M.D Francesca Montagnani

Ph.D Francesca De Paolis

Elisa Beghetto

Nicola Gargano

Use of recombinant chimeric antigens for serodiagnosis of Mycoplasma pneumoniae infection

Keywords: Mycoplasma pneumoniae, diagnosis, recombinant antigens, enzyme-linked immunosorbent assay, ELISA Mycoplasma pneumoniae, diagnosis, recombinant antigens, enzyme-linked immunosorbent assay, ELISA

Purpose. We have evaluated the diagnostic utility of three antigenic regions of the Mycoplasma pneumoniae P1, P30, and MNP456 gene products in order to replace the soluble, wholecell bacterial extract in serological assays. Antigenic regions, being previously identified B cell epitopes, were used individually or assembled in a recombinant chimeric antigen by genetic engineering. Methods. Paired serum samples from 47 patients with M. pneumoniae infection and from 39 subjects with a clinical picture of atypical pneumonia but without a defined diagnosis of M. pneumoniae infection, were included. Immunoglobulin G (IgG) antibodies against epitopes carried by recombinant antigens were measured by performing recombinant enzyme-linked immunosorbent assays (Rec-ELISA's). Rec-ELISA's results were compared to those obtained by a commercial assay using the wholecell Mycoplasma antigen. Results. Our study demonstrates that all IgG Rec-ELISA's using recombinant antigens have better sensitivity with respect to the commercial assay. Furthermore, we show that the use of chimeric antigens improve the performance of the assays. Conclusions. The use of recombinant antigens is effective in distinguishing M. pneumoniae-infected from uninfected individuals and shows that immunoassays based on recombinant antigens could provide the basis for standardized commercial tests for the serodiagnosis of M. pneumoniae diseases.

To explain the choice of B cell epitopes, the following sentences have been included at the end of the Introduction section: "We have previously shown the identification and characterization of immunodominant B-cell epitopes of M. pneumoniae antigens using the phage-display technology [START_REF] Beghetto | Discovery of new Mycoplasma pneumoniae antigens by use of a whole genome lambda-display library[END_REF]. We show here the usefulness of recombinant proteins containing epitopes of the P1, P30 and MPN456 antigens in M. pneumoniae serodiagnosis." With regards to the recently described P1 variants, it should be noted that the B cell epitope employed in our work (residues 1307-1340) is located in a different region with respect to the variant-specific antigen regions described by Dumke and collaborators (Dumke et al., Int. J. Med. Microbiol. 2008, 298:483-491).

Materials and Methods: Line 5 -37, cloning of the chimeric antigen EC12. Location of the antigenic regions used: please refer to figure 1. PCR programs used? DNA extraction method? PCR reaction mixture? Please provide more details on pGEX-P30. Where does is come from? Cloning method? Briefly.

As requested by the reviewer, more details about chimeric antigen cloning have been added in the Methods section.

Pag 5 Line 46. bacterial cells. Which ones were used? Pag 5 line 52. Should be: Luria Bertani medium and gene expression was induced using,..... Bacteria were centrifuged,.... The bacterial strain (E. coli AD202), the LB medium and other details have been specified. Pag 6 line 8. Purification of proteins? Checked for remaining LPS as antibodies against LPS of other bacteria could cross react in the ELISA's.

It should be noted that the absence of immunoreactivity of recombinant antigens with negative control sera (collected from subjects with S. pneumoniae infection) demonstrates that bacterial contaminants, including LPS, were not present in purified proteins. Pag 6 Serum samples. Groups are rather small. We disagree with reviewer's observation since in our retrospective analysis most of the patients that meet selection criteria (hospitalized in one Centre during years 1999-2005, clinical symptoms compatible with atypical pneumonia, paired serum samples available) were included.

Group B on page 6 line 56. pneumonia group with diagnosis of M. pneumoniae negative with available methods or diagnosis not performed. Should you not exclude the ones where the diagnosis was not performed as they could be positive?

We wish to thank reviewer #2 for this observation. However, we would like to highlight that this analysis has been performed to better compare the sensitivity of Rec-ELISA's versus the whole bacterial extract (the commercial ELISA) and other diagnostic tools. Thus, we decided to include also the ones where the diagnosis was not performed.

Pag 13 line 5. Gold standard not available. What about Western blotting although less sensitive often regarded as serological gold standard?

To avoid any debate about ELISA and Western blotting, the sentence of gold-standard in page 13 has been deleted in the revised manuscript. Line 15 pag 13. By the commercial ELISA instead of with Pag 13, line 44-50. Grammatically incorrect Pag 13 line 57: use as compared to instead of 'than'

The terms and the grammatical error have been corrected in accordance with the suggestions.

REVIEWER #3

Firstly, the control group was small paediatric patients.

It should be noted that the control group was chosen accordingly to the mean age of other groups. Since both groups A and B were mainly composed of paediatric patients, we thought that the best control group should be composed by paediatric patients. As a consequence, every group was comparable to each other.

Secondly, atypical CAP group was ill-defined. Thirdly, those with supposed M. pneumoniae CAP had elevated IgG titers, but IgM (acute) cold agglutinin titers not included. Although it was not the focus of their report, their conclusion would be more clinically relevant if they included data on key clinical findings of M. pneumoniae CAP i.e., loose stools, temp of less than 102 F wihout relative bradycardia, sore throat, etc. not included.

The retrospective nature of the study makes it difficult to meet the suggestions by reviewer #3. In fact, cold agglutinin titers are not routinely required by hospital clinicians (at least in Italian hospitals). Also, cold agglutinin titers were not assessed due to the low sensitivity and specificity of such analysis when frozen serum samples need to be employed. We agree that specific key clinical findings were not available for all patients. However, since the focus of our report was the development of laboratory diagnostic tools, it seemed more suitable to screen patients by ill-definition and then to group them by laboratory data (serology and/or PCR). Thus, due to the main scope of our work (that is the evaluation of recombinant antigen immunoassays in order to replace the soluble, whole-cell bacterial extract in serological assays), a comparison of laboratory techniques seemed enough, in our opinion, to reach a valid conclusion.

RE: Ms. No. EJCMID-D-10-00080

"Use of recombinant chimeric antigens for serodiagnosis of Mycoplasma pneumoniae infection" Dear Prof. Van Belkum, Thank you very much for the comments to our paper. The manuscript has now been revised in accordance with the suggestions of the reviewers. We complied with all points raised and we believe that our manuscript is now more clear and complete.

Best regards, Francesca

Montagnani ------------------------------------------------------------------------------------------------------------------------ REVIEWER #2
1) Abstract lines 5-12. Sentence should be: We have evaluated,.....gene products in order to replace tjhe soluble, whole -cell bacterial extract in serological assays. Antigenic regions, being previously identified B cell epitopes, were used individually or assembled in a recombinant chimeric antigen by genetic engineering.

The sentence has been changed in accordance to reviewer's suggestion.

2) Line 20. Immunoglobulin G (IgG) antibodies. Which serum dilutions used in the different tests including the CF test? The sensitivity of the different recombinants tests and the commercial ELISA could have been evaluated by testing a serial dilution of well-known positive sera. I can't find this in the paper. It would give us an objective idea on the sensitivity of those ELISA's.

The serum dilutions used in the different tests, including the CF test, has been indicated in the methods section. To compare the sensitivity of the different ELISA's (recombinants versus commercial assay), the dilution employed by the commercial test (1:100 -information added in the methods section) has been used as the reference standard.

3) What about cross-reactivity against other respiratory pathogens. How sure are the authors on the specificity of the test. Have they tested sera of patients infected with other respiratory pathogens or even of patients infected with other non-respiratory mycosplasma species?

We agree with the reviewer observation. For this reason serum samples from patients infected with S. pneumoniae have been used as negative controls and none of them did react with the recombinant antigens. Unfortunately, blood samples from patients with other non-respiratory mycoplasma species were not available at the time of the study. 24-25: better: (Rec-ELISA's). Rec-ELISA results were compared to,....assay using the whole .... According to reviewer's suggestion, the term "Rec-ELISAs" has been changed in "Rec-ELISA's" throughout the manuscript.

4) Abstract line

5) Results in the abstract; have performance characteristics significantly,....this is vague, please talk about sensitivity and specificity of the tests rather than about performance characteristics

The sentence has been changed in: "Our study demonstrates that all IgG Rec-ELISA's using recombinant antigens have better sensitivity with respect to the commercial assay." 6) Abstract: line 31-32, omit the word 'corresponding'; line 44 and throughout the text, use recombinant antigens rather than recombinant products. Introduction: line 20 pag 4, recombinant 'antigens' can not be challenged.

The text has been changed in accordance with the suggestions and the term "challenged" has been replaced with "tested".

Please mention already in the intro why those regions were chosen (refer to B cell epitopes). How was the B cell epitope mapping performed? Did the authors take in account the recently described P1 variants?

To explain the choice of B cell epitopes, the following sentences have been included at the end of the Introduction section: "We have previously shown the identification and characterization of immunodominant B-cell epitopes of M. pneumoniae antigens using the phage-display technology [START_REF] Beghetto | Discovery of new Mycoplasma pneumoniae antigens by use of a whole genome lambda-display library[END_REF]. We show here the usefulness of recombinant proteins containing epitopes of the P1, P30 and MPN456 antigens in M. pneumoniae serodiagnosis."

With regards to the recently described P1 variants, it should be noted that the B cell epitope employed in our work (residues 1307-1340) is located in a different region with respect to the variant-specific antigen regions described by Dumke and collaborators (Dumke et As requested by the reviewer, more details about chimeric antigen cloning have been added in the Methods section.

Pag 5 Line 46. bacterial cells. Which ones were used? Pag 5 line 52. Should be: Luria Bertani medium and gene expression was induced using,..... Bacteria were centrifuged,.... The bacterial strain (E. coli AD202), the LB medium and other details have been specified. Pag 6 line 8. Purification of proteins? Checked for remaining LPS as antibodies against LPS of other bacteria could cross react in the ELISA's.

It should be noted that the absence of immunoreactivity of recombinant antigens with negative control sera (collected from subjects with S. pneumoniae infection) demonstrates that bacterial contaminants, including LPS, were not present in purified proteins.

Pag 6 Serum samples. Groups are rather small. We disagree with reviewer's observation since in our retrospective analysis most of the patients that meet selection criteria (hospitalized in one Centre during years 1999-2005, clinical symptoms compatible with atypical pneumonia, paired serum samples available) were included.

Group B on page 6 line 56. pneumonia group with diagnosis of M. pneumoniae negative with available methods or diagnosis not performed. Should you not exclude the ones where the diagnosis was not performed as they could be positive?

We wish to thank reviewer #2 for this observation. However, we would like to highlight that this analysis has been performed to better compare the sensitivity of Rec-ELISA's versus the whole bacterial extract (the commercial ELISA) and other diagnostic tools. Thus, we decided to include also the ones where the diagnosis was not performed.

Pag 13 line 5. Gold standard not available. What about Western blotting although less sensitive often regarded as serological gold standard?

To avoid any debate about ELISA and Western blotting, the sentence of gold-standard in page 13 has been deleted in the revised manuscript. The terms and the grammatical error have been corrected in accordance with the suggestions.

REVIEWER #3

Firstly, the control group was small paediatric patients.

It should be noted that the control group was chosen accordingly to the mean age of other groups. Since both groups A and B were mainly composed of paediatric patients, we thought that the best control group should be composed by paediatric patients. As a consequence, every group was comparable to each other.

Secondly, atypical CAP group was ill-defined. Thirdly, those with supposed M. pneumoniae CAP had elevated IgG titers, but IgM (acute) cold agglutinin titers not included. Although it was not the focus of their report, their conclusion would be more clinically relevant if they included data on key clinical findings of M. pneumoniae CAP i.e., loose stools, temp of less than 102 F wihout relative bradycardia, sore throat, etc. not included.

The retrospective nature of the study makes it difficult to meet the suggestions by reviewer #3. In fact, cold agglutinin titers are not routinely required by hospital clinicians (at least in Italian hospitals). Also, cold agglutinin titers were not assessed due to the low sensitivity and specificity of such analysis when frozen serum samples need to be employed.

We agree that specific key clinical findings were not available for all patients. However, since the focus of our report was the development of laboratory diagnostic tools, it seemed more suitable to screen patients by ill-definition and then to group them by laboratory data (serology and/or PCR). Thus, due to the main scope of our work (that is the evaluation of recombinant antigen immunoassays in order to replace the soluble, whole-cell bacterial extract in serological assays), a comparison of laboratory techniques seemed enough, in our opinion, to reach a valid conclusion.

INTRODUCTION

M. pneumoniae is the leading cause of atypical pneumonia in children and young adults; it is responsible for 10-20% of cases of community acquired pneumonia, giving rise to extrapulmonary complications in 25% of infected patients [START_REF] Waites | Mycoplasma pneumoniae and its role as a human pathogen[END_REF][START_REF] Baum | Mycoplasma pneumoniae and atypical pneumonia[END_REF][START_REF] Sánchez-Vargas | Mycoplasma pneumoniae -an emerging extra-pulmonary pathogen[END_REF].

Infected individuals show flu-like symptoms, but characteristically there is an insidious onset with chronic recovery [START_REF]Mycoplasma pneumoniae respiratory disease symposium: summation and significance[END_REF][START_REF] Baum | Mycoplasma pneumoniae and atypical pneumonia[END_REF]. Misdiagnosis and subsequent inappropriate antibiotic treatment pose a serious threat to infected individuals [START_REF] Levine | The clinical spectrum of Mycoplasma pneumoniae infections[END_REF][START_REF] Tully | Mycoplasma[END_REF]. Although clinicians may suspect infections during epidemics, an aetiological diagnosis cannot be formulated on the basis of clinical appearance, since other bacterial and viral infections often share symptoms and clinical features [START_REF] Saikku | Atypical respiratory pathogens[END_REF][START_REF] Baum | Mycoplasma pneumoniae and atypical pneumonia[END_REF].

Diagnosis of M. pneumoniae infection can be established by isolating bacteria or

detecting mycoplasma-specific DNA sequences and immunoglobulins in body fluids and sera from infected individuals, respectively. Serological methods play a predominant role in routine clinical practice, mainly due to the ease of specimen collection and the widespread availability of serologic tests [START_REF] Daxboeck | Laboratory diagnosis of Mycoplasma pnemoniae infection[END_REF].

Host antibody response to M. pneumoniae infection can be detected after 1 week of illness, but it generally peaks three to six weeks after acute infection, followed by a gradual decay of antibody production over months to years [START_REF] Waites | Mycoplasma pneumoniae and its role as a human pathogen[END_REF][START_REF] Atkinson | Epidemiology, clinical manifestations, pathogenesis and laboratory detection of Mycoplasma pneumoniae infections[END_REF]. As a result of the long incubation period, the antibody response is often evident by the time symptoms appear. A simultaneous testing for both IgG and IgM in paired serum sample collected 2 to 3 weeks apart is usually required to achieve a proper diagnosis.

Serodiagnosis of M. pneumoniae infection can be performed by use of complement

fixation tests (CF), indirect immunofluorescent assays (IFA) and enzyme-linked immunoassays (EIA) to detect the presence of the various classes of anti-Mycoplasma immunoglobulins (IgG, IgM) [START_REF] Atkinson | Epidemiology, clinical manifestations, pathogenesis and laboratory detection of Mycoplasma pneumoniae infections[END_REF]. All of the commercially available assays use the whole M. pneumoniae soluble extract or sub-cellular fractions as the antigen. However, these assays have limited sensitivity and specificity since cross-reactions of the antigen with antibodies to other Mycoplasma species and other bacterial pathogens may occur [START_REF] Waites | Mycoplasma pneumoniae and its role as a human pathogen[END_REF]. Also, the available assays may vary in their ability to detect serum immunoglobulins [START_REF] Beersma | Evaluation of 12 commercial tests and the complement fixation test for Mycoplasma pneumoniae-specific immunoglobulin G (IgG) and IgM antibodies, with PCR used as the "gold standard[END_REF][START_REF] Yoo | Evaluation of four commercial IgG-and IgMspecific enzyme immunoassays for detecting Mycoplasma pneumoniae antibody: comparison with particle agglutination assay[END_REF], due to the lack of a purified standardized antigen or standard methods for preparing the antigen.

The aim of this study was to improve the performance of enzyme-linked immunoassays for the serodiagnosis of M. pneumoniae infection using recombinant productsantigens, which were challengedchallengetested with sera from a cohort of patients with respiratory tract diseases clinically compatible with "atypical" bacterial aetiology. We have previously shown the identification and characterization of immunodominant B-cell epitopes of M. pneumoniae antigens using the phage-display technology [START_REF] Beghetto | Discovery of new Mycoplasma pneumoniae antigens by use of a whole genome lambda-display library[END_REF]. We show here the usefulness of recombinant proteins containing epitopes of the P1, P30 and MPN456 antigens in M. pneumoniae serodiagnosis.

MATERIALS AND METHODS

Cloning the chimeric antigen EC12

The DNA coding for the antigenic regions of M. pneumoniae P1 (residues 1307-1340), P30 (residues 194-241) and MPN456 (residues 765-846) open reading frames [START_REF] Beghetto | Discovery of new Mycoplasma pneumoniae antigens by use of a whole genome lambda-display library[END_REF] were used for the construction of the chimeric antigen EC12. DNA fragments of P1 and MPN456 were amplified by PCR from M. pneumoniae DNA, FH strain (DNA commercially available and purchased from Abionline, USA), using specific oligonucleotides as follows: P1-for, 5'-GGGGATCCTTCACATTGGCAGTGTGCTTAG-3'; P1-rev, 5'-AGGCTACCGCCACCAGAACTATTCCCACCACCTCCA-3'; MPN456-for 5'-GTTCTGGTGGCGGTAGCCTGTTAGCTGCGAGTCAAAA-3'; MPN456-rev 5'-ACTAGTGCTACCGCCACCAGATGCTTTAGCATCACCAAAGTTC-3'. PCR conditions were 30 sec at 94°C, 30 sec at 55°C and 60 sec at 72°C for 30 cycles. PCR products of P1 and MPN456 fragments were purified, mixed and then used as templates for DNA amplification using oligonucleotides P1-for and MPN456-rev. The PCR protocol was 30 sec at 94°C, 30 sec at 55°C and 60 sec at 72°C for 25 cycles. The resulting DNA was purified by standard methods and cloned into the plasmid pGEX-P30 [START_REF] Beghetto | Discovery of new Mycoplasma pneumoniae antigens by use of a whole genome lambda-display library[END_REF], which containeding the antigenic region of the P30 protein antigen (amino acids 194-241) fused at the N-terminus of the GST protein [START_REF] Beghetto | Discovery of new Mycoplasma pneumoniae antigens by use of a whole genome lambda-display library[END_REF], to generate the plasmid pGEX-EC12 (schematically shown in Fig. 1).

Purification of recombinant antigens

Four M. pneumoniae antigens were used: P1, P30, MPN456 [START_REF] Beghetto | A combination of antigenic regions of Toxoplasma gondii microneme proteins induce protective immunity against oral infection with parasite cysts[END_REF] and EC12.

Recombinant antigens were expressed in bacterial cells as fusion proteins with glutathione Stransferase (GST) and purified by affinity chromatography as described previously [START_REF] Beghetto | A combination of antigenic regions of Toxoplasma gondii microneme proteins induce protective immunity against oral infection with parasite cysts[END_REF].

Briefly, recombinant Escherichia coli strain AD202 were grown in Luria Bertani (LB) medium and gene expression was induced with using isopropyl-β-D-thiogalactopyranoside (IPTG). Bacteria were, centrifuged at 2000 x g for 30 min at 4°C, and then suspended in STE buffer (10 mM Tris-HCl [pH 8.0], 150 mM NaCl) containing 100 μg mL -1 of lysozyme and protease inhibitors (Boehringer, Germany). The mixture was sonicated, and Triton X-100 was added to a final concentration of 1%. After centrifugation at 10,000 x g for 30 min at 4°C, the supernatant was incubated with glutathione-Sepharose beads (BD Biosciences, USA), and the GST-fusion proteins were eluted following the manufacturer's instructions and stored at -20°C.

The recombinant antigens were efficiently expressed and purified in large amounts from the cytoplasm of bacterial cells, with a purity >90% and a yield of purified proteins of 5-15 mg per liter of bacterial cultures.

Serum samples

One hundred eighty-four serum samples collected from 98 patients hospitalized for pulmonary diseases at the Clinic of Infectious Diseases of Siena University, Italy, during years 1999 to 2005, were used. A clinical picture compatible with atypical pneumonia [START_REF] Baum | Mycoplasma pneumoniae and atypical pneumonia[END_REF] was defined by the presence of the following features: insidious onset, malaise, fever, cough ≥ 1 week; signs of interstitial pneumonia ± wheezing; extra-pulmonary manifestations compatible with Mycoplasma infection (e.g. arthralgia, skin eruption); radiological findings suggestive of interstitial involvement ± minimal pleuritis; lack of responsiveness to previous beta-lactams therapy.

Accordingly to the results obtained by clinical and diagnostic examinations, serum samples were divided into three groups as follows:

Whole-cell Mycoplasma antigen immunoassays

Analysis of Mycoplasma-specific IgG antibodies were done by the whole-cell, Mycoplasma antigen assay ELISA-IgG (IBL Hamburg, Germany), according to the manufacturer's instructions (e.g. sera diluted 1:100). All serum samples were analyzed in a blinded fashion.

Recombinant protein enzyme immunoassays (Rec-ELISA)

Maxisorb plates (Nunc) were adsorbed with recombinant proteins at a concentration of 10 μg mL -1 in 50 mM NaHCO 3 , pH 9.6. Each protein (GST-P1, GST-P30, GST-MPN456 and GST-EC12) was coated separately onto individual microtiter wells. After incubation overnight at 4°C, plates were blocked with 5% non-fat dry milk and 0.05% Tween 20 in PBS (blocking buffer) and subsequently incubated for 1 h at 37°C with serum samples diluted 1:100 in blocking buffer. The plates were washed with 0.05% Tween-20 in PBS, and antihuman IgG alkaline phosphatase-conjugated antibodies (Sigma-Aldrich) were added to each well. Finally, incubating plates with the chromogenic substrate p-nitrophenylphosphate (pNPP; Sigma-Aldrich) revealed the enzymatic activity. The results were recorded as the difference between the absorbance (optical density, OD) at 405 nm and that at 620 nm, as detected by an automated ELISA reader (Labsystem Multiskan, Finland).

For each serum, the assay was done in duplicate and average values were calculated. For every GST-fusion product, the cut-off value was determined as the mean plus three times the standard deviation of the absorbance readings obtained from the Mycoplasma-negative sera (group C).

Statistical analysis

The sensitivity, specificity, positive and negative predictive values of the enzymelinked immunosorbent assays are given. Proportions were compared by the chi-square test or the Fisher exact test (two tailed), with a p value <0.05 considered statistically significant.

RESULTS

In a retrospective study, 184 sera from 98 patients hospitalized for pneumonia were analyzed. Paired sera from 86 cases of atypical pneumonia were available and were further divided into two groups, as follows: group (A), from 47 subjects with a defined diagnosis of M. pneumoniae infection, group (B), from 39 subjects without a defined diagnosis of M. pneumoniae infection but with a clinical picture compatible with atypical pneumonia. Twelve sera from 12 patients affected by Streptococcus pneumoniae pneumonia with negative results for M. pneumoniae-specific serology and PCR analysis were also assayed as negative controls (group C).

To set up an immunoassay based on recombinant productsantigens, three regions of the M. pneumoniae P1, P30 and MPN456 gene products, which had been shown previously to contain B-cell epitopes [START_REF] Beghetto | Discovery of new Mycoplasma pneumoniae antigens by use of a whole genome lambda-display library[END_REF], were chosen for the investigation. The antigen fragments were produced in E. coli as GST fusion products (namely GST-P1, GST-P30 and GST-MPN456). Also, the antigenic regions of P1, P30 and MPN456 proteins were assembled to give the chimeric antigen GST-EC12 (Figure 1).

The immunoreactivity of GST-P1, GST-P30, GST-MPN456 and GST-EC12 with IgG antibodies in sera from patients with a defined diagnosis of M. pneumoniae infection (group A) was determined. Each of the antigens was coated separately in individual wells and tested in the IgG Rec-ELISAsRec-ELISA's with paired sera from the 47 subjects of group A (Table 1). Overall, the proportion of IgG-reactive sera were 54%, 48%, 25% and 57% by use of recombinant GST-P1, GST-P30, GST-MPN456 and GST-EC12 antigens, respectively. None of the Mycoplasma-negative sera reacted with the GST fusion products. GST-MPN456, GST-P1, GST-P30 and GST-EC12 specifically reacted with IgG antibodies present in at least one of the paired sera from 40%, 87%, 81% and 94% of infected patients, respectively. Also, 40 out of 47 patients (85%) developed specific IgG antibodies against at least two distinct M. pneumoniae antigens along hospitalization. Sera from group A patients were also analyzed with a commercial IgG ELISA assay, which employs the whole cell extract of M. pneumoniae as the antigen (Table 1). A low proportion of IgG-positive sera was observed (19%), allowing to obtain a correct diagnosis of M. pneumoniae infection in only 17 out of 47 infected patients (36.2%). Also, only 12 out of 59 serum samples having specific anti-Mycoplasma antibodies, as assessed by the CF test, were found to be positive with the commercial ELISA (patients A1-A39), allowing a positive diagnosis in only 12 out of 39 subjects (31%).

The performance of IgG assays employing the whole cell extract or recombinant antigens for the serological diagnosis of M. pneumoniae infection is summarized in Table 2.

All of the assays based on recombinant antigens showed a significantly better performance when compared to the commercial test. In particular, the assay based on the chimeric antigen EC12 revealed a significantly higher sensitivity with respect to the commercial assay (93.6% versus 36.2 %, respectively; p < 0.001).

Next, the immunoreactivity of recombinant antigens was assessed with IgG antibodies in sera from patient with a clinical picture of atypical pneumonia but without a defined diagnosis of M. pneumoniae infection (group B), and the results of Rec-ELISAsRec-ELISA's were compared with those obtained using the commercial assay. Detailed results of all IgG assays performed with paired sera collected from the 39 patients of group B are shown in Table 3. Overall, 23 out of 78 sera specifically reacted with at least one recombinant antigen, allowing serological diagnosis of M. pneumoniae infection in 13 patients (33%). In particular, a constant IgG titre towards GST-EC12 and other recombinant antigens was revealed in 5 patients (B2, B28-B31), while an increase of IgG titre towards single antigens and/or EC12 was revealed in 3 patients (B32-B34) with a negative diagnosis of M. pneumoniae infection on the basis of the CF assay and in 5 patients (B35-B39) who were not checked for M. pneumoniae infection. By contrast, none of the sera displayed a positive result when the commercial assay was used.

DISCUSSION

M. pneumoniae infection occurs endemically and epidemically worldwide, hence it should be considered in the differential diagnosis of upper and lower respiratory tract diseases [START_REF] Waites | Mycoplasma pneumoniae and its role as a human pathogen[END_REF][START_REF] Baum | Mycoplasma pneumoniae and atypical pneumonia[END_REF][START_REF] Atkinson | Epidemiology, clinical manifestations, pathogenesis and laboratory detection of Mycoplasma pneumoniae infections[END_REF]. Moreover, M. pneumoniae colonization is believed to be strongly implicated in the pathogenesis and exacerbation of asthma, and it seems of certain concern in other chronic diseases [START_REF] Atkinson | Epidemiology, clinical manifestations, pathogenesis and laboratory detection of Mycoplasma pneumoniae infections[END_REF][START_REF] Johnson | Distribution of Mycoplasma pneumoniae and Mycoplasma salivarium in the synovial fluid of arthritis patients[END_REF][START_REF] Kraft | Detection of Mycoplasma pneumoniae in the airways of adults with chronic asthma[END_REF][START_REF] Kraft | Mycoplasma pneumoniae and Chlamydia pneumoniae in asthma: effect of clarithromycin[END_REF][START_REF] Ramirez | Relationship between rheumatoid arthritis and Mycoplasma pneumoniae: a case-control study[END_REF][START_REF] Roediger | Intestinal mycoplasma in Crohn's disease[END_REF][START_REF] Sutherland | Asthma and atypical bacterial infection[END_REF]. The clinical management of this "atypical" pathogen requires aimed therapy but a standardized and rapid diagnostic test is lacking, due to inherent limitations of current microbiological, molecular and serological assays [START_REF] Atkinson | Epidemiology, clinical manifestations, pathogenesis and laboratory detection of Mycoplasma pneumoniae infections[END_REF].

The present study evaluated the diagnostic utility of serological assays employing recombinant proteins, composed by distinct antigenic regions of M. pneumoniae gene products. The main objective of our work was, therefore, the provision of "artificial antigens" and their use for the development of selective diagnostic immunoassays. Three distinct antigenic regions of M. pneumoniae proteins [START_REF] Beghetto | Discovery of new Mycoplasma pneumoniae antigens by use of a whole genome lambda-display library[END_REF] were used individually (P1, P30 and MNP456) or further assembled into a chimeric product (EC12). By using a standard bacterial expression system, all recombinant antigens were purified under native condition in large amounts and were found to have good solubility and elevated purification yield, which are fundamental prerequisites for the commercial use of recombinant productsantigens.

The immunoreactivity of EC12 as well as the single antigenic regions composing the chimera was assessed with IgG antibodies present in sera from patient hospitalized for M. pneumoniae infection. All IgG assays employing recombinant antigens showed excellent sensitivity and specificity, with the EC12-based assay reaching the best sensitivity (93.6%), based on the available sera used in this study. The results of the chimeric antigen immunoassay were in agreement with those obtained using the CF assay since 92% of the patients who received a positive diagnosis by the CF analysis were found to be positive in the EC12 Rec-ELISA.

Because a "gold-standard" for the serological diagnosis of Mycoplasma infection is currently unavailable, theThe results of the CF test were also compared to those obtained with a commercial assay which utilizes the whole M. pneumoniae cell extract as the antigen.

Surprisingly, only 31% of subjects having specific anti-Mycoplasma antibodies, as measured by the CF test, were found to be positive with by the commercial ELISA.

The immunoreactivity of recombinant antigens was then investigated with sera from patients without a defined etiological diagnosis of M. pneumoniae infection but with a clinical picture compatible with atypical pneumonia. Noteworthy, the presence of IgG towards EC12 was revealed in serum samples from six patients who received a negative diagnosis of M. pneumoniae infection by the CF test. Moreover, IgG antibodies against the recombinant antigens were detected in sera from seven patients who were not checked for M. pneumoniae infection. By contrast, none of these sera positively reacted with the commercial ELISA, thus further highlighting the better sensitivity of assays based on recombinant antigens.

Previous studies have demonstrated the clinical usefulness of recombinant antigens for the serological diagnosis of M. pneumoniae infection in humans [START_REF] Chaudhry | Expression and immunological characterization of the carboxy-terminal region of the P1 adhesin protein of Mycoplasma pneumoniae[END_REF][START_REF] Drasbek | Immune response to Mycoplasma pneumoniae P1 and P116 in patients with atypical pneumonia analyzed by ELISA[END_REF][START_REF] Duffy | Indirect enzyme-linked immunosorbent assay for detection of immunoglobulin G reactive with a recombinant protein expressed from the gene encoding the 116-kilodalton protein of Mycoplasma pneumoniae[END_REF][START_REF] Dumke | Characterisation of subtype-and variantspecific antigen regions of the P1 adhesin of Mycoplasma pneumoniae[END_REF][START_REF] Varshney | Cloning, expression, and immunological characterization of the P30 protein of Mycoplasma pneumoniae[END_REF]. Also, the use of recombinant productsantigens has been evaluated for different types of infections, particularly those regarding veterinary Mycoplasma species [START_REF] Abdelmoumen Mardassi | A recombinant antigen-based ELISA for the simultaneous differential serodiagnosis of Mycoplasma gallisepticum, Mycoplasma synoviae and Mycoplasma meleagridis infections[END_REF][START_REF] Büyüktanir | A recombinant PvpA protein-based diagnostic prototype for rapid screening of chicken Mycoplasma gallisepticum infections[END_REF][START_REF] Fusco | Development of a sensitive and specific enzyme-linked immunosorbent assay based on recombinant antigens for rapid detection of antibodies against Mycoplasma agalactiae in sheep[END_REF]. However, the composition of the protein cocktail representative of the M. pneumoniae antigenic repertoire and usable for antibody detection in humans remains an open question requiring further investigations. The present study demonstrates that the use of recombinant antigens is effective in distinguishing M. pneumoniae-infected individuals from uninfected individuals and that assays with such antigens have improved performance characteristics than compared to those using the whole-cell bacterial antigen, thus providing the basis of standardized assays for the serodiagnosis of M. pneumoniae diseases.

Line 15 pag 13 .

 13 By the commercial ELISA instead of with Pag 13, line 44-50. Grammatically incorrect Pag 13 line 57: use as compared to instead of 'than'

Formatted:

  Font: Not Italic Formatted: Font: Not Italic Formatted: Font: Italic  group A (n=94), was composed of samples from 42 paediatric patients (males, 52.4%; median age, 10 years; interquartile range [IQR], 8-12) and 5 adults (males, 20%; median age, 21 years; IQR, 20-28) who had a clinical picture compatible with atypical pneumonia and a defined diagnosis of M. pneumoniae infection by the CF test and/or PCR assays on pharyngeal swab or sputum;  group B (n=78), was composed of samples from 35 paediatric patients (males, 60%; median age 4 years, IQR 3-6) and 4 adults (males, 25%; median age, 24 years; IQR, 22-30), who had a clinical picture compatible with atypical pneumonia, but with: (i) a M. pneumoniae infection not previously defined (available methods with negative results or not performed); (ii) a direct or indirect diagnostic investigation for Chlamydia pneumoniae and respiratory viruses with negative results; (iii) no evidence of infection by other pulmonary bacterial pathogens (e.g. Streptococcus pneumoniae, Haemophilus influenzae);  group C (n=12), was composed of control sera from 12 paediatric patients affected by Streptococcus pneumoniae pneumonia and that resulted negative for M. pneumoniaespecific serology and PCR analysis. Two sera were collected from each patient of groups (A) and (B); the first sample at the time of recovery and the second after dismissing (usually 2-3 weeks after hospitalization).

Table 1

 1 Immunoreactivity of recombinant antigens with IgG antibodies in sera from patients with M.

	pneumoniae infection (group A)
	Patient	Age (years)	Serum sample	Days from first sample PCR	a	IgG CF b	IgG ELISA	c	IgG Rec-ELISA	d
									GST-P1 GST-P30 GST-MP456 GST-EC12
	A1	11	A1a A1b	+ 12	n.d.		NEG 1/128	0.075 0.222	0.191 2.942	0.077 0.972	0.081 2.469	0.173 2.774
	A2	15	A2a A2b	+ 21	n.d.		NEG 1/128	0.121 0.538	0.092 0.760	0.106 2.990	0.094 0.092	0.125 2.809
	A3	13	A3a A3b	+ 17	n.d.		<1/512 0.175 >1/102 0.202	1.308 2.354	0.579 1.174	0.122 0.567	0.815 1.713
	A4	12	A4a A4b	+ 15	n.d.		4 NEG >1/102	0.084 0.104	0.060 0.045	0.066 0.048	0.060 0.052	0.124 0.103
	A5	12	A5a A5b	+ 17	n.d.		4 <1/16 1/512	0.188 0.468	0.055 2.257	0.064 0.398	0.071 2.923	0.161 2.942
	A6	10	A6a A6b	+ 36	n.d.		NEG 1/64	0.100 0.564	0.161 1.949	0.202 2.958	0.140 0.088	0.185 2.459
	A7	10	A7a A7b	+ 16	n.d.		1/256 1/1024 0.317 0.075	0.073 0.435	0.140 1.174	0.071 0.063	0.129 0.626
	A8	12	A8a A8b	+ 38	n.d.		NEG 1/64	0.073 0.639	0.073 0.351	0.075 2.123	0.069 0.084	0.120 0.613
	A9	9	A9a		n.d.		NEG	0.105	0.064	0.066	0.069	0.128
			A9b	+ 17			1/128	0.395	1.508	2.400	0.888	1.040
	A10	11	A10a		n.d.		1/512	0.301	0.477	0.140	0.076	0.294
			A10b	+ 15			1/512	0.468	1.032	1.737	0.137	1.175
	A11	7	A11a		n.d.		NEG	0.562	0.112	0.124	0.093	0.157
			A11b	+ 13			1/128	0.269	0.864	1.253	0.070	0.962
	A12	5	A12a		n.d.		>1/102	0.379	2.906	3.013	0.083	2.992
			A12b	+ 12			>1/102	0.443	2.954	2.880	0.085	2.846
	A13	10	A13a A13b	+ 24	n.d.		4 NEG 4 1/1024 0.234 0.077	0.056 2.527	0.055 0.115	0.059 0.990	0.110 1.832
	A14	20	A14a		n.d.		1/256	0.151	0.075	0.309	0.058	0.309
			A14b	+ 16			>1/102	0.348	0.338	2.213	0.347	1.365
	A15	2	A15a A15b	+ 20	n.d.		NEG 4 1/32	0.179 0.201	0.093 0.543	0.076 1.045	0.069 0.085	0.136 0.934
	A16	1.3	A16a		n.d.		1/512	0.086	0.054	0.052	0.053	0.118
			A16b	+ 11			>1/102	0.126	0.519	0.683	0.061	1.181
	A17	13	A17a A17b	+ 14	n.d.		1/32 4 >1/102	0.146 0.033	0.105 0.055	0.127 0.116	0.115 0.074	0.171 0.238
	A18	10	A18a A18b	+ 13	n.d.		>1/102 4 >1/102	0.138 0.431	0.368 1.193	0.281 1.053	0.294 0.456	0.587 2.263
	A19	12	A19a A19b	+ 16	n.d.		4 1/64 4 1/128	0.240 0.219	0.077 0.333	0.240 1.115	0.062 0.080	0.209 1.022
	A20	15	A20a		n.d.		NEG	0.133	0.068	0.090	0.062	0.123
			A20b	+ 15			1/128	0.226	0.354	1.312	0.094	1.418
	A21	7	A21a		n.d.		NEG	0.045	0.095	0.092	0.088	0.125
			A21b	+ 14			1/32	0.041	0.189	0.109	2.952	2.938
	A22	10	A22a		n.d.		NEG	0.028	0.066	0.063	0.059	0.054
			A22b	+ 13			1/152	0.229	0.332	0.157	0.092	0.229
	A23	14	A23a		n.d.		1/32	0.096	0.088	0.166	0.077	0.126

a PCR, Polymerase Chain Reaction on sputum and/or pharyngeal swab. b CF, Complement Fixation test expressed in serum dilution. c Cut off value for the commercial IgG ELISA was 0.358.

Table 2

 2 Diagnostic performance of the IgG ELISAs with sera from M. pneumoniae infected

	individuals
	IgG assay	No. of positive diagnosis a	Sensitivity (%)	Specificity (%)	PPV b (%)	NPV b (%)	p c
	GST-P1	41	87.2	100	100	66.7	< 0.001
	GST-P30	38	80.9	100	100	57.1	< 0.001
	GST-MP456 19	40.4	100	100	30.0	0.005
	GST-EC12	44	93.6	100	100	80.0	< 0.001
	IBL assay	17	36.2	100	100	27.3	0.125

a Patients with at least one positive result by analysing paired sera.

b PPV, positive predictive value; NPV, negative predictive value. c p values were determined by two-tailed Fisher exact test by comparison of the results for sera from patients with M. pneumoniae infection (n=47) and sera from patients with pneumococcal pneumonia (n=12)

Table 3

 3 Immunoreactivity of recombinant antigens with sera from patients with a suspected M.Bold-face type. value greater than cut off; n.d.. not determined; NEG. negative.

	pneumoniae infection (group B)
	Patient	Age (years)	Serum sample	Days from first sample	IgG CF a	IgG ELISA	b	IgG Rec-ELISA	c
							GST-P1	GST-P30 GST-MP456 GST-EC12
	B1	4	B1a		n.d.	0.040	0.058	0.062	0.100	0.069
			B1b	+ 12		0.041	0.062	0.063	0.098	0.071
	B2	5	B2a		NEG	0.050	0.062	0.107	0.056	0.220
			B2b	+ 10		0.055	0.089	0.114	0.070	0.277
	B3	4	B3a		NEG	0.092	0.103	0.111	0.091	0.081
			B3b	+ 12		0.066	0.104	0.097	0.067	0.068
	B4	3	B4a		n.d.	0.053	0.091	0.091	0.084	0.078
			B4b	+ 14		0.049	0.063	0.066	0.121	0.058
	B5	6	B5a		NEG	0.041	0.086	0.075	0.057	0.052
			B5b	+ 16		0.092	0.071	0.081	0.033	0.066
	B6	4	B6a		NEG	0.046	0.135	0.092	0.090	0.157
			B6b	+ 11		0.039	0.097	0.061	0.051	0.082
	B7	4	B7a		NEG	0.044	0.057	0.056	0.051	0.052
			B7b	+ 15		0.079	0.057	0.050	0.051	0.047
	B8	2.3	B8a		n.d.	0.046	0.055	0.052	0.059	0.047
			B8b	+ 30		0.048	0.049	0.046	0.049	0.052
	B9	2	B9a		NEG	0.041	0.081	0.069	0.066	0.069
			B9b	+ 16		0.041	0.068	0.057	0.066	0.058
	B10	1.7	B10a		n.d.	0.039	0.062	0.058	0.062	0.206
			B10b	+ 26		0.065	0.076	0.066	0.065	0.168
	B11	8	B11a		NEG	0.050	0.059	0.072	0.042	0.063
			B11b	+ 11		0.075	0.073	0.084	0.053	0.062
	B12	3	B12a		n.d.	0.041	0.113	0.080	0.063	0.091
			B12b	+ 18		0.036	0.117	0.097	0.063	0.071
	B13	3	B13a		n.d.	0.038	0.112	0.086	0.067	0.077
			B13b	+ 31		0.039	0.102	0.086	0.062	0.063
	B14	5	B14a		n.d.	0.043	0.089	0.081	0.087	0.064
			B14b	+ 15		0.041	0107	0.077	0.080	0.067
	B15	4	B15a		n.d.	0.159	0.123	0.117	0.119	0.108
			B15b	+ 12		0.045	0.103	0.088	0.072	0.082
	B16	5	B16a		n.d.	0.039	0.079	0.070	0.068	0.066
			B16b	+ 9		0.035	0.067	0.064	0.059	0.063
	B17	23	B17a		n.d.	0.077	0.058	0.084	0.107	0.132
			B17b	+ 17		0.056	0.055	0.074	0.086	0.112
	B18	1.6	B18a		n.d.	0.037	0.097	0.079	0.091	0.104
			B18b	+ 8		0.045	0.086	0.074	0.069	0.087
	B19	2	B19a		n.d.	0.054	0.068	0.063	0.065	0.057
			B19b	+ 12		0.038	0.084	0.066	0.059	0.059
	B20	5	B20a		n.d.	0.034	0.097	0.077	0.061	0.060
			B20b	+ 34		0.037	0.093	0.072	0.067	0.077
	B21	2	B21a		n.d.	0.063	0.118	0.069	0.064	0.072
			B21b	+ 13		0.041	0.133	0.066	0.102	0.111
	B22	5	B22a		n.d.	0.048	0.089	0.072	0.078	0.064
			B22b	+ 24		0.037	0.059	0.064	0.069	0.066
	B23	3	B23a		n.d.	0.031	0.065	0.071	0.070	0.064
			B23b	+ 10		0.039	0.078	0.083	0.050	0.062

a CF. Complement Fixation test. b Cut off value for the commercial IgG ELISA was 0.358. c Cut off values for IgG Rec-ELISAsRec-ELISA's were 0.232 (GST-P1). 0.268 (GST-P30).

d Cut off values for the IgG Rec-ELISAsRec-ELISA's were 0.232 (GST-P1), 0.268 (GST-P30), 0.209 (GST-MPN456), 0.219 (GST-EC12).Bold-face type, value greater than cut off; n.d., not determined; NEG, negative.

ACKNOWLEDGMENTS

We are grateful to Carla Cellesi for very helpful suggestion and advice during this work. We acknowledge Luca Bruno and Andrea Spadoni for skillful technical assistance.

Results. Our study demonstrates that all IgG Rec-ELISA's using recombinant antigens have better sensitivity with respect to the commercial assay.Our study demonstrates that all IgG Rec-ELISAs using recombinant antigens have performance characteristicssensitivity significantly better than those of the corresponding commercial assay. Furthermore, we show that the use of chimeric antigens improve the performance of the assays.

Conclusions. The use of recombinant antigens is effective in distinguishing M. pneumoniaeinfected from uninfected individuals and shows that immunoassays based on recombinant productsantigens could provide the basis for standardized commercial tests for the serodiagnosis of M. pneumoniae diseases.

CONFLICT OF INTEREST

The authors declare that they have no conflict of interest.

.


Dr L Poirel

Mourad Drissi

Pauline D Mugnier

Z Baba

Patrice Nordmann

Z Baba Ahmed

Carbapenemase-producing Acinetobacter baumannii, Algeria

Keywords: Carbapenemase-producing Acinetobacter baumannii, Algeria oxacillinase, OXA-58, carbapenemase, Acinetobacter baumannii

Sir,

Acinetobacter baumannii is increasingly reported as a nosocomial pathogen [START_REF] Peleg | Acinetobacter baumannii: emergence of a successful pathogen[END_REF].

Carbapenem resistance is now observed worldwide in A. baumannii isolates, leading to limited therapeutic options. Several mechanisms are responsible for resistance to carbapenems in A. baumannii [START_REF] Peleg | Acinetobacter baumannii: emergence of a successful pathogen[END_REF].

The carbapenem-hydrolyzing β-lactamases in A. baumannii are either metallo-β-lactamases (MBLs) or oxacillinases (carbapenem-hydrolyzing class D β-lactamases [CHDLs]) [START_REF] Poirel | Carbapenem resistance in Acinetobacter baumannii: mechanisms and epidemiology[END_REF]. Four major subgroups of acquired CHDLs have been identified in A. baumannii, being OXA-23, OXA-40, OXA-58, and OXA-143 β-lactamase groups in addition to the naturally-occurring OXA-51 ß-lactamase [START_REF] Poirel | Carbapenem resistance in Acinetobacter baumannii: mechanisms and epidemiology[END_REF][START_REF] Poirel | Diversity, epidemiology, and genetics of class D ßlactamases[END_REF]. The bla OXA-58 gene has been identified worldwide, but mostly from Europe [START_REF] Poirel | Diversity, epidemiology, and genetics of class D ßlactamases[END_REF].

In this study, the molecular basis of the carbapenem resistance in Acinetobacter spp. was investigated in human isolates collected from April to November 2008 at the University Hospital of Tlemcen (Algeria). A total of 12 Acinetobacter spp. isolates have been recovered from seven different patients (urine, broncheal and rectal specimens) hospitalized at the intensive care unit (ICU). In addition, 4 isolates recovered from the environnemental screening performed in that ICU ward were included. MICs of ticarcillin, ticarcillin plus clavulanic acid (4 mg/L), piperacillin, piperacillin plus tazobactam (4 mg/L), ceftazidime, cefepime, aztreonam, imipenem, gentamicin, tobramycin, amikacin and ciprofloxacin were determined by the agar dilution technique in Mueller Hinton medium. Results were interpreted according to the guidelines of the Clinical and Laboratory Standards Institute [START_REF]Performance standards for antimicrobial susceptibility testing; 18th informational supplement M100-S18[END_REF].

All isolates were multidrug resistant, including resistance to the broad-spectrum cephalosporins, aztreonam, imipenem (MIC of 16 mg/L), fluoroquinolones, and aminoglycosides. All the 16 isolates studied here showed identical pulsed-field gel electrophoresis patterns, suggesting that they were closely related. The presence of bla OXA carbapenemase genes was searched by PCR using previously described primer pairs for the bla OXA-23 , bla OXA-40 , and bla OXA-58 genes, representative of the four OXA clusters [START_REF] Poirel | Genetic structures at the origin of acquisition and expression of the carbapenem-hydrolyzing oxacillinase gene bla OXA-58 in Acinetobacter baumannii[END_REF]. All of these carbapenem-resistant strains were positive for the bla OXA-58 gene, suggesting that ßlactamase OXA-58 significantly contributed to the imipenem resistance in these Acinetobacter strains. The bla OXA-58 amplicons were fully sequenced, demonstrating 100% identity to the bla OXA-58 sequence, although the OXA-58 point-mutant derivative OXA-97 had been recently identified in Tunisia [START_REF] Poirel | Carbapenem-resistant Acinetobacter baumannii isolates from Tunis producing the OXA-58-like carbapenem-hydrolyzing oxacillinase OXA-97[END_REF]. Plasmid extraction followed by electro-transformation into an A. baumannii recipient strain as described revealed that the bla OXA-58 gene was located on a plasmid that transferred a decreased susceptibility to carbapenems but not to ceftazidime. Genetic investigations showed that the bla OXA-58 gene was bracketed by insertion sequences (ISAba3-bla OXA-58 -ISAba3), which were likely at the origin of its acquisition and expression [START_REF] Poirel | Genetic structures at the origin of acquisition and expression of the carbapenem-hydrolyzing oxacillinase gene bla OXA-58 in Acinetobacter baumannii[END_REF]. The plasmid mediated bla OXA-58 gene described in this study could contribute to a rapid spread of carbapenem resistance in Acinetobacter in our hospital. This work constitutes the first evidence of an outbreak situation involving CHDL-producing A. baumannii strains in Algeria.

Funding

Transparency declaration

  This work was funded mostly by a grant from the INSERM (U914), and by grants from the European Community (TROCAR, HEALTH-F3-2008-223031), and from the Ministère de l'Education Nationale et de la Recherche (UPRES-EA3539), Université Paris XI, France.