Macrolide resistance determination and molecular typing of Mycoplasma pneumoniae by pyrosequencing

Spuesens, Emiel B. M.; Hoogenboezem, Theo; Sluijter, Marcel; Hartwig, Nico G.; Rossum, Annemarie M. C. van; Vink, Cornelis

doi:10.1016/j.mimet.2010.06.004

Cited by 63 publications

(64 citation statements)

References 19 publications

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“…Although these preliminary typing results are promising, further studies that include a larger number of isolates are needed to confirm these findings. Moreover, MALDI-TOF MS allowed for not only the identification of M. pneumoniae isolates but also their adhesin P1 typing in a single step, without the need of additional molecular typing techniques (28,43,44). However, no direct correlation was found between MALDI-TOF MS and MLVA typing.…”

Section: Discussionmentioning

confidence: 99%

Identification and Subtyping of Clinically Relevant Human and Ruminant Mycoplasmas by Use of Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry

et al. 2013

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g Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) recently emerged as a technology for the identification of bacteria. In this study, we aimed to evaluate its applicability to human and ruminant mycoplasmal identification, which can be demanding and time-consuming when using phenotypic or molecular methods. In addition, MALDI-TOF MS was tested as a subtyping tool for certain species. A total of 29 main spectra (MSP) from 10 human and 13 ruminant mycoplasma (sub)species were included in a mycoplasma MSP database to complete the Bruker MALDI Biotyper database. After broth culture and protein extraction, MALDI-TOF MS was applied for the identification of 119 human and 143 ruminant clinical isolates that were previously identified by antigenic or molecular methods and for subcultures of 73 ruminant clinical specimens that potentially contained several mycoplasma species. MALDI-TOF MS resulted in accurate (sub)species-level identification with a score of >1.700 for 96% (251/262) of the isolates. The phylogenetically closest (sub)species were unequivocally distinguished. Although mixtures of the strains were reliably detected up to a certain cellular ratio, only the predominant species was identified from the cultures of polymicrobial clinical specimens. For typing purposes, MALDI-TOF MS proved to cluster Mycoplasma bovis and Mycoplasma agalactiae isolates by their year of isolation and genome profiles, respectively, and Mycoplasma pneumoniae isolates by their adhesin P1 type. In conclusion, MALDI-TOF MS is a rapid, reliable, and cost-effective method for the routine identification of high-density growing mycoplasmal species and shows promising prospects for its capacity for strain typing.

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Section: Discussionmentioning

confidence: 99%

Identification and Subtyping of Clinically Relevant Human and Ruminant Mycoplasmas by Use of Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry

et al. 2013

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“…Methods such as PCR-restriction fragment length polymorphism (RFLP) analysis of the P1 gene (4,18) and real-time PCR followed by high-resolution melt analysis (HRM) targeting the region of M. pneumoniae repetitive elements 2 and 3 (RepMp2/3) of the P1 gene (19,20) were used to genotype M. pneumoniae into two subtypes (PCR-RFLP) and variants of subtypes 1 and 2 (PCR-HRM). Both sequencing (9) and pyrosequencing (6,21,22) techniques have also been developed to differentiate subtypes of M. pneumoniae. Recently, Degrange et al (6) developed a multiple-locus variable-number tandem-repeat (VNTR) analysis (MLVA) method for M. pneumoniae based on whole-genome analysis that was able to differentiate 26 distinct VNTR types.…”

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confidence: 99%

Multilocus Variable-Number Tandem-Repeat Analysis of Mycoplasma pneumoniae Clinical Isolates from 1962 to the Present: a Retrospective Study

et al. 2012

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In this study, we evaluated a recently developed multilocus variable-number tandem-repeat (VNTR) analysis (MLVA) method for the molecular typing of Mycoplasma pneumoniae. The method is based on GeneScan analysis of five VNTR loci throughout the genome which define a specific genotype based on the number of tandem repeats within each locus. A retrospective analysis of 154 M. pneumoniae clinical isolates collected over the last 50 years and a limited (n ‫؍‬ 4) number of M. pneumoniae-positive primary specimens acquired by the CDC was performed using MLVA. Eighteen distinct VNTR types were identified, including two previously unidentified VNTR types. Isolates from several M. pneumoniae community outbreaks within the United States were also analyzed to examine clonality of a specific MLVA type. Observed in vitro variability of the Mpn1 VNTR locus prompted further analysis, which showed multiple insertions or deletions of tandem repeats within this locus for a number of specimens and isolates. To our knowledge, this is the first report showing variation within the Mpn1 locus, thus affecting precise and reliable classification using the current MLVA typing system. The superior discriminatory capability of MLVA provides a powerful tool for greater resolution of M. pneumoniae strains and could be useful during outbreaks and epidemiological investigations. Mycoplasma pneumoniae is one of the most common bacterial etiologies of community-acquired pneumonia (CAP), representing 15 to 20% of cases in some studies (28). It is also a significant cause of community-wide outbreaks which are reported to occur in 3-to 7-year intervals with various incidence rates (10,12,28). Upper and lower respiratory tract symptoms are often mild and self-limiting; however, occasional extrapulmonary complications may develop, sometimes resulting in death (5,7,23).Strain subtyping is an important epidemiological tool for surveillance and outbreak investigations. Until recently, molecular typing of M. pneumoniae had been hindered by its minimal and highly uniform genome. Molecular typing was often restricted to a single gene encoding the P1 protein. Methods such as PCR-restriction fragment length polymorphism (RFLP) analysis of the P1 gene (4, 18) and real-time PCR followed by high-resolution melt analysis (HRM) targeting the region of M. pneumoniae repetitive elements 2 and 3 (RepMp2/3) of the P1 gene (19,20) were used to genotype M. pneumoniae into two subtypes (PCR-RFLP) and variants of subtypes 1 and 2 (PCR-HRM). Both sequencing (9) and pyrosequencing (6, 21, 22) techniques have also been developed to differentiate subtypes of M. pneumoniae. Recently, Degrange et al. (6) developed a multiple-locus variable-number tandem-repeat (VNTR) analysis (MLVA) method for M. pneumoniae based on whole-genome analysis that was able to differentiate 26 distinct VNTR types. This procedure takes advantage of the variation in the copy numbers of tandemly repeated sequences within five different loci within the genome and has proven to be highly discriminatory (...

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“…In the Western Hemisphere, lower rates of macrolide resistance have been reported (<10%), however, several epidemics with notable complications have occurred (8)(9)(10)(11). We report the detection of macrolide resistance in M. pneumoniae in Israel.…”

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confidence: 84%

“…This was initially reported in children; however, a surge of resistance in adults was recently reported (2,4,7). Macrolide-resistant M. pneumoniae has also been suggested to be associated with a longer course of disease (2,4).In the Western Hemisphere, lower rates of macrolide resistance have been reported (<10%), however, several epidemics with notable complications have occurred (8)(9)(10)(11). We report the detection of macrolide resistance in M. pneumoniae in Israel.…”

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confidence: 88%

Macrolide Resistance inMycoplasma pneumoniae, Israel, 2010

Averbuch¹,

Hidalgo-Grass²,

Moses³

et al. 2011

Emerg. Infect. Dis.

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Macrolide resistance in Mycoplasma pneumoniae is often found in Asia but is rare elsewhere. We report the emergence of macrolide-resistant M. pneumoniae in Israel and the in vivo evolution of such resistance during the treatment of a 6-year-old boy with pneumonia.M ycoplasma pneumoniae is a leading respiratory pathogen in both pediatric (1,2) and adult (1,3) populations. Macrolides are considered the fi rst line of therapy and are almost the only treatment for children. In recent years, alarming rates of M. pneumoniae with macrolide resistance (<90%) have occurred in eastern Asia, including the People's Republic of China, Japan, and Korea (2,4-7). This was initially reported in children; however, a surge of resistance in adults was recently reported (2,4,7). Macrolide-resistant M. pneumoniae has also been suggested to be associated with a longer course of disease (2,4).In the Western Hemisphere, lower rates of macrolide resistance have been reported (<10%), however, several epidemics with notable complications have occurred (8)(9)(10)(11). We report the detection of macrolide resistance in M. pneumoniae in Israel. The StudyA previously healthy 6-year-old boy was hospitalized after 2 weeks with fever up to 40°C. At onset of illness, a diagnosis of pharyngitis was made. Streptococcus pyogenes was isolated from his throat, and amoxicillin was prescribed without any clinical response. Later, a clinical diagnosis of sinusitis was made, and amoxicillin-clavulanate was prescribed. A chest radiograph done at that time reportedly showed no abnormalities. Laboratory investigation before admission showed leukocytosis of 19,600 cells/mm 3 with 2,200 monocytes/mm 3 and 7,600 neutrophils/mm 3 ; L-lactate dehydrogenase (LDH) was 1,854 U/L (reference value up to 600 U/L).Ten days after the beginning of his illness, his fever decreased for 2 days and then reappeared, together with cough, resulting in hospitalization. At admission, pneumonia of the right middle and lower lobe was confi rmed by chest radiograph. Laboratory tests showed leukocytes within normal ranges, erythrocyte sedimentation rate (ESR) 80/h, and C-reactive protein (CRP) 15 mg/L (reference range up to 0.5 mg/L). Treatment with penicillin was started without clinical improvement. Azithromycin (10 mg/kg/d) was added on the third day. After receiving this treatment, his leukocytes increased to 20,000 cells/mm 3 with ESR 97/h and CRP 22.5 mg/L. The β-lactam coverage was switched to cefuroxime and later to ceftriaxone because no response was observed. Chest ultrasound showed a small pleural effusion. Bronchoscopy showed thick mucus secretions; respiratory specimens tested were negative for respiratory syncytial virus, infl uenza viruses A and B, parainfl uenza virus, human metapneumovirus, and adenovirus, as were results of urine tests for Legionella spp. and blood tests for pneumococcal antigen and cryptococcal antigen.Throat swab specimens were collected and DNA extracted by boiling. Samples were positive for M. pneumoniae by real-time PCR based on the detection of ...

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Macrolide resistance determination and molecular typing of Mycoplasma pneumoniae by pyrosequencing

Cited by 63 publications

References 19 publications

Identification and Subtyping of Clinically Relevant Human and Ruminant Mycoplasmas by Use of Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry

Identification and Subtyping of Clinically Relevant Human and Ruminant Mycoplasmas by Use of Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry

Multilocus Variable-Number Tandem-Repeat Analysis of Mycoplasma pneumoniae Clinical Isolates from 1962 to the Present: a Retrospective Study

Macrolide Resistance inMycoplasma pneumoniae, Israel, 2010

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