Mediated Macrolide Resistance Research Articles

Emerging erm(B)-Mediated Macrolide Resistance Associated with Novel Multidrug Resistance Genomic Islands in Campylobacter.

The rapid dissemination of the macrolide resistance gene erm(B) will likely compromise the efficacy of macrolides as the treatment of choice for campylobacteriosis. More importantly, erm(B) is always associated with several multidrug resistance genomic islands (MDRGIs), which confer resistance to multiple other antimicrobials. Continuous monitoring of the emergence of erm(B) and analysis of its associated genetic environments are crucial for our understanding of macrolide resistance in Campylobacter In this study, 290 Campylobacter isolates (216 Campylobacter coli isolates and 74 Campylobacter jejuni isolates) were obtained from 1,039 fecal samples collected in 2016 from pigs and chickens from three regions of China (344 samples from Guangdong, 335 samples from Shanghai, and 360 samples from Shandong). Overall, 74 isolates (72 C. coli isolates and 2 C. jejuni isolates) were PCR positive for erm(B). Combined with data from previous years, we observed a trend of increasing prevalence of erm(B) in C. coli Pulsed-field gel electrophoresis analyses suggested that both clonal expansion and horizontal transmission were involved in the dissemination of erm(B) in C. coli, and three novel types of erm(B)-associated MDRGIs were identified among the isolates. Furthermore, 2 erm(B)-harboring C. jejuni isolates also contained an aminoglycoside resistance genomic island and a multidrug-resistance-enhancing efflux pump, encoded by RE-cmeABC Antimicrobial susceptibility testing showed that most of the isolates were resistant to all clinically important antimicrobial agents used for the treatment of campylobacteriosis. These findings suggest that the increasing prevalence of erm(B)-associated MDRGIs might further limit treatment options for campylobacteriosis.

The Current State of Macrolide Resistance in Campylobacter spp.: Trends and Impacts of Resistance Mechanisms.

Campylobacter spp., especially Campylobacter jejuni and C. coli, are leading bacterial foodborne pathogens worldwide. In the United States, an estimated 0.8 million cases of campylobacteriosis occur annually, mostly involving C. jejuni Campylobacteriosis is generally self-limiting, but in severe cases, treatment with antibiotics may be mandated. The increasing incidence of fluoroquinolone resistance in Campylobacter has rendered macrolides such as erythromycin and azithromycin the drugs of choice for human campylobacteriosis. The prevalence of macrolide resistance in C. jejuni remains low, but macrolide resistance can be common in C. coli Substitutions in the 23S rRNA gene, specifically A2075G, and less frequently A2074C/G, remain the most common mechanism for high-level resistance to macrolides. In C. jejuni, resistance mediated by such substitutions is accompanied by a reduced ability to colonize chickens and other fitness costs, potentially contributing to the low incidence of macrolide resistance. Interestingly, similar fitness impacts have not been noted in C. coli Also noteworthy is a novel mechanism first reported in 2014 for a C. coli isolate from China and mediated by erm(B) harbored on multidrug resistance genomic islands. The incidence of erm(B) appears to reflect clonal expansion of certain strains, and whole-genome sequencing has been critical to the elucidation of erm(B)-associated macrolide resistance in Campylobacter spp. With the exception of one report from Spain, erm(B)-mediated macrolide resistance has been restricted to Campylobacter spp., mostly C. coli, of animal and human origin from China. If erm(B)-mediated macrolide resistance does not confer fitness costs in C. jejuni, the range of this gene may expand in C. jejuni, threatening to compromise treatment effectiveness for severe campylobacteriosis cases.

Molecular Characteristics of Group B Streptococci Isolated from Adults with Invasive Infections in Japan.

Streptococcus agalactiae (group B streptococcus) isolates (n = 443) obtained from Japanese adults with invasive infections between April 2010 and March 2013 were analyzed for capsular serotype, multilocus sequence type (ST), antibiotic susceptibility, and resistance genes. Among these cases, bacteremia without primary focus was the most common variety of infection (49.9%), followed by cellulitis (12.9%) and pneumonia (9.0%). Concerning patient age (18 to 59, 60 to 69, 70 to 79, 80 to 89, and 90 years old or older), the incidence of pneumonia increased in patients in their 70s and 80s (P < 0.001), while younger patients (18 to 59 and 60 to 69 years old) were more likely to have abscesses (P < 0.05). The mortality rate was 10.2% for all ages. The most common capsular serotype was Ib (39.5%), followed by V (16.0%), III (13.8%), VI (9.5%), and Ia (8.6%). The main ST of serotype Ib strains was ST10, which belonged to clonal complex 10 (88.0%). The predominant clonal complexes of serotypes V and III, respectively, were 1 (78.9%) and 19 (75.4%). Among these isolates, 9 strains (2.0%) were identified as group B streptococci with reduced penicillin susceptibility, reflecting amino acid substitutions in penicillin-binding protein 2X (PBP2X). In addition, 19.2% of all strains possessed mef(A/E), erm(A), or erm(B) genes, which mediate macrolide resistance, while 40.2% of strains were resistant to quinolones resulting from amino acid substitutions in GyrA and ParC. Our data argue strongly for the continuous surveillance of microbial characteristics and judicious antibiotic use in clinical practice.

Associations between capsular serotype, multilocus sequence type, and macrolide resistance in Streptococcus agalactiae isolates from Japanese infants with invasive infections.

SUMMARY Streptococcus agalactiae (group B streptococcus; GBS) isolates (n = 150) from infants with invasive infections between 2006 and 2011 were analysed for capsular serotype, multilocus sequence type, and antibiotic susceptibility. In cases with late-onset disease (n = 115), primary meningitis was predominant (62.6%), but represented only 39.1% in cases with early-onset disease (n = 23). The most common serotype was III (58.7%), followed by Ia (21.3%) and Ib (12.7%). Sequence types (STs) of serotype III strains included ST17 (50.0%), ST19 (26.1%), ST335 (18.2%), ST27 (4.5%), and ST1 (1.1%). Predominant STs of serotypes Ia and Ib were ST23 (81.3%) and ST10 (84.2%), respectively. No penicillin-resistant strains were detected, but 22·0% of strains had mef(A/E), erm(A), or erm(B) genes, which mediate macrolide resistance. A new ST335, possessing an mef(A/E) gene belonging to clonal complex 19 gradually increased in frequency. Improved prevention of invasive GBS infections in infants requires timely identification, and ultimately vaccine development.

Effects of Clarithromycin at Sub-Minimum Inhibitory Concentrations on Early &lt;i&gt;erm&lt;/i&gt;B Gene Expression, Metabolic Activity and Growth of an &lt;i&gt;erm&lt;/i&gt;(B)-Expressing Macrolide-Resistant Strain of &lt;i&gt;Streptococcus pneumoniae&lt;/i&gt;

Aim: To investigate the effects of exposure of a macrolide-resistant [erm (B)-expressing] strain of Streptococcus pneumoniae (strain 2507) to clarithromycin (0.5 and 5 mg/L) added at the outset and 6 hours after initiation of culture on early gene expression, energy metabolism, and growth. Methods: Bacterial growth was determined by turbidometric and colony counting procedures, energy metabolism by measurement of ATP, while analysis of gene expression was performed using reverse transcription-PCR and sequencing. Results: Addition of clarithromycin, at either concentration, at the outset of culture, caused transient suppression of growth of 10 - 12 hours duration, while delayed addition of antibiotic (during the logarithmic phase) resulted in an abrupt halt in growth followed by recovery. These inhibitory effects of clarithromycin on bacterial growth were associated with up-regulation of expression of erm(B), decreased ATP and protein synthesis, and were unaffected by inclusion of either catalase (500 and 1000 kunits/L), or competence-stimulating peptide (CSP-1, 0.5 mg/L). The inhibitory effects could, however, be overcome by pre-exposure of the bacteria to the antibiotic. Moreover, clarithromycin appeared to potentiate the antimicrobial actions of ceftriaxone, at sub-MIC concentrations, for strain 2507. Conclusions: Unlike several other common bacterial pathogens, the full expression of erm(B)-mediated macrolide resistance by the pneumococcus has a slow onset, which is associated with transient susceptibility to macrolides and inhibition of growth.

Synthesis, activity and pharmacokinetics of novel antibacterial 15-membered ring macrolones

Synthesis, antibacterial activity and pharmacokinetic properties of a novel class of macrolide antibiotics—macrolones—derived from azithromycin, comprising oxygen atom(s) in the linker and either free or esterified quinolone 3-carboxylic group, are reported. Selected compounds showed excellent antibacterial potency towards key erythromycin resistant respiratory pathogens. However, the majority of compounds lacked good bioavailability. The isopropyl ester, compound 35, and a macrolone derivative with an elongated linker 29 showed the best oral bioavailability in rats, both accompanied with an excellent overall microbiology profile addressing inducible and constitutive MLSb as well as efflux mediated macrolide resistance in streptococci, while compound 29 is more potent against staphylococci.

Molecular characterization of spa type t127, sequence type 1 methicillin-resistant Staphylococcus aureus from pigs

The aim of this study was to provide molecular characterization of methicillin-resistant Staphylococcus aureus (MRSA) spa type t127, sequence type (ST) 1 isolates, detected in a European baseline survey in holdings of breeding pigs, to determine phenotypic and genotypic drug resistance and to compare the results with those obtained from a collection of t127, ST1 MRSA and methicillin-susceptible S. aureus (MSSA) clinical isolates. Twenty-four t127, ST1 MRSA from dust sampled in different breeding holdings in Italy, Spain and Cyprus were studied, along with 2 t127, ST1 MRSA from fattening pigs and 11 human t127, ST1 MRSA and MSSA. Genotyping was performed using multilocus sequence typing (MLST), spa typing and PFGE. SCCmec elements were characterized by multiplex-PCR and resistance and pathogenicity genes by PCR and microarray. PFGE patterns separated a porcine cluster (PC) from a human cluster (HC), with 75% similarity. The PC carried SCCmec cassette type V, while all isolates of the HC carried SCCmec cassette type IVa. Kanamycin resistance mediated by aadD, fluoroquinolone and erm(A)-mediated macrolide resistance and the absence of the sakA gene were features of the PC only. All isolates of both clusters were positive for LukE-LukD and LuF-LukS-HlgA leukotoxin genes and one human MSSA harboured Panton-Valentine leucocidin genes. Despite differences in the host-specific genetic features, the possibility of PC transmission to humans cannot be excluded. MRSA spa type t127, ST1 from pigs possesses several virulence and resistance genes towards major classes of antimicrobials and may represent a serious therapeutic challenge in case of invasive infections in humans.

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.

Increase in Pneumococcus Macrolide Resistance, United States

During year 6 (2005-2006) of the Prospective Resistant Organism Tracking and Epidemiology for the Ketolide Telithromycin surveillance study, 6,747 Streptococcus pneumoniae isolates were collected at 119 centers. The susceptibility of these isolates to macrolides was compared with data from previous years. Macrolide resistance increased significantly in year 6 (35.3%) from the stable rate of approximately 30% for the previous 3 years (p<0.0001). Macrolide resistance increased in all regions of the United States and for all patient age groups. Rates were highest in the south and for children 0-2 years of age. Lower-level efflux [mef(A)]-mediated macrolide resistance decreased in prevalence to approximately 50%, and highly resistant [erm(B) + mef(A)] strains increased to 25%. Telithromycin and levofloxacin susceptibility rates were >99% and >98%, respectively, irrespective of genotype. Pneumococcal macrolide resistance in the United States showed its first significant increase since 2000. High-level macrolide resistance is also increasing.

Novel mutations in ribosomal proteins L4 and L22 that confer erythromycin resistance in Escherichia coli

L4 and L22, proteins of the large ribosomal subunit, contain globular surface domains and elongated ‘tentacles’ that reach into the core of the large subunit to form part of the lining of the peptide exit tunnel. Mutations in the tentacles of L4 and L22 confer macrolide resistance in a variety of pathogenic and non-pathogenic bacteria. In Escherichia coli, a Lys-to-Glu mutation in L4 and a three-amino-acid deletion in the L22 had been reported. To learn more about the roles of the tentacles in ribosome assembly and function, we isolated additional erythromycin-resistant E. coli mutants. Eight new mutations mapped in L4, all within the tentacle. Two new mutations were identified in L22; one mapped outside the tentacle. Insertion mutations were found in both genes. All of the mutants grew slower than the parent, and they all showed reduced in vivo rates of peptide-chain elongation and increased levels of precursor 23S rRNA. Large insertions in L4 and L22 resulted in very slow growth and accumulation of abnormal ribosomal subunits. Our results highlight the important role of L4 and L22 in ribosome function and assembly, and indicate that a variety of changes in these proteins can mediate macrolide resistance.

Macrolide resistance mechanisms among Streptococcus pneumoniae isolated over 6 years of Canadian Respiratory Organism Susceptibility Study (CROSS) (1998 2004)

Resistance to macrolides in Streptococcus pneumoniae arises primarily due to Erm(B) or Mef(A). Erm(B) typically confers high-level resistance to macrolides, lincosamides and streptogramin B (MLS(B) phenotype), whereas Mef(A) confers low-level resistance to macrolides only (M phenotype). The purpose of this study was to investigate the incidence of macrolide resistance mechanisms in Canadian isolates of S. pneumoniae obtained between 1998 and 2004. Furthermore, the genetic relatedness, serotype distribution and antibiotic susceptibility profile among S. pneumoniae isolates with dual erythromycin ribosomal methylase [Erm(B)] and efflux pump [Mef(A)] were analysed. A total of 865 macrolide-resistant (erythromycin MIC > or = 1 mg/L) S. pneumoniae isolates were collected from the Canadian Respiratory Organism Susceptibility Study (CROSS) from 1998 to 2004. The presence of erm(B) and mef(A) was determined for each isolate by PCR; mutations in the genes coding for L4 and L22 ribosomal proteins and for 23S rRNA were identified by DNA sequencing. Each isolate containing both erm(B)- and mef(A)-mediated macrolide resistance was genotyped by PFGE and serotyped using the Quellung reaction with antisera. Of the 865 isolates studied, 404 (46.7%) were mef(A)-positive, 371 (42.9%) were erm(B)-positive, 50 (5.8%) were positive for both mef(A) and erm(B) and 40 (4.6%) were negative for both mef(A) and erm(B). Of the macrolide-resistant isolates negative for both mef(A) and erm(B), 22 (2.5%) contained 23S rRNA A2058G, A2059G or A2059C mutations, 7 (0.8%) contained 23S rRNA A2058G or A2059G mutations along with an S20N mutation in L4 ribosomal protein, and 1 isolate contained an E30K ribosomal protein mutation alone. Of the macrolide-resistant strains positive for both mef(A) and erm(B), 36 (72%) were multidrug-resistant (macrolide-, penicillin- and trimethoprim/sulfamethoxazole-resistant), 39 (78%) isolates belonged to serotype 19A or 19F and 36 (72%) belonged to one clonal complex (> or =80% genetic relatedness) genetically related to the Taiwan 19F-14 clone. The prevalence of efflux-based macrolide resistance in S. pneumoniae in Canada remained steady between 1998 and 2004. Macrolide resistance due to erm(B) decreased over the same time period, with a rapid increase in isolates with both erm(B) and mef(A) macrolide resistance.

Molecular epidemiology of multiresistant Streptococcus pneumoniae with both erm(B)- and mef(A)-mediated macrolide resistance.

Of a total of 1043 macrolide-resistant Streptococcus pneumoniae isolates collected from 24 countries as part of PROTEKT 1999-2000, 71 isolates tested positive for both the mef(A) and erm(B) genes. Of 69 isolates subjected to further molecular investigations, all were resistant to tetracycline, 63 (91.3%) were resistant to penicillin, and 57 (82.6%) were resistant to trimethoprim-sulfamethoxazole. One isolate was also fluoroquinolone resistant, and another was resistant to quinupristin-dalfopristin. The ketolide telithromycin retained activity against all of the isolates. Of the 69 of these 71 isolates viable for further testing, 46 were from South Korea, 13 were from the United States, 8 came from Japan, and 1 each came from Mexico and Hungary. One major clonal complex (59 [85.5%] of 69 isolates) was identified by serotyping (with 85.5% of the isolates being 19A or 19F), pulsed-field gel electrophoresis, and multilocus sequence typing. The remaining isolates were less clonal in nature. Representative isolates were shown to carry the mobile genetic elements Tn1545 and mega, were negative for Tn1207.1, had tetracycline resistance mediated by tet(M), and contained the mef(E) variant of mef(A). All isolates were positive for mel, a homologue of the msr(A) efflux gene. These clones are obviously very efficient at global dissemination, and hence it will be very important to monitor their progress through continued surveillance. Telithromycin demonstrated high levels of activity (MIC for 90% of the strains tested, 0.5 micro g/ml; MIC range, 0.06 to 1 micro g/ml) against all isolates.

In vitro activities of new ketolide, telithromycin, and eight other macrolide antibiotics against Streptococcus pneumoniae having mefA and ermB genes that mediate macrolide resistance

The comparative in vitro activity of a new ketolide, telithromycin (TEL), and eight other macrolide-lincosamide antibiotics (MLS) against 215 strains, of Streptococcus pneumoniae including penicillin-resistant isolates (PRSP), was determined by the agar dilution method. These strains were isolated from patients with pneumonia, otitis media, and purulent meningitis between 1995 and 1997. Two genes, mefA and ermB, that encode MLS resistance in the strains were identified by polymerase chain reaction (PCR). Of the strains, 30.2% (n = 65) had the mefA gene, 37.7% (n = 81) had the ermB gene, and 1.4% (n = 3) had both resistant genes. The minimum inhibitory concentration (MIC90s) of TEL and 16-membered ring MLS for strains having the mefA gene were 0.063-0.25 microg/ml, which were the same level as those for MLS-susceptible strains. On the other hand, the strains with the mefA gene showed low-level resistance to 14- and 15-membered ring MLS, with MIC90s ranging from 1 to 4 microg/ml. Only the MIC90 of TEL at 0.5 microg/ml, for strains having the ermB gene was superior to that of the 14-, 15-, and 16-membered ring MLS (MIC90, > or =64 microg/ml). TEL also showed excellent activity against PRSP having abnormal pbp1a, pbp2x, and pbp2b genes. Most strains having the mefA and ermB genes were serotyped to 3, 6, 14, 19, and 23. These results suggest that TEL may be a useful chemotherapeutic agent for respiratory tract infections caused by S. pneumoniae.

Isolation and characterization of two plasmids that mediate macrolide resistance in Escherichia coli: transferability and molecular properties.

Escherichia coli BM2506 is highly resistant to macrolide antibiotics; it produces macrolide 2'-phosphotransferase II [MPH(2')II] which inactivates such drugs. We investigated the localization and the transfer of the macrolide-resistance determinant that encoded the mphB gene for MPH(2')II in strain BM2506. Although we detected no clear band of plasmid DNA after agarose gel electrophoresis, transformation analysis using satellite DNA that corresponded to plasmid DNA after CsCl-ethidium bromide gradient centrifugation and restriction analysis of plasmid DNA in transformants showed that strain BM2506 harbored two plasmids, pTZ3721 (84 kb) and pTZ3723 (24 kb), that specified resistance to macrolides, ampicillin, streptomycin, tetracycline and sulfonamide and to macrolides and ampicillin, respectively. Southern hybridization showed that the mphB gene hybridized to both plasmids. Furthermore, pTZ3721 was transferred by conjugation to another strain of E. coli and pTZ3723 was mobilized with a self-transferable plasmid RP1 to other strains of E. coli. Therefore, it appears that the mphB gene is located on two plasmids in BM2506 and can be transferred to other strains of E. coli by conjugation or mobilization.