Macrolide Resistance Mutations Research Articles

P2.094 The Contribution of Macrolide Resistance Mutations to Failure of Azithromycin Treatment in Mycoplasma Genitalium Infection

BackgroundCurrent recommended treatment for Mycoplasma genitalium (Mg) is azithromycin. Macrolide resistance mutations (MRM), predominantly on the 23SrRNA gene of Mg, have been found to be associated with failure of azithromycin....

P3.273 Molecular Typing and Detection of Macrolide Resistance Mutations in T. Pallidum Strains from Cali, Colombia

BackgroundCali is a syphilis-endemic area, with a very high prevalence of gestational syphilis (14.7/1000 live births) and congenital syphilis (7.7/1000 live births). Molecular typing techniques are useful for studying bacterial...

P162 Utility of a novel dichromatic real-time PCR detection method of macrolide resistant mutations of Mycoplasma pneumoniae

P162 Utility of a novel dichromatic real-time PCR detection method of macrolide resistant mutations of Mycoplasma pneumoniae

Two mutations associated with macrolide resistance in Treponema pallidum: increasing prevalence and correlation with molecular strain type in Seattle, Washington.

Although azithromycin promised to be a safe and effective single-dose oral treatment of early syphilis, azithromycin treatment failure has been documented and is associated with mutations in the 23S rDNA of corresponding Treponema pallidum strains. The prevalence of strains harboring these mutations varies throughout the United States and the world. We examined T. pallidum strains circulating in Seattle, Washington, from 2001 to 2010 to determine the prevalence of 2 mutations associated with macrolide resistance and to determine whether these mutations were associated with certain T. pallidum strain types. Subjects were enrolled in a separate ongoing study of cerebrospinal fluid abnormalities in patients with syphilis. T. pallidum DNA purified from blood and T. pallidum strains isolated from blood or cerebrospinal fluid were analyzed for two 23S rDNA mutations and for the molecular targets used in an enhanced molecular stain typing system. Nine molecular strain types of T. pallidum were identified in Seattle from 2001 to 2010. Both macrolide resistance mutations were identified in Seattle strains, and the prevalence of resistant T. pallidum exceeded 80% in 2005 and increased through 2010. Resistance mutations were associated with discrete molecular strain types of T. pallidum. Macrolide-resistant T. pallidum strains are highly prevalent in Seattle, and each mutation is associated with discrete strain types. Macrolides should not be considered for treatment of syphilis in regions where prevalence of the mutations is high. Combining the resistance mutations with molecular strain typing permits a finer analysis of the epidemiology of syphilis in a community.

Two cases of macrolide resistance in Mycoplasma pneumoniae acquired during the treatment period

Two cases of macrolide resistance in Mycoplasma pneumoniae acquired during the treatment period

Transmission and Selection of Macrolide Resistant Mycoplasma genitalium Infections Detected by Rapid High Resolution Melt Analysis

Background Mycoplasma genitalium (MG) causes urethritis, cervicitis and pelvic inflammatory disease. The MG treatment failure rate using 1 g azithromycin at an Australian Sexual Health clinic in 2007–9 was 31% (95%CI 23–40%). We developed a rapid high resolution melt analysis (HRMA) assay targeting resistance mutations in the MG 23S rRNA gene, and validated it against DNA sequencing by examining pre- and post-treatment archived samples from MG-infected patients.Methodology/Principal FindingsAvailable MG-positive pre-treatment (n = 82) and post-treatment samples from individuals with clinical treatment failure (n = 20) were screened for 23S rRNA gene mutations. Sixteen (20%) pre-treatment samples possessed resistance mutations (A2058G, A2059G, A2059C), which were significantly more common in patients with symptomatic azithromycin-treatment failure (12/26; 44%) than in those clinically cured (4/56; 7%), p<0.001. All 20 patients experiencing azithromycin-failure had detectable mutations in their post-treatment samples. In 9 of these cases, the same mutational types were present in both pre- and post-treatment samples indicating transmitted resistance, whilst in 11 of these cases (55%), mutations were absent in pre-treatment samples indicating likely selection of resistant isolates have occurred. HRMA was able to detect all mutational changes determined in this study by DNA sequencing. An additional HRMA assay incorporating an unlabelled probe was also developed to detect type 4 single-nucleotide polymorphisms found in other populations, with a slightly lower sensitivity of 90%.Conclusions/SignificanceTreatment failure is associated with the detection of macrolide resistance mutations, which appear to be almost equally due to selection of resistant isolates following exposure to 1 g azithromycin and pre-existing transmitted resistance. The application of a rapid molecular assay to detect resistance at the time of initial detection of infection allows clinicians to shorten the time to initiate effective second line treatment. This has the potential to reduce transmission of resistant strains and to avoid sequelae associated with persistent untreated infection.

Evaluation of Macrolide Resistance and Enhanced Molecular Typing of Treponema pallidum in Patients with Syphilis in Taiwan: a Prospective Multicenter Study

Studies of macrolide resistance mutations and molecular typing using the newly proposed enhanced typing system for Treponema pallidum isolates obtained from HIV-infected patients in the Asia-Pacific region are scarce. Between September 2009 and December 2011, we conducted a survey to detect T. pallidum using a PCR assay using clinical specimens from patients with syphilis at six major designated hospitals for HIV care in Taiwan. The T. pallidum strains were genotyped by following the enhanced molecular typing methodology, which analyzed the number of 60-bp repeats in the acidic repeat protein (arp) gene, T. pallidum repeat (tpr) polymorphism, and the sequence of base pairs 131 to 215 in the tp0548 open reading frame of T. pallidum. Detection of A2058G and A2059G point mutations in the T. pallidum 23S rRNA was performed with the use of restriction fragment length polymorphism (RFLP). During the 2-year study period, 211 clinical specimens were obtained from 136 patients with syphilis. T. pallidum DNA was isolated from 105 (49.8%) of the specimens, with swab specimens obtained from chancres having the highest yield rate (63.2%), followed by plasma (49.4%), serum (35.7%), and cerebrospinal fluid or vitreous fluid (18.2%) specimens. Among the 40 fully typed specimens, 11 subtypes of T. pallidum were identified. Subtype 14f/f (18 isolates) was the most common isolates, followed by 14f/c (3), 14b/c (3), and 14k/f (3). Among the isolates examined for macrolide resistance, none had the A2058G or A2059G mutation. In conclusion, we found that type 14 f/f was the most common T. pallidum strain in this multicenter study on syphilis in Taiwan and that none of the isolates exhibited 23S rRNA mutations causing resistance to macrolides.

Ribosomal Mutations as the Main Cause of Macrolide Resistance in Campylobacter jejuni and Campylobacter coli

The aim of this study was to examine macrolide resistance mutations in Campylobacter species. In 76 strains studied, point mutation A to G at position 2059 of the 23S rRNA gene was detected in 30 of the 33 erythromycin-resistant strains. An amino acid insertion in the ribosomal protein L22 was found in one resistant strain without a 23S rRNA mutation. The A2059G mutation is the main cause of macrolide resistance in Campylobacter species.

The physiologic and phenotypic alterations due to macrolide exposure in Campylobacter jejuni

Physiologic and phenotypic alterations in the context of antibiotic resistance have been extensively studied in some bacteria. However there are not enough data addressing these alterations due to macrolide resistance in Campylobacter jejuni. The present study examined the fitness cost imposed by different macrolide resistance mutations and the phenotypic alterations due to exposure to macrolides in C. jejuni. C. jejuni was induced with different macrolide agents to obtain different macrolide resistance mutations. The results revealed that the mutations significantly imposed defect variations on the doubling time and the relative fitness in the resistant strains when competed against the susceptible strain. Furthermore macrolides through induction or exposure to sub-MIC concentrations impaired the motility of C. jejuni in 0.4% MH agar plates. Electron microscope analysis revealed the absence of flagellar filaments from strains exposed to macrolides. SDS-PAGE demonstrated that macrolides have no effect on protein synthesis and immunoblotting analysis further confirmed that flagellin was fully synthesized within C. jejuni strains exposed to macrolides. Nevertheless C. jejuni strains exposed to macrolides demonstrated defect in their excreted flagellin into the supernatant compared to strains not exposed to macrolides. Accordingly we speculated that macrolides inhibited flagellar filament formation in the strains exposed to macrolides via affecting the secretion of flagellin without affecting the amount synthesized within the bacteria. Taken together these findings demonstrated that different macrolide resistance mutations imposed different fitness costs and exposure to macrolides resulted in phenotypic alterations such as inhibition of flagellar filament formation and loss of motility in C. jejuni.

O3-S1.02 Deciphering the code of Treponema pallidum in the UK: implications for treatment and prevention

BackgroundThe efficacy and convenience of single-dose oral azithromycin meant it became the preferred treatment for syphilis in many countries and a suitable alternative to penicillin in many more. However, two...

Induced Macrolide Resistance in Mycoplasma genitalium Isolates from Patients with Recurrent Nongonococcal Urethritis

Mycoplasma genitalium accounts for as much as 15 to 20% of cases of male nongonococcal urethritis (NGU) (7). It is also implicated in female cervicitis and increasingly so in pelvic inflammatory disease (3). Since it is a fastidious microorganism that is very difficult to culture, nucleic acid amplification tests (NAAT) have become invaluable in its detection and microbiological evaluation. Currently recommended treatments of NGU include multidoses of doxycycline (100 mg twice daily for 7 days) or a single dose of azithromycin (1 g). With regard to M. genitalium-positive NGU, a recent randomized study has shown multidose treatment with doxycycline to be inferior to single-dose azithromycin (cure rates of 45% versus 87%, respectively) (8). However, even single-dose azithromycin can be associated with treatment failure rates of up to 28% (2). An extended 5-day course of azithromycin (500 mg on day 1, followed by 250 mg on days 2 to 5) has a much higher cure rate of up to 96% (1) but becomes less efficacious when used after initial treatment failure with single-dose azithromycin (6). Jensen et al. (4) have shown that macrolide resistance in M. genitalium can be induced by insufficient therapy with single-dose azithromycin and found the resistance to be strongly associated with three single-base mutations (A2058C, A2058G, A2059G) in region V of the 23S rRNA gene (4). They developed a conventional PCR to detect all of these mutations. In September 2009, we started M. genitalium NAAT testing of genital samples from selected patients with recurrent NGU at the Christchurch Sexual Health Centre, Christchurch, New Zealand. The PCR was performed as described by Jensen et al. (5) with the 2× Universal TaqMan gene expression master mix (Applied Biosystems, Life Technologies, Foster City, CA). Between September 2009 and October 2010, 52 samples were collected and M. genitalium was detected in 11 (21%) samples. The nucleic acid extracts were stored at −80°C until October 2010, when we retrospectively performed PCR and DNA sequencing on these extracts to look for the single-base mutations mentioned above, using the method described by Jensen et al. (4). Of the 11 nucleic acid extracts, we were able to amplify and sequence the 23S rRNA genes of 9 extracts. PCR of the other 2 nucleic acid extracts did not yield an amplicon upon repeat testing, probably due to nucleic acid degradation during storage. Of the 9 sequenced nucleic acid extracts, 4 extracts contained the A2059G mutation, whereas none contained mutations at position 2058. We retrospectively reviewed the notes for the relevant patients (those with extracts containing the mutation) and found that all four patients had previously been treated multiple times for sexually transmitted infections. We were able to determine that three of these patients had received single-dose azithromycin between 34 and 58 days preceding the positive test for M. genitalium; the fourth patient had also received single-dose azithromycin in the few weeks preceding a positive test, but we were unable to determine the exact time frame. All four patients had received an extended course of azithromycin following the diagnosis of M. genitalium urethritis: two experienced treatment failure, whereas the other two were subsequently cured, based on the fact that M. genitalium DNA was no longer detectable. Of the 5 patients whose M. genitalium extracts did not contain any of the macrolide resistance mutations (of the 9 extracts whose rRNA genes we were able to amplify and sequence), 3 had experienced successful empirical therapy with single-dose azithromycin (2 patients) or extended-dose azithromycin (1 patient); 1 patient was lost to follow up. The remaining 1 patient had received treatment with single-dose azithromycin, but upon subsequent testing, M. genitalium DNA was detectable and in this case contained the A2059G mutation, and the patient was included in our group above. This nicely demonstrates the induction of macrolide resistance by single-dose azithromycin treatment. To our knowledge, this is the first report from New Zealand describing macrolide resistance in M. genitalium. Despite a very limited number of samples, we have evidence that the A2059G mutation conferring induced macrolide resistance is present at a high frequency in M. genitalium extracts from patients with recurrent NGU at the Christchurch Sexual Health Centre. This brings into question whether or not single-dose azithromycin should be the first-line treatment of choice for M. genitalium-positive urethritis. At the Christchurch Sexual Health Centre, the current practice is to prescribe an extended course of azithromycin for all cases of M. genitalium-positive urethritis (primarily because moxifloxacin is currently not readily accessible in New Zealand). We also note with interest that in two patients with the resistant isolate, infection was still cleared with an extended course of azithromycin. This would suggest that macrolide resistance conferred by the A2059G mutation was not absolute. Clearly, the subject of macrolide resistance in M. genitalium requires more study.

Nested PCR-Linked Capillary Electrophoresis and Single-Strand Conformation Polymorphisms for Detection of Macrolide-Resistant Mycoplasma pneumoniae in Beijing, China

Mycoplasma pneumoniae is usually susceptible to macrolides, but macrolide-resistant strains have been found frequently in recent years. Mutations in domain V of the 23S rRNA gene of M. pneumoniae interfere with the binding of macrolides to rRNA and mediate macrolide resistance. In this study, we developed a rapid and inexpensive method that combines nested PCR (nPCR), single-strand conformation polymorphisms (SSCPs), and capillary electrophoresis (CE) to detect macrolide-resistant mutants directly from throat swabs. nPCR was used to specifically amplify M. pneumoniae 23S rRNA gene fragments containing mutations, and amplicons were analyzed by CE-SSCP for macrolide resistance mutations, with results confirmed by sequencing. From January to December 2009, 665 throat swabs were collected in Beijing, China, yielding 110 samples that tested positive for M. pneumoniae by nPCR and serological testing. We randomly selected 64 positive throat swabs for CE-SSCP analysis. The A2063G mutation was found in 57 samples, and a coexisting T2611C mutation was identified in 1 sample. An A2063T mutation was identified in 1 sample. The total mutation rate was 91%. All mutant samples identified by nPCR-CE-SSCP were sequenced. The nPCR-CE-SSCP method could identify macrolide-resistant mutants directly from clinical samples. This is the first report of an nPCR-CE-SSCP assay for the detection of dominant mutations that confer macrolide resistance on M. pneumoniae. This approach would allow clinicians to choose appropriate therapy rapidly and could be used as a screening method for genetic mutations related to antibiotic resistance.

A method for rapid detection of Mycoplasma pneumoniae and its macrolide resistance mutation

Objective To develop a method for rapid detection of Mycoplasma pneumoniae and its macrolide resistance mutation. Methods The primers and cycling probe sets were designed to detect two single nucleotide mutation, A2063G and A2064G, in the 23s rRNA gene of Mycoplasma pneumoniae. By using recombinant plasmids containing 23s rRNA gene fragments, 102 Mycoplasma pneumoniae clinical isolates from 2005 to 2008, and 136 nasopharyngeal suction specimens from pediatric patients with low respiratory tract infections in Shanghai Children's Hospital from November to December in 2009 were investigated to determine the specificity and the sensitivity of the CycleavePCR method. PCR amplification and sequence analysis of 23S rRNA genes were performed for all Mycoplasma pneumoniae strains and Mycoplasma pneumoniae positive specimens to confirm the results of the CycleavePCR method. Results Of 102 clinical isolates, 83 was resistant to erythromycin and sequence results show that all macrolide-resistant Mycoplasma pneumoniae strains harbored an A2063G ( 82/83 ) or A2064G ( 1/83 ) transition mutation in 23S rRNA genes. Twelve was Mycoplasma pneumoniae detected positive by CycleavePCR in 136nasopharyngeal suction specimens. The CycleavePCR results were consistent with those of routine PCR and sequencing. There was no signal production from other bacterial species. Sensitivity and specificity were 100%. The detection limit of the CycleavePCR was 10 plasmid copies per reaction. Experiment can be done within 1.5 h. Conclusion A novel method is developed to detect erythromycin-resistant strains harboring A2063G and A2064G transition mutation in the 23s rRNA gene using CycleavePCR. Key words: Mycoplasma pneumoniae; Macrolides; Drug resistance, bacterial; Mutation; Polymerase chain reaction

Detection and Quantification of Macrolide Resistance Mutations at Positions 2058 and 2059 of the 23S rRNA Gene by Pyrosequencing

A pyrosequencing method for detection and quantification of macrolide resistance mutations at positions 2058 and 2059 (Escherichia coli numbering) of the 23S rRNA gene is described. The method was developed and tested for Streptococcus pneumoniae, Streptococcus pyogenes, Mycobacterium avium, Campylobacter jejuni, and Haemophilus influenzae.

Antimicrobial resistance of Campylobacter jejuni and C coli isolates in Japan

Campylobacter jejuni and Campylobacter coli are foodborne enteric pathogens of human beings (Allos 2001), with poultry meat being a major source of Campylobacter infection (Park 2002). Recently, increasing levels of resistance in C jejuni and C coli to macrolide and fluoroquinolone antibiotics have become a worldwide problem as these are the therapeutic agents used for Campylobacter infections in humans (Aarestrup and Engberg 2001). The aims of this study were to investigate the antimicrobial resistance of Campylobacter isolates from human beings and poultry meat in Kanagawa, Japan, and to detect point mutations related to macrolide and quinolone resistance by using a line probe assay (MQ-LiPA), designed for the rapid and easy simultaneous detection of macrolide resistance mutations in 23S rDNA and quinolone resistance mutations in gyrA (Niwa and others 2003). In total, 249 isolates were used in this study: 193 human Campylobacter strains and 56 poultry meat strains were isolated in Kanagawa prefecture from 1979 to 2001 and from 1997 to 2000, respectively, and identified by biological and biochemical tests in the Kanagawa Prefectural Public Health Laboratory. All strains were cultured on Mueller-Hinton (MH) agar plates (Oxoid) at 42°C under microaerobic conditions (85 per cent nitrogen, 5 per cent oxygen, 10 per cent carbon dioxide) for 24 hours. The following antimicrobial agents were tested: nalidixic acid (Sigma), ofloxacin (Sigma), erythromycin (Sigma), tetracycline (Wako), ampicillin (Sigma), gentamicin (Sigma) and phosphomycin (Wako). Minimum inhibitory concentrations (MICs) were determined by an agar dilution method using MH agar plates. All MIC plates were inoculated with approximately 104 colony-forming units of each strain per spot using a multipoint inoculator. A control plate without antibiotics was also incubated at the end of each procedure. The plates were incubated for 24 hours at 42°C under microaerobic conditions. The MIC was defined as the lowest concentration producing no visible growth. The breakpoints were used according to the recommendations of the National Committee for Clinical Laboratory Standards (NCCLS 2002) except for ofloxacin and phosphomycin. No criteria were shown for ofloxacin or phosphomycin in the NCCLS document, so a breakpoint of 8 mg/litre was used for ofloxacin (Sanchez and others 1994) and 128 mg/litre for phosphomycin (Gomez-Garces and others 1995). The line probe assay (MQ-LiPA) was performed by the method described by Niwa and others (2003). This method is based on PCR and reverse hybridisation to a number of oligonucleotide probes immobilised as parallel lines on a nylon membrane strip. Oligonucleotide probes were tailed with dTTP (Gibco BRL). Poly (dT)-tailed probes and biotinylated DNA used as a colour development control were immobilised as parallel lines on the nylon membrane strip by ultraviolet exposure. The PCR was performed using primers designed according to the sequences of 23S rDNA and gyrA. The results of the antimicrobial susceptibility tests for the C jejuni and C coli isolates are shown in Table 1. In human isolates, the frequency of strains resistant to the quinolones, nalidixic acid and ofloxacin, increased remarkably, while tetracycline-resistant strains decreased during the study periods 1979 to 1989 and 1990 to 2001. Quinolone-resistant Campylobacter strains were observed from 1977 in both human and poultry meat isolates in this study. In Japan, many kinds of fluoroquinolones were introduced from the second half of the 1980s and the first half of the 1990s in the medical and veterinary fields, respectively. Poultry meat is a main source of Campylobacter infection in Japan (Ono and Yamamoto 1999) and various other countries (Padungton and Kaneene 2003), and the increase in the incidence of fluoroquinolone resistance in human isolates appears to be related to that in poultry. There was a marked decrease in erythromycin-resistant strains in human C jejuni isolates between the two study periods (1979 to 1989 and 1990 to 2000). The resistance level of all erythromycin-resistant C jejuni strains was low (MIC 8 to 16 mg/litre), while high-level (MIC ≥128 mg/litre) erythromycin-resistant strains were observed only in C coli. Low-level erythromycin-resistant strains might not be truly resistant, because the amount of carbon dioxide in the atmosphere increases the MICs of erythromycin (Rautelin and others 1991). Range of MIC (mg/litre) Profile by MQ-LiPA* Nalidixic acid Ofloxacin Erythromycin

Binding site of macrolide antibiotics on the ribosome: new resistance mutation identifies a specific interaction of ketolides with rRNA.

Macrolides represent a clinically important class of antibiotics that block protein synthesis by interacting with the large ribosomal subunit. The macrolide binding site is composed primarily of rRNA. However, the mode of interaction of macrolides with rRNA and the exact location of the drug binding site have yet to be described. A new class of macrolide antibiotics, known as ketolides, show improved activity against organisms that have developed resistance to previously used macrolides. The biochemical reasons for increased potency of ketolides remain unknown. Here we describe the first mutation that confers resistance to ketolide antibiotics while leaving cells sensitive to other types of macrolides. A transition of U to C at position 2609 of 23S rRNA rendered E. coli cells resistant to two different types of ketolides, telithromycin and ABT-773, but increased slightly the sensitivity to erythromycin, azithromycin, and a cladinose-containing derivative of telithromycin. Ribosomes isolated from the mutant cells had reduced affinity for ketolides, while their affinity for erythromycin was not diminished. Possible direct interaction of ketolides with position 2609 in 23S rRNA was further confirmed by RNA footprinting. The newly isolated ketolide-resistance mutation, as well as 23S rRNA positions shown previously to be involved in interaction with macrolide antibiotics, have been modeled in the crystallographic structure of the large ribosomal subunit. The location of the macrolide binding site in the nascent peptide exit tunnel at some distance from the peptidyl transferase center agrees with the proposed model of macrolide inhibitory action and explains the dominant nature of macrolide resistance mutations. Spatial separation of the rRNA residues involved in universal contacts with macrolides from those believed to participate in structure-specific interactions with ketolides provides the structural basis for the improved activity of the broader spectrum group of macrolide antibiotics.

A ketolide resistance mutation in domain II of 23S rRNA reveals the proximity of hairpin 35 to the peptidyl transferase centre.

Ketolides represent a new generation of macrolide antibiotics. In order to identify the ketolide-binding site on the ribosome, a library of Escherichia coli clones, transformed with a plasmid carrying randomly mutagenized rRNA operon, was screened for mutants exhibiting resistance to the ketolide HMR3647. Sequencing of the plasmid isolated from one of the resistant clones and fragment exchange demonstrated that a single U754A mutation in hairpin 35 of domain II of the E. coli 23S rRNA was sufficient to confer resistance to low concentrations of the ketolide. The same mutation also conferred erythromycin resistance. Both the ketolide and erythromycin protected A2058 and A2059 in domain V of 23S rRNA from modification with dimethyl sulphate, whereas, in domain II, the ketolide protected, while erythromycin enhanced, modification of A752 in the loop of the hairpin 35. Thus, mutational and footprinting results strongly suggest that the hairpin 35 constitutes part of the macrolide binding site on the ribosome. Strong interaction of ketolides with the hairpin 35 in 23S rRNA may account for the high activity of ketolides against erythromycin-resistant strains containing rRNA methylated at A2058. The existence of macrolide resistance mutations in the central loop of domain V and in hairpin 35 in domain II together with antibiotic footprinting data suggest that these rRNA segments may be in close proximity in the ribosome and that hairpin 35 may be a constituent part of the ribosomal peptidyl transferase centre.

Macrolide and aminoglycoside antibiotic resistance mutations in the bacillus subtilis ribosome resulting in temperature-sensitive sporulation.

Mutants of Bacillus subtilis resistant to various macrolide antibiotics have been isolated and characterized with respect to their sporulation phenotype and the electrophoretic mobility of their ribosomal proteins (r-proteins). Two types of major alterations of r-protein L17, one probably due to a small deletion, are found among mutants exhibiting high-level macrolide resistance. These mutants are all temperature-sensitive for sporulation (Spots). Low-level resistance to some macrolides is found to be associated with minor alterations in r-protein L17. These mutations do not cause a defective sporulation phenotype. All of the macrolide resistance mutations map at the same locus within the Str-Spc region of the B. subtilis chromosome. Hence, changes in a single ribosomal protein can result in different sporulation phenotypes. Mutants resistant to the aminoglycoside antibiotics neomycin and kanamycin have been isolated. Approximately 5% of these are Spots. Representative mutations, neo162 and kan25, cause concomitant drug resistance and sporulation temperature-sensitivity and map a single-site lesions in the Str-Spc region of the chromosome. Strains bearing neo162 or kan25 are equally cross-resistant to streptomycin or spectinomycin. These mutations define a new B. subtilis drug resistance locus at which mutation can cause defective sporulation.