Christine Bley

Mark Van Der Linden

Ralf René Reinert
email: reinert@rwth-aachen.de

mef(A) is the predominant macrolide resistance determinant in Streptococcus pneumoniae and Streptococcus pyogenes in Germany

Keywords:

In this study, macrolide-resistant Streptococcus pneumoniae and Streptococcus pyogenes isolates from Germany were carefully characterised by susceptibility testing, phenotyping, polymerase chain reaction (PCR) and sequencing of macrolides resistance genes, and multilocus sequence typing (MLST). Of 2045 S. pneumoniae and 352 S. pyogenes isolates, 437 (21.4%) and 29 (8.2%), respectively, were found to be macrolide-resistant. Among the S. pneumoniae isolates, the most prevalent resistance marker was mef(A) (57.7%) followed by erm(B) (27.0%) and mef(E) (11.2%). Of note, the dual resistance mechanism mef(E)+erm(B) was found in a relatively high proportion (4.1%) of pneumococcal isolates. Among the S. pyogenes isolates, 31.0% carried mef(A), 34.5% erm(B) and 13.8% erm(A).

Dissemination of a single clone [mef(A)-positive England 14 -9] has significantly contributed to the emergence of macrolide resistance among pneumococci in Germany.

A c c e p t e d M a n u s c r i p t

Introduction

Streptococcus pneumoniae is a major cause of many childhood diseases such as respiratory tract infections, acute otitis media, community-acquired pneumonia, meningitis and sepsis [START_REF] Musher | Infections caused by Streptococcus pneumoniae: clinical spectrum, pathogenesis, immunity, and treatment[END_REF]. Streptococcus pyogenes (Group A Streptococcus) is responsible for the majority of cases of pharyngitis but can also cause much more severe infections such as necrotizing fasciitis and toxic shock syndrome [START_REF] Reinert | Macrolide-resistant Streptococcus pneumoniae and Streptococcus pyogenes in the pediatric population in Germany during 2000-2001[END_REF].

Resistance of S. pneumoniae and S. pyogenes to macrolides has become an increasing problem worldwide owing to spread of the resistance genes mef(A), subclasses mef(A) and mef(E), as well as erm(A) and erm(B) [START_REF] Reinert | Clinical efficacy of ketolides in the treatment of respiratory tract infections[END_REF][START_REF] Van Bambeke | Multidrug-resistant Streptococcus pneumoniae infections: current and future therapeutic options[END_REF]. Streptococci can acquire macrolide resistance through various mechanisms, for example efflux and methylation. The efflux mechanism consists of a pump encoded by the mef genes mef(A) and mef(E), and an ATP-binding cassette protein encoded by the mel gene, which reduces the intracellular antibiotic concentration to subtoxic levels [START_REF] Montanari | Phenotypes and genotypes of erythromycin-resistant pneumococci in Italy[END_REF][START_REF] Zhong | The role of efflux in macrolide resistance[END_REF], and is associated with the M-phenotype [START_REF] Gay | Structure and dissemination of a chromosomal insertion element encoding macrolide efflux in Streptococcus pneumoniae[END_REF][START_REF] Santagati | Characterization of a genetic element carrying the macrolide efflux gene mef(A) in Streptococcus pneumoniae[END_REF][START_REF] Grosso | Macrolide efflux genes mef(A) and mef(E) are carried by different genetic elements in Streptococcus pneumoniae[END_REF][START_REF] Leclercq | Resistance to macrolides and related antibiotics in Streptococcus pneumoniae[END_REF][START_REF] Grosso | The mef(E)-carrying genetic element (MEGA) of Streptococcus pneumoniae: insertion sites and association with other genetic elements[END_REF]. The sequences of the mef(A) and mef (E) genes are very similar but they are located on different genetic elements. mef(A) is located on the transposon Tn1207.1, whereas mef(E) is located on the so-called MEGA (macrolide efflux gene assembly) element. The second macrolide resistance mechanism is a 23S rRNA methylase that mediates ribosomal modification of the macrolide-binding site and is encoded by the erm genes located on conjugative transposons such as Tn1545 and Tn2010. Is associated with the MLS B phenotype, which infers multidrug resistance to 14-, 15-and 16-membered ring macrolides, A c c e p t e d M a n u s c r i p t 4 lincosamides and streptogramin B [START_REF] Montanari | Phenotypes and genotypes of erythromycin-resistant pneumococci in Italy[END_REF][START_REF] Cochetti | New Tn916-related elements causing erm(B)-mediated erythromycin resistance in tetracycline-susceptible pneumococci[END_REF][START_REF] Seppala | A novel erythromycin resistance methylase gene (ermTR) in Streptococcus pyogenes[END_REF]. erm(B) has been described in S. pneumoniae, and erm(B) and erm(A) in S. pyogenes [START_REF] Roberts | Nomenclature for macrolide and macrolide-lincosamide-streptogramin B resistance determinants[END_REF].

Other mechanisms of macrolide resistance include mutations in domain V of 23S rRNA as well as modifications of ribosomal proteins L4 and L22, which are located in the direct vicinity of the macrolide-binding site [START_REF] Santagati | Characterization of a genetic element carrying the macrolide efflux gene mef(A) in Streptococcus pneumoniae[END_REF][START_REF] Grosso | Macrolide efflux genes mef(A) and mef(E) are carried by different genetic elements in Streptococcus pneumoniae[END_REF][START_REF] Leclercq | Resistance to macrolides and related antibiotics in Streptococcus pneumoniae[END_REF][START_REF] Canu | Diversity of ribosomal mutations conferring resistance to macrolides, clindamycin, streptogramin, and telithromycin in Streptococcus pneumoniae[END_REF].

The purpose of this study was (i) to analyse the macrolide resistance gene distribution in a large collection of clinical streptococcal isolates and (ii) to analyse the clonal distribution of these isolates and their contribution to the recent emergence of macrolide resistance in Germany.

Material and methods

Study design and bacterial isolates

Invasive streptococcal isolates are sent to the German National Reference Center for Streptococci (GNRCS) (Aachen, Germany) as part of active prospective surveillance by more than 300 microbiology laboratories throughout Germany [START_REF] Imohl | Association of serotypes of Streptococcus pneumoniae with age in invasive pneumococcal disease[END_REF]. pneumoniae by optochin susceptibility and bile solubility testing. Optochin susceptibility testing was carried out in a 5% CO 2 atmosphere on sheep blood agar.

Non

For bile solubility testing, a bacterial suspension in 1 mL of 0.85% NaCl (McFarland standard 1.0) with four drops of 10% sodium desoxycholate was prepared. Following incubation for 2 h at 35 C, complete lysis was taken as a positive result.

Streptococcus pyogenes isolates were identified by -haemolysis on sheep blood agar, Lancefield grouping using a commercially available agglutination technique (Slidex ® Strepto Kit; bioMérieux, Marcy-l'Étoile, France) and the pyrrolidonyl arylamidase test [START_REF] Reinert | Molecular characterisation of macrolide resistance mechanisms of Streptococcus pneumoniae and Streptococcus pyogenes isolated in Germany, 2002-2003[END_REF].

Susceptibility testing

All strains were tested for antibiotic MICs using the broth microdilution method as recommended by the Clinical and Laboratory Standards Institute (CLSI) [START_REF]Performance standards for antimicrobial susceptibility testing; eighteenth informational supplement[END_REF]. The following breakpoints (g/mL) used were for susceptible, intermediate and resistant: clarithromycin, ≤0.25, 0.5 and ≥1; penicillin (parenteral), ≤0.06,and ≥0.12 [meningitis, isolates from cerebrospinal fluid (CSF)]/≤2, 4 and ≥8 (non-meningitis, isolates from sources other than CSF); clindamycin, ≤0.25, 0.5 and ≥1; tetracycline, ≤2, 4 and ≥8; and levofloxacin ≤2, 4 and ≥8. Wesel, Germany) and 5% lysed horse blood. The final inoculum was 5 10 5 colonyforming units/mL. Incubation was at 37 C for 24 h in ambient air. Streptococcus pneumoniae ATCC 49619 was used as a quality control strain. For interpretation, CLSI criteria were applied [START_REF]Performance standards for antimicrobial susceptibility testing; eighteenth informational supplement[END_REF]. In this study, co-resistance is defined as a macrolideresistant strain that shows resistance to one of the other antibiotics also tested in this study. To define the macrolide resistance phenotype, disks of erythromycin (15 g), clindamycin (2 g) and rokitamycin (30 g) (Oxoid Ltd., Basingstoke, UK) were placed 10-15 mm apart on Mueller-Hinton agar with 5% sheep blood (Oxoid). For phenotyping of S. pyogenes isolates, the same method was applied with disks of 30 g erythromycin, 10 g clindamycin and 30 g rokitamycin. The agar had been inoculated with a swab containing 0.5 McFarland bacterial suspensions [START_REF] Giovanetti | Phenotypes and genotypes of erythromycin-resistant Streptococcus pyogenes strains in Italy and heterogeneity of inducibly resistant strains[END_REF].

DNA isolation and detection of macrolide resistance genes by polymerase chain reaction (PCR)

Strains were cultivated on blood agar (5% sheep blood). Chromosomal DNA was isolated using a standardised procedure (DNeasy Blood & Tissue Kit; QIAGEN, Hilden, Germany). The distinction between mef(A) and mef(E) was made using a multiplex PCR [START_REF] De La Pedrosa | Polyclonal population structure of Streptococcus pneumoniae isolates in Spain carrying mef and mef plus erm(B)[END_REF] with the following primers: mefAF, 5'-AATACAACAATTGGATT- Gold ® DNA Polymerase (Applied Biosystems, Darmstadt, Germany). Initial denaturation was at 94 C for 12 min, followed by 30 cycles at denaturation at 94 C for 1 min, annealing at 52 C for 1 min and elongation at 72 C for 1 min. Final elongation was performed at 72 C for 10 min. To detect erm(B), the primers ermB up (5'-GAAAGGTACTCAACCAAATA-3') and ermB down (5'-AGTAACGGTACTTAAATTGTTTAC-3') were used with PCR conditions as described by Sutcliffe et al. [START_REF] Sutcliffe | Detection of erythromycinresistant determinants by PCR[END_REF]. For detection of the erm(A) gene in S. pyogenes, PCR with the following primers was performed: ermA up, 5'-TCTCCTTGCCGGTTATAA-3'; and ermA down, 5'-ATCAATTAAGACAGGTGCTGAAGC-3' [START_REF] Seppala | A novel erythromycin resistance methylase gene (ermTR) in Streptococcus pyogenes[END_REF]. A positive reference control strain was included in all PCR experiments. Products were run on a 1% agarose gel and were visualised by ethidium bromide staining.

Sequencing of macrolides resistance genes

DNA sequencing of four S. pneumoniae isolates, randomly selected from each of the resistance genotypes/phenotypes [mef(A), mef(E), erm(B) and mef(E)+erm(B)] was performed on an ABI 710 capillary sequencer (Applied Biosystems) according to the manufacturer's instructions using the PCR products and the same primers as used for the PCRs in order to confirm the PCR results. Resulting sequences were compared with the GenBank database using the program BLAST (http://www.ncbi.nlm.nih.gov/BLAST).

A c c e p t e d

M a n u s c r i p t 8

Typing

Strains were serotyped using the Neufeld Quellung reaction with type and factor sera provided by the Statens Serum Institut (Copenhagen, Denmark) [START_REF] Neufeld | Über die Agglutination der Pneumokokken und über die Theorie der Agglutination[END_REF]. All 437 macrolide-resistant S. pneumoniae isolates were available for serotyping. Multilocus sequence typing (MLST) of a randomly selected subgroup (166/437 isolates) of all pneumococcal isolates was performed as previously described [START_REF] Enright | A multilocus sequence typing scheme for Streptococcus pneumoniae: identification of clones associated with serious invasive disease[END_REF]. Briefly, internal fragments of the aroE, gdh, gki, recP, spi, xpt and ddl genes were amplified by PCR from chromosomal DNA with the described primer pairs. A special allelic profile is provided by the alleles at each of the seven loci, and their sequence type (ST) is defined. Allelic profiles were compared with each other and with other isolates in the pneumococcal MLST database using software available at http://www.mlst.net.

Clusters of related STs were grouped into clonal complexes (CCs) using the program eBURST (http://www.mlst.net).

Results

Bacterial strains

Susceptibility testing

Among the 2045 pneumococcal isolates, the following rates of resistance were observed for all isolates/invasive isolates ( showed co-resistance (Table 1).

Genotypes, phenotypes, co-resistance and dual resistance mechanism

Of the 437 macrolide-resistant pneumococcal isolates, 252 (57.7%) carried the mef(A) gene, 49 (11.2%) carried the mef(E) gene, 118 (27%) carried the erm(B) gene and 18 (4.1%) carried mef(E)+erm(B) (see Table 2). All mef(A)-and mef(E)positive S. pneumoniae isolates showed the M-phenotype. All erm(B)-positive isolates exhibited the MLS B phenotype with clarithromycin MICs up to 32 g/mL.

Clarithromycin MICs for M-phenotype strains (2-16 g/mL) were slightly lower compared with MLS B phenotype strains. Serotypes for 430 macrolide-resistant isolates could be determined, whereas 7 isolates were non-typeable (NT). MLST was performed for a randomly selected subgroup (166/437 isolates) of isolates, comprising 31% (n = 79), 39% (n = 46), 55% (n = 27) and 78% (n = 14) of the isolates containing mef(A), erm(B), mef(E) and dual mef(E)+erm(B), respectively.

Of the 118 isolates harbouring erm(B), 87.2% and 94.1% were resistant to tetracycline and clindamycin, respectively; and the 18 isolates with mef(E)+erm(B) had co-resistance rates of 83.3% and 77.7% to tetracycline and clindamycin, respectively. Co-resistance rates to tetracycline and clindamycin were 24.5% and 2.0%, respectively, for the 49 mef(E)-positive isolates and 1.2% and 0.4% for the When applying the non-susceptibility breakpoint of ≥0.12 g/mL for all S. pneumoniae isolates, isolates carrying mef(E), erm(B) or mef(E)+erm(B) showed higher MICs for penicillin than mef(A)-carrying isolates. mef(A) isolates rarely have a penicillin MIC ≥ 0.12 g/mL (0.8% of cases), whereas 20-25% of mef(E)-positive isolates (both CSF and non-CSF), 40-50% of erm(B)-positive isolates, and 33% (CSF) and 73% (non-CSF) of mef(E)+erm(B)-positive isolates have a penicillin MIC ≥0.12 g/mL. Interestingly, 18 (4.1%) of the macrolide-resistant S. pneumoniae isolates showed the dual resistance mechanism mef(E)+erm(B), 9 of which were from non-invasive sites with an MLS B phenotype and MICs for clarithromycin ranging from 16 g/mL to 32 g/mL. Various combinations of STs and serotypes were found for the 14 mef(E)+erm(B) isolates (Table 3) where MLST (ST), with CC and serotype information, was available: ST15, CC9, serotype 14 (n = 1); ST135, CC135, serotype 6B (n = 1); ST143, CC156, serotype 6B (n = 1), serotype 14 (n = 1); ST156, CC156, serotype 14 (n = 1); ST162, CC156, serotype 9V (n = 1); ST199, CC199, serotype 15B (n = 1); ST271, CC271, serotype 7F (n = 1), serotype 19F (n = 1); ST306, (n = 1), 15B (n = 1), 15C (n = 1) and 17F (n = 1). MLST data were available for 46 of the erm(B)-positive isolates, among which the most prevalent combination was ST135/serotype 6B (n = 4); other combinations were ST315/serotype 6B (n = 3) and ST193/serotype 19A (n = 2) (Table 3).

The 29 macrolide-resistant S. pyogenes showed the following distribution of resistance determinants: mef(A) (n = 9); erm(A) (n = 4); and erm(B) (n = 10). No resistance determinant could be detected in six isolates. There were no isolates harbouring a dual resistance mechanisms. Among the S. pyogenes, all mef(A)positive isolates had an M-phenotype, whilst all erm(B) and erm(A)-positive isolates exhibited the MLS B phenotype (Table 2).

Sequencing of macrolide resistance determinants

For each of the four possible combinations of resistance determinants [mef(A), mef(E), erm(B), and mef(E)+erm(B)] in S. pneumoniae, sequencing of the macrolide resistance gene(s) of one randomly selected isolate was performed The sequences were obtained and compared with GenBank (http://www.ncbi.nlm.nih.gov/BLAST).

The sequences were identical to those available in the database, thus confirming that the isolates indeed carried the two different macrolide resistance markers

mef(A) and mef(E), as well as the combination of mef(E)+erm(B).

A c c e p t e d M a n u s c r i p t 14 Primers for the multiplex PCR were designed based on a multiple sequence alignment of sequences belonging to both genes, revealing areas with nucleotide differences between the two genes and thus allow differentiation between mef(A) and mef(E). The primers are located within the genes.

Discussion

Macrolide resistance among streptococci has become an increasing problem worldwide [START_REF] Reinert | Clinical efficacy of ketolides in the treatment of respiratory tract infections[END_REF][START_REF] Van Bambeke | Multidrug-resistant Streptococcus pneumoniae infections: current and future therapeutic options[END_REF]. In Germany, macrolide resistance rates in S. pneumoniae have further increased from 15.3% in 2000 [START_REF] Reinert | Emergence of macrolide and penicillin resistance among invasive pneumococcal isolates in Germany[END_REF] to 21.4% in 2005/2006 as analysed in the present study, and now in 2010 reach a level comparable with those reported from Mediterranean countries such as Spain (34.3%) and Italy (24.3%) [START_REF] De La Pedrosa | Polyclonal population structure of Streptococcus pneumoniae isolates in Spain carrying mef and mef plus erm(B)[END_REF]. In contrast, S. pyogenes isolates included in the present study showed a macrolide resistance rate of 8.2%, which is significantly lower than the resistance rates reported from Spain (34%) and Italy (35%) [START_REF] Pérez-Trallero | Spectrum of antibiotic resistance of the Spain 14 -5 Streptococcus pneumoniae clone over a 22 year period[END_REF][START_REF] Cornaglia | Resistance of Streptococcus pyogenes to erythromycin and related antibiotics in Italy. The Italian Surveillance Group for Antimicrobial Resistance[END_REF] and even somewhat lower than in earlier reports from Germany, with a 14% resistance level reported among children up to 16 years of age with community-acquired infections in 2004 [START_REF] Reinert | Molecular characterisation of macrolide resistance mechanisms of Streptococcus pneumoniae and Streptococcus pyogenes isolated in Germany, 2002-2003[END_REF], or a rate of 19.1% (adults) and 11.8% (children) reported from Bavaria in 2003 [START_REF] Sauermann | Phenotypes of macrolide resistance of group A streptococci isolated from outpatients in Bavaria and susceptibility to 16 antibiotics[END_REF].

In Germany, macrolide resistance in S. pneumoniae is mainly due to mef genes. In all previous studies performed in Germany, no distinction between mef(A) and mef(E) determinants was made [START_REF] Reinert | Molecular epidemiology of macrolide-resistant Streptococcus pneumoniae isolates in Europe[END_REF]. In some Mediterranean countries and Canada, mef(E) appears to be the most common efflux gene variant [START_REF] De La Pedrosa | Polyclonal population structure of Streptococcus pneumoniae isolates in Spain carrying mef and mef plus erm(B)[END_REF][START_REF] Wierzbowski | Molecular epidemiology and prevalence of macrolide efflux genes mef(A) and mef(E) in Streptococcus pneumoniae obtained in Canada from 1997 to 2002[END_REF]. In Germany, mef(A) is the most common macrolide resistance gene.

A c c e p t e d M a n u s c r i p t

Macrolide-resistant S. pneumoniae strains were increasingly found to be co-resistant to other compounds, such as tetracycline (31%), clindamycin (29%) and penicillin (2% meningitis/2% non-meningitis). In Germany, multidrug-resistant (MDR) isolates predominantly exhibit the MLS B phenotype and carry erm(B). Multiple antibiotic resistance is widespread in Europe and an increase in MDR S. pneumoniae and S. pyogenes strains has also been observed in other European countries such as Spain [START_REF] Van Bambeke | Multidrug-resistant Streptococcus pneumoniae infections: current and future therapeutic options[END_REF][START_REF] De La Pedrosa | Polyclonal population structure of Streptococcus pneumoniae isolates in Spain carrying mef and mef plus erm(B)[END_REF][START_REF] Imohl | Epidemiology of invasive Streptococcus pyogenes disease in Germany during 2003-2007[END_REF].

Streptococcus pneumoniae isolates carrying mef(E), erm(B) or mef(E)+erm(B)

showed higher MICs for penicillin than mef(A)-carrying isolates. This probably reflects the greater clonal diversity among the mef(E)-and erm(B)-positive isolates.

Obviously, the chance of acquiring macrolide resistance elements in low-affinity penicillin-binding protein strains is then higher.

Of note, this is the first study to report a relatively high level of isolates with the dual resistance mechanism [both erm(B) and mef(A), subclass mef(E) gene] from Germany. Similar isolates were first described in South Africa in 2001 [START_REF] Mcgee | Serotype 19F multiresistant pneumococcal clone harboring two erythromycin resistance determinants (erm(B) and mef(A)) in South Africa[END_REF] and in the last decade have been increasingly reported from the USA, Canada, South Korea, China, South Africa, Japan, Mexico and Hungary [START_REF] Farrell | Emergence and spread of Streptococcus pneumoniae with erm(B) and mef(A) resistance[END_REF]. In the present study, which differentiated between mef(A) subclasses mef(A) and mef(E), only isolates carrying the mef(E) gene, but not the mef(A) gene, with erm(B) were observed, underscoring the different genetic background of mef(E) and mef(A). Moreover, two isolates belonged to CC271, which is the most predominant clone in the USA, where >90% A c c e p t e d M a n u s c r i p t 16 of isolates with the dual resistance mechanism were clonally related to the MDR international clone Taiwan 19F -14 (CC271) [START_REF] Farrell | Emergence and spread of Streptococcus pneumoniae with erm(B) and mef(A) resistance[END_REF]. This cross-resistance could be related to the presence of conjugative transposons such as Tn2010 described for these isolates [START_REF] Grosso | The macrolide resistance genes erm(B) and mef(E) are carried by Tn2010 in dual-gene Streptococcus pneumoniae isolates belonging to clonal complex CC271[END_REF]. Apart from the erm(B) gene, Tn2010 also harbours a tetracycline resistance gene. The MEGA element, which encodes the mef(E) gene, can be inserted in Tn2010 but also in various other insertion sites on the chromosome [START_REF] Gay | Structure and dissemination of a chromosomal insertion element encoding macrolide efflux in Streptococcus pneumoniae[END_REF][START_REF] Grosso | The mef(E)-carrying genetic element (MEGA) of Streptococcus pneumoniae: insertion sites and association with other genetic elements[END_REF][START_REF] De La Pedrosa | Polyclonal population structure of Streptococcus pneumoniae isolates in Spain carrying mef and mef plus erm(B)[END_REF]. Of note, four of the isolates with the dual resistance mechanism exhibited the M-phenotype and remained sensitive to the lincosamide clindamycin in repeated experiments, indicating that the erm(B) gene in these isolates has no functional activity.

In the past there has been discussion regarding the nomenclature of the mef genes.

The first mef gene described in S. pyogenes was mef(A). Later, a homologous mef gene was found in S. pneumoniae and was named mef(E) [START_REF] Klaassen | Molecular detection of the macrolide efflux gene: to discriminate or not to discriminate between mef(A) and mef(E)[END_REF]. After both genes were found in both species and their sequences were found to be 90% identical, Roberts et al. [START_REF] Roberts | Nomenclature for macrolide and macrolide-lincosamide-streptogramin B resistance determinants[END_REF] suggested to rename all the genes mef(A) and to refer to the two variants as subclasses mef(A) and mef(E). However, since the mef(A) subclasses mef(A) and mef(E) are carried by different genetic elements, which are inserted at different sites into the chromosome, they showed different clonal dispersion. Owing to these differences, it was suggested to maintain the distinction between the two genes [START_REF] Klaassen | Molecular detection of the macrolide efflux gene: to discriminate or not to discriminate between mef(A) and mef(E)[END_REF]. The findings of the present paper clearly support the view that both genes have a different genetic background as documented by their association with different CCs and further support the view of Klaassen et al. [START_REF] Klaassen | Molecular detection of the macrolide efflux gene: to discriminate or not to discriminate between mef(A) and mef(E)[END_REF].

A c c e p t e d M a n u s c r i p t

In conclusion, macrolide resistance in S. pneumoniae, but not S. pyogenes, is further increasing in Germany. In pneumococci, macrolide resistance is mainly caused by the spread of a single clone (England 14

  -invasive isolates are sent to GNRCS as part of several local surveillance studies. Between January 2005 and December 2006, GNRCS received a total of 2045 (1887 invasive and 158 non-invasive) S. pneumoniae and 352 (227 invasive and 125 non-invasive) S. pyogenes isolates. The present study focuses on characterisation of 437 macrolide-resistant S. pneumoniae [minimal inhibitory A c c e p t e d M a n u s c r i p t 5 concentration (MIC) for clarithromycin >1 g/mL] and 29 macrolide-resistant S. pyogenes isolates. Strain identification by local laboratories was confirmed at GNRCS for S.

A c c e p t e d M a n u s c r i p t 6

 6 Microtitre plates (Sensititre NLMMCS10; TREK Diagnostic Systems Ltd., East Grinstead, UK) contained penicillin G, cefotaxime, clarithromycin, clindamycin, tetracycline and levofloxacin with cation-adjusted Mueller-Hinton broth (Oxoid,

3 '

 3 ; mefEF, 5'-AAGGAGTTGTGGTTCTGA-3'; and mefR, 5'-CCAATGATTTACACCGATT-3'. The PCR mixture for 25 L contained 15 mM Tris-A c c e p t e d M a n u s c r i p t 7 HCl, 50 mM KCl (pH 8.0), 25 mM MgCl 2 , 100 M of each nucleotide, 0.15 mM of mefAF and mefEF primer and 0.25 mM of the mefR primer plus 1.5 U of AmpliTaq

  In total, 437 (21.4%) of 2045 S. pneumoniae and 29 (8.2%) of 352 S. pyogenes were found to be macrolide-resistant. Of the 437 pneumococci, 210 and 227 were isolated from individuals in the age groups 0 to <16 years and ≥16 years, respectively. Streptococcus pneumoniae isolates were obtained from blood (n = 304), CSF (n = 71), nasopharyngeal swabs (n = 31), pleural fluid (n = 6), tracheal secretion or A c c e p t e d M a n u s c r i p t 9 sputum (n = 12), other normally sterile body sides (n = 7) and other sources such as urine, superficial wound swabs or bronchial secretion (n = 6). Streptococcus pyogenes isolates were from nasopharyngeal swabs (n = 19), blood (n = 7), punctures (n = 2) and bronchial secretion (n = 1).

  A c c e p t e d M a n u s c r i p t 10 study. erm(B)-positive isolates exhibiting the MLS B phenotype had the highest rates of co-resistance to -lactams (S. pneumoniae), clindamycin and tetracycline. In contrast, mef(A)-and mef(E)-positive isolates exhibiting the M-phenotype only rarely

A c c e p t e d M a n u s c r i p t 11 252 9 ( 9 .

 1199 mef(A)-positive isolates. In addition, 8 (42.1%) of 19 erm(B)-positive isolates from CSF, 1 (20.0%) of 5 mef(E)-positive isolates from CSF and 1 (33.3%) of 3 mef(E)+erm(B)-positive isolates from CSF were resistant to penicillin. Lastly, 1%) of the 99 erm(B)-positive isolates from sources other than CSF (nonmeningitis patients) and 1 (6.7%) of 15 mef(E)+erm(B)-positive isolates from sources other than CSF were intermediate resistant to penicillin, with an MIC of 4 g/mL.

  1 (n = 1); SLV 306, CC306, serotype 1 (n = 1); ST310, CC393, serotype 19F (n = 1); ST2432, CC271, serotype 14 (n = 1); and ST3563, CC242, serotype 23F (n = 1). Two of the 18 mef(E)+erm(B)-positive isolates (MLS B phenotype) were co-resistant to penicillin G (one meningitis and one non-meningitis), 15 to clindamycin and 15 to tetracycline. Four of the isolates with a dual resistance mechanism exhibited the M-phenotype, eight isolates had the constitutive MLS B (cMLS B ) phenotype and six had the inducible MLS B (iMLS B ) phenotype, as shown in repeated experiments (Table 2). Of note, the majority of mef(A)-positive S. pneumoniae isolates (237/252; 94.0%) belonged to serotype 14. Other serotypes included 1 (n = 1), 6A (n = 1), 6B (n = 1), 7F (n = 2), 9N (n = 1), 9V (n= 4), 19F (n = 2) and 23F (n = 3). Of the 79 mef(A)positive S. pneumoniae isolates with MLST data available, 69 were ST9 in combination with serotype 14, indicating clonal relatedness [mef(A)-positive England 14 -9]. Other combinations were ST15/serotype 14, ST138/serotype 14, ST191/serotype 14, ST350/serotype 14, DLV 9/serotype 14, SLV 1775/serotype 14, ST162/serotype 9V, ST176/serotype 6B, ST306/serotype 1 and ST2031/serotype 23F. Among the erm(B)-positive isolates, serotypes 6B (n = 32), 19F (n = 20), 19A (n = 19) and 14 (n = 16) were the most predominant, in addition to 23F (n = 7), NT (n = A c c e p t e d M a n u s c r i p t 13 5), 9V (n = 4), 24F (n = 4), 6A (n = 2), 15A (n = 2), 1 (n = 1), 3 (n = 1), 4 (n = 1), 10A

  -9) carrying the mef(A) gene. For the first time, isolates with a dual macrolide resistance mechanism were observed in Germany. Moreover, this study underscores that a differentiation between mef(A) and mef(E) gene variants provides a better insight into the spread of macrolide resistance among S. pneumoniae and S. pyogenes in different geographical locations. A c c e p t e d M a n u s c r i p t A c c e p t e d M a n u s c r i p t A c c e p t e d M a n u s c r i p t A c c e p t e d M a n u s c r i p t A c c e p t e d M a n u s c r i p t

  constitutive macrolide-lincosamide-streptogramin B (MLS B ) phenotype; iMLS B , inducible MLS B phenotype. a No resistance gene could be detected in four S. pyogenes isolates from adults and two isolates from children.

A c c e p t e d M a n u s c r i p t 1 3

 13 Table Clonal distribution and macrolide resistance markers among 166 randomly selected Streptococcus pneumoniae isolates in Germany, E) (n = 6) 9V (n = 5), nt (n = 1) mef(E)+erm(B)

  from a normally sterile body side)/non-

	invasive isolates: clarithromycin, 21.4/20.6/30.6% (intermediate resistance to
	clarithromycin 2.6/7.7/20.4%); penicillin G, 3.6/0.0/0.0% (non-meningitis strains)
	(intermediate resistance to penicillin G for non-meningitis strains 4.8/1.6/10.2%), and
	0.8/14.1% (meningitis strains from CSF) (no isolates with intermediate resistance);
	cefotaxime, 0.9/0.0/0.0% (non-meningitis) (intermediate resistance to cefotaxime
	1.0/1.3/12.3%), and 0.3/0.6% (meningitis) (intermediate resistance to cefotaxime
	0.0/1.3%); clindamycin, 7.5/6.4/20.1%; tetracycline, 10.2/8.8/27.2%; and
	levofloxacin, 0.6/0.0/0.0%. There were no isolates with intermediate resistance to
	clindamycin, tetracycline or levofloxacin.
	Among the 352 S. pyogenes isolates, all isolates were susceptible to penicillin G,
	cefotaxime or levofloxacin. Moreover, 8.2% of the isolates were resistant to
	clarithromycin, 2.5% to clindamycin and 12.7% to tetracycline. Of the macrolide
	(clarithromycin)-resistant isolates of S. pneumoniae and S. pyogenes isolates, 36%

and 21%, respectively, were found to be co-resistant to other antibiotics tested in this

1 Table 1

 11 Minimal inhibitory concentrations (MICs) and co-resistance among macrolide-resistant isolates of Streptococcus pneumoniae (S.p.) MICs for 50% and 90% of the isolates, respectively; NS, non-susceptibility; CLA R , clarithromycin-resistant; intermediate;

	Edited Table 1
	in Germany, 2005--2006 Resistance genotype (no. of isolates) Clarithromycin MIC 50/90 (g/mL) MIC range (g/mL) mef(A) (252) 8/16 2-16 mef(E) (49) 8/16 2-16 mef(E)+erm(B) (18) 32/32 2-32 erm(B) (118) 32/32 2-32 A c c e p t e d % NS of all 2045 S.p. 12.3 57.7 0.015/0.015 0.015-Penicillin % of all CLA R S.p. (n = 437) MIC 50/90 MIC range 0.06 2.4 11.2 0.015/0.25 0.015-2 0.9 4.1 0.25/2 0.015-4 5.8 27.0 0.06/2 0.015-4 2 A c c e p t e d M MIC 50/90 , R, resistant. a The following breakpoints (g/mL) were used (susceptible/intermediate/resistant): penicillin (parenteral), ≤0.06, -and ≥0.12 Clindamycin Tetracycline % NS (I+R) of 437 CLA R S.p. (meningitis/non-meningitis) a MIC 50/90 MIC range % NS of 437 CLA R S.p. a MIC 50/90 MIC range 0/0 0.12/0.12 0.12-0.12 0.4 0.25/0.5 0.06-32 20.0/0 0.12/0.12 0.12-32 2.0 0.5/16 0.25-16 33.3/6.7 16/32 0.12-32 77.7 16/32 0.12--64 42.1/9.1 32/32 0.5-32 94.1 32/32 0.25-32 (meningitis)/≤2, 4 and ≥8 (non-meningitis); clindamycin, ≤0.25, 0.5 and ≥1; tetracycline, ≤2, 4 and ≥8; and levofloxacin, ≤2, 4 and ≥8 a n u s c r i p t (all macrolide-resistant isolates were susceptible to levofloxacin). M a n u s c r i p t	% NS of 437 CLA R S.p. a 1.2 24.5 83.3 87.2
	1

Table 2

 2 Phenotypes and genotypes of 437 macrolide-resistant Streptococcus pneumoniae

	and 29 macrolide-resistant Streptococcus pyogenes in Germany, 2005-2006
	S. pneumoniae (No. of	Phenotype No. of isolates with genotype:
	isolates)		mef(A) mef(E) mef(E)+erm(B) erm(B)
	Adults, invasive disease	M	125	21	0	0
	(207)	cMLS B	0	0	1	47
		iMLS B	0	0	1	12
		Total	125	21	2	59
	Children, invasive disease	M	122	19	2	0
	(181)	cMLS B	0	0	4	28
		iMLS B	0	0	1	5
		Total	122	19	7	33
	Adults, non-invasive	M	2	5	1	0
	disease (20)	cMLS B	0	0	1	5
		iMLS B	0	0	2	4
		Total	2	5	4	9
	Children, non-invasive	M	3	4	1	0
	disease (29)	cMLS B	0	0	2	11
		iMLS B	0	0	2	6
		Total	3	4	5	17
	S. pyogenes (No. of		mef(A) mef(E) erm(B)	erm(A)
	isolates)
	Adults (19) a

Acknowledgments

The authors thank all participating laboratories for providing the streptococcal isolates as well as the clinicians for providing clinical data.

Funding

This work was supported in part by the Robert Koch Institute and the German Ministry of Health (grant no. 1369-235). This study was supported by research grants from the National Reference Center for Streptococci (Aachen, Germany).

Competing interests

MvdL has received research grants from Wyeth/Pfizer and GSK and acts as a consultant for Wyeth/Pfizer, Sanofi-Aventis and GSK. RRR has been a Novartis


F Scarpa
email: fscarpa@ditec.unige.it

L A Tagliafico

G Tagliafico

Keywords: ATE Solar assisted, Renewable energy, Heat pump, Direct expansion, Water heater

A direct expansion integrated solar assisted heat pump (ISAHP) is compared to a traditional flat plate solar panel for low temperature (45 °C) water heating applications. The (simulated) comparison is accomplished assuming both the devices are energy supplemented with an auxiliary standard gas burner, to provide the typical heat duty of a four-member family. Literature dynamical models of the systems involved have been used to calculate the main performance figures in a context of actual climatic conditions and typical stochastic user demand.

The paper highlights new heat pump control concepts, needed when maximum energy savings are the main goal of the apparatus for given user demand. Simulations confirm the high collector efficiency of the ISAHP when its panel/evaporator works at temperature close to the ambient one. The device, with respect to a flat plate solar water heater, shows a doubled performance, so that it can do the same task just using an unglazed panel with roughly half of the surface.

Introduction

A strong innovation is needed in energy production technologies in order to make renewable energy resources affordable and competitive with traditional ones and to adhere to the lines settled by the Kyoto protocol [START_REF]Kyoto Protocol to the United Nations Framework Convention on Climatic Change[END_REF] and subsequent international agreements. In the last years, many improvements have been carried out in the field of air conditioning, heating and refrigeration, thanks to the spreading of heat pump systems, generally reversible, that allow water heating and air heating or cooling to be performed with a significant energy saving with respect to traditional electrical and gas systems [START_REF] Swardt | A performance comparison between an air-source and a ground-source reversible heat pump[END_REF][START_REF] Sarkar | Transcritical CO 2 heat pump systems: exergy analysis including heat transfer and fluid flow effects[END_REF]. Scientific research is being concentrated in the development and improvement of these technologies, integrating them with other ones (sea water heat exchangers, geothermal pipes, absorption systems).

On the side of solar heaters, several applications are available in the field of low-temperature (around 45°C) water heaters, while some developments are envisaged in the field of solar absorption cooling plants [START_REF] Garcia-Casals | Solar Absorption cooling in Spain: Perspectives and outcomes from the simulation of recent installations[END_REF] and solar assisted heat pumps (SAHP). These last can use traditional solar panels to pre-heat a water tank to be coupled to electrical heat pumps [START_REF] Yumrutas | Experimental investigation of thermal performance of a solar assisted heat pump system with an energy storage[END_REF], also with intermediate energy storage [START_REF] Çomakly | A Thermodynamic model of a solar assisted heat pump system with energy storage[END_REF][START_REF] Zalba | Review on thermal energy storage with phase change: materials, heat transfer analysis and applications[END_REF], or gas burners [START_REF] Fasulo | Comparison between a simple collector accumulator and conventional accumulator[END_REF] or absorption systems [START_REF] Sozen | Development and testing of a prototype of absorption heat pump system operated by solar energy[END_REF][START_REF] Ozgener | Experimental performance analysis of a solar assisted ground-source heat pump greenhouse heating system[END_REF]. Some industrial attempts are now available on the market [START_REF] Huang | Performance evaluation method of solar-assisted heat pump water heater[END_REF].

A possible alternative to exploit at best the available solar radiation is to directly use solar panels as evaporators in a vapor compression heat pump cycle. Recently, the use of fine-tuned control techniques in order to obtain "optimal performance" from a system is quickly diffusing also in the field of vapor compression refrigeration technology [START_REF] Dong | Multivariable Control of Vapor Compression Systems[END_REF] ,thus matching the need for continuous regulation asked by these modern devices [START_REF] Chaturvedi | Thermal performance of a variable capacity direct expansion solar-assisted heat pump[END_REF].

Also for this reason there is a growing interest around ISAHP (Integrated Solar Assisted Heat Pump)

technology. We use the term ISAHP to specify that the traditional vapor compression heat pump is integrated into a solar panel, directly used as the evaporator of the system. These systems are also known as "direct expansion solar assisted heat pumps" or DX-SAHP.

Various studies on ISAHP models are available. Some of them, e.g. [START_REF] Chaturvedi | Thermal performance of a variable capacity direct expansion solar-assisted heat pump[END_REF], refers to variable speed compressors, others [START_REF] Huang | Long-term performance of solar-assisted heat pump water heater[END_REF][START_REF] Huang | Heat-pipe enhanced solar-assisted heat pump water heater[END_REF] propose the simpler household refrigeration technology to be used as the heat pump system. For the variable capacity compressors configuration different fluids are being tried [START_REF] Chata | Analysis of a direct expansion solar assisted heat pump using different refrigerants[END_REF].

The aim of this work, once a proper dynamic model for the ISAHP is developed and tuned, is to analyze its performance and compare it to a traditional thermal solar panel (TSP) under variable climatic conditions and typical stochastic user demand. The comparison is made using an ISAHP bare solar panel with half the active surface area than that of the one of the TSP device (indeed preliminary tests revealed a great increase of the ISAHP plate collector efficiency due to its lower working temperature).

Performances are analyzed using actual weather conditions (environmental temperature and solar radiation) occurred in Genoa, Italy, during the year 2004. A stochastic model of water variable consumption (sanitary water) has been provided as well, in order to make the dynamic boundary conditions of the systems as realistic as possible.

Basic ISAHP assembly

Figure 1 shows the simplified schematic of the traditional thermal solar panel(TSP) plant, Fig. 1A, and its evolution, the ISAHP, Fig. 1B. The basic function is the production of sanitary water at the desired temperature (e.g. 45 °C), using also some thermal energy by means of an auxiliary burner, when needed.

The two drawings are practically equal except for the presence of the compressor and the expansion valve in the ISAHP system, which obviously operates with a proper refrigerant fluid. In the traditional panel the temperature T pan of the irradiated plate is, during the active phase, higher than the temperature T stg of the water contained in the storage tank. As a consequence, a great amount of energy is wasted to the surroundings by means of both radiation and convection. On the other hand, in the innovative device the compressor (along with the expansion valve) allows a definite temperature difference between the plate and the heat exchanger (a condenser) to be held. In this way the panel can be maintained near the thermal equilibrium with the surroundings, and heat transfer losses are minimized. Furthermore, the panel structure is simplified down to a bare flat metal plate since nor glass cover nor expensive vacuum solutions are any more required to decrease convection and irradiation losses.

Another advantage of the ISAHP is its potential ability to deliver useful thermal power also during the night and in the cold months, performance that is precluded to traditional TSP.

Since the panel temperature is also a function of solar radiation G, and this last is variable during the day, a Variable Capacity Compressor (VCC) is needed to take out at any time the right heat rate from the panel, thus keeping T pan near the environmental one. More sophisticated control techniques, with respect to the brute on-off regulation, are therefore required to manage the process complexity of this innovative system during operation.

Device models

The formulation of the dynamic model for refrigeration devices presented in [START_REF] Tagliafico | A compact dynamic model for vapor compression refrigerated systems[END_REF] has been adapted to the ISAHP. The scheme of Figure 2(A) focuses on the relevant energy transfer rates occurring in the ISAHP device:

q in : thermal power from the surroundings to the solar panel (solar irradiation and other convective heat transfers); -q evap : the heat transferred to the operating fluid, equal to q in in steady state conditions; -q cond : thermal power from the operating fluid to the condenser solid body, equal to q out in steady state conditions;

q out : thermal power to the water reservoir.

q stg : heat delivered from the storage tank, based on user demand. It is supplemented by q aux in an instantaneous gas burner if water temperature in the reservoir is less than 45 °C. Otherwise (T stg >45°C) a proper mixing with tap water occurs.

q d : thermal power wasted to the surroundings trough the tank walls.

q u : user request.

The model is based on a simplified lumped description of a system operating along an inverse cycle. The cycle works between thermal sources with finite thermal capacities as shown in Figure 2(A), the external power, Pc, being provided by means of a VCC. The approach just accounts for the differential equations governing the time-temperature history of the different devices involving heat transfer (with the refrigerant fluid and with the outside) while neglecting the dynamics of physical phenomena having time constants below a few minutes. The general validity of this approach was proven experimentally in [START_REF] Tagliafico | A compact dynamic model for vapor compression refrigerated systems[END_REF] and briefly reported as Appendix.

The model of the traditional solar plant, TSP, is very close to the ISAHP one. The inverse cycle is replaced by a water circulation loop operated by a hydraulic pump, working between two finite thermal capacity sources (the solar panel and the water storage tank). In the other parts and components of the plant (storage tank and gas burner) the same equations of the ISAHP model were used.

Usual forced convection heat transfer calculations have been performed for the water loop, (instead of ISAHP condenser and evaporator sections), including them in the dynamic TSP system analysis.

The TSP solar panel is conceived as a thin absorber sheet (4 m 2 active surface) to which a selective coating is applied. As usual for TSP, coil tubes are placed in an insulated casing with a glass cover for reducing convection and irradiation heat losses. The calculated pump power consumption for water circulation (m H2O =120 kg•h -1 ) resulted to be negligible.

Concerning the collector efficiencies, both the unglazed active surface (2 m 2 ) of the ISAHP and the single glazed one of the TSP are characterized by efficiency curves comparable to data available in literature [START_REF] Kalogirou | Solar thermal collectors and applications[END_REF]. All the operating and geometrical data used in the calculation are available in Table 1.

The resulting set of differential dynamic equations where solved by means of standard software packages [START_REF]Simulink®-Simulation and Model-Based Design[END_REF].

Working conditions

Simulations were performed using, over a period of one year, actual climatic conditions (environmental temperature and solar radiation) occurred in Genoa in the year 2004 according to [22]. Figure 3 shows, as an example, the environmental temperature and the solar radiation during October.

A four-member typical family has been selected as "the user" and the behavior of the ISAHP and the other TSP device has been investigated regarding their ability to deliver about 250 l/day of sanitary water (e.g., about 4 showers) with a temperature of 45 °C. The energy saving for both systems, the ISAHP and the TSP, is calculated with respect to an instantaneous gas burner (combustion efficiency 0.87), which has been assumed as a reference. The same burner is also employed for energy integration purposes in both the solar heating setups.

The systems include a 300 liters water storage tank and are controlled by means of very simple operating criteria. The pump of the TSP is "on" only when the water temperature in the panel is higher than that in the tank (T pan >T stg ) but lower than an imposed maximum temperature of 90 °C. Temperature dead bands of 3 °C, ∆T L , and 10°C, ∆T H , for the lower and the higher set points respectively have been implemented.

The ISAHP device, instead, employs a variable capacity compressor and its power is controlled in open loop, making it nearly proportional to the solar incident radiation. In this case, a time dead band of 15 minutes has been adopted to avoid frequent on/off switches (e.g., rapid passage of clouds) to provide reliable operation for long time. Again, the maximum temperature in the storage tank has been limited as in the TSP device for safety reasons.

When the water at the outlet of the storage tank has a temperature lower than 45 °C, an instantaneous gas burner is used as extra energy supplier. On the other hand, when the water from the storage tank has a temperature exceeding 45 °C, a proper mixing with cold tap water is assumed (see Table 2 for the values of T tap ).

Due to our regulation choice the temperature of the panel is always greater than that of the surroundings, thus condensation phenomena are unlikely to happen and were not considered in the simulation.

The user load is represented by approximately 250 l/day of 45 °C water (i.e. around 1000 MJ/month).

We simulate this load as a stochastic process that behaves like a typical family making random use of sanitary water for hygienic purposes, e.g. to have a shower. The use of this approach has to be emphasized since it provides results different from those coming out of standard "duty cycle load" methods (e.g., [START_REF] Anderson | Effect of load pattern on solar-boosted heat pump water heater performance[END_REF]).

The "one shower process" works as follows: a couple of random numbers representing time intervals are drawn from two different uniform distributions. The first number is in the range from 2 up to 9 minutes and represents the shower duration. The second number is the time gap between two showers. It is in the range 0 -9 hours between 2:00 and 22:00 o'clock and in the range 0 -24 hours otherwise. With these values the water request history appears to follow a realistic behavior and the global energy demand approaches quite well the total value of 1000 MJ for one month.

Due to the stochastic nature of the simulation, proper confidence bounds must be added to each numeric result. We make this in a MonteCarlo fashion, calculating the sample mean and variance from various (100 runs) simulated experiments and then reporting the 95% confidence bounds of all the calculated parameters.

Performance indicators

In what follows the definitions of the main figures of merit used to quantify and compare the performance of the investigated devices are reported. To take into account the random mechanism applied to simulate the user demand and the variations of the environmental conditions (solar irradiation, temperature, clouds, and so on) energy data were calculated from integration over a time period of one month.

Coefficient of performance of the thermodynamic cycle (Heat Pump mode)

1 -         ⋅ = ∫ ∫ month c month cond d P d q COP τ τ (3)
It is the mean COP of the inverse cycle, based on monthly energy balances.

Second law efficiency (HP subsystem)

The ratio between the work spent a Carnot inverse machine and the one spent by the actual heat pump subsystem, when operated between the same thermal sources and with the same instantaneous thermal transfer toward the storage tank;

1 1 -         ⋅         - = ∫ ∫ month c month stg pan cond II d P d T T q τ τ η T [K] (4)

Exergetic efficiency (HP subsystem)

The ratio between the desired exergy transfer and the exergy input to the HP [START_REF] Moran | Availability Analysis: A Guide to Efficient Energy Use[END_REF].

1 1 1 -         ⋅                 - +         - = ∫ ∫ month c month pan e evap stg e cond Ex d P d T T q T T q τ τ η T [K] (5)
It will coincide with the previous index when T pan =T e

Solar panel efficiency

It is the ratio between the energy entering the solar collector (the evaporator for the ISAHP) and the incident irradiated energy;

1 -         ⋅ = ∫ ∫ month month in pan GAd d q τ τ η (6)

Renewable energy exploitation parameters

Applying, at steady state, global first law energy balance to the system of Figure 2, several energy flow rates can be observed and the corresponding performance parameters defined (in what follows all thermal and mechanical powers are considered positive for a simpler reading). Since aux d c in u q q P q q + -+ = [START_REF] Zalba | Review on thermal energy storage with phase change: materials, heat transfer analysis and applications[END_REF] the energy used "for free" since coming from renewable sources (not only solar, if ISAHP operates also when T pan <T e ) will be:

d in c aux u free q q P q q q - = - - = (8)
These relations should be better used on time-integrated, energy-based mean values, referred to reasonable long time periods, so we introduce the following;

"for free" fraction

The ratio between free (from the environment) and useful user energy:

1 -         ⋅ = ∫ ∫ month u month free free d q d q τ τ ξ (9)
(it is similar to solar fraction);

Energy fraction for the Compressor

The ratio between compressor energy consumption and useful thermal energy for the user

1 -         ⋅ = ∫ ∫ month u month c comp d q d P τ τ ξ (10)

Saved Primary Energy index

It makes a comparison between the actual consumption of primary energy resources and the one needed in the case of gas burner alone (assumed as a reference) [START_REF] Huang | Performance evaluation method of solar-assisted heat pump water heater[END_REF] where some suitable values for the combustion efficiency η burn of the burner and of the conversion efficiency from primary-to-electric energy, η el , is to be assumed. In the present paper the conventional values η burn =0.87 [START_REF] Eec | The Boiler Efficiency Directive: Council Directive on Efficiency Requirements for New Hot-Water Boilers Fired with Liquid or Gaseous Fuels[END_REF] and η el =0.36 have been assumed, this last being the global average conversion efficiency for all fossil fuels [START_REF]Energy Efficiency Indicators for Public Electricity Production from Fossil Fuels[END_REF]. For the TSP the saved primary energy index and the "for free" index will have the same numerical values. Concerning ISAHP, the index defined in Eq. ( 11) also gives information about actual money saving, since the ratio between the two conversion efficiency parameters, / el burn η η , has similar values (at least in Italy) to the ratio between the final prices of gas and electric energy in end uses.

∫ ∫ ⋅ + - = - = month u month el burn c aux gas p ISAHP p gas p spe d q d P q E E E τ τ η η ξ ) / ( 1 _ _ _

Tuning of the control strategy

As said, a variable capacity compressor is employed to better fit the variability of the weather conditions.

In particular the supplied power is made roughly proportional to the solar incident radiation. The right proportionality factor has been chosen according to a preliminary analysis of the relationship between Saved Primary Energy index, spe ξ , values and COP values during four representative months as depicted in Figure 4.

It is confirmed that there is no advantage (from the primary energy saving point of view) in operating the system at COP values lower than 3, since all that is gained from environmental (free) sources is duly paid in terms of compressor electric energy consumption. The value of this operative constrain is well known and can be inferred, in a roughly way, also from Eq. 7. Neglecting thermal dispersions from the water storage and in steady state conditions, it emerges that COP has to be greater than el burn η η / (2.4 in this study) to provide useful operations, i.e. spe ξ > 0, in respect to those of a gas burner alone. On the other side, the abrupt decline of the curves when COP>20 has to be ascribed to the lack of control ability of the compressor below 1600 rpm, and to the utilized time dead bands to avoid compressor damages.

For all the months, excluding July, a maximum of the COP is found for values around 7. In any case the maximum shape is rather flat giving us some flexibility in the final choice to be implemented in the control algorithm. Higher COP values mean lower average compressor speed with some issues about lubrication and bearing reliability, therefore it appears reasonable to tune the control law to always maintain the COP at values around 8. In this way we give up edge performances in favor of greater system reliability.

Results and discussion

For the sake of simplicity, all the parameters are presented only with reference to four different representative months; January, April, July and October. Table 2 shows the performance of the devices using the defined indices, along with their confidence bounds, plus a few integral quantities.

Month by month, the main average weather conditions (external temperature, irradiation power, rain time fraction) are given along with the user load info, such as the monthly energy consumption, the average daily water demand and the temperature of the cold tap water. Also the time averaged (over one month) temperatures of the panel and of the stored water are given, differentiating between global averages and averages taken only during active working periods, i.e. with running compressor/pump. These last are meaningful in the characterization of the average working point of the devices. To avoid the risk of an oversimplified model, we include in the analysis the monitoring of second law efficiencies as defined in Eq. 4 and 5. thus confirming the model is able to keep the main characteristics of "real world" devices.

Figure 5 shows instantaneous collector efficiency, pan η , values sampled during the simulation along with reference literature curves. The reported values correspond to an arbitrary day of July for both ISAHP and TSP. Dashed lines refer to the assumed reference [START_REF] Kalogirou | Solar thermal collectors and applications[END_REF]. On this side, Table 2 shows a clear supremacy of the ISAHP with averaged values of pan η in the order 0.6-0.8 against a value of 0.3 for the traditional panel. This confirms ISAHP ability to keep the panel temperature low, in order to reduce heat losses toward the surroundings, thus achieving high collector efficiency. In the introduction we stressed how this ISAHP feature corroborate the choice to utilize a strongly reduced active surface comparing different devices (ISAHP and TSP) able to satisfy the same user request.

Among the various performance parameters introduced in the previous paragraph, we focus our attention on the saved primary energy index, spe ξ , and on the free energy index, free ξ . They are reported, for convenience, also in Figure 6. As expected, despite the halved surface, the ISAHP assures about the same performance than the TSP. In particular it offers better performance in the cold months, during which the need of thermal power is usually more important. Only in July the TSP shows a better value for both spe ξ (0.81 versus 0.63 of the ISAHP) and free ξ (0.81 versus 0.80 of the ISAHP).

Averaging data over a year basis, it results for the TSP : free ξ ≡ spe ξ = 0.48 while the ISAHP shows free ξ = 0.56 and spe ξ =0.44. So, the ISAHP maintains the promise to deliver about the same useful heat power of the TSP using just half of the active solar panel surface. Moreover, it uses a simpler and less expensive structure: Table 1 reports the innovative panel featuring a weight of 18 kg against 80 kg of the traditional one (ordinary production of a big Italian manufacturer). As a consequence also the installation is simplified with an additional cost decrease.

The obtained values of COP need some comments since they appear, at a glance, too much high (around 8), especially if we consider that these are average values, not peak ones. Ordinary air and water heat pumps exhibit COP values some how in the range 2.5-4.5 and, generally speaking, we are unfamiliar with values greater than 5 when dealing with inverse cycles, irrespective of their nature. Nevertheless, opposite to traditional heat pumps, ISAHP evaporator temperature is not linked to the ambient one and the opportunity to increase the panel temperature to minimize compressor work has to be exploited as suggested by the simulation results.

However, literature data about solar heat pumps does not seem to confirm this operative choice, and COP values beyond 7 [START_REF] Li | Experimental performance analysis on a direct-expansion solar-assisted heat pump water heater[END_REF] are rarely found. In our opinion this is probably due to the control criterion often adopted for these systems, which asks for the collector temperature to be maintained near or below the environmental one. Many Authors indeed, e.g. [START_REF] Lu | Solar Heat Pumps Systems for Domestic Hot Water[END_REF][START_REF] Huang | Performance Characteristics of Integral Type Solar Assisted Heat Pumps[END_REF], stress the feature of collecting energy not only from solar irradiation but also from the environment and ambient air (including humidity condensation).

Although this characteristic of the solar assisted heat pumps is one of the key points with respect to TSP, it has to be exploited during night or, anyway, only in case of low solar irradiation, since it belongs to system flexibility rather than to system "performance". As a consequence the results of this study strongly suggest that the right balance for a good "performance" is towards low compressor energy consumption, rather than low collector thermal losses.

The main reason for this apparent discrepancy is due to the kind of investigation made in this study, which refers to the tuning and test of the system when external gas burner device is used for thermal aid, rather than using an electric boosted one [START_REF] Lu | Solar Heat Pumps Systems for Domestic Hot Water[END_REF] or a standalone system [START_REF] Chyng | Performance analysis of a solar-assisted heat pump water heater[END_REF]. It seems reasonable that in a different operative context optimal performance is achieved by means of different working set points.

If we had for instance replaced the gas burner used in the present ISAHP device by means of an instantaneous electrical heater, hypothetically able to deliver the same thermal power of the gas burner, we could assume 36 . 0 = = el burn η η to convert auxiliary energy into primary energy consumption. In such a case, free spe ξ ξ = . So, to maximize primary saved energy we have to maximize in q , the power collected by the panel, and this is accomplished by means of low collector temperature both to increase collector efficiency and to draw energy also from the environment ( e pan T T < ). This working condition, which requires to increase q in against q aux , also causes high storage temperatures, driving toward low COP values as a preferable choice. A very different picture, compared to our gas burner configuration.

We conclude by noting another positive feature of the ISAHP device, i.e. its small electric power consumption. Indeed, owing to the high values of COP, the compressor energy fraction is, on average, around 10% of the total useful energy delivered to the user. The combined use of photovoltaic devices, along with electric energy storage, appears thus feasible even when electric power sources are not available. This is, in our opinion, an important issue for future studies.

Duty cycles vs. stochastic loads

It frequently happens in current practice that standard duty load cycles, e.g. [START_REF]Performance test for Solar plus supplementary Systems[END_REF], are used to measure or estimate conventional solar panel performance, with measuring techniques well consolidated mainly for steady state at nearly optimal operating conditions. So, significant differences arise between actual data collected during the operating life of the apparatus and expected parameters based on standard duty load cycles. Usually in a duty cycle the interval between water usages are well and regularly spaced, so that the storage tank has enough time to receive energy from the solar panel system, that is to recharge itself. On the contrary, user requests are not so well placed in the real world. It happens, for instance, that two persons have their showers consecutively, with no or little time gap in between. In this case the reservoir temperature will drop down and the gas burner shall supply more energy, with a worsening of both free ξ and spe ξ . An increase of the storage tank size is only of limited usefulness and this is not surprising since a standard load of 0.2 kg/s of 45°C hot water represents a thermal power of about 25kW (T tap =15 °C).

To better quantify this concept the simulations were repeated using a deterministic load, that is assuming the duration of a shower to be always equal to 5.5 minutes and the wait time fixed at 4.5 or 12 hours (daytime and night-time respectively). In other words, we substitute the function RND in the programmed stochastic process with its average value, that is 0.5. The results reported in Table 3 show an overestimation of both free ξ and spe ξ in respect of the results previously described. This behavior is particularly marked during July, where differences of about 5÷10% can be noted. This kind of results, worth of further study, rises some doubts about the use of standardized hot water consumption pattern load such as the aforementioned ISO 9459-3, as reported in [START_REF] Kulkarni | Determination of design space and optimization of solar water heating systems[END_REF]. In the authors' opinion such load profiles can be used only as a starting point for the design and construction of solar panel systems, while more realistic stochastic loads are needed to develop detailed analyses and optimized control strategies, especially when multi-control multi-variable systems like ISAHP are involved.

Conclusions

Two contending devices have been modeled and tested in a simulated environment, which provides realistic energy demand of a four-member family and whether conditions representative of temperate climates. From these tests several performance parameters have been calculated assuming as a reference the standard instantaneous gas burner solution.

The considered devices are an integrated direct expansion solar assisted heat pump (ISAHP, 2 m 2 ) and a traditional flat plate collector solar assembly (TSP, 4 m 2 ). They are both boosted by means of an auxiliary gas burner in order to assure the needed heat duty (45 °C hot water).

The results show that the ISAHP plant can replace the traditional solar water heater using only around half of its active collector surface. Furthermore, since the ISAHP panel can be maintained at a temperature nearer the environmental one, it can be used simply bare, without the pricey technologies (glass cover, insulation, vacuum, etc.) commonly used to minimize the heat losses of a TSP. The innovative solar panel configuration is therefore lighter and definitely less expensive than the traditional one, also from the installation cost point of view.

The required electric energy is very small, so that ISAHP configurations can be envisaged with an integrated photovoltaic surface able to operate, like the natural circulation TSP, also in absence of electric energy supply.

To obtain the reported results a variable capacity compressor is needed and the necessity to operate at high COP value is emerged. High COP values were obtained with low compressor speed, thus increasing the risk for lubrication related problems. However high panel temperatures are needed to fully exploit the energy potential of solar assisted heat pumps. In other words, the key is toward low compressor energy consumption, rather than low thermal losses of the solar collector.

This conclusion is valid only when a gas burner is assumed as a reference heat source and boosting device; different operating conditions (and conclusions) should follow if electrical water heaters were assumed as reference heating means and boosting devices.

The ISAHP has shown its ability to thermally decouple the solar collector from the storage tank with low electric power consumption, thus encouraging the studies towards higher temperature applications (60 °C for heating purposes or even 80 °C for cooling systems coupled to absorption refrigerators). Finally, the low compressor energy fraction drives to further studies in the field of combined use of photovoltaic devices, along with electric energy storage, thus opening for ISAHP applications were electric power sources are not available.

h h m q r cond - = & (A.2) expansion valve ( ) 4 3 0 h h m r - = & (A.3) evaporator ( ) 4 1 h h m q r evap - = & (A.4)
where P c is the power (work) done on the refrigerant by the compressor, q cond is the power (heat) discharged from the refrigerant to the warm region (the condenser inside the water tank) while q evap enters the refrigerant coming from the cold region (the evaporator). r m & is the refrigerant mass flow rate; h 1 h 2 h 3 and h 4 represent specific enthalpy values at the relevant points numbered in Figure 2(B).

To avoid misunderstanding, we have used a notation in which all the energy transfers (work and heat) are assumed to be positive in the direction suggested by the arrows.

A.3 -Dynamic equations

The differential transient equations of the condenser and the evaporator, together with the dynamics of the storage tank (fully mixed model), were described by means of lumped unsteady energy balance equations by neglecting, as said, the fluid-wall temperature gradient in respect to the wall-outside gradients (T cond =T 3 , T evap =T 1 ): (  ) [START_REF] Garcia-Casals | Solar Absorption cooling in Spain: Perspectives and outcomes from the simulation of recent installations[END_REF]

in out p y T T c m q - = & A.

-Constitutive and other relations

Finally, we make use of the analytical description of the fluid enthalpy in correspondence of the saturation lines (Eqs. A.8,A.9), of the relations for the calculation of the actual enthalpy at the compressor outlet (Eqs. A.10, A.11), and of the expression for the refrigerant mass flow rate (Eq. A.12).

)

( 1 1 T h h l = (A.8) ) ( 3 3 T h h v = (A.9) ) , ( T T f h s = (A.10) ( ) ( ) 1 2 1 2 h h h h id c - - = η (A.11) ) ( 60 1 T v V m c v r η ω = & (A.12)
Where V C is the compressor displacement. Isentropic, η c , and volumetric, η v , compressor efficiencies have been adapted from the performance map data of a major manufacturer [START_REF] Danfoss | Compressor, Collection of Datasheets[END_REF], relative to the variable speed drive compressor TLV6K:

              - =
        -         +       - = p p p p c ω η (A.14)
The model, after a tuning stage, has been tested against an isahp prototype actually under construction at our department. These very preliminary tests, both static and dynamic, show a good agreement between simulated and measured behavior, although some differences are still present. Table 1. Main geometrical and thermal data of the heating systems. See the nomenclature section for the meaning of each symbol. [START_REF] Kalogirou | Solar thermal collectors and applications[END_REF], respectively for bare panels and for single-glazed panels. Figure 6. Synthetic view of two main figures of merit from table 2; Free used energy fraction, free ξ (left) and Saved primary energy index, spe ξ .

Tables

ISAHP TSP

  heat transfer equations are provided in then form ( )

Figure 1 .

 1 Figure 1. Simplified schematic of a solar panel assembly together with auxiliary means. (A) TSP -Traditional; (B) ISAHP device -the water loop has been replaced by a refrigerant vapor compression system.

Figure 2 .

 2 Figure 2. Block diagram of the ISAHP device. Thermal transfers in evidence.

Figure 3 .

 3 Figure 3. Genoa (Italy) October 2004, raw data. Environmental temperature (A) and solar irradiation (B).

Figure 4 .

 4 Figure 4. Saved primary energy index, spe ξ , as a function of the COP for the four representative months.

Figure 5 .

 5 Figure 5. Instantaneous collector efficiency, pan η , values during an arbitrary July day. Dots represent the

Figure 1 .

 1 Figures

Figure 2 .

 2 Figure 2. Block diagram of the ISAHP device with thermal transfers in evidence (A). Thermodynamic cycle in the enthalpy-pressure plane, no pressure losses (B).

Figure 3 .

 3 Figure 3. Genoa (Italy) October 2004 row data. Environmental temperature (A) and solar irradiation (B).

Figure 5 .

 5 Figure 5. Instantaneous collector efficiency, pan η , values during an arbitrary July day. Dots represent the calculated data for ISAHP and TSP devices on the basis of simulation results. Dashed lines refer to literature data[START_REF] Kalogirou | Solar thermal collectors and applications[END_REF], respectively for bare panels and for single-glazed panels.

Figure A1 .

 A1 Figure A1. Diagram of the heating/mixing logic and associated expressions for the involved heat exchanges

  Table 2 puts in evidence values of II η and Ex η around 0.35, typical for this kind of systems,

Table 2 .

 2 Performance comparison results. Four representative months. The behavior of the 2 m 2 innovative panel ISAHP is reported side by side with that of a 4 m 2 traditional thermal panel. All data are averaged over 100 different simulations. When applicable, the confidence associated to these results is reported in brackets (as 95% confidence bounds).

	panel/evaporator
	A, m 2	2	4
	M, kg	18	80
	KA, WK -1	30	35
	Mc, JK -1	16400 7200
	α ε		0.93 0.4	0 0.81 0.1
	compressor
	Vcc, cm 3	5.7	-
	ω, η v η c	rpm 1600-4500 -Eq. A13 -Eq. A14 -
	storage heat
	exchanger
	Mc, JK -1	2000	2000
	KA, WK -1	100	50
	storage tank
	M, kg	300	300
	K, Wm -1 K -1	0.06	0.06
	S, m	0.05	0.05

Table 3 .

 3 Performance comparison of the results obtained with different thermal load simulation methods. Free used energy fraction, free

	ξ	and saved primary

Acknowledgements

The present work was supported by Genuense Atheneum.

Appendix -Lumped dynamic model

In this appendix, the simplified dynamic lumped model used to simulate the direct expansion solar assisted heat pump is synthesized.

A.1 Main assumptions

With reference to the plant sketch of Figure 2(A) and the corresponding thermodynamic cycle of the refrigerant fluid of Figure 2(B), a model is proposed to describe the dynamic behavior of the systems.

Only the dynamics of the thermal inertias associated to the evaporator/panel, the condenser and storage tank will be described, while all the phenomena characterized by time constants smaller than few seconds will be systematically neglected.

In particular the following simplifying hypotheses were assumed:

-evaporation and condensation processes of the fluid are described only by means of steady-state first law balances; -the refrigerant fluid temperature is assumed to be the same of that of the involved heat exchanger body (convective thermal resistances inside tubes have been neglected).

-the whole thermal capacity has been concentrated in the material of the heat exchangers, -the expansion valve is simply described by its isenthalpic process, -the dynamics of the reciprocating VCC system (conceived as a hermetic volumetric compressor operated by an asynchronous electrical motor) and of the associated regulation system (inverter) are not considered.

The performance of the refrigeration cycle is simulated by means of steady state, off-design calculations based on the thermodynamics of the refrigerant fluid, developed in a MATLAB Function code and coupled to a properly adapted NIST REFPROP database [START_REF] Lemmon | [END_REF] for the calculation of the thermophysical properties.

A.2 -Control volume balance equations

The inlet-outlet energy balance equations, in steady state, for each component of the vapor-compression system turn out to be (kinetic and gravitational terms neglected as usual): compressor ( )