Erythromycin Resistance Gene Erm Research Articles

Microbial community of municipal drinking water in Hangzhou using metagenomic sequencing

While traditional culture-dependent methods can effectively detect certain microorganisms, the comprehensive composition of the municipal drinking water (DW) microbiome, including bacteria, archaea, and viruses, remains unknown. Metagenomic sequencing has opened the door to accurately determine and analyze the entire microbial community of DW, providing a comprehensive understanding of DW species diversity, especially in the context of public health concerns during the COVID-19 era. In this study, we found that most of the culturable bacteria and some fecal indicator bacteria, such as Escherichia coli and Pseudomonas aeruginosa, were non-culturable using culture-dependent methods in all samples. However, metagenomic analysis showed that the predominant bacterial species in the DW samples belonged to the phyla Proteobacteria and Planctomycetes. Notably, the genus Methylobacterium was the most abundant in all water samples, followed by Sphingomonas, Gemmata, and Azospirilum. While low levels of virulence-associated factors, such as the Esx-5 type VII secretion system (T7SS) and DevR/S, were detected, only the erythromycin resistance gene erm(X), an rRNA methyltransferase, was identified at low abundance in one sample. Hosts corresponding to virulence and resistance genes were identified in some samples, including Mycobacterium spp. Archaeal DNA (Euryarchaeota, Crenarchaeota) was found in trace amounts in some DW samples. Viruses such as rotavirus, coxsackievirus, human enterovirus, and SARS-CoV-2 were negative in all DW samples using colloidal gold and real-time reverse transcription polymerase chain reaction (RT‒PCR) methods. However, DNA encoding a new order of reverse-transcribing viruses (Ortervirales) and Herpesvirales was found in some DW samples. The metabolic pathways of the entire microbial community involve cell‒cell communication and signal secretion, contributing to cooperation between different microbial populations in the water. This study provides insight into the microbial community and metabolic process of DW in Hangzhou, China, utilizing both culture-dependent methods and metagenomic sequencing combined with bioinformatics tools during the COVID-19 pandemic era.

Genomic Island-Mediated Horizontal Transfer of the Erythromycin Resistance Gene erm(X) among Bifidobacteria.

Antibiotic resistance is a serious medical issue driven by antibiotic misuse. Bifidobacteria may serve as a reservoir for antibiotic resistance genes (ARGs) that have the potential risk of transfer to pathogens. The erythromycin resistance gene erm(X) is an ARG with high abundance in bifidobacteria, especially in Bifidobacterium longum species. However, the characteristics of the spread and integration of the gene erm(X) into the bifidobacteria genome are poorly understood. In this study, 10 tetW-positive bifidobacterial strains and 1 erm(X)-positive bifidobacterial strain were used to investigate the transfer of ARGs. Conjugation assays found that the erm(X) gene could transfer to five other bifidobacterial strains. Dimethyl sulfoxide (DMSO) and vorinostat significantly promoted the transfer of the erm(X) from strain Bifidobacterium catenulatum subsp. kashiwanohense DSM 21854 to Bifidobacterium longum subsp. suis DSM 20211. Whole-genome sequencing and comparative genomic analysis revealed that the erm(X) gene was located on the genomic island BKGI1 and that BKGI1 was conjugally mobile and transferable. To our knowledge, this is the first report that a genomic island-mediated gene erm(X) transfer in bifidobacteria. Additionally, BKGI1 is very unstable in B. catenulatum subsp. kashiwanohense DSM 21854 and transconjugant D2TC and is highly excisable and has an intermediate circular formation. In silico analysis showed that the BKGI1 homologs were also present in other bifidobacterial strains and were especially abundant in B. longum strains. Thus, our results confirmed that genomic island BKGI1 was one of the vehicles for erm(X) spread. These findings suggest that genomic islands play an important role in the dissemination of the gene erm(X) among Bifidobacterium species. IMPORTANCE Bifidobacteria are a very important group of gut microbiota, and the presence of these bacteria has many beneficial effects for the host. Thus, bifidobacteria have attracted growing interest owing to their potential probiotic properties. Bifidobacteria have been widely exploited by the food industry as probiotic microorganisms, and some species have a long history of safe use in food and feed production. However, the presence of antibiotic resistance raises the risk of its application. In this study, we analyzed the transfer of the erythromycin resistance gene erm(X) and revealed that the molecular mechanism behind the spread of the gene erm(X) was mediated by genomic island BKGI1. To the best of our knowledge this is the first report to describe the transfer of the gene erm(X) via genomic islands among bifidobacteria. This may be an important way to disseminate the gene erm(X) among bifidobacteria.

Genome Comparison of Erythromycin Resistant Campylobacter from Turkeys Identifies Hosts and Pathways for Horizontal Spread of erm(B) Genes.

Pathogens in the genus Campylobacter are the most common cause of food-borne bacterial gastro-enteritis. Campylobacteriosis, caused principally by Campylobacter jejuni and Campylobacter coli, is transmitted to humans by food of animal origin, especially poultry. As for many pathogens, antimicrobial resistance in Campylobacter is increasing at an alarming rate. Erythromycin prescription is the treatment of choice for clinical cases requiring antimicrobial therapy but this is compromised by mobility of the erythromycin resistance gene erm(B) between strains. Here, we evaluate resistance to six antimicrobials in 170 Campylobacter isolates (133 C. coli and 37 C. jejuni) from turkeys. Erythromycin resistant isolates (n = 85; 81 C. coli and 4 C. jejuni) were screened for the presence of the erm(B) gene, that has not previously been identified in isolates from turkeys. The genomes of two positive C. coli isolates were sequenced and in both isolates the erm(B) gene clustered with resistance determinants against aminoglycosides plus tetracycline, including aad9, aadE, aph(2″)-IIIa, aph(3′)-IIIa, and tet(O) genes. Comparative genomic analysis identified identical erm(B) sequences among Campylobacter from turkeys, Streptococcus suis from pigs and Enterococcus faecium and Clostridium difficile from humans. This is consistent with multiple horizontal transfer events among different bacterial species colonizing turkeys. This example highlights the potential for dissemination of antimicrobial resistance across bacterial species boundaries which may compromise their effectiveness in antimicrobial therapy.

A novel erythromycin resistance plasmid from Bacillus sp. strain HS24, isolated from the marine sponge Haliclona simulans.

A better understanding of the origin and natural reservoirs of resistance determinants is fundamental to efficiently tackle antibiotic resistance. This paper reports the identification of a novel 5.8 kb erythromycin resistance plasmid, from Bacillus sp. HS24 isolated from the marine sponge Haliclona simulans. pBHS24B has a mosaic structure and carries the erythromycin resistance gene erm(T). This is the first report of an erythromycin resistance plasmid from a sponge associated bacteria and of the Erm(T) determinant in the genus Bacillus.

Selective pressure affects transfer and establishment of a Lactobacillus plantarum resistance plasmid in the gastrointestinal environment

A Lactobacillus plantarum strain recently isolated from French raw-milk cheese was tested for its ability to transfer a small plasmid pLFE1 harbouring the erythromycin resistance gene erm(B) to Enterococcus faecalis. Mating was studied in vitro and in different gastrointestinal environments using gnotobiotic rats as a simple in vivo model and streptomycin-treated mice as a more complex model. Transfer and establishment of transconjugants in the intestine were investigated with and without selective pressure. Compared with the relatively low transfer frequency of approximately 5.7 x 10(-8) transconjugants/recipient obtained in vitro by filter mating, a surprisingly high number of transconjugants (10(-4) transconjugants/recipient) was observed in gnotobiotic rats even without antibiotic treatment. When erythromycin was administered, a transfer rate of approximately 100% was observed, i.e. the recipient population turned completely into transconjugants (3 x 10(9) cfu/g faeces). Additionally, the time to reach a stable transconjugant population level was much faster in the erythromycin-treated gnotobiotic rats (1 day) than in the untreated animals (4-5 days). Transconjugants persisted in the gut in relatively stable numbers at least 12 days after termination of antibiotic treatment. In the streptomycin-treated mice, no transfer was observed either with or without erythromycin treatment. The overall results imply that the gastrointestinal tract may comprise a more favourable environment for antibiotic resistance transfer than conditions provided in vitro. However, the indigenous gut microbiota severely restricts transfer, thus minimizing the number of detectable transfer events. Treatment with erythromycin strongly favoured transfer and establishment of pLFE1.

Resistance of potential probiotic lactic acid bacteria and bifidobacteria of African and European origin to antimicrobials: Determination and transferability of the resistance genes to other bacteria

Probiotic bacteria and starter cultures of Lactobacillus, Weissella and Bifidobacterium of African and European origins were studied and compared for their susceptibility to antimicrobials. The study included, for all isolates, determination of MICs (Minimal Inhibitory Concentration) for 24 antimicrobials, detection of resistance genes by PCR reactions using specific primers and sequencing of positive amplicons. The ability of Lb. reuteri from Africa to transfer the erythromycin resistance gene erm(B) to closely related bacteria was investigated by conjugation. Variations were observed and high levels of intrinsic resistance were found among the tested species. Positive amplicons were obtained for resistance genes encoding aminoglycoside ( aph(3′) -III, aadA, aadE) and tetracycline ( tet(S)) from isolates from Europe and macrolide ( erm(B)) from an isolate from Africa. However, only the erm(B) gene found in Lb. reuteri L4: 12002 from Africa contained a homologous sequence to previously published sequences. This gene could be transferred in vitro to enterococci. Higher prevalence of phenotypic resistance for aminoglycoside was found in isolates from Europe.

Demonstration of in vivo transfer of doxycycline resistance mediated by a novel transposon

The aim of this study was to investigate the transfer of bacterial doxycycline resistance between oral bacteria in subjects receiving systemic doxycycline for the treatment of periodontitis. Streptococci were cultured before and after treatment from the subgingival plaque of two patients with periodontitis, genotyped and investigated for the presence of antimicrobial resistance determinants and conjugative transposons. In one subject, a strain of Streptococcus sanguinis resistant to doxycycline was a minor component of the pre-treatment streptococcal flora but dominated post-treatment. In a second subject, a strain of Streptococcus cristatus, which was sensitive to doxycycline before treatment, was found to have acquired a novel conjugative transposon during treatment, rendering it resistant to doxycycline and erythromycin. The novel transposon, named CTn6002, was sequenced and found to be a complex element derived in part from Tn916, and an unknown element which included the erythromycin resistance gene erm(B). A strain of Streptococcus oralis isolated from this subject pre-treatment was found to harbour CTn6002 and was therefore implicated as the donor. This is the first direct demonstration of transfer of antimicrobial resistance carried on a conjugative transposon between oral bacteria during systemic antimicrobial treatment of periodontitis in humans.

Horizontal Transfer of Erythromycin Resistance from Clostridium difficile to Butyrivibrio fibrisolvens

This study demonstrates for the first time the in vitro transfer of the erythromycin resistance gene erm(B) between two obligate anaerobes, the human spore-forming pathogen Clostridium difficile and the rumen commensal Butyrivibrio fibrisolvens, suggesting that this event might occur also in the natural environment.

Generating Novel Polyene Antifungal Drugs

In this issue of Chemistry & Biology, Francisco Malpartida and colleagues [1] report the formation of novel polyene amide derivatives upon transformation of the producer strain with SCP2*-derived vectors carrying the erythromycin resistance gene ermE. This unexpected finding provides a new tool for generating antifungal drugs by biotransformation.

Phenotypic and molecular characterization of tetracycline- and erythromycin-resistant strains of Streptococcus pneumoniae.

Sixty-five clinical isolates of Streptococcus pneumoniae, all collected in Italy between 1999 and 2002 and resistant to both tetracycline (MIC, >or=8 microg/ml) and erythromycin (MIC, >or=1 microg/ml), were investigated. Of these strains, 11% were penicillin resistant and 23% were penicillin intermediate. With the use of the erythromycin-clindamycin-rokitamycin triple-disk test, 14 strains were assigned to the constitutive (cMLS) phenotype of macrolide resistance, 44 were assigned to the partially inducible (iMcLS) phenotype, 1 was assigned to the inducible (iMLS) phenotype, and 6 were assigned to the efflux-mediated (M) phenotype. In PCR assays, 64 of the 65 strains were positive for the tetracycline resistance gene tet(M), the exception being the one M isolate susceptible to kanamycin, whereas tet(K), tet(L), and tet(O) were never found. All cMLS, iMcLS, and iMLS isolates had the erythromycin resistance gene erm(B), and all M phenotype isolates had the mef(A) or mef(E) gene. No isolate had the erm(A) gene. The int-Tn gene, encoding the integrase of the Tn916-Tn1545 family of conjugative transposons, was detected in 62 of the 65 test strains. Typing assays showed the strains to be to a great extent unrelated. Of 16 different serotypes detected, the most numerous were 23F (n = 13), 19A (n = 10), 19F (n = 9), 6B (n = 8), and 14 (n = 6). Of 49 different pulsed-field gel electrophoresis types identified, the majority (n = 39) were represented by a single isolate, while the most numerous type included five isolates. By high-resolution restriction analysis of PCR amplicons with four endonucleases, the tet(M) loci from the 64 tet(M)-positive pneumococci were classified into seven distinct restriction types. Overall, a Tn1545-like transposon could reasonably account for tetracycline and erythromycin resistance in the vast majority of the pneumococci of cMLS, iMcLS, and iMLS phenotypes, whereas a Tn916-like transposon could account for tetracycline resistance in most M phenotype strains.

Molecular epidemiology of pneumococcal carriage among children with upper respiratory tract infections in Hanoi, Vietnam.

To investigate the molecular epidemiology of pneumococcal nasopharyngeal carriage in Hanoi, Vietnam, we studied 84 pneumococcal strains retrieved from children with upper respiratory tract infections. Serotypes 23F (32%), 19F (21%), 6B (13%), and 14 (10%) were found most often. A significant number of strains were antibiotic resistant. Fifty-two percent of the strains were (intermediate) resistant to penicillin, 87% were (intermediate) resistant to co-trimoxazole, 76% were resistant to tetracycline, 73% were resistant to erythromycin, and 39% were (intermediate) resistant to cefotaxime. Seventy-five percent were resistant to three or more classes of antibiotics. A high degree of genetic heterogeneity among the penicillin resistance genes was observed. In addition, the tetracycline resistance gene tet(M) and the erythromycin resistance gene erm(B) were predominantly observed among the isolates. Molecular analysis of the 84 isolates by restriction fragment end labeling (RFEL) revealed 35 distinct genotypes. Twelve of these genotypes represented a total of eight genetic clusters with 61 isolates (73%). The two largest clusters contained 24 and 12 isolates, and the isolates in those clusters were identical to the two internationally spreading multidrug-resistant clones Spain 23F-1 and Taiwan 19F-14, respectively. The remaining RFEL types were Vietnam specific, as they did not match the types in our reference collection of 193 distinct RFEL types from 16 countries. Furthermore, 57 of the 61 horizontally spreading isolates (93%) in the eight genetic clusters were covered by the seven-valent conjugate vaccine, whereas this vaccine covered only 43% of the isolates with unique genotypes. According to the serotype distribution of the nasopharyngeal pneumococcal isolates, this study suggests a high potential benefit of the seven-valent pneumococcal conjugate vaccine for children in Hanoi.

Construction and characterization of a Streptomyces rimosus recA mutant: the RecA-deficient strain remains viable.

Although previously reported attempts to construct recA null mutants in Streptomyces spp. have been unsuccessful, we have used the suicide plasmid pErmdeltaRecA to inactivate the recA gene in Streptomyces rimosus by gene disruption. pErmdeltaRecA carries the erythromycin resistance gene ermE and a 451-bp fragment of the S. rimosus recA gene (encoding amino acids 2-151). An erythromycin-resistant clone with single plasmid integration into the recA gene on the chromosome was analyzed in detail. This clone possesses one inactive copy of recA which lacks the entire promoter region and the ATG start codon, and a second, truncated gene that encodes only first 151 amino acids of the RecA protein. This S. rimiosus rec A mutant can therefore be considered a completely RecA-deficient strain. The mutant strain is highly sensitive to UV light. Introduction of a plasmid carrying the wild type S. rimosus recA gene completely restored the UV resistance of the recA mutant to wild-type levels. recA genes encoding RecA proteins with short deletions at the C-terminus (21 and 51 amino acids) could not fully rescue the UV sensitivity of the S. rimosus recA strain, when introduced in the same way.

Mutation and cloning of eryG, the structural gene for erythromycin O-methyltransferase from Saccharopolyspora erythraea, and expression of eryG in Escherichia coli

A mutant strain derived by chemical mutagenesis of Saccharopolyspora erythraea (formerly known as Streptomyces erythreus) was isolated that accumulated erythromycin C and, to a lesser extent, its precursor, erythromycin D, with little or no production of erythromycin A or erythromycin B (the 3"-O-methylation products of erythromycin C and D, respectively). This mutant lacked detectable erythromycin O-methyltransferase activity with erythromycin C, erythromycin D, or the analogs 2-norerythromycin C and 2-norerythromycin D as substrates. A 4.5-kilobase DNA fragment from S. erythraea originating approximately 5 kilobases from the erythromycin resistance gene ermE was identified that regenerated the parental phenotype and restored erythromycin O-methyltransferase activity when transformed into the erythromycin O-methyltransferase-negative mutant. Erythromycin O-methyltransferase activity was detected when the 4.5-kilobase fragment was fused to the lacZ promoter and introduced into Escherichia coli. The activity was dependent on the orientation of the DNA relative to lacZ. We have designated this genotype eryG in agreement with Weber et al. (J.M. Weber, B. Schoner, and R. Losick, Gene 75:235-241, 1989). It thus appears that a single enzyme catalyzes all of the 3"-O-methylation reactions of the erythromycin biosynthetic pathway in S. erythraea and that eryG codes for the structural gene of this enzyme.