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ABSTRACTIn recent years, Mycoplasma pneumoniaestrains that are clinically resistant to macrolide antibiotics have occasionally been encountered in Japan. Of 76 strains of M. pneumoniaeisolated in three different areas in Japan during 2000 to 2003, 13 strains were erythromycin (ERY) resistant. Of these 13 strains, 12 were highly ERY resistant (MIC, ≥256 μg/ml) and 1 was weakly resistant (MIC, 8 μg/ml). Nucleotide sequencing of domains II and V of 23S rRNA and ribosomal proteins L4 and L22, which are associated with ERY resistance, showed that 10 strains had an A-to-G transition at position 2063 (corresponding to 2058 in Escherichia colinumbering), 1 strain showed A-to-C transversion at position 2063, 1 strain showed an A-to-G transition at position 2064, and the weakly ERY-resistant strain showed C-to-G transversion at position 2617 (corresponding to 2611 in E. colinumbering) of domain V. Domain II and ribosomal proteins L4 and L22 were not involved in the ERY resistance of these clinical M. pneumoniaestrains. In addition, by using our established restriction fragment length polymorphism technique to detect point mutations of PCR products for domain V of the 23S rRNA gene of M. pneumoniae, we found that 23 (24%) of 94 PCR-positive oral samples taken from children with respiratory infections showed A2063G mutation. These results suggest that ERY-resistant M. pneumoniaeinfection is not unusual in Japan.

Inactivation, one of the mechanisms of resistance to macrolide, lincosamide and streptogramin (MLS) antibiotics, appears to be fairly rare in clinical isolates in comparison with target site modification or efflux. However, inactivation is one of the major mechanisms through which macrolide-producing organisms avoid self-damage during antibiotic biosynthesis. The inactivation mechanisms for MLS antibiotics in pathogens are mainly hydrolysis, phosphorylation, glycosylation, reduction, deacylation, nucleotidylation, and acetylation. The ere (erythromycin resistance esterase) and mph (macrolide phosphotransferase) genes were originally found in Escherichia coli. Subsequently, Wondrack et al. (Wondrack, L.; Massa, M.; Yang, B.V.; Sutcliffe, J. Antimicrob. Agents Chemother., 1996, 40, 992) reported ere-like activity in Staphylococcus aureus. In addition, a variant of erythromycin esterase was found in Pseudomonas sp. from aquaculture sediment by Kim et al. (Kim, Y.H.; Cha, C.J.; Cerniglia, C.E. FEMS Microbiol. Lett., 2002, 210, 239). Although the mph genes, including mph(K), were first characterized in E. coli, a recent study revealed that S. aureus and Stenotrophomonas maltophilia have mph(C). The mph(C) has a low G+C content, like mph(B), and has high homology with mph(B), but not with mph(A) or mph(K). Consequently, the mph(C) and ere(B) genes seem to have originated from Gram-positive bacteria and been transferred between Gram-positive and Gram-negative bacteria. In this chapter, the genes and the mechanisms involved in the inactivation of MLS antibiotics by antibiotic-producing bacteria are reviewed.

Some patients with Mycoplasma pneumoniaeinfection are clinically resistant to antibiotics such as erythromycin, clarithromycin, or clindamycin. We isolated M. pneumoniaefrom such patients and found that one of three isolates showed a point mutation in the 23S rRNA gene. Furthermore, 141 EM‐sensitive clinical isolates of M. pneumoniaewere cultured in broth medium containing 100 μg/ml of erythromycin (EM). Among 11 EM‐resistant strains that grew in the medium, point mutations in the 23S rRNA were found in 3 strains at A2063G, 5 strains at A2064G and 3 strains at A2064C. The relationship between the point mutation pattern of these EM‐resistant strains and their resistance phenotypes to several macrolide antibiotics was investigated.

ABSTRACTMycoplasma penetransis a recently identified mycoplasma, isolated from urine samples collected from human immunodeficiency virus (HIV)-infected patients. Its presence is significantly associated with HIV infection. The major antigen recognized during natural and experimental infections is an abundant P35 lipoprotein which, upon extraction, segregates in the Triton X-114 detergent phase and is the basis of M. penetrans-specific serological assays. We report here that the P35 antigen undergoes spontaneous and reversible phase variation at high frequency, leading to heterogeneous populations of mycoplasmas, even when derived from a clonal lineage. This variation was found to be determined at the transcription level, and although this property is not unique among the members of the class Mollicutes, the mechanism by which it occurs in M. penetransdiffers from those previously described for other Mycoplasmaspecies. Indeed, the P35 phase variation was due neither to a p35gene rearrangement nor to point mutations within the gene itself or its promoter. The P35 phase variation in the different variants obtained was concomitant with modifications in the pattern of other expressed lipoproteins, probably due to regulated expression of selected members of a gene family which was found to potentially encode similar lipoproteins. M. penetransvariants could be selected on the basis of their lack of colony immunoreactivity with a polyclonal antiserum against a Triton X-114 extract, strongly suggesting that the mechanisms involved in altering surface antigen expression might allow evasion of the humoral immune response of the infected host.

The process of attachment of Mycoplasma hominisand M. oraleto HAIN‐55 cells, derived from normal embryonic human lung, was investigated quantitatively. The attachment reached its maximum within about 2–4 hr at 37 C and increased linearly as a function of the number of organisms present in the system. The relative attachment efficiency of M. hominiswas approximately 1% under our experimental conditions. Trypsin and EDTA were effective in detaching particles of M. hominisand M. oralefrom the surfaces of HAIN‐55 cells. Therefore it was suggested that some proteinaceous substance and salt bridges might be involved in the attachment of these mycoplasmas to HAIN‐55 cells.

The behavior of Mycoplasma hominisin normal human embryonic lung fibroblast (HAIN‐55) cell cultures was investigated. Multiplication patterns of cell‐associated mycoplasmas and of extracellular mycoplasmas in the HAIN‐55 cultures depended upon the size of the inoculum. This relationship did not vary with the number of days the cells had been cultured, nor with the number of HAIN‐55 cell passages. The maximum mycoplasmal growth was obtained with inoculum sizes of 105to 106colony‐forming units (CFU)/ml. The recovery of mycoplasmas decreased rapidly with inoculum size beyond 107CFU/ml, and growth of the HAIN‐55 cells was inhibited. Growth of the cells was also inhibited by the addition of the cytoplasmic fraction of Mycoplasma hominis.

It was demonstrated that a 67 kilodalton (kDa) protein of Mycoplasma pneumoniaeis a main cross‐reactive antigen with similar molecular weight protein of Mycoplasma genitaliumby Western blot analysis using monoclonal antibody to 67 kDa protein of M. pneumoniaeand hyperimmune rabbit sera directed against each mycoplasma strain.

A strain of Mycoplasma hominislacking a major membrane protein of 120 kDa was isolated from a Vero cell culture. This strain showed very slow growth rate and formed nipple‐less colonies on agar medium.