Accepted Article
Received Date : 09-Dec-2013 Revised Date : 07-Apr-2014 Accepted Date : 10-Apr-2014 Article type
: Original Article - E-only
Genotyping, Local Prevalence, and International Dissemination of β-Lactamase-Producing Kingella kingae Strains
Romain Basmaci1,2,3, Stéphane Bonacorsi1,2,3, Philippe Bidet1,2,3, Nataliya V. Balashova4, Jenny Lau4, Carmen Muñoz-Almagro5, Amadeu Gene5, Pablo Yagupsky6
1: IAME, UMR 1137, INSERM, F-75018 Paris, France 2: IAME, UMR 1137, Univ Paris Diderot, Sorbonne Paris Cité, F-75018 Paris, France 3: AP-HP, Laboratoire de Microbiologie, Hôpital Robert-Debré, F-75019 Paris, France 4: Department of Oral Biology, Rutgers School of Dental Medicine, Newark, New Jersey 07103, USA. 5: Departamento de Microbiología Molecular, Hospital Universitario Sant Joan de Déu, Barcelona, Spain. 6: Clinical Microbiology Laboratory, Soroka University Medical Center, Ben-Gurion University of the Negev, Beer-Sheva, Israel.
This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/1469-0691.12648 This article is protected by copyright. All rights reserved.
Accepted Article
Running Title: β-Lactamase-Producing K. kingae Strains
Key words: Kingella kingae, β-lactamase, clones, genotypes, dissemination
Corresponding author: Stéphane Bonacorsi. Mailing address: Service de Microbiologie, Hôpital Robert-Debré, 48 Boulevard Sérurier, 75019 Paris, France. Phone: 33 (0)1 40 03 57 92. Fax: 33 (0)1 40 03 24 50. e-mail: [email protected]
ABSTRACT β-lactamase production has been sporadically reported in the emerging Kingella kingae pathogen but the phenomenon has not been studied in-depth. We investigated the prevalence of β-lactamase production among K. kingae isolates from different geographic origins and genetically characterized β-lactamase-producing strains. 778 isolates from Iceland, the USA, France, Israel, Spain and Canada were screened for βlactamase production and, if positive, were characterized by PFGE and MLST genotyping, as well as rtxA, por, blaTEM and 16S rRNA sequencing. β-lactamase was identified in invasive strains from Iceland (n=4/14, 28.6%), the USA (n=3/15, 20.0%) and Israel (n=2/190, 1.1%) and in carriage strains in the USA (n=5/17, 29.4%) and Israel (n=66/429, 15.4%). No French, Spanish or Canadian isolates were β-lactamase producers. Among β-lactamase producers, a perfect congruency between the different typing methods was observed. Surprisingly, all US and Icelandic β−lactamase-producing isolates were almost indistinguishable, belonged to the major international invasive PFGE clone K/MLST ST-6, but differed
from
the
four
genetically
unrelated
Israeli β−lactamase-producing
Representative strains of different genotypes produced TEM-1 enzyme.
This article is protected by copyright. All rights reserved.
clones.
Accepted Article
K. kingae β-lactamase-producers exhibit a clear clonal distribution and have dissimilar invasive potential. Presence of the enzyme in isolates belonging to the major worldwide invasive clone K/ST-6 highlights the possible spread of β-lactam resistance, and emphasizes the importance of routine testing all K. kingae clinical isolates for β-lactamase production.
INTRODUCTION β-lactamase production is a survival strategy shared by many pathogens of respiratory origin to colonize and persist on the human mucosal surfaces. Organisms such as Staphylococcus aureus, Moraxella catarrhalis or Haemophilus influenzae, which are frequently carried on the upper respiratory tract, are repeatedly exposed to antimicrobial drugs that exert a strong selective pressure upon the resident flora [1], resulting in high rates of antibiotic resistance. Kingella kingae, an emerging aetiology of bacteremia and bone and joint infections in young children, is also a normal component of the upper respiratory tract microbiota [2-5]. The prevalence of the organism in the oropharynx reaches 10-12% in 12-24-month-old children [6], coinciding with the age of increased antibiotic consumption [7] and enhanced vulnerability to invasive K. kingae infections [2, 3, 5]. Although K. kingae is usually susceptible to antibiotics that are empirically administered to children with suspected bacteremia or skeletal system infections, β-lactamase production has been sporadically detected in Iceland since 1995 [8, 9], the USA since 1993 [9-11], and Israel since 2001 [12] (Figure 1).
Typing of K. kingae isolates by molecular methods, such as multilocus sequence typing (MLST), pulsed field gel electrophoresis (PFGE), single locus polymorphisms of the rtxA gene encoding the RTX toxin [13] and the por gene that encodes the bacterial porin [9], has revealed remarkable genomic heterogeneity in the species. To date, 37 MLST sequence types (STs) and 74 PFGE clones have been identified, as well as 18 rtxA and 11 different por alleles [9, 13-15]. Using
This article is protected by copyright. All rights reserved.
Accepted Article
these molecular tools, a study was conducted to characterize β-lactamase-producing strains of K. kingae and investigate the prevalence of β-lactamase production among isolates from different geographic origins.
METHODS
Source of the Kingella kingae isolates employed in the study. Strains isolated from healthy carriers (n=447) and patients (n=331) with a variety of invasive diseases (bacteremia, skeletal system infections and endocarditis) were screened for βlactamase production. Geographic origins and periods of isolation are provided in the Table 1. Among β-lactamase producers, all the US and Icelandic isolates, as well as the two invasive and 33 of 66 carriage strains from Israel were further characterized by molecular methods. Knowing that these 66 β-lactamase producing Israeli isolates derived from respiratory carriers were previously genotyped by PFGE and that all belonged to only four clones (A, F, T and Ψ), the 33 carriage β-lactamase-producers were randomly selected among 66 representing these four clones [12].
Antibiotic susceptibility testing. β-lactamase production was assessed by the nitrocefin method (Becton Dickinson & Co., Sparks, MD). Minimal inhibitory concentrations (MICs) were determined by the E-test method (BioMérieux, Marcy-l’Etoile, France) on Mueller-Hinton agar with added 5% sheep-blood, as previously described [16].
This article is protected by copyright. All rights reserved.
Accepted Article
Pulsed-field gel electrophoresis typing. PFGE typing was performed as previously described [12, 14, 15]. Restriction patterns were interpreted according to the criteria proposed by Tenover et al. [17].
Multilocus sequencing typing. Detailed description of the MLST method has been published elsewhere [13]. The ST of all examined strains is available on the Pasteur Institute of Paris website http://www.pasteur.fr/recherche/genopole/PF8/mlst/Kingella kingae.html. The genomic relatedness of typeable β-lactamase-producing organisms and previously characterized K. kingae strains [13] was investigated by comparing allelic profiles using the minimum spanning tree method employing the BioNumerics software (version 7.1, AppliedMaths, Belgium).
RTX-toxin gene sequencing. Amplification, sequencing and polymorphism of the rtxA gene were previously described [13, 18] and rtxA gene alleles sequences are available in Genbank (accession number from JQ340459 to JQ340476) [12].
Phylogeny by 16S rRNA gene sequencing. Sequencing of the 16S rRNA gene of several representative organisms of K. kingae clone was performed as described by Weisburg et al. [19]. The relatedness between these isolates and other species within the Kingella genus (K. oralis ATCC 51147, K. potus CIP 108935, K. denitrificans ATCC 33394) was expressed as a dendrogram, constructed by the neighbor-joining method using the MEGA 3.1 software.
Sequencing of β-lactamase gene (blaTEM). For the Israeli isolates, amplification of β-lactamase gene was performed using multiplex PCR, targeting TEM, SHV and OXA-1-like genes, as previously described [20]. Amplification products were sequenced by a Beckman-Coulter
This article is protected by copyright. All rights reserved.
Accepted Article
Genomics instrument (Takeley, Essex, United Kingdom) using primers MultiTSO-T_for and MultiTSO-T_rev previously described [20]. The β-lactamase gene of the US and Icelandic isolates has been previously characterized and published [9].
Porin gene typing. The por gene amplification, sequencing, and typing were performed as previously described [9].
Statistical analysis. Statistical analyses were performed using GraphPad Prism version 5.01 for Windows (GraphPad Software, San Diego, California, USA). Categorical variables were compared with the Chi-square test or the Fisher’s exact test as appropriate. Continuous variables were compared with the Mann-Whitney U test or Student t-test according to their homogeneity. A pvalue
Received Date : 09-Dec-2013 Revised Date : 07-Apr-2014 Accepted Date : 10-Apr-2014 Article type
: Original Article - E-only
Genotyping, Local Prevalence, and International Dissemination of β-Lactamase-Producing Kingella kingae Strains
Romain Basmaci1,2,3, Stéphane Bonacorsi1,2,3, Philippe Bidet1,2,3, Nataliya V. Balashova4, Jenny Lau4, Carmen Muñoz-Almagro5, Amadeu Gene5, Pablo Yagupsky6
1: IAME, UMR 1137, INSERM, F-75018 Paris, France 2: IAME, UMR 1137, Univ Paris Diderot, Sorbonne Paris Cité, F-75018 Paris, France 3: AP-HP, Laboratoire de Microbiologie, Hôpital Robert-Debré, F-75019 Paris, France 4: Department of Oral Biology, Rutgers School of Dental Medicine, Newark, New Jersey 07103, USA. 5: Departamento de Microbiología Molecular, Hospital Universitario Sant Joan de Déu, Barcelona, Spain. 6: Clinical Microbiology Laboratory, Soroka University Medical Center, Ben-Gurion University of the Negev, Beer-Sheva, Israel.
This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/1469-0691.12648 This article is protected by copyright. All rights reserved.
Accepted Article
Running Title: β-Lactamase-Producing K. kingae Strains
Key words: Kingella kingae, β-lactamase, clones, genotypes, dissemination
Corresponding author: Stéphane Bonacorsi. Mailing address: Service de Microbiologie, Hôpital Robert-Debré, 48 Boulevard Sérurier, 75019 Paris, France. Phone: 33 (0)1 40 03 57 92. Fax: 33 (0)1 40 03 24 50. e-mail: [email protected]
ABSTRACT β-lactamase production has been sporadically reported in the emerging Kingella kingae pathogen but the phenomenon has not been studied in-depth. We investigated the prevalence of β-lactamase production among K. kingae isolates from different geographic origins and genetically characterized β-lactamase-producing strains. 778 isolates from Iceland, the USA, France, Israel, Spain and Canada were screened for βlactamase production and, if positive, were characterized by PFGE and MLST genotyping, as well as rtxA, por, blaTEM and 16S rRNA sequencing. β-lactamase was identified in invasive strains from Iceland (n=4/14, 28.6%), the USA (n=3/15, 20.0%) and Israel (n=2/190, 1.1%) and in carriage strains in the USA (n=5/17, 29.4%) and Israel (n=66/429, 15.4%). No French, Spanish or Canadian isolates were β-lactamase producers. Among β-lactamase producers, a perfect congruency between the different typing methods was observed. Surprisingly, all US and Icelandic β−lactamase-producing isolates were almost indistinguishable, belonged to the major international invasive PFGE clone K/MLST ST-6, but differed
from
the
four
genetically
unrelated
Israeli β−lactamase-producing
Representative strains of different genotypes produced TEM-1 enzyme.
This article is protected by copyright. All rights reserved.
clones.
Accepted Article
K. kingae β-lactamase-producers exhibit a clear clonal distribution and have dissimilar invasive potential. Presence of the enzyme in isolates belonging to the major worldwide invasive clone K/ST-6 highlights the possible spread of β-lactam resistance, and emphasizes the importance of routine testing all K. kingae clinical isolates for β-lactamase production.
INTRODUCTION β-lactamase production is a survival strategy shared by many pathogens of respiratory origin to colonize and persist on the human mucosal surfaces. Organisms such as Staphylococcus aureus, Moraxella catarrhalis or Haemophilus influenzae, which are frequently carried on the upper respiratory tract, are repeatedly exposed to antimicrobial drugs that exert a strong selective pressure upon the resident flora [1], resulting in high rates of antibiotic resistance. Kingella kingae, an emerging aetiology of bacteremia and bone and joint infections in young children, is also a normal component of the upper respiratory tract microbiota [2-5]. The prevalence of the organism in the oropharynx reaches 10-12% in 12-24-month-old children [6], coinciding with the age of increased antibiotic consumption [7] and enhanced vulnerability to invasive K. kingae infections [2, 3, 5]. Although K. kingae is usually susceptible to antibiotics that are empirically administered to children with suspected bacteremia or skeletal system infections, β-lactamase production has been sporadically detected in Iceland since 1995 [8, 9], the USA since 1993 [9-11], and Israel since 2001 [12] (Figure 1).
Typing of K. kingae isolates by molecular methods, such as multilocus sequence typing (MLST), pulsed field gel electrophoresis (PFGE), single locus polymorphisms of the rtxA gene encoding the RTX toxin [13] and the por gene that encodes the bacterial porin [9], has revealed remarkable genomic heterogeneity in the species. To date, 37 MLST sequence types (STs) and 74 PFGE clones have been identified, as well as 18 rtxA and 11 different por alleles [9, 13-15]. Using
This article is protected by copyright. All rights reserved.
Accepted Article
these molecular tools, a study was conducted to characterize β-lactamase-producing strains of K. kingae and investigate the prevalence of β-lactamase production among isolates from different geographic origins.
METHODS
Source of the Kingella kingae isolates employed in the study. Strains isolated from healthy carriers (n=447) and patients (n=331) with a variety of invasive diseases (bacteremia, skeletal system infections and endocarditis) were screened for βlactamase production. Geographic origins and periods of isolation are provided in the Table 1. Among β-lactamase producers, all the US and Icelandic isolates, as well as the two invasive and 33 of 66 carriage strains from Israel were further characterized by molecular methods. Knowing that these 66 β-lactamase producing Israeli isolates derived from respiratory carriers were previously genotyped by PFGE and that all belonged to only four clones (A, F, T and Ψ), the 33 carriage β-lactamase-producers were randomly selected among 66 representing these four clones [12].
Antibiotic susceptibility testing. β-lactamase production was assessed by the nitrocefin method (Becton Dickinson & Co., Sparks, MD). Minimal inhibitory concentrations (MICs) were determined by the E-test method (BioMérieux, Marcy-l’Etoile, France) on Mueller-Hinton agar with added 5% sheep-blood, as previously described [16].
This article is protected by copyright. All rights reserved.
Accepted Article
Pulsed-field gel electrophoresis typing. PFGE typing was performed as previously described [12, 14, 15]. Restriction patterns were interpreted according to the criteria proposed by Tenover et al. [17].
Multilocus sequencing typing. Detailed description of the MLST method has been published elsewhere [13]. The ST of all examined strains is available on the Pasteur Institute of Paris website http://www.pasteur.fr/recherche/genopole/PF8/mlst/Kingella kingae.html. The genomic relatedness of typeable β-lactamase-producing organisms and previously characterized K. kingae strains [13] was investigated by comparing allelic profiles using the minimum spanning tree method employing the BioNumerics software (version 7.1, AppliedMaths, Belgium).
RTX-toxin gene sequencing. Amplification, sequencing and polymorphism of the rtxA gene were previously described [13, 18] and rtxA gene alleles sequences are available in Genbank (accession number from JQ340459 to JQ340476) [12].
Phylogeny by 16S rRNA gene sequencing. Sequencing of the 16S rRNA gene of several representative organisms of K. kingae clone was performed as described by Weisburg et al. [19]. The relatedness between these isolates and other species within the Kingella genus (K. oralis ATCC 51147, K. potus CIP 108935, K. denitrificans ATCC 33394) was expressed as a dendrogram, constructed by the neighbor-joining method using the MEGA 3.1 software.
Sequencing of β-lactamase gene (blaTEM). For the Israeli isolates, amplification of β-lactamase gene was performed using multiplex PCR, targeting TEM, SHV and OXA-1-like genes, as previously described [20]. Amplification products were sequenced by a Beckman-Coulter
This article is protected by copyright. All rights reserved.
Accepted Article
Genomics instrument (Takeley, Essex, United Kingdom) using primers MultiTSO-T_for and MultiTSO-T_rev previously described [20]. The β-lactamase gene of the US and Icelandic isolates has been previously characterized and published [9].
Porin gene typing. The por gene amplification, sequencing, and typing were performed as previously described [9].
Statistical analysis. Statistical analyses were performed using GraphPad Prism version 5.01 for Windows (GraphPad Software, San Diego, California, USA). Categorical variables were compared with the Chi-square test or the Fisher’s exact test as appropriate. Continuous variables were compared with the Mann-Whitney U test or Student t-test according to their homogeneity. A pvalue
