# A modified multilocus sequence typing protocol to genotype Kingella kingae from oropharyngeal swabs without bacterial isolation

Nawal El Houmami, Janek Bzdrenga, Jean-Christophe Pons, Philippe Minodier, Guillaume Andre Durand, Anis Oubraham, Dimitri Ceroni, Pablo Yagupsky, Didier Raoult, Philippe Bidet, et al.

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Nawal El Houmami, Janek Bzdrenga, Jean-Christophe Pons, Philippe Minodier, Guillaume Andre Durand, et al.. A modified multilocus sequence typing protocol to genotype Kingella kingae from oropharyngeal swabs without bacterial isolation. BMC Microbiology, 2017, 17, pp.200. 10.1186/s12866-017-1104-5. [hal-01730990](https://hal.science/hal-01730990)

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# A modified multilocus sequence typing protocol to genotype *Kingella kingae* from oropharyngeal swabs without bacterial isolation

Nawal El Houmami<fnref n="1" />*, Janek Bzdrenga², Jean-Christophe Pons<fnref n="1" />, Philippe Minodier³, Guillaume André Durand<fnref n="1" />, Anis Oubraham<fnref n="1" />, Dimitri Ceroni⁴, Pablo Yagupsky⁵, Didier Raoult<fnref n="1" />, Philippe Bidet⁶ and Pierre-Edouard Fournier<fnref n="1" />

## Abstract

**Background:** Outbreaks of *Kingella kingae* infection are an emerging public health concern among daycare attendees carrying epidemic clones in the oropharynx. However, genotyping of such epidemic clones from affected cases is limited by the low performance of current methods to detect *K. kingae* from blood samples and lack of specimens available from infected sites. We aimed at developing a modified multilocus sequence typing (MLST) method to genotype *K. kingae* strains from oropharyngeal samples without prior culture. We designed in silico MLST primers specific for *K. kingae* by aligning whole nucleotide sequences of *abcZ*, *adk*, *aroE*, *cpn60*, *recA*, and *gdh/zwf* genes from closely related species belonging to the *Kingella* and *Neisseria* genera. We tested our modified MLST protocol on all *Kingella* species and *N. meningitidis*, as well as 11 oropharyngeal samples from young children with sporadic (n = 10) or epidemic (n = 1) *K. kingae* infection.

**Results:** We detected *K. kingae*-specific amplicons in the 11 oropharyngeal samples, corresponding to sequence-type 6 (ST-6) in 6 children including the epidemic cases, ST-25 in 2 children, and 3 possible novel STs (ST-67, ST-68, and ST-69). No amplicon was obtained from other *Kingella* species and *N. meningitidis*.

**Conclusions:** We herein developed a specific MLST protocol that enables genotyping of *K. kingae* by MLST directly from oropharyngeal samples. This discriminatory tool, with which we identified the first *K. kingae* outbreak caused by ST-6 in Europe, may be used in further epidemiological investigations.

**Keywords:** *Kingella kingae*, MLST, Pediatrics, Outbreaks, Bone and joint infections

## Background

Outbreaks of *Kingella kingae* infections are emerging as a public health issue in daycare facilities [1-3]. Defined as the occurrence of at least two epidemiologically connected cases of *K. kingae* infections within a 1 month-period, they are characterized by a high attack rate and spread of a virulent clone among children aged from 6 to 36 months sharing the same classroom, and causing a variety of osteoarticular and

soft tissue infections, and occasionally endocarditis [1-3]. Epidemiological investigation of these events implies isolation and genotypic characterization of the strain causing the outbreak. At the same time, asymptomatic daycare center attendees and staff may be colonized by this virulent strain and, thus, deemed to be at risk to develop an invasive infection and/or to serve as reservoirs and sources of further dissemination of the disease [1, 2]. However, not all colonizing strains are capable of penetrating the epithelial layer and invading the bloodstream, and it is currently recognized that worldwide outbreaks are caused by a limited number of particularly invasive clones [2-4]. Epidemiological investigations revealed that only *K*.

<footnote>* Correspondence: nawal.elho@gmail.com; nawal.el-houmami@etu.univ-amu.fr</footnote>

<footnote n="1">¹Aix-Marseille Univ, UM63, CNRS 7278, IRD 198, Inserm 1095, Assistance Publique – Hôpitaux de Marseille, URMITE, Institut Hospitalo-Universitaire Méditerranée Infection, 19-21 Boulevard Jean Moulin, 13385 Marseille, France
Full list of author information is available at the end of the article</footnote>
*kingae* clones belonging to the hypervirulent sequence types 6 (ST-6), ST-14, ST-23, ST-25, and ST-66 have caused in the past few years outbreaks in the USA, Israel and France [2–4].

Since *K. kingae* is notoriously difficult to recover in culture, real-time polymerase chain reaction (PCR) assays have been developed during the last 10 years and gained increasing acceptance for the diagnosis of *K. kingae* infections [1, 3, 5]. These culture-independent methods exhibit higher sensitivity compared to conventional cultures, shorten the time of detection from days to a few hours, enable the diagnosis in patients being administered antibiotics, as well as identification of asymptomatic *K. kingae* carriers [2, 5]. When no surgical specimen is available and blood cultures are negative, alternative strategies have been developed [1, 5, 6]. Notably, the presence of an oropharyngeal *K. kingae* carriage in children under the age of four with sporadic osteoarticular infection was demonstrated to have a 90.5% positive predictive value for *K. kingae* infection [6]. On this point, it was demonstrated that *K. kingae* clones carried in the oropharynx of children with *K. kingae* infection are genotypically identical to those detected within infected sites [7].

Although the apparent increase in reported cases of *K. kingae* infections can be partly explained by improved isolation methods and better recognition of this emerging pathogen, the drawback of molecular detection tests is that, until now, they did not enable typing of the colonizing organisms and, thus, did not distinguish between individuals carrying non-invasive *K. kingae* strains and those colonized by the strain which caused the outbreak. We herein report the development of a modified multilocus sequence typing protocol (MLST) which enables to genotype *K. kingae* in oropharyngeal samples with no prior culture. This method was applied in clinics and successfully used to investigate an outbreak of invasive *K. kingae* infection that occurred in a daycare facility in 2016 in the Marseille area (France).

# Methods

## Development of a novel specific MLST typing tool for K. Kingae

We started with the analysis of MLST primers previously described in the Institut Pasteur MLST *K. kingae* database [8], and we observed a lack of in silico primer specificity between the *Kingella* and *Neisseria* genera. Therefore, we designed specific MLST primers for *K. kingae* by using the following criteria:

1) maximizing mismatches against other *Kingella* and *Neisseria* species, especially at the 3' end;

2) maximizing consensus between distinct *K. kingae* sequence types;

3) selecting hybridization temperatures close to 58 °C (Additional file 1: Figure S1). We designed thereafter a modified MLST method for *K. kingae*, consisting in PCR amplification and sequencing of 6 housekeeping genes, namely *abcZ*, *adk*, *aroE*, *cpn60*, *recA*, and *gdh/zwf* (Table 1). We first aligned the whole nucleotide sequences of the 6 above-mentioned housekeeping genes from fourty *K. kingae* strains, as well as those from closely-related species including, *K. negevensis* Sch538T [9], *K. denitrificans* ATCC 33394T, *K. oralis* ATCC 51147T, *N. meningitidis* Z2491, *N. lactamica* 020–06, and *N. elongata* subs. *N. elongata* subs. *glycolytica* ATCC 29315T.

Finally, we tested this novel *K. kingae* MLST protocol on 11 oropharyngeal samples that had previously been tested positive for *K. kingae* by specific real-time PCR targeting the *cpn60* gene [10], and on DNA from *K. denitrificans* CIP 103803, *K. oralis* CIP 103473, *K. potus* CIP 108935, *K. negevensis* Sch538T, and *N. meningitidis* CSUR P782.

# Results

*K. kingae*-specific amplicons were detected by Sanger sequencing in all tested oropharyngeal specimens corresponding to ST-6 in 6, ST-25 in 2, and possible novel STs in 3, but in none of the strains from others *Kingella* species and *N. meningitidis* (Table 2). A few single nucleotide polymorphisms (SNPs) were detected in some alleles for 7 specimens. In these cases, the highest peak of the chromatogram was selected to determine the dominant sequence (Fig. 1). Given that only one copy of each reference housekeeping gene was found in the *K. kingae* KWG1 genome, the only strain for which the whole genome was sequenced using the highly reliable Pacific Biosciences SMRT technology [11], we postulate that *K. kingae* clones belonging to different STs may coexist in the oropharynx of these individuals where one clone dominated.

This method was then applied in clinics in 2016. The study was approved by the Ethics committee of the IHU Mediterranee-Infection under reference number 2016-024. From June to July 2016, an outbreak of *K. kingae* osteoarticular infection involving two infants (aged 17 and 19 months) who shared the same classroom was identified in a daycare facility in southern France. The first patient sustained a left ankle arthritis and the second a first metatarsophalangeal joint's arthritis. Both had presented with herpangina, fever, and peri-oral rash in the 2 previous weeks. Blood cultures were negative and no joint fluid was surgically collected in either case. Both children
<table><caption>**Table 1** PCR protocol for specific *Kingella kingae* multilocus sequence typing</caption><thead><tr><th>Primer design</th><th>Primers name</th><th>Primers</th><th>Primer length (bp)</th><th>Amplicon length (bp)</th></tr></thead><tbody><tr><td rowspan="2">Gene<br>abcZ</td><td>abcZ_Kki_Fwd</td><td>CGCAAGAAAGCGTGTTTGAC</td><td>20</td><td>532</td></tr><tr><td>abcZ_Kki_Rev</td><td>CAATTCCTGCGCCTTTTTCTC</td><td>21</td><td></td></tr><tr><td rowspan="2">adk</td><td>adk_Kki_Fwd</td><td>CACACAAGCGCAATTTATTACG</td><td>22</td><td>491</td></tr><tr><td>adk_Kki_Rev</td><td>AAACTTCGGTTTGTTTCGTGATAT</td><td>23</td><td></td></tr><tr><td rowspan="2">aroE</td><td>aroE_Kki_Fwd</td><td>CAAATCCCCACAAATTCATCAATG</td><td>24</td><td>621</td></tr><tr><td>aroE_Kki_Rev</td><td>AACGCGGTGGGCTGGTTC</td><td>18</td><td></td></tr><tr><td rowspan="2">cpn60</td><td>cpn60_Kki_Fwd</td><td>CATGGGCGCACAAATGGTT</td><td>19</td><td>467</td></tr><tr><td>cpn60_Kki_Rev</td><td>CAAACAACAACACAAATGGGC</td><td>21</td><td></td></tr><tr><td rowspan="2">recA</td><td>recA_Kki_Fwd</td><td>GACGGCAGCCACCAAGAC</td><td>21</td><td>456</td></tr><tr><td>recA_Kki_Rev</td><td>TCCTGCCAGTTTACGCAAG</td><td>19</td><td></td></tr><tr><td rowspan="2">gdh/zwf</td><td>gdh/zwf_Kki_Fwd</td><td>GAGCGCGGCGAGTTTTAT</td><td>18</td><td>671</td></tr><tr><td>gdh/ zwf_Kki_Rev</td><td>CAGTTGTCCAAAATTGGCATG</td><td>21</td><td></td></tr><tr><td colspan="5">10x PCR Buffer 1x</td></tr><tr><td colspan="5">25 mM MgCl<sub>2</sub> 2.0 mM</td></tr><tr><td colspan="5">dNTP mix (10 mM of each) 200 µM of each dNTP</td></tr><tr><td colspan="5">Forward primer 0,1 µM</td></tr><tr><td colspan="5">Reverse primer 0,1 µM</td></tr><tr><td colspan="5">HotStarTaq DNA Polymerase 2.5 units/ reaction</td></tr><tr><td colspan="5">Distilled water variable</td></tr><tr><td colspan="5">Template DNA &lt; 0,5 µg</td></tr><tr><td colspan="5">Total volume 50 µl</td></tr><tr><td colspan="5">PCR protocol</td></tr><tr><td rowspan="2">Cycle step</td><td>3 step-protocol</td><td></td><td rowspan="2">Cycles</td><td rowspan="2"></td></tr><tr><td>Temperature</td><td>Time</td></tr><tr><td>Initial denaturation</td><td>95 °C</td><td>15 min</td><td>1</td><td></td></tr><tr><td>Denaturation</td><td>95 °C</td><td>1 min</td><td></td><td></td></tr><tr><td>Annealing</td><td>58 °C</td><td>30 s</td><td>35</td><td></td></tr><tr><td>Elongation</td><td>72 °C</td><td>1 min 30 s</td><td></td><td></td></tr><tr><td>Final elongation</td><td>72 °C</td><td>10 min</td><td>1</td><td></td></tr></tbody></table>

recovered with no sequelae after receiving intravenous cefamandole followed by oral amoxicillin. An oropharyngeal sample from the second case was collected prior to antibiotic therapy. Detection of *K. kingae* using specific real-time PCR was positive in this specimen. By using our modified MLST typing tool, we unambiguously identified *K. kingae* belonging to ST-6 composed of *abcZ-5*, *adk-2*, *aroE-4*, *cpn60-5*, *gdh/zwf-5*, and *recA-1* alleles.

Discussion

We herein developed a specific MLST method enabling to genotype *K. kingae* in oropharyngeal samples without requiring prior strain isolation. Given the fastidious nature of the species and the increasing use of molecular techniques for investigating epidemics or

sporadic infections, such an improved genotyping tool is relevant. Indeed, it was previously demonstrated that the presence of an oropharyngeal invasive *K. kingae* carriage in children under four with sporadic osteoarticular infections had a 90.5% positive predictive value for *K. kingae* infection [6]. Regarding this matter, it is important to note that, of the eleven *K. kingae* outbreaks in daycare centers that have been reported to date [2, 3], only 30% of children (10/33) underwent surgical procedures to obtain synovial fluid or tissue samples. This may be explained by the fact that most infected sites during *K. kingae* outbreaks were located within small joints located in hands, wrists and feet, ankles, as well as bony sites rich in growth cartilage such as epiphysis of long bones and spine [2]. Since these are regions where joint fluids
<table><caption>**Table 2** Specific multilocus sequence typing (MLST) for *Kingella kingae* performed by Sanger sequencing method on DNA directly extracted from 11 oropharyngeal specimens (=11 children) with no prior bacterial isolation allowed to detect *K. kingae* clones belonging to ST-6 in 6 children, ST-25 in 2, and possible new STs in 3, namely ST-67, ST-68, and ST-69</caption><thead><tr><th>No.</th><th>Age (mo)</th><th>Year</th><th>Syndrome</th><th>Country/region</th><th>abcZ</th><th>adk</th><th>aroE</th><th>cpn60</th><th>gdh/zwf</th><th>recA</th><th>ST</th><th>STc</th></tr></thead><tbody><tr><td>1572468</td><td>17</td><td>2016</td><td>OAI</td><td>France</td><td>5</td><td>2</td><td>4</td><td>5</td><td>5</td><td>1</td><td>6</td><td>6</td></tr><tr><td>1980738</td><td>16</td><td>2016</td><td>OAI</td><td>France</td><td>5</td><td>2</td><td>4</td><td>5*</td><td>5</td><td>1</td><td>6</td><td>6</td></tr><tr><td>1956884</td><td>18</td><td>2016</td><td>OAI</td><td>France</td><td>5</td><td>2</td><td>4</td><td>5</td><td>5</td><td>1</td><td>6</td><td>6</td></tr><tr><td>1882247</td><td>16</td><td>2016</td><td>OAI</td><td>France</td><td>5</td><td>2</td><td>4</td><td>5</td><td>5</td><td>1</td><td>6</td><td>6</td></tr><tr><td>1815589</td><td>12</td><td>2016</td><td>OAI</td><td>France</td><td>5*</td><td>2</td><td>4*</td><td>5</td><td>5*</td><td>1*</td><td>6</td><td>6</td></tr><tr><td>6847254</td><td>8</td><td>2016</td><td>OAI</td><td>France</td><td>5</td><td>2</td><td>4*</td><td>5*</td><td>5</td><td>1*</td><td>6</td><td>6</td></tr><tr><td>1541670</td><td>11</td><td>2016</td><td>OAI</td><td>France</td><td>7</td><td>2</td><td>6</td><td>2</td><td>2</td><td>2</td><td>25</td><td>25</td></tr><tr><td>0990626</td><td>28</td><td>2015</td><td>OAI</td><td>France</td><td>7*</td><td>2</td><td>6</td><td>2</td><td>2</td><td>2</td><td>25</td><td>25</td></tr><tr><td>1822057</td><td>7</td><td>2016</td><td>OAI</td><td>France</td><td>1</td><td>2</td><td>6</td><td>2*</td><td>1*</td><td>14*</td><td>69</td><td>...</td></tr><tr><td>1730798</td><td>33</td><td>2013</td><td>AC</td><td>French Guiana</td><td>5</td><td>2</td><td>3*</td><td>2</td><td>2</td><td>2</td><td>68</td><td>...</td></tr><tr><td>1746575</td><td>8</td><td>2013</td><td>AC</td><td>French Guiana</td><td>5*</td><td>2</td><td>6*</td><td>11*</td><td>9*</td><td>1*</td><td>67</td><td>...</td></tr></tbody></table>

are uncommonly sampled and epiphyseal bone, vertebrae, or intervertebral disks specimens are rarely obtained, many cases remain unconfirmed [1–3] but are still treated since *K. kingae* clones carried in the oropharynx of children with *K. kingae* infection are genotypically identical to those detected within infected sites [7]. In two *K. kingae* outbreaks involving four children in Israel, no suspected cases could be formally confirmed [1, 2, 12]. In this peculiar context, the genotype of epidemic clones was obtained from *K. kingae* oropharyngeal isolates cultivated from either presumed cases or from healthy classmates sharing the same classroom [1, 12].

Blood cultures and even PCR on blood specimens are really disappointing; although skeletal system infections result from the blood-borne dissemination of the bacterium, the prerequisite bacteremic episode is short and most of the time, when a localized infection has been established, the pathogen has usually been cleared from the blood.

Moreover, given that 60% of epidemic cases are not microbiologically confirmed during *K. kingae* outbreaks, and that *K. kingae* may be difficult to isolate from polymicrobial samples even on appropriate culture media [2], this specific *K. kingae* MLST tool may be helpful when oropharyngeal swabs are the only biological samples available for genotyping, as was the case in this report. Clones belonging to ST-6 are among the most invasive and disseminated worldwide and the main cause of *K. kingae* outbreaks in Israel [2, 4]. To the best of our knowledge, we here report the first *K. kingae* outbreak caused by ST-6 in Europe. Therefore, this genotype appears to be responsible for 50% of outbreaks worldwide.

## Conclusions

This modified, specific *K. kingae* MLST tool demonstrated a high discriminatory power and may be used in further epidemiological investigations for sporadic and epidemic *K. kingae* infections.

<figure><img src="image_3.png" /><figcaption>**Fig. 1** Chromatograms illustrating single nucleotide polymorphisms that were detected in nucleotide position 150 of the allele *abcZ-5* and those identified in nucleotide 336 of the allele *gdh/zwf-5* from oropharyngeal sample No. 1815589. In these cases, the highest peak was selected to determine the dominant clone. The nucleotide positions refer to the corresponding allele reference numbers provided in the Institut Pasteur database (http://bigsdb.pasteur.fr/perl/bigsdb/bigsdb.pl?db=pubmlst_kingella_seqdef_public&page=downloadAlleles)</figcaption></figure>

<footnote>*: detection of SNPs OAI osteoarticular infections, AC asymptomatical carriage, mo: months ...: not defined
Data referring to the second epidemic case of the Châteauneuf-Grasse *K. kingae* outbreak 2016 are indicated in bold</footnote>
**Additional file**

**Additional file 1: Figure S1.** MAFFT alignment of MLST genomic regions of the *abcZ*, *adk*, *aroE*, *cpn60*, *gdh/zwf*, *recA* genes from the 40 *Kingella kingae* strains that were used in this study, and those from 6 closely related *Kingella* and *Neisseria* species. Only each distinct variant of *K. kingae* sequence types is represented. MAFFT alignment and figures were performed by using Geneious 10.2.3 (Biomatters). (PPTX 24674 kb)

**Abbreviations**

CIP: Collection de l'institut pasteur; CSUR: Collection de souches de l'unité des rickettsies; DNA: Deoxyribonucleic acid; IHU: Institut hospitalo-universitaire; MLST: Multilocus sequence typing; OAI: Osteoarticular infection; PCR: Polymerase chain reaction; SMRT: Single molecule real time; SNP: Single nucleotide polymorphism; ST: Sequence type; STc: Sequence type complex

**Acknowledgements**

Not applicable.

**Funding**

This work was supported by the Ethics committee of the IHU Méditerranée Infection under reference number 2016-024.

**Availability of data and materials**

The datasets supporting the conclusions of this article are indicated within the article and its additional files.

**Authors' contributions**

NEH, PM, and PEF conceptualized the study. NEH and JB designed the modified MLST protocol. NEH and PM collected clinical samples. NEH, JCP, AO, GD, and JB collected data and carried out the initial analyses. NEH drafted the initial manuscript that was critically revised by PEF, PY, PM, DR, and DC. All authors approved the final manuscript as submitted.

**Ethics approval and consent to participate**

The study was approved by the Ethics committee of the IHU Méditerranée-Infection under reference number 2016-024.

**Consent for publication**

Not applicable.

**Competing interests**

The authors declare that they have no competing interests.

**Author details**

¹Aix-Marseille Univ, UM63, CNRS 7278, IRD 198, Inserm 1095, Assistance Publique – Hôpitaux de Marseille, URMITE, Institut Hospitalo-Universitaire Méditerranée Infection, 19-21 Boulevard Jean Moulin, 13385 Marseille, France.
²University of Grenoble Alpes, CEA, CNRS, IBS, F-38000, Grenoble, France.
³Department of Pediatric Emergency Medicine, North Hospital, Aix-Marseille University, Marseille, France.
⁴Département de l'enfant et de l'adolescent, HUG-Hôpital des Enfants, Geneva, Switzerland.
⁵Clinical Microbiology Laboratory, Soroka University Medical Center, Beer-Sheva, Israel.
⁶Laboratoire de Microbiologie, Hôpital Robert Debré, Assistance Publique – Hôpitaux de Paris, Université Paris Diderot, Sorbonne Paris Cité, Inserm, IAME, UMR 1137, Paris, France.

Received: 26 May 2017 Accepted: 5 September 2017

Published online: 21 September 2017

**References**

1. Yagupsky P, El Houmami N, Fournier PE. Outbreaks of invasive *Kingella kingae* infections in daycare facilities: approach to investigation and management. J Pediatr. 2017;182:14-20.

2. El Houmami N, Minodier P, Dubourg G, Mirand A, Jouve JL, Basmaci R, et al. Patterns of *Kingella kingae* disease outbreaks. Pediatr Infect Dis J. 2016;35:340-6.

3. El Houmami N, Cointat V, Mirand A, Fouilloux V, Bakour S, Minodier P, et al. An outbreak of *Kingella kingae* infections complicating a severe hand, foot, and mouth disease outbreak in Nice, France, 2016. Pediatr Infect Dis J. 2017;36:530-2.

4. Basmaci R, Bidet P, Yagupsky P, Muñoz-Almagro C, Balashova NV, Doit C, et al. Major intercontinentally distributed sequence types of *Kingella kingae* and development of a rapid molecular typing tool. J Clin Microbiol. 2014;52:3890-7.

5. El Houmami N, Minodier P, Dubourg G, Martin-Laval E, Lafont E, Jouve JL, et al. An outbreak of *Kingella kingae* infections associated with hand, foot and mouth disease/herpangina virus outbreak in Marseille, France, 2013. Pediatr Infect Dis J. 2015;34:246-50.

6. Ceroni D, Dubois-Ferriere V, Cherkaoui A, Gesuele R, Combescure C, Lamah L, et al. Detection of *Kingella kingae* osteoarticular infections in children by oropharyngeal swab PCR. Pediatrics. 2013;131:e230-5.

7. Basmaci R, Ilharreborde B, Bidet P, Doit C, Lorrot M, Mazda K, et al. Isolation of *Kingella kingae* in the oropharynx during *K. kingae* arthritis in children. Clin Microbiol Infect. 2012;18:E134-6.

8. Basmaci R, Yagupsky P, Ilharreborde B, Guyot K, Porat N, Chomton M, et al. Multilocus sequence typing and *rtxA* toxin gene sequencing analysis of *Kingella kingae* isolates demonstrates genetic diversity and international clones. PLoS One. 2012;7:e38078.

9. El Houmami N, Bakour S, Bzdrenga J, Rathored J, Seligmann H, Robert C, et al. Isolation and characterization of *Kingella negevensis* sp. nov., a novel *Kingella* species detected in a healthy paediatric population. Int J Syst Evol Microbiol. 2017;67:2370-6.

10. Levy PY, Fournier PE, Fenollar F, Raoult D. Systematic PCR detection in culture-negative osteoarticular infections. Am J Med. 2013;126:1143.e25-33.

11. Bidet P, Basmaci R, Guglielmini J, Doit C, Jost C, Birgy A, et al. Genome analysis of *Kingella kingae* strain KWG1 reveals how a β-Lactamase gene inserted in the chromosome of this species. Antimicrob Agents Chemother. 2015;60:703-8.

12. Yagupsky P, Ben-Ami Y, Trefler R, Porat N. Outbreaks of invasive *Kingella kingae* infections in closed communities. J Pediatr. 2016;169:135-9.

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