged to ST-17 (allelic profile: 1-5-4-11-9-3-47), which is identical except for a change in the allele number of ddl from 16 to 47.


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Table 1. Distribution of the 93 S. pneumoniae Spain14-5 clone isolates according to their pbp2b RFLP profile, BOX-PCR, multilocus typing and antibiotic-resistance patterns
 
All 93 isolates and the reference strain were then studied by PCR-RFLP assay to differentiate between the two alleles (Figure 2). Using the PCR-RFLP assay, eight isolates and the reference strain had allele 16 at the ddl locus (ddl-16) and the remaining 85 isolates had allele 47 (ddl-47). As there are currently three different STs documented at www.MLST.net (ST12, ST18 and ST19) that have ddl-16, the eight isolates with ddl-16 revealed by PCR-RFLP and the reference strain were fully characterized by MLST and all corresponded to ST18. On the other hand, of the S. pneumoniae serotype 14 isolates described at www.MLST.net with ddl-47, only one belonged to ST17. In contrast, in this study, each of the 26 isolates with ddl-47 revealed by PCR-RFLP and studied by MLST corresponded to ST17. No discrepancies between the PCR-RFLP assay and sequencing were observed in the 32 isolates with ddl-47 revealed by PCR-RFLP in which sequencing of the ddl gene was carried out. Therefore, the remaining isolates with ddl-47 in which only the ddl allele was studied by PCR-RFLP were assigned to ST17.



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Figure 2. PCR-RFLP profiles of the ddl allele 47 (lanes 1 and 5) and allele 16 (lanes 2 and 6) after digestion with the restriction enzymes AccI (lanes 1–3) and AluI (lanes 5–7). Lanes 3 and 7: PCR-RFLP profile of the ddl gene of the reference strain ATCC 700902. Lanes 4 and 8: molecular weight control (100 bp ladder).

 
RFLP analysis of the pbp2b gene of the 93 isolates of the Spain14-5 clone and the reference strain revealed four different pbp2b-RFLP types (Figure 1). The first isolate of this clone, isolated in Gipuzkoa in 1981, showed a pbp2b-RFLP type arbitrarily named ‘type a’ that differed from the RFLP types of the other isolates of this clone. Eight isolates as well as the reference strain (all of which had allele 16 at the ddl locus) showed the same pbp2b-RFLP type arbitrarily named ‘type b’. Of the remaining 84 isolates that had allele 47 at the ddl locus, two different pbp2b-RFLP types were found: 79 isolates had pbp2b-RFLP ‘type c’ and five had type ‘d’.

Antibiotic resistance

The penicillin MIC for all isolates of the Spain14-5 clone ranged from 1 to 4 mg/L. The amoxicillin MIC for all isolates was always equal to or greater than the penicillin MIC (Table 2). Thirty-seven isolates showed a high level of resistance to amoxicillin (8–16 mg/L). The MICs (mg/L) for the first isolate of the Spain14-5 clone from Gipuzkoa (April 1981) were: penicillin 2, amoxicillin 2, cefotaxime 1, erythromycin >128, chloramphenicol 16, tetracycline 32, and trimethoprim–sulfamethoxazole 0.5/9.5.


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Table 2. MICs (mg/L) for 93 S. pneumoniae Spain14-5 isolates from Gipuzkoa, Spain (1981–2002)
 
In addition to showing resistance to penicillin, chloramphenicol and trimethoprim–sulfamethoxazole (Table 2), 86 of the 93 isolates of the Spain14-5 clone were also resistant to erythromycin. Before 1992, only five of the 10 (50%) S. pneumoniae isolates of this clone were erythromycin-resistant whereas from 1992 to 2002, all but two of the 83 (97.6%) isolates were resistant to this antibiotic. All erythromycin-resistant isolates of the Spain14-5 clone showed the MLSB phenotype of resistance (resistance to macrolides, lincosamides and streptogramin B antibiotics) and all had the erm(B) macrolide resistance gene.

All isolates of this clone were chloramphenicol-resistant and 91 of 93 were tetracycline non-susceptible (MIC >= 4 mg/L); the presence of the cat and the tet(M) genes was detected in all 93 isolates. The two tetracycline-susceptible isolates had a tetracycline MIC of 2 mg/L and both had the tet(M) gene. The tet(O) gene was not detected in any of the isolates.

Only the first isolate of this clone was trimethoprim–sulfamethoxazole-susceptible (MIC = 0.5/9.5 mg/L). The dihydrofolate reductase genes of the reference strain, of the trimethoprim–sulfamethoxazole-susceptible isolate and of five trimethoprim–sulfamethoxazole-resistant isolates were amplified, and sequenced and the amino acid sequences were deduced and compared with the sequence of S. pneumoniae ATCC 49619.22 The five resistant clinical isolates (trimethoprim–sulfamethoxazole MICs >=2/38 mg/L) and the ATCC strain 700902 (the reference strain of the clone, trimethoprim–sulfamethoxazole MIC 2/38 mg/L) shared five amino acid substitutions (Glu-20 -> Asp, Lys-60 -> Gln, Asp-92 -> Ala, Ile-100 -> Leu, and Leu-135 -> Phe). The only amino acid substitution found in the susceptible isolate was Asp-92 -> Ala.

Of the 93 isolates of the Spain14-5 clone, 61 (65.6%) were levofloxacin-susceptible (levofloxacin MIC <= 2 mg/L; ciprofloxacin MIC <= 2 mg/L), one was levofloxacin-intermediate (levofloxacin MIC = 4 mg/L, ciprofloxacin MIC = 4 mg/L) and 31 (33.3%) were levofloxacin-resistant (levofloxacin and ciprofloxacin MIC >= 8 mg/L). Until 1999, only two levofloxacin-resistant isolates of the Spain14-5 clone (2/35; 5.4%) were isolated, both in 1993. From 1999 to 2002, 29/58 (50%) isolates of this clone were resistant to levofloxacin. All levofloxacin- and ciprofloxacin-resistant isolates had a point mutation at Ser-79 in parC and at Ser-81 in the gyrA gene. The levofloxacin-intermediate isolate showed no point mutation in parC and one point mutation at Ser-81 in the gyrA gene.

The two levofloxacin-resistant isolates of the Spain14-5 clone isolated in 1993 were isolated from the sputum of two different HIV-infected patients. These two isolates that have previously been described,18 were also erythromycin- and clindamycin-resistant and were the only two rifampicin-resistant isolates of this clone (rifampicin MIC > 4 mg/L). Both pertained to ST17 and had the same mutation at the rpoB gene.


    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Antibiotic resistance in S. pneumoniae, the leading pathogen implicated in community-acquired pneumonia, is a health problem in most countries throughout the world. Although almost all serotypes can acquire resistance to penicillin,7 the association of penicillin and multidrug resistance with particular serotypes is a general finding25 and several multidrug-international clones have recently been described.6 Of the four Spanish multidrug-resistant international clones, the dissemination of the Spain14-5 clone to other countries seemed to be less successful than that of the other three clones.913,2628 In a recent survey in the USA, only three out of 672 penicillin-resistant S. pneumoniae isolates belonged to the Spain14-5 clone and none was found between 1997 and 2000.5 In other parts of the world, this clone is also rarely found among penicillin-resistant isolates or among serotype 14 invasive isolates. 2931 It now seems clear that antibiotic usage is not always necessary for rapid expansion of clones,31 as the successful international spread of some multiresistant clones may vary depending on the properties of the clone.

In our region, the incidence of this clone was negligible until 1989 when its detection was limited to one to six isolates yearly for 10 years. Most of the Spain14-5 isolates found were respiratory tract isolates, a source from which more resistant pneumococci are usually isolated.32 The low incidence of this clone confirmed its circulation among the population and, as occurred in 1999, it can cause outbreaks of reactivation of chronic obstructive pulmonary disease among the elderly. This finding revealed the ability of this clone to disseminate in a small area. Since that year, its annual incidence returned to previous levels.

A frequent event among pneumococci is that the expression of certain capsular types can be altered as a consequence of the horizontal transfer of capsular genes.33 For other purposes, we used PFGE to study more than 150 penicillin non-susceptible S. pneumoniae isolated in the same period that belonged to more than 10 different serotypes (data not shown) and found no relation with the Spain14-5 clone. Although genes specifying the capsular type can be replaced by genetic transformation,34 we did not find any isolates expressing other capsular types that showed a PFGE pattern similar to that of the Spain14-5 clone. Another genetic characteristic found in the isolates of this clone was their low degree of genetic diversity through time, as PFGE revealed that most of the isolates had a similarity of >85% and that most had exactly the same PFGE pattern. Although PFGE is a typing technique that reflects small variations in the overall bacterial genome,35 only slight differences were found in four of the 93 isolates of this clone.

The S. pneumoniae Spain14-5 clone was first detected in our area in 1981 and is, to the best of our knowledge, the oldest strain belonging to this clone described anywhere. It was an ST17 isolate, had a unique pbp2b-RFLP pattern and was already multiresistant. It was the only trimethoprim–sulfamethoxazole-susceptible isolate of this clone but was erythromycin-resistant. The reference strain of this clone is trimethoprim–sulfamethoxazole-resistant and erythromycin-susceptible. All these findings indicate that this first isolate of the Spain14-5 clone was an intermediate strain in the evolution of this particular clone.

An association between penicillin-resistance and particular ddl alleles has been described for S. pneumoniae isolates as the recombinational exchanges at the pbp2b gene have extended in many cases into or through the ddl gene.36 We also found a relationship between pbp2b profiles and alleles at the ddl locus in isolates of the Spain14-5 clone, as almost all the isolates with the ddl allele 47 had the same pbp2b profile. The finding of isolates with identical PFGE profiles but different pbp genes is relatively frequent in other penicillin non-susceptible S. pneumoniae isolates.37

Throughout the study period, the isolates of this clone have not only maintained their spectrum of multidrug resistance but have incorporated a high level of resistance to fluoroquinolones. Most of the Spain14-5 S. pneumoniae isolates in our area showed resistance to erythromycin owing to the presence of the erm(B) gene. All ST17 (ddl allele 47) variants of the Spain14-5 clone were resistant to erythromycin probably as a result of a more active spread of this variant of the Spain14-5 clone, which included strains with the MLSB phenotype of macrolide resistance. Possibly, isolates of this clone would be better represented if the reference strain were a ST17 erythromycin-resistant isolate. Among the S. pneumoniae isolated in hospitals located in different regions of Spain between 1990 and 1996, a parallel increase in both serotype 14 and erythromycin-resistant isolates was observed.38 Isolates belonging to the most common clones usually share identical susceptibility patterns, but regional selective pressure in different parts of the world can add resistance to erythromycin and extended-spectrum cephalosporins.26

The finding that the two tetracycline-susceptible isolates harboured the tet(M) gene encoding tetracycline resistance but did not express it was surprising. The tetracycline MICs for these two isolates were 2 mg/L, which is on the breakpoint recommended by the NCCLS.16 Only the first isolate of this clone was trimethoprim–sulfamethoxazole-susceptible and it had only a single amino acid substitution at dhfr, which was not associated with resistance to trimethoprim.21 The remaining five trimethoprim–sulfamethoxazole clinical isolates studied, as well as the reference strain, all of which were resistant to trimethoprim–sulfamethoxazole, had several mutations in the dhfr gene, known to confer resistance to this antibiotic.

From a molecular point of view, the main genetic characteristics of this clone over the 22 year period were the lack of horizontal transfer of capsular genes, the overall homogeneity found by the three genotyping techniques employed (PFGE, BOX-PCR and MLST) and the less successful intercontinental spread to date compared with the three other Spanish major clones, Spain23F-1, Spain6B-2 and Spain9V-3.2628 Study of antibiotic resistance revealed an increase in the spectrum of antibiotic resistance in isolates of this clone over time. Nearly all the isolates studied harboured the well known determinants of antibiotic resistance [cat, tet(M), erm(B) genes] or had the mutations known to confer antibiotic resistance in the genes encoding the corresponding antibiotic targets (point mutations in the dhfr, rpoB and parC and gyrA genes). Since 1999, nearly half of the isolates were fluoroquinolone-resistant and all but one isolate were ST17 erythromycin-resistant.


    Acknowledgements
 
We are grateful to the pneumococcal MLST database, held at Imperial College London, funded by the Wellcome Trust, for the free availability of the data included in the database.

The Spanish Pneumococcal Infection Study Network (G03/103). General Coordination: Roman Pallares (rpallares{at}bell.ub.es). Participants and Centres: Ernesto García (Centro de Investigaciones Biológicas, Madrid); Julio Casal, Asunción Fenoll, Adela G. de la Campa (Centro Nacional de Microbiología, Instituto de Salud Carlos III, Madrid); Emilio Bouza (Hospital Gregorio Marañón, Madrid); Fernando Baquero (Hospital Ramón y Cajal, Madrid); Francisco Soriano, José Prieto (Fundación Jiménez Díaz y Facultad de Medicina de la Universidad Complutense, Madrid); Roman Pallares, Josefina Liñares (Hospital Universitari de Bellvitge, Barcelona); Javier Garau, Javier Martínez Lacasa (Hospital Mutua de Terrassa, Barcelona); Cristina Latorre (Hospital Sant Joan de Deu, Barcelona); Emilio Pérez-Trallero, María Ercibengoa (Hospital Donostia, San Sebastián); Juan García de Lomas (Hospital Clínico, Valencia); Ana Fleites (Hospital Central de Asturias).


    Footnotes
 
* Corresponding author. Tel: +34-94-30-07046; Fax: +34-94-30-07063; E-mail: mikrobiol{at}terra.es Back

§ Participants in the Spanish Pneumococcal Infection Study Network are listed in the Acknowledgements. Back


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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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