Pyogenic flexor tenosynovitis due to Kingella kingae in an infant.

Pyogenic flexor tenosynovitis is relatively common but is seldom reported in young children. Kingella kingae is increasingly recognised as a causative agent. We report on an infant who presented with a palmar deep space infection and pyogenic flexor tenosynovitis caused by K. kingae K. kingae is a fastidious, often culture-negative, organism which has been increasingly recognised as a cause of paediatric orthopaedic infections, including flexor tenosynovitis. Clinical suspicion should be heightened, and antibiotic coverage broadened in the setting of a positive physical examination and negative blood cultures.

Acquisition, co-option, and duplication of the rtx toxin system and the emergence of virulence in Kingella.

The bacterial genus Kingella includes two pathogenic species, namely Kingella kingae and Kingella negevensis, as well as strictly commensal species. Both K. kingae and K. negevensis secrete a toxin called RtxA that is absent in the commensal species. Here we present a phylogenomic study of the genus Kingella, including new genomic sequences for 88 clinical isolates, genotyping of another 131 global isolates, and analysis of 52 available genomes. The phylogenetic evidence supports that the toxin-encoding operon rtxCA was acquired by a common ancestor of the pathogenic Kingella species, and that a preexisting type-I secretion system was co-opted for toxin export. Subsequent genomic reorganization distributed the toxin machinery across two loci, with 30-35% of K. kingae strains containing two copies of the rtxA toxin gene. The rtxA duplication is largely clonal and is associated with invasive disease. Assays with isogenic strains show that a single copy of rtxA is associated with reduced cytotoxicity in vitro. Thus, our study identifies key steps in the evolutionary transition from commensal to pathogen, including horizontal gene transfer, co-option of an existing secretion system, and gene duplication.

Kingella kingae RtxA toxin interacts with sialylated gangliosides.

The membrane-damaging RTX family cytotoxin RtxA is a key virulence factor of the emerging pediatric pathogen Kingella kingae, but little is known about the mechanism of RtxA binding to host cells. While we have previously shown that RtxA binds cell surface glycoproteins, here we demonstrate that the toxin also binds different types of gangliosides. The recognition of gangliosides by RtxA depended on sialic acid side groups of ganglioside glycans. Moreover, binding of RtxA to epithelial cells was significantly decreased in the presence of free sialylated gangliosides, which inhibited cytotoxic activity of the toxin. These results suggest that RtxA utilizes sialylated gangliosides as ubiquitous cell membrane receptor molecules on host cells to exert its cytotoxic action and support K. kingae infection.

Whole-Genome Sequencing Reveals Differences among Kingella kingae Strains from Carriers and Patients with Invasive Infections.

As a result of the increasing use of sensitive nucleic acid amplification tests, Kingella kingae is being recognized as a common pathogen of early childhood, causing medical conditions ranging from asymptomatic oropharyngeal colonization to bacteremia, osteoarthritis, and life-threatening endocarditis. However, the genomic determinants associated with the different clinical outcomes are unknown. Employing whole-genome sequencing, we studied 125 international K. kingae isolates derived from 23 healthy carriers and 102 patients with invasive infections, including bacteremia (n = 23), osteoarthritis (n = 61), and endocarditis (n = 18). We compared their genomic structures and contents to identify genomic determinants associated with the different clinical conditions. The mean genome size of the strains was 2,024,228 bp, and the pangenome comprised 4,026 predicted genes, of which 1,460 (36.3%) were core genes shared by >99% of the isolates. No single gene discriminated between carried and invasive strains; however, 43 genes were significantly more frequent in invasive isolates, compared to asymptomatically carried organisms, and a few showed a significant differential distribution among isolates from skeletal system infections, bacteremia, and endocarditis. The gene encoding the iron-regulated protein FrpC was uniformly absent in all 18 endocarditis-associated strains but was present in one-third of other invasive isolates. Similar to other members of the Neisseriaceae family, the K. kingae differences in invasiveness and tropism for specific body tissues appear to depend on combinations of multiple virulence-associated determinants that are widely distributed throughout the genome. The potential role of the absence of the FrpC protein in the pathogenesis of endocardial invasion deserves further investigation. IMPORTANCE The wide range of clinical severities exhibited by invasive Kingella kingae infections strongly suggests that isolates differ in their genomic contents, and strains associated with life-threatening endocarditis may harbor distinct genomic determinants that result in cardiac tropism and severe tissue damage. The results of the present study show that no single gene discriminated between asymptomatically carried isolates and invasive strains. However, 43 putative genes were significantly more frequent among invasive isolates than among pharyngeal colonizers. In addition, several genes displayed a significant differential distribution among isolates from bacteremia, skeletal system infections, and endocarditis, suggesting that the virulence and tissue tropism of K. kingae are multifactorial and polygenic, depending on changes in the allele content and genomic organization. Further analysis of these putative genes may identify genomic determinants of the invasiveness of K. kingae and its affinity for specific body tissues and potential targets for a future protective vaccine.

[An infant with septic arthritis due toKingella kingae]. / Zuigeling met septische artritis doorKingella kingae.

BACKGROUND: Kingellakingae is the most frequently found pathogen in septic arthritis in children ≤ 4 years. In contrast to more well-known pathogens, K. kingae usually causes mild arthritis symptoms without high fever or elevated infection markers. In the current (general practitioners') guidelines on septic arthritis in children too little attention is given to the indolent symptoms caused by K. kingae. This could cause a delay in the diagnosis and treatment of children with K. kingae arthritis. CASE DESCRIPTION: An 11 months old boy presented to the general practitioner with 6 days of general malaise, upper airway symptoms and a painful, swollen left knee without fever or preceding trauma. Ultrasound of the knee was normal. Blood samples showed mildly elevated infection markers. K. kingae DNA was isolated using an oropharyngeal PCR and the diagnosis K. kingae septic arthritis was made. Antimicrobial therapy was initiated and resulted in a full recovery. CONCLUSION: In children ≤ 4 years with joint symptoms a septic arthritis caused by K. kingaeshould be considered, even if in the absence of evident symptoms of infection.

In vitro Activity of Ceftaroline Against an International Collection of Kingella kingae Isolates Recovered From Carriers and Invasive Infections.

BACKGROUND: Improvements in blood culture techniques and molecular-based diagnostics have led to increased recognition of Kingella kingae as an invasive human pathogen causing bacteremia, septic arthritis, osteomyelitis and endocarditis in young children. Serious disease and potentially life-threatening complications of infection due to K. kingae necessitate timely identification and appropriate antimicrobial therapy. Ceftaroline is a fifth-generation broad spectrum cephalosporin that possesses activity against Gram-negative and Gram-positive pathogens similar to third-generation cephalosporins, but also includes methicillin-resistant Staphylococcus aureus . This study reports the in vitro activity of ceftaroline and comparator agents against an international collection of K. kingae isolates. METHODS: A collection of 308 K. kingae isolates was obtained primarily from children with bacteremia, endocarditis, osteoarticular infections or from asymptomatic pediatric carriers. Isolates were tested for antibiotic susceptibility using Clinical and Laboratory Standard Institute broth microdilution methodology and screened for ß-lactamase production using a nitrocefin chromogenic test. RESULTS: Ceftaroline inhibited all K. kingae isolates at ≤0.06 mg/L (MIC 50/90 , 0.015/0.03 mg/L). Ceftaroline MICs were similar to results with ceftriaxone (MIC 50/90 , 0.015/0.015 mg/L), meropenem (MIC 50/90 , 0.015/0.015 mg/L) and ampicillin-sulbactam (MIC 50/90 , 0.06/0.06 mg/L). Ceftaroline MICs were slightly lower than MICs for cefuroxime and amoxicillin/clavulanate (MIC 50/90 , 0.06/0.12 mg/L). MICs were high for clindamycin (MIC 50/90 , 2/4 mg/L) and oxacillin (MIC 50/90 , 4/8 mg/L). Sixteen isolates (5.2%) yielded a positive nitrocefin test indicating production of ß-lactamase; ceftaroline demonstrated equivalent MICs against ß-lactamase - positive and ß-lactamase - negative strains (MIC 50/90 , 0.015/0.3 mg/L). CONCLUSIONS: The potent activity of ceftaroline against this large international collection of K. kingae isolates supports further clinical evaluation in children.

Clinical and Biologic Characteristics of Kingella kingae -Induced Septic Arthritis of the Knee in Young Children.

BACKGROUND AND OBJECTIVES: Septic arthritis of the knee is presumed to be the most frequent form of Kingella kingae -induced osteoarticular infection. This study aimed to report on the clinical course, biological parameters, and results of microbiological investigations among children with K. kingae -induced septic arthritis of the knee. It also assessed the modified Kocher-Caird criteria's ability to predict K. kingae -induced septic arthritis of the knee. METHODS: The medical charts of 51 children below 4 years old with confirmed or highly probable K. kingae -induced arthritis of the knee were reviewed. Data were gathered on the five variables in the commonly-used Kocher-Caird prediction algorithm (body temperature, refusal to bear weight, leukocytosis, erythrocyte sedimentation rate, and C-reactive protein level). RESULTS: Patients with K. kingae -induced arthritis of the knee usually presented with a mildly abnormal clinical picture and normal or near-normal serum levels of acute-phase reactants. Data on all five variables were available for all the children: 7 children had zero predictors; 8, 20, 12, and 4 children had 1, 2, 3, and 4 predictors, respectively; no children had 5 predictors. This gave an average of 1.96 predictive factors and a subsequent probability of ≤ 62.4% of infectious arthritis in this pediatric cohort. CONCLUSIONS: Because the clinical features of K. kingae -induced arthritis of the knee overlap with many other conditions affecting this joint, the Kocher-Caird prediction algorithm is not sensitive enough to effectively detect K. kingae -induced septic arthritis of the knee. Excluding K. kingae -induced arthritis of the knee requires performing nucleic acid amplification assays on oropharyngeal swabs and joint fluid from those young children presenting with effusion of the knee, even in the absence of fever, leukocytosis, or a high Kocher-Caird score.

The Kingella kingae PilC1 MIDAS Motif Is Essential for Type IV Pilus Adhesive Activity and Twitching Motility.

Kingella kingae is an emerging pathogen that has recently been identified as a leading cause of osteoarticular infections in young children. Colonization with K. kingae is common, with approximately 10% of young children carrying this organism in the oropharynx at any given time. Adherence to epithelial cells represents the first step in K. kingae colonization of the oropharynx, a prerequisite for invasive disease. Type IV pili and the pilus-associated PilC1 and PilC2 proteins have been shown to mediate K. kingae adherence to epithelial cells, but the molecular mechanism of this adhesion has remained unknown. Metal ion-dependent adhesion site (MIDAS) motifs are commonly found in integrins, where they function to promote an adhesive interaction with a ligand. In this study, we identified a potential MIDAS motif in K. kingae PilC1 which we hypothesized was directly involved in mediating type IV pilus adhesive interactions. We found that the K. kingae PilC1 MIDAS motif was required for bacterial adherence to epithelial cell monolayers and extracellular matrix proteins and for twitching motility. Our results demonstrate that K. kingae has co-opted a eukaryotic adhesive motif for promoting adherence to host structures and facilitating colonization.

A multicentre evaluation and expert recommendations of use of the newly developed BioFire Joint Infection polymerase chain reaction panel.

Septic arthritis is a serious condition with significant morbidity and mortality, routinely diagnosed using culture. The FDA has recently approved the rapid molecular BioFire® Joint Infection Panel (BJIP) for synovial fluid. We aimed to evaluate the BJIP compared to culture and its potential use in patient management. A multicentre retrospective evaluation of BJIP was conducted in the UK and Ireland. Positive percent agreement (PPA) and negative percent agreement (NPA) were calculated between the BJIP and routine culture. A multidisciplinary team (MDT) discussion addressing the optimal or potential case use of the assay practice was facilitated. Three hundred ninety-nine surplus synovial fluid samples (~ 70% from native joints) from eight centres were processed using BJIP in addition to routine culture. An increased yield of positive results was detected using BJIP compared to routine culture (98 vs 83), giving an overall PPA of 91.6% and overall NPA of 93% for the BJIP compared to culture results. The BJIP detected resistant markers and additional organisms that could influence antibiotic choices including Neisseria gonorrhoeae and Kingella kingae. The MDT agreed that the assay could be used, in addition to standard methods, in adult and children patients with specialist advice use based on local needs. Rapid results from BJIP were assessed as having potential clinical impact on patient management. Organisms not included in the panel may be clinically significant and may limit the value of this test for PJI.

Surface Anchoring of the Kingella kingae Galactan Is Dependent on the Lipopolysaccharide O-Antigen.

Kingella kingae is a leading cause of bone and joint infections and other invasive diseases in young children. A key K. kingae virulence determinant is a secreted exopolysaccharide that mediates resistance to serum complement and neutrophils and is required for full pathogenicity. The K. kingae exopolysaccharide is a galactofuranose homopolymer called galactan and is encoded by the pamABC genes in the pamABCDE locus. In this study, we sought to define the mechanism by which galactan is tethered on the bacterial surface, a prerequisite for mediating evasion of host immune mechanisms. We found that the pamD and pamE genes encode glycosyltransferases and are required for synthesis of an atypical lipopolysaccharide (LPS) O-antigen. The LPS O-antigen in turn is required for anchoring of galactan, a novel mechanism for association of an exopolysaccharide with the bacterial surface. IMPORTANCE Kingella kingae is an emerging pediatric pathogen and produces invasive disease by colonizing the oropharynx, invading the bloodstream, and disseminating to distant sites. This organism produces a uniquely multifunctional exopolysaccharide called galactan that is critical for virulence and promotes intravascular survival by mediating resistance to serum and neutrophils. In this study, we established that at least some galactan is anchored to the bacterial surface via a novel structural interaction with an atypical lipopolysaccharide O-antigen. Additionally, we demonstrated that the atypical O-antigen is synthesized by the products of the pamD and pamE genes, located downstream of the gene cluster responsible for galactan biosynthesis. This work addresses how the K. kingae exopolysaccharide can mediate innate immune resistance and advances understanding of bacterial exopolysaccharides and lipopolysaccharides.

Simplified antibiotic treatment for paediatric osteoarticular infections achieved good outcomes.

AIM: Early diagnosis of osteoarticular infections (OAI) in children and effective treatment prevents complications. The objective of this study was to evaluate effectiveness and safety of shortened intravenous antibiotic treatment of OAI. Incidence, diagnostics and pathogens of paediatric OAI were assessed. METHODS: This retrospective study included all paediatric OAI admissions to The Children's Hospital Iceland in 2006-2020. The treatment was evaluated by dividing the study cohort into two groups. The simplified treatment group received intravenous antibiotics for less than 7 days. The longer intravenous group received intravenous antibiotics for a minimum of 7 days. RESULTS: In total, 205 cases of OAI were included: 106 osteomyelitis, 83 septic arthritis and 16 with both. Age standardised incidence was 17 per 100,000 children and decreased over the study period (p = 0.004). A pathogen was identified in 37% (75/205) of cases of which 65% (49/75) were Staphylococcus aureus and 12% (9/75) Kingella kingae. Simplified treatment was not associated with increased risk of complications. CONCLUSION: This study supports claims that simplified treatment for OAI is safe and effective. Further simplification of treatment might be viable. For uncertain reasons the incidence of OAI was decreasing in Iceland, predominantly in young children.

Oral Versus Intravenous Antibiotics for Pediatric Osteoarticular Infection: When and to Whom?

BACKGROUND: Osteoarticular infections (OAIs) are typically treated initially with intravenous antibiotics. The objective of this study was to evaluate whether an exclusive oral treatment in selected children may be appropriate. METHODS: The Spanish Network of Osteoarticular Infections is a nationwide multicenter registry comprising 37 hospitals in Spain. The registry prospectively includes clinical characteristics and outcome of children with OAI. One of the hospitals from RioPed offers oral treatment to children meeting certain criteria. Patients were classified into 2 groups. Group 1: management with initial intravenous antibiotic therapy. Group 2: patients exclusively treated with oral antibiotics. A comparison between the 2 groups was performed. RESULTS: We compared 893 children who initially received intravenous antibiotics (group 1) with 64 children who received exclusively oral therapy (group 2). Patients from group 2 were younger (33.9 vs. 20.3 months; P = 0.001), had a lower percentage of Staphylococcus aureus (23.3% vs. 3.1%; P < 0.001), a higher proportion of Kingella kingae (12.1% vs. 28.1%; P = 0.001), higher erythrocyte sedimentation rate/C-reactive protein (CRP) ratio (1.4 interquartile range 0.6-3.6 vs. 3.3 interquartile range 1.7-5.7; P < 0.001) and showed lower rate of fever (63% vs. 48.8%; P = 0.024) than in group 1. Complications were not found in group 2. CONCLUSIONS: An exclusively oral administration could be a safe option in selected patients with OAI. Low-risk criteria are proposed: good general condition, no underlying disease, 6 months to 3 years old, appropriate oral tolerance, C-reactive protein <80 mg/L, erythrocyte sedimentation rate/C-reactive protein ratio ≥0.67, no skin injury, no recent surgery, no cervical spondylodiscitis and no local complications at onset.

First report of Kingella kingae infection in a paediatric population in Accra, Ghana.

Introduction: Kingella kingae is recognized as a frequent source of childhood bacteremia and the commonest agent of skeletal system infections in children 6 months - 4 years old. Several factors, including difficulty in detecting this fastidious organism in routine laboratory assays, result in underdiagnosis of the infections. Species-specific nucleic acid amplification assays, however, significantly improve the detection of K. kingae in blood samples. The aim of this study was to detect K. kingae infection in young children in Accra, Ghana. Methods: a cross-sectional based study was carried out in three hospitals in Accra. Children with febrile illness and directed by a clinician for blood culture were recruited. Blood samples collected were analysed by culture and polymerase chain reaction (PCR), using universal prokaryotic and K. kingae rtxA primers. Results: blood samples from 232 children (mean age 20.10 ± 12.57 months) were analysed. Bacteremia (72.4%) was the highest clinical diagnosis particularly in the 12-24 months age group. Only 7 (3.1%) samples showed bacterial growth and were negative for Kingella. PCR with universal prokaryotic primers succeeded in 223 (96.1%) out of 232 samples. PCR with K. kingae rtxA toxin primers was positive for 12 (5.4%) samples, all diagnosed as bacteremia, out of the 223 samples. Eleven (91.7%) out of the 12 K. kingae PCR positives were culture-negative. Conclusion: Kingella kingae was detected only by PCR specific for the K. kingae rtxA toxin. Kingella kingae may be a potential cause of bacteremia and hence febrile illness in young children living in Accra, Ghana.

Primer brote documentado de artritis séptica por Kingella kingae en una guardería de España / First documented outbreak of arthritis caused by Kingella kingae in a Spanish childcare center

IntroducciónRecientemente, Kingella kingae (K. kingae) se ha descrito como el principal agente causal de infecciones osteoarticulares entre los 6 meses y 2 años de vida. Más excepcional es su presentación en forma de clúster de infección invasiva por K. kingae. Se describe la investigación del primer brote de 3casos de artritis séptica causada por K. kingae documentado en España en una guardería de Roses, Girona.Pacientes y métodosEn diciembre del 2015, se realizó frotis faríngeo a todos los niños de la misma clase de la guardería. La muestra estaba compuesta por 9 lactantes (rango de edad: 16-23 meses), que incluía los 3casos índice. El estudio microbiológico se realizó mediante cultivo y RT-PCR específicos a K. kingae. Se administró amoxicilina y rifampicina profilácticas a todos los que presentaron colonización por K. kingae. Después de finalizar la profilaxis, se tomó un nuevo frotis faríngeo para confirmar la erradicación.ResultadosSe detectó K. kingae por RT-PCR en los 3casos índices y 5/6 compañeros de clase. Los cultivos fueron negativos en todos los casos. Después de recibir profilaxis, 3lactantes aún presentaban positividad a K. kingae en RT-PCR.ConclusionesK. kingae puede causar brotes de enfermedad invasiva en comunidades cerradas. Para una adecuada investigación, se requiere un mayor conocimiento de su existencia, así como una mejoría de la sensibilidad de las pruebas diagnósticas. En nuestra experiencia, la administración de profilaxis antibiótica puede erradicar parcialmente la colonización orofaríngea por K. kingae. Después de la profilaxis no se detectaron nuevos casos.

BackgroundRecently, Kingella kingae (K. kingae) has been described as the most common agent of skeletal system infections in children 6 months-2 years of age. More exceptional is the clinical presentation in clusters of invasive K. kingae infections. We describe the investigation of the first outbreak of 3cases of arthritis caused by K. kingae documented in Spain detected in a daycare center in Roses, Girona.Patients and methodsIn December of 2015 surveillance throat swabs obtained from all attendees from the same class of the index daycare center were assessed to study the prevalence of K. kingae colonization. The sample was composed of 9 toddlers (range: 16-23 months of age). Investigation was performed by culture and K. kingae-specific RT-PCR. Combined amoxicillin-rifampicin prophylaxis was offered to all attendees who were colonized by K. kingae. Following antimicrobial prophylaxis, a new throat swab was taken to confirm bacterial eradication.ResultsK. kingae was detected by RT-PCR throat swabs in the 3index cases and 5of the 6daycare attendees. Cultures were negative in all cases. After administration of prophylactic antibiotics, 3toddlers were still positive for K. kingae-specific RT-PCR.ConclusionsClusters of invasive K. kingae infections can occur in daycare facilities and closed communities. Increased awareness and use of sensitive detection methods are needed to identify and adequately investigate outbreaks of K. kingae disease. In our experience, the administration of prophylactic antibiotics could result in partial eradication of colonization. No further cases of disease were detected after prophylaxis.

Endocarditis por Kingella kingae en un paciente adulto / Kingella kingae endocarditis in an adult pacient

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Kingella kingae PilC1 and PilC2 are adhesive multifunctional proteins that promote bacterial adherence, twitching motility, DNA transformation, and pilus biogenesis.

The gram-negative bacterium Kingella kingae is a leading cause of osteoarticular infections in young children and initiates infection by colonizing the oropharynx. Adherence to respiratory epithelial cells represents an initial step in the process of K. kingae colonization and is mediated in part by type IV pili. In previous work, we observed that elimination of the K. kingae PilC1 and PilC2 pilus-associated proteins resulted in non-piliated organisms that were non-adherent, suggesting that PilC1 and PilC2 have a role in pilus biogenesis. To further define the functions of PilC1 and PilC2, in this study we eliminated the PilT retraction ATPase in the ΔpilC1ΔpilC2 mutant, thereby blocking pilus retraction and restoring piliation. The resulting strain was non-adherent in assays with cultured epithelial cells, supporting the possibility that PilC1 and PilC2 have adhesive activity. Consistent with this conclusion, purified PilC1 and PilC2 were capable of saturable binding to epithelial cells. Additional analysis revealed that PilC1 but not PilC2 also mediated adherence to selected extracellular matrix proteins, underscoring the differential binding specificity of these adhesins. Examination of deletion constructs and purified PilC1 and PilC2 fragments localized adhesive activity to the N-terminal region of both PilC1 and PilC2. The deletion constructs also localized the twitching motility property to the N-terminal region of these proteins. In contrast, the deletion constructs established that the pilus biogenesis function of PilC1 and PilC2 resides in the C-terminal region of these proteins. Taken together, these results provide definitive evidence that PilC1 and PilC2 are adhesins and localize adhesive activity and twitching motility to the N-terminal domain and biogenesis to the C-terminal domain.

First documented outbreak of arthritis caused by Kingella kingae in a Spanish childcare center.

BACKGROUND: Recently, Kingella kingae (K. kingae) has been described as the most common agent of skeletal system infections in children 6 months-2 years of age. More exceptional is the clinical presentation in clusters of invasive K. kingae infections. We describe the investigation of the first outbreak of 3 cases of arthritis caused by K. kingae documented in Spain detected in a daycare center in Roses, Girona. PATIENTS AND METHODS: In December of 2015 surveillance throat swabs obtained from all attendees from the same class of the index daycare center were assessed to study the prevalence of K. kingae colonization. The sample was composed of 9 toddlers (range: 16-23 months of age). Investigation was performed by culture and K. kingae-specific RT-PCR. Combined amoxicillin-rifampicin prophylaxis was offered to all attendees who were colonized by K. kingae. Following antimicrobial prophylaxis, a new throat swab was taken to confirm bacterial eradication. RESULTS: K. kingae was detected by RT-PCR throat swabs in the 3 index cases and 5 of the 6 daycare attendees. Cultures were negative in all cases. After administration of prophylactic antibiotics, 3 toddlers were still positive for K. kingae-specific RT-PCR. CONCLUSIONS: Clusters of invasive K. kingae infections can occur in daycare facilities and closed communities. Increased awareness and use of sensitive detection methods are needed to identify and adequately investigate outbreaks of K. kingae disease. In our experience, the administration of prophylactic antibiotics could result in partial eradication of colonization. No further cases of disease were detected after prophylaxis.