Detection and susceptibility testing of Mycoplasma amphoriforme isolates from patients with respiratory tract infections

Research Notes

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determinants in regard to Enterobacteriaceae remains to be determined. For Aeromonas spp. to act as a reservoir of qnr genes, they must be capable of acquiring these determinants from their progenitors [13] and of transferring this genetic information to Enterobacteriaceae. Because Aeromonas are ubiquitous in a wide range of environments, they might act as important vectors for the transfer of plasmid-mediated quinolone resistance [14].

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14. Rhodes G, Parkhill J, Bird C et al. Complete nucleotide sequence of conjugative tetracycline resistance plasmid pFBAOT6, a member of a group of INcU plasmids with global ubiquity. Appl Environ Microbiol 2004; 70: 7497–7510.

Detection and susceptibility testing of Mycoplasma amphoriforme isolates from patients with respiratory tract infections S. Pereyre1, H. Renaudin1, A. Touati2, A. Charron1, O. Peuchant1, A. Bon Hassen2, C. Be´be´ar1 and C. M. Be´be´ar1 1) Laboratoire de Bacte´riologie EA 3671, Mycoplasma and Chlamydia

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Infections in Humans, Universite´ Victor Segalen Bordeaux 2 and CHU de

The authors declare that they have no conflicts of interest in relation to this work.

Bordeaux, Bordeaux, France and 2) Service des Laboratoires, Centre

References

Abstract

1. Jacoby GA. Mechanisms of resistance to quinolones. Clin Infect Dis 2005; 41 (suppl 2): 120–126. 2. Cattoir V, Poirel L, Aubert C, Soussy CJ, Nordmann P. Unexpected occurrence of plasmid-mediated quinolone resistance determinants in environmental Aeromonas spp. Emer Infect Dis 2008; 14: 231–233. 3. Clinical and Laboratory Standards Institute. Performance for antimicrobial susceptibility testing; 17th informational supplement M100-S17. Wayne, PA: The Institute, 2007. 4. Clinical and Laboratory Standars Institute. Performance for antimicrobial susceptibility testing; 18th informational supplement M100-S18. Wayne, PA: The Institute, 2007. 5. Borrell N, Acinas S, Figueras MJ, Martinez-Murcia AJ. Identification of Aeromonas clinical isolates by restriction fragment length polymorphism of PCR-amplified 16S rRNA genes. J Clin Microbiol 1997; 35: 1671–1674. 6. Ghatak S, Agarwal RK, Bhilegaonkar KN. Species identification of clinically important Aeromonas spp. by restriction fragment of 16S rDNA. Lett Appl Microbiol 2007; 44: 550–554. 7. Yan˜ez MA, Catala´n V, Apra´iz D, Figueras MJ, Martı´nez-Murcia AJ. Phylogenetic analysis of members of the genus Aeromonas based on gyrB gene sequences. Int J Syst Evol Microbiol 2003; 53: 875–883. 8. Cattoir V, Poirel L, Rotimi V, Soussy CJ, Nordmann P. Multiplex PCR for detection of plasmid-mediated quinolone resistance qnr genes in ESBL-producing enterobacterial isolates. J Antimicrob Chemother 2007; 60: 394–397. 9. Goni-Urriza M, Arpin C, Capdepuy M, Dubois V, Caumette P, Quentin C. Type II topoisomerase quinolone resistance-determining regions of Aeromonas caviae, A. hydrophila, and A. sobria complexes and mutations associated with quinolone resistance. Antimicrob Agents Chemother 2002; 46: 350–359. 10. Martı´nez-Martı´nez L, Eliecer Cano M, Manuel Rodrı´guez-Martı´nez J, Calvo J, Pascual A. Plasmid-mediated quinolone resistance. Expert Rev Anti Infect Ther 2008; 6: 685–711. 11. Pica RC, Poirel L, Demarta A et al. Plasmid-mediated quinolone resistance in Aeromonas allosaccharophila recovered from a Swiss lake. J Antimicrob Chemother 2008; 62: 948–950. 12. Sa´nchez-Ce´spedes J, Blasco MD, Marti S et al. Plasmid-mediated QnrS2 determinant from a clinical Aeromonas veronii isolate. Antimicrob Agents Chemother 2008; 52: 2990–2991. 13. Young HK. Antimicrobial resistance spread in aquatic environments. J Antimicrob Chemother 1993; 31: 627–635.

National de Greffe de Moelle Osseuse, Tunis, Tunisia

Three isolates of Mycoplasma amphoriforme, a new Mycoplasma species rarely described to date, were obtained from respiratory tract specimens from two children and one adult with respiratory tract infections. Molecular methods were required to distinguish them from Mycoplasma pneumoniae. MICs of macrolides, tetracyclines and fluoroquinolones were identical to those for M. pneumoniae, except for that of ciprofloxacin, which was slightly more potent against M. amphoriforme. M. amphoriforme could possibly have been involved in one case of severe respiratory infection with sepsis, but further studies are needed to specify its role as a potential respiratory tract pathogen.

Keywords: Human mycoplasma, molecular detection, Mycoplasma amphoriforme, respiratory tract infection, susceptibility testing Original Submission: 14 April 2009; Revised Submission: 24 June 2009; Accepted: 3 July 2009 Editor: D. Raoult Article published online: 23 September 2009 Clin Microbiol Infect 2010; 16: 1007–1009 10.1111/j.1469-0691.2009.02993.x

Corresponding author and reprint requests: S. Pereyre, Laboratoire de Bacte´riologie EA 3671, Mycoplasma and Chlamydia Infections in Humans, Universite´ Victor Segalen Bordeaux 2, 146 rue Le´o Saignat, 33076 Bordeaux Cedex, France E-mail: [email protected]

ª2009 The Authors Journal Compilation ª2009 European Society of Clinical Microbiology and Infectious Diseases, CMI, 16, 1005–1030

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Clinical Microbiology and Infection, Volume 16 Number 7, July 2010

Mycoplasma amphoriforme is a novel human Mycoplasma species belonging to the phylogenetic group Pneumoniae [1]. Only 12 isolates have been reported, mainly from respiratory tract specimens of patients with immunological disorders and productive cough [2,3]. The mode of acquisition and the pathogenic role of this species have not yet been established. However, this organism is unlikely to be a common commensal of the upper respiratory tract, as it has not been detected in healthy persons or asymptomatic patients with primary antibody deficiency [2]. Two difficulties could be encountered in the diagnosis and treatment of this species. First, M. amphoriforme may be easily confused with Mycoplasma pneumoniae, the main respiratory tract pathogenic mycoplasma species [1]. Second, it has been suggested that this Mycoplasma species could be resistant to antibiotics that are generally active against mycoplasmas [2]. We report the detection and the susceptibility testing of three M. amphoriforme clinical strains, isolated in France and in Tunisia. A bronchoalveolar lavage sample and two throat swabs were grown in Hayflick modified broth medium supplemented with glucose [4]. Broths with a colour change were plated on Hayflick agar medium, and the DNA was extracted from yellow broths using the MagNA Pure extraction system (Roche, France). A real-time PCR targeting the M. pneumoniae P1 adhesin gene was performed on DNA extracts as previously described [5]. DNA extracts were amplified with primers GPO1 and MGSO [6], targeting a conserved fragment of the Mycoplasma 16S rRNA genes, and PCR products were sequenced. Finally, DNA extracts were amplified with primers amph-F and amph-R, specific to the M. amphoriforme 16S rRNA, as previously described [1]. MICs of macrolides and related antibiotics, tetracyclines and fluoroquinolones were determined for the three clinical isolates by a broth dilution method [4]. The three respiratory tract specimens led to a colour change of the broth medium from red to yellow, after 15–20 days, indicating acidification and bacterial growth. Plated on agar medium, two of the three broths led to bubble-shaped

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colonies resembling typical M. pneumoniae colonies. However, the M. pneumoniae-specific real-time PCR remained negative on all broths. Amplification and sequencing of a 16S rRNA gene fragment led to approximately 550-bp sequences presenting identities ranging from 98.7% to 99.7% with the 16S rRNA gene from the M. amphoriforme A39 reference strain. Moreover, a 550-bp amplification product was obtained with the PCR targeting the M. amphoriforme 16S rRNA gene [1]. Thus, the identification of three M. amphoriforme clinical isolates, namely Ma3663, Ma4526 and G75, was confirmed. The Ma3663 isolate was cultured from a throat swab of a previously healthy 35-year-old adult hospitalized in 2004 in an intensive-care unit of the Bordeaux University Hospital (France) for undocumented severe sepsis with a cutaneous rash and a bilateral interstitial syndrome on the chest radiograph. Except for M. amphoriforme, no other bacterial or viral agent was found in his respiratory or blood specimens. The Ma4526 isolate, as well as 4 · 106 CFU/mL Haemophilus influenzae, were cultured from the bronchoalveolar lavage fluid of a French 2-year-old girl hospitalized in 2007 at the same hospital for an exacerbation of cystic fibrosis. The G75 strain and commensal oral bacteria were isolated from a throat swab of a Tunisian 2-year-old boy with a history of b-thalassaemia major, consulting in 2006 for an undocumented productive cough with fever in the Bone Marrow Transplant Centre of Tunis (Tunisia). None of the three patients had received any antimycoplasmal antibiotic treatment before or during the course of their infectious syndrome. Moreover, M. pneumoniae serology findings remained negative for all patients. MICs for the three isolates are presented in Table 1, in comparison with those for the M. pneumoniae M129 susceptible reference strain. The three isolates were susceptible to antibiotics usually active against mycoplasmas. MICs of macrolides and related antibiotics, tetracyclines and fluoroquinolones were about the same as those for M. pneumoniae, except for that of ciprofloxacin, which was more potent against M. amphoriforme, with MICs four times lower than

TABLE 1. MICs (mg/L) of macrolides and related antibiotics, tetracyclines and fluoroquinolones for three Mycoplasma amphoriforme clinical isolates in comparison with those obtained for the Mycoplasma pneumoniae M129 susceptible reference strain MIC Mycoplasmal strains M. amphoriforme Ma3663 Ma 4526 G75 M. pneumoniae M129

ERY

AZM

JOS

CLI

PRI

TEL

TET

DOX

CIP

LEV

MOX

0.03 0.015 0.015

0.001 0.001 0.001

0.008 0.008 0.008

0.2 0.5 0.5

0.06 0.06 0.12

0.001 0.002 0.002

0.12 0.12 0.12

0.12 0.12 0.12

0.25 0.25 0.25

0.25 0.25 0.12

0.06 0.03 0.06

0.015

0.001

0.015

0.5

0.12

0.001

0.25

0.12

1

0.5

0.06

ERY, erythromycin A; AZM, azithromycin; JOS, josamycin; CLI, clindamycin; PRI, pristinamycin; TEL, telithromycin; TET, tetracycline; DOX, doxycycline; CIP, ciprofloxacin; LEV, levofloxacin; MOX, moxifloxacin.

ª2009 The Authors Journal Compilation ª2009 European Society of Clinical Microbiology and Infectious Diseases, CMI, 16, 1005–1030

Research Notes

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that of M. pneumoniae (Table 1). The ciprofloxacin MICs of 0.25 mg/L for these M. amphoriforme clinical isolates contrasted with the ciprofloxacin MIC of 1.5 mg/L obtained for the M. amphoriforme reference strain A39 with an agar dilution method [2]. Unfortunately, this strain was not available for this difference to be checked. As previously suggested [1], M. amphoriforme could be easily missed and confused with M. pneumoniae by culture, as both of them hydrolyse glucose and lack the characteristic ‘fried egg’ appearance of many mycoplasmas. In consequence, molecular methods were required to distinguish them. M. amphoriforme was previously described as an opportunist that was probably pathogenic in immunodeficient patients and did not belong to the commensal respiratory flora [1,3]. Its pathogenic role was reinforced by the study of its detergent-insoluble structure, which was similar to the electrondense core of the M. pneumoniae attachment organelle required for virulence [7]. In our study, two children had an underlying immunodepressive disease. However, the clinical significance of the detection of M. amphoriforme was uncertain in both cases, as the microbiological information was incomplete, and both children recovered without receiving an antibiotic active against mycoplasmas. In contrast, the adult with severe sepsis was a previously healthy person without any immunological disorder or antecedents. As no other bacterial or viral agent was found, M. amphoriforme could possibly be considered to be involved in his severe infectious syndrome, which resulted in a 2-month stay in an intensive-care unit. Unfortunately, the detection of the mycoplasmal origin was too late for antibiotic treatment active against mycoplasmas to be started. In conclusion, M. amphoriforme was isolated in respiratory specimens from three patients with respiratory tract infections, but molecular methods were required to distinguish it from M. pneumoniae. This species was susceptible to antibiotics that are generally active against mycoplasmas, and was slightly more susceptible to ciprofloxacin than M. pneumoniae. In this study, M. amphoriforme could possibly be involved in one case of severe respiratory infection with sepsis. Moreover, to add to the epidemiological knowledge of this species and to specify its role as a potential respiratory pathogen, further studies on its detection in respiratory tract specimens with molecular methods are needed.

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References 1. Pitcher DG, Windsor D, Windsor H et al. Mycoplasma amphoriforme sp. nov., isolated from a patient with chronic bronchopneumonia. Int J Syst Evol Microbiol 2005; 55: 2589–2594. 2. Webster D, Windsor H, Ling C, Windsor D, Pitcher D. Chronic bronchitis in immunocompromised patients: association with a novel Mycoplasma species. Eur J Clin Microbiol Infect Dis 2003; 22: 530–534. 3. Waites K, Talkington D. New developments in human diseases due to mycoplasmas. In: Blanchard A, Browning GF, eds. Mycoplasmas molecular biology pathogenicity and strategies for control. Wymondham: Horizon Bioscience, 2005; 289–354. 4. Waites KB, Be´be´ar CM, Roberston JA, Talkington DF, Kenny GE, eds. Cumitech 34, Laboratory diagnosis of mycoplasmal infections. Washington, DC: American Society for Microbiology, 2001. 5. Touati A, Benard A, Ben Hassen A, Be´be´ar CM, Pereyre S. Evaluation of five commercial real-time PCR assays for the detection of Mycoplasma pneumoniae in respiratory tract specimens. J Clin Microbiol 2009; 47: 2269–2271. 6. van Kuppeveld FJ, van der Logt JT, Angulo AF et al. Genus- and species-specific identification of mycoplasmas by 16S rRNA amplification. Appl Environ Microbiol 1992; 58: 2606–2615. 7. Hatchel JM, Balish RS, Duley ML, Balish MF. Ultrastructure and gliding motility of Mycoplasma amphoriforme, a possible human respiratory pathogen. Microbiology 2006; 152: 2181–2189.

First international spread and dissemination of the virulent Queensland community-associated methicillin-resistant Staphylococcus aureus strain M. J. Ellington1, M. Ganner1, M. Warner2, E. Boakes1, B. D. Cookson1, R. L. Hill2 and A. M. Kearns1 1) Laboratory of Healthcare Associated Infection and 2) Antibiotic Resistance Monitoring and Reference Laboratory, Centre for Infections, Health Protection Agency, London, UK

Abstract We report the first international spread and dissemination of ST93-SCCmecIV (Queensland clone) methicillin-resistant Staphylococcus aureus (MRSA), previously identified in communities and hospitals in Australia. Ten highly genetically related MRSA isolates and one methicillin-susceptible S. aureus (MSSA) isolate were identified in England between 2005 and June 2008. The demography and clinical features were typical for communityassociated-MRSA. One female with MRSA infection died from necrotizing pneumonia. Travel between Australia and the UK,

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and some onward transmission, suggested that both importation and clonal dissemination of this strain had occurred, albeit to a

All authors declare that they have no conflicts of interest, financial or other.

small extent. Nosocomial transmission was not detected, but we remain vigilant for further importations and/or spread.

ª2009 The Authors Journal Compilation ª2009 European Society of Clinical Microbiology and Infectious Diseases, CMI, 16, 1005–1030