# High Intraspecific Genetic Diversity ofNocardia brasiliensis, a Pathogen Responsible for Cutaneous Nocardiosis Found in France: Phylogenetic Relationships by Usingsod andhsp65 Genes

Authors: D. Kosova-Maali; E. Bergeron; Y. Maali; T. Durand; J. Gonzalez; D. Mouniée; H. Sandoval Trujillo; P. Boiron; M.-C. Salinas-Carmona; V. Rodriguez-Nava
Journal: BioMed Research International
 (2018)

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## Abstract

This study aims at genetic characterization and phylogenetic relationships ofNocardia brasiliensis focusing by using housekeepingrrs,hsp65,andsodA genes.N. brasiliensis is the species responsible for 80% of cases of actinomycetoma, one form of cutaneous nocardiosis which occurs mainly in tropical regions reaching immunocompetent patients in which the disease can lead to amputation. We analyze 36 indigenous cases ofN. brasiliensis that happened in France. Phylogenetic analysis targetingrrs gene showed no robustness at phylogenetic nodes level. However, the use of a concatenation ofhsp65 andsodA genes showed that the tested strains surprisingly ranked in 3 well-defined genotypes. Genotypes 2 and 3 were phylogenetically closer to each other and both diverged from genotype 1 sustained by a high bootstrap of 81%. This last genotype hosts all the cases of pulmonary forms (3), the sole cerebral form, and almost all the cases of immunocompromised patients (3 out of 4). Moreover, excepting one of them, all the strains belonging to this group present a susceptibility to imipenem which is not the case in the other genotypes that rarely count among them strains being susceptible to this drug. The haplotype diversity (Hd) ofhsp65 (0.927) andsodA (0.885) genes was higher than that ofrrs (0.824). For this gene, we obtained 16 polymorphic sites whereas, forhsp65 andsodA genes, up to 27 and 29 were identified, respectively. This study reveals that these two genes have an important genetic discriminatory power for the evaluation of the intraspecies genetic variability ofN. brasiliensisand they may be useful for identification purposes at species level. This study also reveals the possible existence of a new species harbored by genotype 1.


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## Body

## 1. Introduction

Nocardia is a genus belonging to the aerobic actinomycetes group of bacteria which are Gram-positive bacilli and showing branching filamentous forms [1]. They are saprophytic ubiquitous bacteria which can be found in several environments such as fresh water and saltwater, soil, dust, decaying vegetation, and decaying fecal deposits from animals [1]. Nevertheless, these environmental bacteria can be opportunistic pathogens and lead to human infectious diseases called “nocardiosis” [2]. Nocardiosis can be discriminated into two groups: invasive infection, mainly caused byN. asteroides,presenting commonly as pneumonia in patients who are immunocompromised, have underlying chronic lung disease, and are with a possible dissemination to other organs [3], and cutaneous infection via a cut or abraded skin, which can be manifest clinically as (i) abscess and cellulitis, (ii) lymphangitis, (iii) skin infection secondary to dissemination, and (iv) actinomycetoma. This latter group is the most amazing infection due to their severity characterized by the presence of tumefaction, subcutaneous nodules, destructive granulomata, fistulas, and pus [2, 4].N. brasiliensis is the species isolated from the majority (approximately 80%) of cases of cutaneous nocardiosis, especially in actinomycetoma [2]. This species is more commonly isolated in areas with tropical or subtropical climates such as South America, Asia, and Africa. Due to false diagnosis, rural lifestyles, and poor access to care in these countries,N. brasiliensis nocardiosis constitutes a real public health problem that can lead, in the absence of treatment, to amputations and death in young populations. On the basis of epidemiological surveys conducted in France, the number of cases of nocardiosis between 2000 and 2007 according to the French Nocardiosis Observatory (OFN) was 607 withN. farcinica andN. nova being the most frequent species [5]. However, no data currently exists on the phylogenetic relationships between the indigenousN. brasiliensis strains of tropical origin and native strains isolated in France. Routine genus/species identification ofNocardiawas based on macroscopic, microscopic, and biochemical characteristics. The methods described by Boiron et al. [6] were used to determine the decomposition of adenine, casein, hypoxanthine, tyrosine, and xanthine. In addition to the phenotype-based methods, species-level identification is mainly genetically based, nowadays. Classically, 16S rRNA(rrs) gene sequencing is generally used for the species-level identification [7, 8], but it fails to discriminate among some species ofNocardia because it does not have enough polymorphism to differentiate them at the species level. Multilocus sequence analysis (MLSA) using concatenated sequences of several housekeeping genes such as superoxide dismutase A(sodA) and heat shock protein 65(hsp65) has been increasingly used to provide higher accuracy and discriminatory power in the molecular identification ofNocardia spp. [9, 10]. Indeed, a recent study seeking to identify new molecular targets shows that the polymorphism observed in thesodA gene sequence contains variable regions that allow the discrimination of closely relatedNocardia species [9].The aim of the present study was to perform a genetic characterization and assess the phylogenetic relationships ofNocardia brasiliensis focusing on using housekeepingrrs,hsp65, andsodA genes, for 36 autochthonousN. brasiliensisstrains isolated in France and analyzed by the OFN between 2002 and 2012. Phenotypic characterization was also conducted by assessing antimicrobial resistance profiles, metabolic profiles, and culture condition.


## 2. Materials and Methods

### 2.1. Bacterial Strains and Culture Media

A collection of 36 human clinical strains ofN. brasiliensis was studied (Table 1). All strains were identified as such, at species level by the French Nocardiosis Observatory (OFN) by genetic approach. Moreover, sixNocardia reference strains belonging toN. brasiliensis clade [9] were also used:N. brasiliensis ATCC 19296T (unknown),N. altamirensis DSM 44997T (karstic cave),N. boironii DSM 101696T (pus sample),N. iowensis DSM 45197T (garden soil),N. tenerifensis DSM 44704T (rhizosphere), andN. vulneris DSM 45737T (human leg wound). Prior to the assays, strains were cultured 72 hours in Bennett medium (made in the laboratory) aerobically at 37°C.Table 1
Table of clinical strains including the type of tropism observed in host, their correspondingsodA/hsp65 genotypes, and drug phenotypes.
Sample date Nature of sampling Patient record Immunosuppressed GenotypesodA/hsp65 Tropism Pristinamycin Imipenem Amikacin Trimethoprim + sulfamethoxazole 04/2002 Intraoperative tissue 02.56 No G3 Cutaneous R R S S 12/2003 Pus from cutaneous thigh abscess 04.21 Yes G1 Cutaneous R S S S 2004 Pus from cutaneous abscess 04.101 No G3 Cutaneous R R S S 07/2005 Expectoration then LBA 05.64 Yes G1 Lung R S S S 01/2005 Skin biopsy 05.12 No G1 Cutaneous R S S S 07/2005 Phalanx biopsy 05.63 No G3 Cutaneous R R S S 2005 CSF 05.77 No G1 Brain R R S S 11/2005 - 3247 No G3 Unknown R S S S 2007 Cutaneous abscess 07.168 No G3 Cutaneous R R S S 10/2008 Wound of forehead 08.178 No G3 Cutaneous R S S S 11/2008 Subcutaneous abscess 08.188 No G3 Subcutaneous R R S S 10/2008 Elbow abscess 2985 No G3 Cutaneous R R S S 03/2008 - 9044 No G2 Unknown R R S S 03/2009 Pus of leg abscess 09.71 No G2 Cutaneous R S S S 04/2009 Bronchial aspiration 09.106 No G1 Lung R S S S 10/2009 Bronchial aspiration 09.244 Yes G1 Lung R S S S 12/2009 Finger skin 09.280 No G3 Cutaneous R R S S 10/2009 Pus from the lip 10.16 No G3 Cutaneous R R S S 02/2010 Finger abscess 10.35 No G2 Cutaneous R R S S 05/2010 Hand abscess 10.82 No G3 Cutaneous R R S S 05/2010 Toe abscess 10.93 Yes G3 Cutaneous R R S S 09/2010 Leg wound 10.146 No G3 Cutaneous R R S S 11/2010 Pus finger 10.180 No G3 Cutaneous R R S S 11/2010 Leg wound 12786 No G2 Cutaneous R R S S 04/2010 - 14229 No G2 Unknown R S S S 07/2010 Sepsis hand 45762 No G3 Cutaneous R R S S 2011 Pus 11.44 No G2 Cutaneous R R S S 05/2011 Cutaneous abscess 11.73 No G1 Cutaneous R S S S 05/2011 Hand wound 11.80 No G1 Cutaneous R S S S 08/2011 Cutaneous 11.116 No G2 Cutaneous R R S S 2011 Thigh abscess 11.140 No G2 Cutaneous R R S S 09/2011 Knee wound 11.151 No G3 Cutaneous R R S S 2011 Hand wound 11.172 No G3 Cutaneous R R S S 12/2011 Cutaneous abscess 11.189 No G3 Cutaneous R R S S 2012 - 12.08 No G1 Unknown R S S S 02/2012 Cutaneous abscess 12.28 No G3 Cutaneous R R S S


### 2.2. Growth Test on Culture Media

From 0.5 McF bacterial suspension, bacterial growth was evaluated on three culture media: (i) bromocresol purple (BCP) (Biomérieux, Marcy l’étoile), (ii) Bennett (made in the laboratory), and (iii) Middlebrook (Biomérieux, Marcy l’étoile). One hundred microliters from bacterial suspension standardized was inoculated on the different plate of culture media. The plates were incubated at 37°C and the observations were performed at 48, 72, and 96 hours.


### 2.3. Antimicrobial Susceptibility

The susceptibility of the isolates to different antimicrobials was determined by disk diffusion method with a panel of 31 antibiotics (Biorad, Marnes-la-Coquette France) on Muller Hinton E medium (Biomérieux, Marcy l’étoile, France). Susceptibility testing was done with amikacin 30μg, gentamycin 15 μg, tobramycin 10 μg, ciprofloxacin 5 μg, levofloxacin 5 μg, moxifloxacin 5 μg, minocycline 30 μg, doxycycline 30 μg, tigecycline 15 μg, cefotaxime 30 μg, ceftriaxone 30 μg, cefepime 30 μg, cefuroxime 30 μg, amoxicillin 25 μg, amoxicillin + clavulanic acid 20/10 μg, ampicillin 10 μg, ertapenem 10 μg, meropenem 10 μg, imipenem 10 μg, vancomycin 30 μg, pristinamycin 15 μg, erythromycin 15 μg, trimethoprim + sulfamethoxazole 1.25/23.75 μg, rifampicin 30 μg, and linezolid 30 μg.From visible colonies, bacterial suspension was done in sterile water, using a cotton swab to obtain a concentration of 0.5 McFarland according to the Clinical and Laboratory Standards Institute standard M24-A2 [11]. Seeding was done according to the swab method. In this latter, the bacterial inoculum was spread on the agar using a sterile cotton swab in three different directions. The disks were dispensed with a dispenser and the plates were incubated at 37°C for 72 hours and read manually according to the thresholds defined in the recommendations of the SFM 2013 [12].


### 2.4. Substrate Degradation

The methods of Boiron et al. [6], Goodfellow et al. [13, 14], and Goodfellow and Lechevalier [15] were used to determine the decomposition of adenine, casein, and uric acid [9]. Clinical strains ofN. brasiliensis and the strains of species belonging to theN. brasiliensis clade (N. brasiliensis, N. altamirensis, N. iowensis, N. tenerifensis,N. boironii, andN. vulneris) were tested [9]. StrainsN. boironii DSM 101696T,N. brasiliensis ATCC 19296T, andN. vulneris DSM 45737T were incubated at 37°C, andN. altamirensis DSM 44997T,N. tenerifensis DSM 44704T, andN. iowensis DSM 45197T were incubated at 28°C [9]. The readings were performed at 3, 7, 10, 14, 17, and 21 days.


### 2.5. Methods of DNA Extraction

DNA extraction fromNocardia strains was performed with achromopeptidase according to the method reported by Rodríguez-Nava et al. [10]. Colonies were picked off with a loop, and one loopful was suspended in 200 μL of sterile water containing a dozen glass beads and vortexed for 5 minutes. The mixture was then incubated for 15 minutes at 70°C. The suspension supplemented with 3.4 μL of achromopeptidase (Sigma, Steinheim, Germany) at 10 U/mL was incubated at 55°C for 15 minutes. The suspensions were then centrifuged for 5 minutes at 13,000 rpm. The supernatants were stored at −20°C until use.


### 2.6. Amplification and Sequencing

Gene rrs. A 606-bp fragment of the rrs gene was amplified with primers Noc1, 5′-GCTTAACACATGCAAGTCG-3′, and Noc2, 5′-GAATTCCAGTCTCCCCTG-3′, and PCR program and reaction mixture were carried out according the recommendations of Rodríguez-Nava et al. [10].Gene hsp65. A 441-bp fragment of thehsp65 gene encoding the 65-kDa heat shock protein was amplified with primers described by Telenti et al. (TB11: 5′-ACCAACGATGGTGTGTCCAT-3′ and TB12: 5′-CTTGTCGAACCGCATACCCT-3′) [16]. PCR program and reaction mixture were carried out according to the recommendations of Sánchez-Herrera et al. [17].Gene sodA. A 440-bp fragment of thesodA gene was amplified and sequenced with primers SodV1 (5′-CAC CAY WSC AAG CAC CA-3′) and SodV2 (5′-CCT TAG CGT TCT GGT ACT G-3′) where Y = C or T, W = A or T, and S = C or G. The amplification was also done according to the recommendations of Sánchez-Herrera et al. [17].All resulting PCR products were sequenced and verified (Biofidal, Lyon, France).The breakpoints for identification based insodA andhsp65 genes are 99% for each one [17, 18]. For therrs gene, a higher breakpoint of 99.6% is used, according to CLSI [19].


### 2.7. Phylogenetic Analysis

Therrs gene sequences which we obtained for the 36 clinical isolates ofN. brasiliensis and the reference strains were aligned manually for the comparative phylogenetic analysis using the Seaview program.MLSA was performed usinghsp65 andsodA sequences of the strains collection. The trimmed aligned sequences were concatenated in the ordersodA-hsp65 to generate an 846 bp sequence using the Seaview program. The Seaview program was also used to infer the evolutionary trees according to the neighbour-joining method [20] and Kimura’s two-parameter model [21]. The robustness of the tree was performed with a bootstrap of 1000 replicates.Taking into account the breakpoints for identification at species level ofsodA andhsp65 genes individually, the breakpoint for concatenated sequence has been also fixed at 99%.


### 2.8. DNA Polymorphism ofrrs,hsp65, andsodA Genes

The number of haplotypes, the haplotype diversity (Hd), the number of polymorphic sites, and other variables were obtained with DnaSP software [22].


## 2.1. Bacterial Strains and Culture Media

A collection of 36 human clinical strains ofN. brasiliensis was studied (Table 1). All strains were identified as such, at species level by the French Nocardiosis Observatory (OFN) by genetic approach. Moreover, sixNocardia reference strains belonging toN. brasiliensis clade [9] were also used:N. brasiliensis ATCC 19296T (unknown),N. altamirensis DSM 44997T (karstic cave),N. boironii DSM 101696T (pus sample),N. iowensis DSM 45197T (garden soil),N. tenerifensis DSM 44704T (rhizosphere), andN. vulneris DSM 45737T (human leg wound). Prior to the assays, strains were cultured 72 hours in Bennett medium (made in the laboratory) aerobically at 37°C.Table 1
Table of clinical strains including the type of tropism observed in host, their correspondingsodA/hsp65 genotypes, and drug phenotypes.
Sample date Nature of sampling Patient record Immunosuppressed GenotypesodA/hsp65 Tropism Pristinamycin Imipenem Amikacin Trimethoprim + sulfamethoxazole 04/2002 Intraoperative tissue 02.56 No G3 Cutaneous R R S S 12/2003 Pus from cutaneous thigh abscess 04.21 Yes G1 Cutaneous R S S S 2004 Pus from cutaneous abscess 04.101 No G3 Cutaneous R R S S 07/2005 Expectoration then LBA 05.64 Yes G1 Lung R S S S 01/2005 Skin biopsy 05.12 No G1 Cutaneous R S S S 07/2005 Phalanx biopsy 05.63 No G3 Cutaneous R R S S 2005 CSF 05.77 No G1 Brain R R S S 11/2005 - 3247 No G3 Unknown R S S S 2007 Cutaneous abscess 07.168 No G3 Cutaneous R R S S 10/2008 Wound of forehead 08.178 No G3 Cutaneous R S S S 11/2008 Subcutaneous abscess 08.188 No G3 Subcutaneous R R S S 10/2008 Elbow abscess 2985 No G3 Cutaneous R R S S 03/2008 - 9044 No G2 Unknown R R S S 03/2009 Pus of leg abscess 09.71 No G2 Cutaneous R S S S 04/2009 Bronchial aspiration 09.106 No G1 Lung R S S S 10/2009 Bronchial aspiration 09.244 Yes G1 Lung R S S S 12/2009 Finger skin 09.280 No G3 Cutaneous R R S S 10/2009 Pus from the lip 10.16 No G3 Cutaneous R R S S 02/2010 Finger abscess 10.35 No G2 Cutaneous R R S S 05/2010 Hand abscess 10.82 No G3 Cutaneous R R S S 05/2010 Toe abscess 10.93 Yes G3 Cutaneous R R S S 09/2010 Leg wound 10.146 No G3 Cutaneous R R S S 11/2010 Pus finger 10.180 No G3 Cutaneous R R S S 11/2010 Leg wound 12786 No G2 Cutaneous R R S S 04/2010 - 14229 No G2 Unknown R S S S 07/2010 Sepsis hand 45762 No G3 Cutaneous R R S S 2011 Pus 11.44 No G2 Cutaneous R R S S 05/2011 Cutaneous abscess 11.73 No G1 Cutaneous R S S S 05/2011 Hand wound 11.80 No G1 Cutaneous R S S S 08/2011 Cutaneous 11.116 No G2 Cutaneous R R S S 2011 Thigh abscess 11.140 No G2 Cutaneous R R S S 09/2011 Knee wound 11.151 No G3 Cutaneous R R S S 2011 Hand wound 11.172 No G3 Cutaneous R R S S 12/2011 Cutaneous abscess 11.189 No G3 Cutaneous R R S S 2012 - 12.08 No G1 Unknown R S S S 02/2012 Cutaneous abscess 12.28 No G3 Cutaneous R R S S


## 2.2. Growth Test on Culture Media

From 0.5 McF bacterial suspension, bacterial growth was evaluated on three culture media: (i) bromocresol purple (BCP) (Biomérieux, Marcy l’étoile), (ii) Bennett (made in the laboratory), and (iii) Middlebrook (Biomérieux, Marcy l’étoile). One hundred microliters from bacterial suspension standardized was inoculated on the different plate of culture media. The plates were incubated at 37°C and the observations were performed at 48, 72, and 96 hours.


## 2.3. Antimicrobial Susceptibility

The susceptibility of the isolates to different antimicrobials was determined by disk diffusion method with a panel of 31 antibiotics (Biorad, Marnes-la-Coquette France) on Muller Hinton E medium (Biomérieux, Marcy l’étoile, France). Susceptibility testing was done with amikacin 30μg, gentamycin 15 μg, tobramycin 10 μg, ciprofloxacin 5 μg, levofloxacin 5 μg, moxifloxacin 5 μg, minocycline 30 μg, doxycycline 30 μg, tigecycline 15 μg, cefotaxime 30 μg, ceftriaxone 30 μg, cefepime 30 μg, cefuroxime 30 μg, amoxicillin 25 μg, amoxicillin + clavulanic acid 20/10 μg, ampicillin 10 μg, ertapenem 10 μg, meropenem 10 μg, imipenem 10 μg, vancomycin 30 μg, pristinamycin 15 μg, erythromycin 15 μg, trimethoprim + sulfamethoxazole 1.25/23.75 μg, rifampicin 30 μg, and linezolid 30 μg.From visible colonies, bacterial suspension was done in sterile water, using a cotton swab to obtain a concentration of 0.5 McFarland according to the Clinical and Laboratory Standards Institute standard M24-A2 [11]. Seeding was done according to the swab method. In this latter, the bacterial inoculum was spread on the agar using a sterile cotton swab in three different directions. The disks were dispensed with a dispenser and the plates were incubated at 37°C for 72 hours and read manually according to the thresholds defined in the recommendations of the SFM 2013 [12].


## 2.4. Substrate Degradation

The methods of Boiron et al. [6], Goodfellow et al. [13, 14], and Goodfellow and Lechevalier [15] were used to determine the decomposition of adenine, casein, and uric acid [9]. Clinical strains ofN. brasiliensis and the strains of species belonging to theN. brasiliensis clade (N. brasiliensis, N. altamirensis, N. iowensis, N. tenerifensis,N. boironii, andN. vulneris) were tested [9]. StrainsN. boironii DSM 101696T,N. brasiliensis ATCC 19296T, andN. vulneris DSM 45737T were incubated at 37°C, andN. altamirensis DSM 44997T,N. tenerifensis DSM 44704T, andN. iowensis DSM 45197T were incubated at 28°C [9]. The readings were performed at 3, 7, 10, 14, 17, and 21 days.


## 2.5. Methods of DNA Extraction

DNA extraction fromNocardia strains was performed with achromopeptidase according to the method reported by Rodríguez-Nava et al. [10]. Colonies were picked off with a loop, and one loopful was suspended in 200 μL of sterile water containing a dozen glass beads and vortexed for 5 minutes. The mixture was then incubated for 15 minutes at 70°C. The suspension supplemented with 3.4 μL of achromopeptidase (Sigma, Steinheim, Germany) at 10 U/mL was incubated at 55°C for 15 minutes. The suspensions were then centrifuged for 5 minutes at 13,000 rpm. The supernatants were stored at −20°C until use.


## 2.6. Amplification and Sequencing

Gene rrs. A 606-bp fragment of the rrs gene was amplified with primers Noc1, 5′-GCTTAACACATGCAAGTCG-3′, and Noc2, 5′-GAATTCCAGTCTCCCCTG-3′, and PCR program and reaction mixture were carried out according the recommendations of Rodríguez-Nava et al. [10].Gene hsp65. A 441-bp fragment of thehsp65 gene encoding the 65-kDa heat shock protein was amplified with primers described by Telenti et al. (TB11: 5′-ACCAACGATGGTGTGTCCAT-3′ and TB12: 5′-CTTGTCGAACCGCATACCCT-3′) [16]. PCR program and reaction mixture were carried out according to the recommendations of Sánchez-Herrera et al. [17].Gene sodA. A 440-bp fragment of thesodA gene was amplified and sequenced with primers SodV1 (5′-CAC CAY WSC AAG CAC CA-3′) and SodV2 (5′-CCT TAG CGT TCT GGT ACT G-3′) where Y = C or T, W = A or T, and S = C or G. The amplification was also done according to the recommendations of Sánchez-Herrera et al. [17].All resulting PCR products were sequenced and verified (Biofidal, Lyon, France).The breakpoints for identification based insodA andhsp65 genes are 99% for each one [17, 18]. For therrs gene, a higher breakpoint of 99.6% is used, according to CLSI [19].


## 2.7. Phylogenetic Analysis

Therrs gene sequences which we obtained for the 36 clinical isolates ofN. brasiliensis and the reference strains were aligned manually for the comparative phylogenetic analysis using the Seaview program.MLSA was performed usinghsp65 andsodA sequences of the strains collection. The trimmed aligned sequences were concatenated in the ordersodA-hsp65 to generate an 846 bp sequence using the Seaview program. The Seaview program was also used to infer the evolutionary trees according to the neighbour-joining method [20] and Kimura’s two-parameter model [21]. The robustness of the tree was performed with a bootstrap of 1000 replicates.Taking into account the breakpoints for identification at species level ofsodA andhsp65 genes individually, the breakpoint for concatenated sequence has been also fixed at 99%.


## 2.8. DNA Polymorphism ofrrs,hsp65, andsodA Genes

The number of haplotypes, the haplotype diversity (Hd), the number of polymorphic sites, and other variables were obtained with DnaSP software [22].


## 3. Results

### 3.1. Growth on Culture Medium

The three culture media allowed the growth of clinical strains ofN. brasiliensis. The Bennett medium showed abundant and rapid growth (48 hours). Middlebrook medium showed strong growth but also it was slightly slower (72 hours). The BCP medium presented interesting results with good rapid growth at 48 hours.N. brasiliensis clade tested type strains showed similar patterns to the clinical strains, except thatN. boironii had a difficult growth on BCP and no growth on Middlebrook; this seems a peculiarity of this species.


### 3.2. Antimicrobial Susceptibilities

Eight out of 31 antibiotic molecules tested were active on all the strains’ collection: linezolid, tigecycline, trimethoprim + sulfamethoxazole, moxifloxacin, amikacin, amoxicillin + clavulanic acid, tobramycin, and gentamycin. Regarding the imipenem and pristinamycin molecules, resistance was observed on the majority of clinical isolates ofN. brasiliensis(Table 1).


### 3.3. Degradation of Substrate

The assimilation test of adenine and uric acid proved negative for all the strains tested of theN. brasiliensis clade including clinical and reference ones. The casein degradation test showed that all clinical strains are able to metabolize casein except the clinical strain 12.28. In addition our result showed that some types of strains such asN. vulneris,N. tenerifensis,N. boironii, andN. iowensis are also able to degrade casein in the same way asN. brasiliensis exceptN. altamirensis. Casein is ultimately a marker that can be used for the phenotypic identification of theN. brasiliensis clade and not theN. brasiliensis species as it has been believed for many years.


### 3.4. Phylogeny

Primers Noc1 and Noc2 amplified the expected 606-bp fragment of therrs gene for all the collection strains. Phylogenetic trees (Figure 1) based uponrrs showed homogeneity within clinical strains ofN. brasiliensis. For this, therrs gene is not relevant to show intraspecies diversity.Figure 1
Phylogenetic distribution ofrrs gene of 36N. brasiliensisclinical strains analyzed in this study using neighbour-joining method, Kimura’s two-parameter model, and bootstrap of 1000. Only values of bootstrap significance greater than 50% (Seaview) were reported.In addition, based upon the concatenation ofsodA andhsp65 housekeeping genes, the phylogenetic tree generated (Figure 2) had several distinct genotypes: (i) genotype 1 containing clinical strains, (ii) genotype 2 harboring some clinical strains, and (iii) genotype 3 harboring some clinical strains andN. brasiliensis ATCC 19296T. For the tropicalN. brasiliensisHUJEG01 strain, it is observed that it does not belong to any of the 3 genotypes and is positioned alone in the tree between genotypes 1 and 2. This distribution of clinical strains ofN. brasiliensis in 3 different genotypes shows an intraspecies diversity rather important. To better understand the polymorphism showed by phylogenetic trees, we studied the percentages of the similarities between the sequences. The average percentages of similarities based on therrs gene (Table 2) range from 99.39% to 99.57% between the clinical strains and the 2 reference strains ofN. brasiliensis (type and tropical strains). According to the CLSI, the similarity percentage needed for identification at species level must be greater than or equal to a threshold of 99.6% [19]. The clinical strains that showed a similarity percentage lower than this threshold for both reference strains ofN. brasiliensis were anyway considered as belonging to this species because no higher similarity percentage was obtained for any other species. In the same way, theN. vulneris type strain was also revealed to be close to clinical strains according to the average of percentage of similarity (98.77%). Between the 2 reference strains ofN. brasiliensis the percentage of similarity is higher, up to 99.82%. The percentages of similarities based on the concatenation of thesodA-hsp65 genes (Table 2) decrease and range now from 97.99% to 99.19% between the clinical strains and the 2 reference strains ofN. brasiliensis. Between the type and reference strains ofN. brasiliensis the percentage of similarity does not reach 99% this time. The comparison of the 3 genotypes between them (based on the representation of each genotype by 3 clinical strains) by usingsodA-hsp65 genes shows that genotypes 2 and 3 are closer to each other (98.97% of similarity). The average of the percentages of similarity between genotypes 1 and 2 were 97.97%. and 98.28% between genotypes 1 and 3. Finally this value goes up to 98.97% between genotypes 2 and 3. This means that the more distant genotypes between them are 1 and 2 and the closer ones are 2 and 3.Table 2
Percentage of similarity expressed in interval and mean forsodA-hsp65 and rrs genes.
N. brasiliensis HUJEG01 N. brasiliensis ATCC 19296T N. vulnerisDSM 45737T sodA-hsp65 rrs sodA-hsp65 rrs sodA-hsp65 rrs N. brasiliensisATCC 19296T Similarity average (%) 98.51 99.82 - - - - N. vulnerisDSM 45737T 98.38 98.73 98.51 98.55 - - N. altamirensisDSM 44997T 95.91 98.24 95.41 98.07 95.91 97.47 N. boironiiDSM 101696T 96.53 98.07 96.28 97.89 96.15 97.29 N. iowensis DSM 45197T 94.91 97.89 95.29 97.71 94.54 97.64 N. tenerifensis DSM 44704T 96.28 96.67 96.03 96.49 95.91 96.03  Genotype 1 Similarity range (%) (97.77–98.38) - (97.77–98.26) - (97.52–98.14) - Similarity average (%) 98.01 - 97.99 - 97.67 -  Genotype 2 Similarity range (%) (98.39–98.88) - (98.88–99.38) - (98.63–98.88) - Similarity average (%) 98.73 - 99.19 - 98.80 -  Genotype 3 Similarity range (%) (98.14–98.88) - (98.26–99.00) - (98.01–98.76) - Similarity average (%) 98.63 - 98.76 - 98.50 -  All clinical strains Similarity range (%) - (98.57–100) - (98.38–99.82) - (98.01–99.09) Similarity average (%) - 99.57 - 99.39 - 98.77Figure 2
Phylogenetic distribution of concatenationsodA-hsp65 genes of 36N. brasiliensisclinical strains analyzed in this study using neighbour-joining method, Kimura’s two-parameter model, and bootstrap of 1000. Only values of bootstrap significance greater than 50% were reported.In parallel, an epidemiological study based on the clinical files was carried out, and the data were presented in Table1. In order to know the link between the genetic diversity and the tropism of the clinical strains, a superposition of data was made between the phylogenetic tree obtained by the concatenation ofsodA andhsp65 and the tropism of the clinical strains (Figure 2). Thus, we can see that in genotypes 2 and 3 we have almost all the clinical strains that have a cutaneous tropism except the 08.188 strain which has a subcutaneous tropism. Regarding genotype 1 it is more heterogeneous with various tropism: (i) pulmonary, (ii) cerebral, and (iii) cutaneous. Regarding the immunocompetence of patients, we have only 4 patients who have immunodepression factors, whose strains are in genotype 1 except the 10.93 strain which is in genotype 3.


### 3.5. Analysis ofrrs,hsp65, andsodA Genes Polymorphism

The 36 clinical strains and 2 reference strains ofN. brasiliensis studied showed (i) forrrs gene 16 polymorphic sites sharing 16 haplotypes and showing a Hd of 0.824; (ii) forhsp65 gene, 27 polymorphic sites and up to 22 different haplotypes with a Hd of 0.927; and, (iii) forsodA gene, up to 29 polymorphic sites sharing 14 haplotypes having a Hd of 0.885 (Table 3).Table 3
DNA polymorphism ofrrs, hsp65, and sodA genes from clinical N. brasiliensis strains isolated in France.
Nocardia species Genes(bp)a Number of haplotypes(Hd, S2, SD)b Number of of polymorphic sites N. brasiliensis   (N=38: 36 clinical strains and 2 reference strains) rrs (569) 16 (0.824, 0.00300, 0.055) 16 hsp65(401) 22 (0.927, 0.00087, 0.029) 27 sodA (406) 14 (0.885, 0.00080, 0.028) 29 aResulting fragment size without the primers sequences; bHd: haplotype (gene) diversity, S2: variance of haplotype diversity, and SD: standard deviation of haplotype diversity.


## 3.1. Growth on Culture Medium

The three culture media allowed the growth of clinical strains ofN. brasiliensis. The Bennett medium showed abundant and rapid growth (48 hours). Middlebrook medium showed strong growth but also it was slightly slower (72 hours). The BCP medium presented interesting results with good rapid growth at 48 hours.N. brasiliensis clade tested type strains showed similar patterns to the clinical strains, except thatN. boironii had a difficult growth on BCP and no growth on Middlebrook; this seems a peculiarity of this species.


## 3.2. Antimicrobial Susceptibilities

Eight out of 31 antibiotic molecules tested were active on all the strains’ collection: linezolid, tigecycline, trimethoprim + sulfamethoxazole, moxifloxacin, amikacin, amoxicillin + clavulanic acid, tobramycin, and gentamycin. Regarding the imipenem and pristinamycin molecules, resistance was observed on the majority of clinical isolates ofN. brasiliensis(Table 1).


## 3.3. Degradation of Substrate

The assimilation test of adenine and uric acid proved negative for all the strains tested of theN. brasiliensis clade including clinical and reference ones. The casein degradation test showed that all clinical strains are able to metabolize casein except the clinical strain 12.28. In addition our result showed that some types of strains such asN. vulneris,N. tenerifensis,N. boironii, andN. iowensis are also able to degrade casein in the same way asN. brasiliensis exceptN. altamirensis. Casein is ultimately a marker that can be used for the phenotypic identification of theN. brasiliensis clade and not theN. brasiliensis species as it has been believed for many years.


## 3.4. Phylogeny

Primers Noc1 and Noc2 amplified the expected 606-bp fragment of therrs gene for all the collection strains. Phylogenetic trees (Figure 1) based uponrrs showed homogeneity within clinical strains ofN. brasiliensis. For this, therrs gene is not relevant to show intraspecies diversity.Figure 1
Phylogenetic distribution ofrrs gene of 36N. brasiliensisclinical strains analyzed in this study using neighbour-joining method, Kimura’s two-parameter model, and bootstrap of 1000. Only values of bootstrap significance greater than 50% (Seaview) were reported.In addition, based upon the concatenation ofsodA andhsp65 housekeeping genes, the phylogenetic tree generated (Figure 2) had several distinct genotypes: (i) genotype 1 containing clinical strains, (ii) genotype 2 harboring some clinical strains, and (iii) genotype 3 harboring some clinical strains andN. brasiliensis ATCC 19296T. For the tropicalN. brasiliensisHUJEG01 strain, it is observed that it does not belong to any of the 3 genotypes and is positioned alone in the tree between genotypes 1 and 2. This distribution of clinical strains ofN. brasiliensis in 3 different genotypes shows an intraspecies diversity rather important. To better understand the polymorphism showed by phylogenetic trees, we studied the percentages of the similarities between the sequences. The average percentages of similarities based on therrs gene (Table 2) range from 99.39% to 99.57% between the clinical strains and the 2 reference strains ofN. brasiliensis (type and tropical strains). According to the CLSI, the similarity percentage needed for identification at species level must be greater than or equal to a threshold of 99.6% [19]. The clinical strains that showed a similarity percentage lower than this threshold for both reference strains ofN. brasiliensis were anyway considered as belonging to this species because no higher similarity percentage was obtained for any other species. In the same way, theN. vulneris type strain was also revealed to be close to clinical strains according to the average of percentage of similarity (98.77%). Between the 2 reference strains ofN. brasiliensis the percentage of similarity is higher, up to 99.82%. The percentages of similarities based on the concatenation of thesodA-hsp65 genes (Table 2) decrease and range now from 97.99% to 99.19% between the clinical strains and the 2 reference strains ofN. brasiliensis. Between the type and reference strains ofN. brasiliensis the percentage of similarity does not reach 99% this time. The comparison of the 3 genotypes between them (based on the representation of each genotype by 3 clinical strains) by usingsodA-hsp65 genes shows that genotypes 2 and 3 are closer to each other (98.97% of similarity). The average of the percentages of similarity between genotypes 1 and 2 were 97.97%. and 98.28% between genotypes 1 and 3. Finally this value goes up to 98.97% between genotypes 2 and 3. This means that the more distant genotypes between them are 1 and 2 and the closer ones are 2 and 3.Table 2
Percentage of similarity expressed in interval and mean forsodA-hsp65 and rrs genes.
N. brasiliensis HUJEG01 N. brasiliensis ATCC 19296T N. vulnerisDSM 45737T sodA-hsp65 rrs sodA-hsp65 rrs sodA-hsp65 rrs N. brasiliensisATCC 19296T Similarity average (%) 98.51 99.82 - - - - N. vulnerisDSM 45737T 98.38 98.73 98.51 98.55 - - N. altamirensisDSM 44997T 95.91 98.24 95.41 98.07 95.91 97.47 N. boironiiDSM 101696T 96.53 98.07 96.28 97.89 96.15 97.29 N. iowensis DSM 45197T 94.91 97.89 95.29 97.71 94.54 97.64 N. tenerifensis DSM 44704T 96.28 96.67 96.03 96.49 95.91 96.03  Genotype 1 Similarity range (%) (97.77–98.38) - (97.77–98.26) - (97.52–98.14) - Similarity average (%) 98.01 - 97.99 - 97.67 -  Genotype 2 Similarity range (%) (98.39–98.88) - (98.88–99.38) - (98.63–98.88) - Similarity average (%) 98.73 - 99.19 - 98.80 -  Genotype 3 Similarity range (%) (98.14–98.88) - (98.26–99.00) - (98.01–98.76) - Similarity average (%) 98.63 - 98.76 - 98.50 -  All clinical strains Similarity range (%) - (98.57–100) - (98.38–99.82) - (98.01–99.09) Similarity average (%) - 99.57 - 99.39 - 98.77Figure 2
Phylogenetic distribution of concatenationsodA-hsp65 genes of 36N. brasiliensisclinical strains analyzed in this study using neighbour-joining method, Kimura’s two-parameter model, and bootstrap of 1000. Only values of bootstrap significance greater than 50% were reported.In parallel, an epidemiological study based on the clinical files was carried out, and the data were presented in Table1. In order to know the link between the genetic diversity and the tropism of the clinical strains, a superposition of data was made between the phylogenetic tree obtained by the concatenation ofsodA andhsp65 and the tropism of the clinical strains (Figure 2). Thus, we can see that in genotypes 2 and 3 we have almost all the clinical strains that have a cutaneous tropism except the 08.188 strain which has a subcutaneous tropism. Regarding genotype 1 it is more heterogeneous with various tropism: (i) pulmonary, (ii) cerebral, and (iii) cutaneous. Regarding the immunocompetence of patients, we have only 4 patients who have immunodepression factors, whose strains are in genotype 1 except the 10.93 strain which is in genotype 3.


## 3.5. Analysis ofrrs,hsp65, andsodA Genes Polymorphism

The 36 clinical strains and 2 reference strains ofN. brasiliensis studied showed (i) forrrs gene 16 polymorphic sites sharing 16 haplotypes and showing a Hd of 0.824; (ii) forhsp65 gene, 27 polymorphic sites and up to 22 different haplotypes with a Hd of 0.927; and, (iii) forsodA gene, up to 29 polymorphic sites sharing 14 haplotypes having a Hd of 0.885 (Table 3).Table 3
DNA polymorphism ofrrs, hsp65, and sodA genes from clinical N. brasiliensis strains isolated in France.
Nocardia species Genes(bp)a Number of haplotypes(Hd, S2, SD)b Number of of polymorphic sites N. brasiliensis   (N=38: 36 clinical strains and 2 reference strains) rrs (569) 16 (0.824, 0.00300, 0.055) 16 hsp65(401) 22 (0.927, 0.00087, 0.029) 27 sodA (406) 14 (0.885, 0.00080, 0.028) 29 aResulting fragment size without the primers sequences; bHd: haplotype (gene) diversity, S2: variance of haplotype diversity, and SD: standard deviation of haplotype diversity.


## 4. Discussion

Nocardia spp. are common soil-inhabiting bacteria that frequently infect humans through traumatic injuries or inhalation routes and cause infections, such as actinomycetoma and nocardiosis, respectively.N. brasiliensis is the main aetiological agent of actinomycetoma in various countries [23]. The input data used in this study highlight the existence of indigenous cases of cutaneous and subcutaneous (such as actinomycetoma) nocardiosis caused byN. brasiliensis in France. Moreover, we can observe thatN. brasiliensis is also responsible for severe cases of disseminated nocardiosis in immunocompromised patients (pulmonary and cerebral cases).To determine whether there is an association between clinical tropism of strains and their genetic profile we performed genetic characterization of 36 indigenous cases ofN. brasiliensis that happened in France.The three culture media allow the growth of clinical strains ofN. brasiliensis. However, on Bennett’s medium more abundant and fast growth (48 hours) was observed. But the downside of this medium is its inaccessibility in the hospital because it is not marketable. Middlebrook medium shows strong growth but also it was slightly slower (72 hours). This medium is very expensive and not accessible to all budgets. However, it is an interesting alternative in isolatingNocardia from a complex sample. It is a selective medium of Mycobacteria, which promotes the growth of someNocardia to the detriment of other external bacteria or commensal flora that may be in the biological sample analyzed. The BCP medium, used routinely in hospitals for Gram-negative bacteria, has interesting results with good fast growth (48 hours). It would therefore be advisable to use it as isolation medium for urgent cases, by the speed of growth.Antibiograms results show resistance of most of the clinical strains to imipenem. This can pose therapeutic problems since it is part of molecules proposed during a phase of a general treatment for nocardiosis [24]. However, all clinical strains ofN. brasiliensis were sensible to SXT and would be an effective molecule during treatments. The sensibility ofN. brasiliensis type strain to this antibiotic has already been observed by Gilquin et al. [9].Our study confirms that all clinical strains ofN. brasiliensis are capable of degrading casein except 12.28 clinical strain. As shown by Seol et al. theN. brasiliensis type strain is able to degrade casein as well [25]. However, the test on the reference strains reveals thatN. vulneris,N. tenerifensis,N. boironii, andN. iowensis are also capable of degrading casein in the same way asN. brasiliensis, as also shown by Gilquin et al. [9]. This type of test is used in some countries without the necessary molecular biology tools to identifyN. brasiliensis. But, now, they must be aware that with this test we target several species of cladeN. brasiliensis. So, it is no longer a criterion of identification proper toN. brasiliensis.Phylogenetic tree based on therrs gene sequence of our collection showed a low genetic diversity resulting in low polymorphism sequence. In addition, we can note thatN. vulneris DSM 45737T, identified as a new species by Lasker et al., present a genetic sequence very close toN. brasiliensis strains [26] with percentages of similarities on average greater than 98%.Analysis of the phylogenetic tree (Figure2) based on the MLSA by the concatenation ofsodA andhsp65 housekeeping genes showed that the isolates are surprisingly classified according to 3 genotypes. These groups were formed upon similarity percentages and existing phylogenetic distances between the sequences of the strains studied. Genotype 1 concerns a well-defined cluster containing 9 clinical strains only which is sustained by a bootstrap of 81%. This genotype hosts all the cases of pulmonary forms (3), the sole cerebral form, and almost all the cases of immunocompromised patients (3 out of 4). Moreover, eight out of twelve strains susceptible to imipenem can be found in this group. There is just one remaining strain in this group not presenting this kind of susceptibility. The reason may be an acquired resistance to this drug due to a previous treatment. This well-defined genotype evokes the possible existence of another species or a strong variability in this case. This may have been caused by environmental pressures in the ecosystem of these isolates which may have resulted in the selection of strains that may have acquired, by mutations or genetic transfer with other microorganisms, new virulence characters different from that of the strain typeN. brasiliensis. Regarding genotypes 2 and 3, they include clinical strains andN. brasiliensis ATCC 19296T type strain. However, the discrimination between these 2 genotypes is less clear than that with genotype 1 because of being in weak bootstrap that is less than 50. The genetic differences do not allow distinguishing them properly and their phenotypic behavior remains similar. Then, it would be interesting to study on another gene capable of generating more divergences, for example,gyrB andrpoB genes, which have already successfully been used for studying the polymorphism of some otherNocardia species [27, 28].Concerning the percentage of similarity between the type and tropical strain ofN. brasiliensis, it is 99.82% according to therrs and goes down to 98.51% with the concatenation ofsodA andhsp65. The fact of highlighting a greater dissimilarity with the concatenation between the type and tropicalN. brasiliensis strain shows the advantage of the use of 2 markers likesodA andhsp65 vis-à-vis therrs. The discriminatory power of these two genes may be explained by the presence of more polymorphic sites (hsp65: 27;sodA: 29) than in the case ofrrs gene (16) and also by having Hd values higher than that ofrrs gene (hsp65: 0.927;sodA: 0.885;rrs: 0.824).It would be interesting to identify the genes involved in the virulence of different genotypes, including those of actinomycetoma. Interesting leads can be considered: (i) as identification of virulence genes expressed using the RNAseq method or (ii) to identify noncoding RNAs [23]. In addition, to genomically distinguishN. brasiliensis andN. vulneris a specific PCR toN. vulneris, using a specific gene of the species, should be developed.



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*Source: 7314054-2018-05-20.xml*