Lincomycin Resistance Research Articles

A Comparative Study on Microbial Air Quality of Radiology and Patient Waiting Rooms of a Full-Equipped Hospital in Aydın Province

Every year many patients die due to hospital-acquired infections. These infections also put the lives of healthcare workers at risk. Additionally, hospitals are one of the main reservoirs of antimicrobial resistance. In this study, the effect of X-rays on species diversity and functions and antimicrobial resistance were observed. Metagenome analysis was performed on air samples taken from the x-ray room (radiation exposure) and the waiting room (non-radiation area) to examine whether the radiation affected the species present in the air. The diversity of microorganisms was analysed based on phylum, genus, and species levels. Functional profiling and resistance screening were also performed. X-ray radiation was found to have a major effect at the phylum level. It was observed that Proteobacteria species almost dominated the microbiome in the x-ray room (99%) while Actinobacteria species dominated the microbiome in the waiting room (84%). No significant differences were observed between the two areas in functional profiling. A total of thirty-eight functions were observed, twenty-four of which were overlapping. Antimicrobial resistance was not as diverse as expected. Only beta-lactam, penicillin, tetracycline, and lincomycin resistance genes were present (2364 reads belonging to four different genes). According to the results, it was observed that X-rays affected the air microbiome, as expected. A decrease in the number of microorganisms was expected, but it was also observed that the dominant microorganism types changed. On the other hand, no significant difference was found in terms of functional profiling and no significant antimicrobial resistance was observed.

Antimicrobial Resistance Characteristics of Fecal Escherichia coli and Enterococcus Species in U.S. Goats: 2019 National Animal Health Monitoring System Enteric Study.

Escherichia coli and Enterococcus species are normal bacteria of the gastrointestinal tract and serve as indicator organisms for the epidemiology and emergence of antimicrobial resistance in their hosts and the environment. Some E. coli serovars, including E. coli O157:H7, are important human pathogens, although reservoir species such as goats remain asymptomatic. We describe the prevalence and antimicrobial resistance of generic E. coli, E. coli O157:H7, and Enterococcus species collected from a national surveillance study of goat feces as part of the National Animal Health Monitoring System (NAHMS) Goat 2019 study. Fecal samples were collected from 4918 goats on 332 operations across the United States. Expectedly, a high prevalence of E. coli (98.7%, 4850/4915) and Enterococcus species (94.8%, 4662/4918) was found. E. coli O157:H7 prevalence was low (0.2%; 10/4918). E. coli isolates, up to three per operation, were evaluated for antimicrobial susceptibility and 84.7% (571/674) were pansusceptible. Multidrug resistance (MDR; ≥3 classes) was uncommon among E. coli, occurring in 8.2% of isolates (55/674). Resistance toward seven antimicrobial classes was observed in a single isolate. Resistance to tetracycline alone (13.6%, 92/674) or to tetracycline, streptomycin, and sulfisoxazole (7.0% 47/674) was the most common pattern. All E. coli O157:H7 isolates were pansusceptible. Enterococcus isolates, up to four per operation, were prioritized by public health importance, including Enterococcus faecium and Enterococcus faecalis and evaluated. Resistance to lincomycin (93.8%, 1232/1313) was most common, with MDR detected in 29.5% (388/1313) of isolates. The combination of ciprofloxacin, lincomycin, and quinupristin resistance (27.1%, 105/388) was the most common pattern detected. Distribution and characteristics of antimicrobial resistance in E. coli and Enterococcus in the U.S. goat population from this study can inform stewardship considerations and public health efforts surrounding goats and their products.

Comparative Genomics of Staphylococcus rostri, an Undescribed Bacterium Isolated from Dairy Mastitis.

This study characterizes 81 S. rostri isolates from bovine mastitis (of which 80 were subclinical). The isolates were first identified as S. microti by MALDI-TOF MS, but later whole genome sequencing analysis allowed reclassification as S. rostri. The isolates were derived from 52 cows and nine dairy herds in Denmark. To describe the pathogenicity of S. rostri, we used whole genome sequencing to infer the distribution of genes associated with virulence, antibiotic resistance, and mobile genetic elements. Also, we performed a core-genome phylogeny analysis to study the genetic relatedness among the isolates. All 81 isolates expressed the same virulence profile comprising two putative virulence genes, clpP and clpC. Three isolates carried a resistance gene encoding streptomycin (str) or lincomycin (lnuA) resistance. The distribution of plasmids suggested the detected antibiotic resistance genes to be plasmid-mediated. Phages were abundant among the isolates, and the single isolate from clinical mastitis acquired a phage disparate from the rest, which potentially could be involved with virulence in S. rostri. The core genome phylogeny revealed a strong genetic intra-herd conservation, which indicates the source of introduction being herd-specific and might further imply the ability of S. rostri to adapt to the bovine niche and spread from cow-to-cow in a contagious manner. With this study, we aim to acquaint clinicians and professionals with the existence of S. rostri which might have been overlooked so far.

Staphylococcus equorum plasmid pKS1030-3 encodes auxiliary biofilm formation and trans-acting gene mobilization systems

The foodborne bacterium Staphylococcus equorum strain KS1030 harbours plasmid pSELNU1, which encodes a lincomycin resistance gene. pSELNU1 undergoes horizontal transfer between bacterial strains, thus spreading antibiotic resistance. However, the genes required for horizontal plasmid transfer are not encoded in pSELNU1. Interestingly, a relaxase gene, a type of gene related to horizontal plasmid transfer, is encoded in another plasmid of S. equorum KS1030, pKS1030-3. The complete genome of pKS1030-3 is 13,583 bp long and encodes genes for plasmid replication, biofilm formation (the ica operon), and horizontal gene transfer. The replication system of pKS1030-3 possesses the replication protein-encoding gene repB, a double-stranded origin of replication, and two single-stranded origins of replication. The ica operon, relaxase gene, and a mobilization protein-encoding gene were detected in pKS1030-3 strain-specifically. When expressed in S. aureus RN4220, the ica operon and relaxase operon of pKS1030-3 conferred biofilm formation ability and horizontal gene transfer ability, respectively. The results of our analyses show that the horizontal transfer of pSELNU1 of S. equorum strain KS1030 depends on the relaxase encoded by pKS1030-3, which is therefore trans-acting. Genes encoded in pKS1030-3 contribute to important strain-specific properties of S. equorum KS1030. These results could contribute to preventing the horizontal transfer of antibiotic resistance genes in food.

Association of Virulence, Biofilm, and Antimicrobial Resistance Genes with Specific Clonal Complex Types of Listeria monocytogenes.

Precise classification of foodborne pathogen Listeria monocytogenes is a necessity in efficient foodborne disease surveillance, outbreak detection, and source tracking throughout the food chain. In this study, a total of 150 L. monocytogenes isolates from various food products, food processing environments, and clinical sources were investigated for variations in virulence, biofilm formation, and the presence of antimicrobial resistance genes based on their Whole-Genome Sequences. Clonal complex (CC) determination based on Multi-Locus Sequence Typing (MLST) revealed twenty-eight CC-types including eight isolates representing novel CC-types. The eight isolates comprising the novel CC-types share the majority of the known (cold and acid) stress tolerance genes and are all genetic lineage II, serogroup 1/2a-3a. Pan-genome-wide association analysis by Scoary using Fisher's exact test identified eleven genes specifically associated with clinical isolates. Screening for the presence of antimicrobial and virulence genes using the ABRicate tool uncovered variations in the presence of Listeria Pathogenicity Islands (LIPIs) and other known virulence genes. Specifically, the distributions of actA, ecbA, inlF, inlJ, lapB, LIPI-3, and vip genes across isolates were found to be significantly CC-dependent while the presence of ami, inlF, inlJ, and LIPI-3 was associated with clinical isolates specifically. In addition, Roary-derived phylogenetic grouping based on Antimicrobial-Resistant Genes (AMRs) revealed that the thiol transferase (FosX) gene was present in all lineage I isolates, and the presence of the lincomycin resistance ABC-F-type ribosomal protection protein (lmo0919_fam) was also genetic-lineage-dependent. More importantly, the genes found to be specific to CC-type were consistent when a validation analysis was performed with fully assembled, high-quality complete L. monocytogenes genome sequences (n = 247) extracted from the National Centre for Biotechnology Information (NCBI) microbial genomes database. This work highlights the usefulness of MLST-based CC typing using the Whole-Genome Sequence as a tool in classifying isolates.

Population Structure and Genomic Characteristics of Australian Erysipelothrix rhusiopathiae Reveals Unobserved Diversity in the Australian Pig Industry.

Erysipelothrix rhusiopathiae is a bacterial pathogen that is the causative agent of erysipelas in a variety of animals, including swine, emus, turkeys, muskox, caribou, moose, and humans. This study aims to investigate the population structure and genomic features of Australian isolates of E. rhusiopathiae in the Australian pig industry and compare them to the broader scope of isolates worldwide. A total of 178 isolates (154 Australian, seven vaccine isolates, six international isolates, and 11 of unknown origin) in this study were screened against an MLST scheme and publicly available reference isolates, identifying 59 new alleles, with isolates separating into two main single locus variant groups. Investigation with BLASTn revealed the presence of the spaA gene in 171 (96%) of the isolates, with three main groups of SpaA protein sequences observed amongst the isolates. Novel SpaA protein sequences, categorised here as group 3 sequences, consisted of two sequence types forming separate clades to groups 1 and 2, with amino acid variants at positions 195 (D/A), 303 (G/E) and 323(P/L). In addition to the newly identified groups, five new variant positions were identified, 124 (S/N), 307 (Q/R), 323 (P/L), 379 (M/I), and 400 (V/I). Resistance screening identified genes related to lincomycin, streptomycin, erythromycin, and tetracycline resistance. Of the 29 isolates carrying these resistance genes, 82% belonged to SpaA group 2-N101S (n = 22) or 2-N101S-I257L (n = 2). In addition, 79% (n = 23) of these 29 isolates belonged to MLST group ST 5. Our results illustrate that Australia appears to have a unique diversity of E. rhusiopathiae isolates in pig production industries within the wider global context of isolates.

Emergence of lnu(C) variant conferring lincomycin resistance in Campylobacter coli of chicken origin

Lincomycin is widely used in respiratory and gastrointestinal infection in veterinary medicine and food animal production. Campylobacter members are vital foodborne pathogens causing campylobacteriosis, and the resistance to lincosamides is seldom reported. To date, only the rRNA methyltransferase Erm(B) has been confirmed to be associated with lincosamides resistance in Campylobacter. In this study, we identified a lnu(C) variant conferring lincomycin resistance in this pathogen of chicken origin. The Lnu(C) encoded by this gene variant showed substitution at position 8 (Asn8Lys), 11 (Phe11Leu) and 112 (Leu112Phe), when compared with the firstly reported Lnu(C) from Streptococcus agalactiae. Cloning of the lnu(C) variant into lincosamide-susceptible Campylobacter jejuni NCTC 11168 confirmed its function in conferring resistance to lincomycin with the 32-fold increased MICs. Sequencing analysis showed that the lnu(C) variant was located within a MTnSag1-like transposon together with insLNU, which is inserted between panB and cj0299 genes on the chromosome. lnu(C) gene was distributed among C. coli globally, and various STs were involved in the dissemination of lnu(C). Although transposition mediated by MTnSag1-like transposon failed to occur, the horizontal transfer mediated by natural transformation and reservoir for resistance genes may facilitate their adaptation to the antimicrobial selection pressure in chickens, which should not be ignored.

Mutations within gene XNR_2147 for TetR-like protein enhance lincomycin resistance and endogenous specialized metabolism of Streptomyces albus J1074.

Streptomyces albus J1074 is one of the most popular heterologous expression platforms among streptomycetes. Identification of new genes and mutations that influence specialized metabolism in this species is therefore of great applied interest. Here, we describe S. albus KO-1304 that was isolated as a spontaneous lincomycin-resistant variant of double rpsLR94G rsmGR15SG40E mutant KO-1295. Besides altered antibiotic resistance profile, KO-1304 exhibited increased antibiotic activity as compared to its parental strains. KO-1304 genome sequencing revealed mutations within gene XNR_2147 encoding putative TetR-like protein. Gene XNR_2146 for efflux protein is the most likely target of repressing action of Xnr_2147. Our data agree with the scenario where lincomycin resistance phenotype of KO-1304 arose from inability of mutated Xnr_2147 protein to repress XNR_2146. Introduction of additional copy of XNR_2146 into wild type strain increased antibiotic activity of the latter, attesting to the practical value of transporter genes for strain improvement.

Transcriptomic analysis of Staphylococcus equorum KM1031 from the high-salt fermented seafood jeotgal under chloramphenicol, erythromycin and lincomycin stresses

Staphylococcus equorum strain KM1031 is resistant to chloramphenicol, erythromycin and lincomycin. To shed light on the genetic factors underlying these antibiotic resistances, we determined the global gene expression profile of S. equorum KM1031 using RNA sequencing. During chloramphenicol, erythromycin and lincomycin treatment, 8.3% (183/2,336), 16.0% (354/2,336), and 2.9% (63/2,336) of S. equorum KM1031 genes exhibited significant differences in expression, respectively. These three antibiotics upregulated genes related to efflux and downregulated genes related to transporters. Antibiotic treatment also upregulated osmoprotectant-related genes involved in salt tolerance. To identify specific genes functionally related to antibiotic resistance, we compared the genome of strain KM1031 with those of three S. equorum strains that are sensitive to these three antibiotics. We identified three genes of particular interest: an antibiotic biosynthesis monooxygenase gene (abm, AWC34_RS01805) related to chloramphenicol resistance, an antibiotic ABC transporter ATP-binding protein gene (msr, AWC34_RS11115) related to erythromycin resistance, and a lincosamide nucleotydyltransferase gene (lnuA, AWC34_RS13300) related to lincomycin resistance. These genes were upregulated in response to the corresponding antibiotic; in particular, msr was upregulated more than fourfold by erythromycin treatment. Finally, the results of RNA sequencing were validated by quantitative real-time PCR. This transcriptomic analysis provides genetic evidence regarding antibiotic stress responses of S. equorum strain KM1031.

Occurrence and Antimicrobial Resistance of Enterococci Isolated from Goat's Milk.

IntroductionEnterococci are widespread, being part of the bacterial flora of humans and animals. The food chain can be therefore considered as the main route of transmission of antibiotic resistant bacteria between the animal and human populations. Milk in particular represents a source from which resistant bacteria can enter the human food chain. The aim of the study was to determine the occurrence and resistance to antimicrobial agents of Enterococcus spp. strains isolated from raw goat’s milk samples.Material and MethodsA total of 207 goat’s milk samples were collected. Samples were cultivated on selective media and confirmed as E. faecium or E. faecalis and screened for selected resistance genes by PCR. Drug susceptibility determination was performed by microdilution on Sensititre EU Surveillance Enterococcus EUVENC Antimicrobial Susceptibility Testing (AST) Plates and Sensititre US National Antimicrobial Resistance Monitoring System Gram Positive CMV3AGPF AST Plates.ResultsEnterococcal strains totalling 196 were isolated, of which 40.8% were E. faecalis and 15.3% were E. faecium. All tested isolates were susceptible to linezolid, penicillin and tigecycline. For most other antimicrobials the prevalence of resistance was 0.5–6.6% while high prevalence of quinupristin/dalfopristin (51.5%), tetracycline (30%) and lincomycin (52%) resistance was observed.ConclusionThis study affords better knowledge concerning the safety of raw goat’s milk in terms of the enterococci possible to isolate from this foodstuff. It seems that enterococci in milk are still mostly susceptible to antimicrobials of major concern as multiply resisted drugs, such as gentamycin and vancomycin. However, the presence of multi-resistant strains in goat milk is cause for apprehension.

Antimicrobial Resistance Genes in Bacteria Isolated From Japanese Honey, and Their Potential for Conferring Macrolide and Lincosamide Resistance in the American Foulbrood Pathogen Paenibacillus larvae.

American foulbrood (AFB) is the most serious bacterial disease of honey bee brood. Spores of the causative agent Paenibacillus larvae are ingested by bee larvae via brood foods and germinated cells proliferate in the larval midgut. In Japan, a macrolide antibiotic, tylosin, is used as the approved prophylactic for AFB. Although tylosin-resistant P. larvae has yet to be found in Japan, it may emerge in the future through the acquisition of macrolide resistance genes from other bacteria, and bacteria latent in brood foods, such as honey, may serve as a source of resistance genes. In this study, to investigate macrolide resistance genes in honey, we attempted to isolate tylosin-resistant bacteria from 53 Japanese honey samples and obtained 209 isolates from 48 samples in the presence of 1 μg/ml of tylosin. All isolates were Gram-positive spore-forming bacteria mainly belonging to genera Bacillus and Paenibacillus, and 94.3% exhibited lower susceptibility to tylosin than Japanese P. larvae isolates. Genome analysis of 50 representative isolates revealed the presence of putative macrolide resistance genes in the isolates, and some of them were located on mobile genetic elements (MGEs). Among the genes on MGEs, ermC on the putative mobilizable plasmid pJ18TS1mac of Oceanobacillus strain J18TS1 conferred tylosin and lincomycin resistance to P. larvae after introducing the cloned gene using the expression vector. Moreover, pJ18TS1mac was retained in the P. larvae population for a long period even under non-selective conditions. This suggests that bacteria in honey is a source of genes for conferring tylosin resistance to P. larvae; therefore, monitoring of bacteria in honey may be helpful to predict the emergence of tylosin-resistant P. larvae and prevent the selection of resistant strains.

Toxinotyping and molecular characterization of antimicrobial resistance in Clostridium perfringens isolated from different sources of livestock and poultry

The present study was designed to understand the presence of antimicrobial resistance among the prevalent toxinotypes of Clostridium perfringens recovered from different animals of Tamil Nadu, India. A total of 75 (10.76%) C. perfringens were isolated from 697 multi-species fecal and intestinal content samples. C. perfringens type A (90.67%), type C (2.67%), type D (4%) and type F (2.67%) were recovered. Maximum number of isolates were recovered from dog (n = 20, 24.10%) followed by chicken (n = 19, 5.88%). Recovered isolates were resistant to gentamicin (44.00%), erythromycin (40.00%), bacitracin (40.00%), and tetracycline (26.67%), phenotypically and most of the isolates were found to be resistant to multiple antimicrobials. Genotypic characterization revealed that tetracycline (41.33%), erythromycin (34.66%) and bacitracin (17.33%) resistant genes were present individually or in combination among the isolates. Combined results of phenotypic and genotypic characterization showed the highest percentage of erythromycin resistance (26.66%) among the isolates. None of the isolates showed amplification for lincomycin resistance genes. The correlation matrix analysis of genotypic resistance showed a weak positive relationship between the tetracycline and bacitracin resistance while a weak negative relationship between the tetracycline and erythromycin resistance. The present study thus reports the presence of multiple-resistance genes among C. perfringens isolates that may be involved in the dissemination of resistance to other bacteria present across species. Further insights into the genome can help to understand the mechanism involved in gene transfer so that measures can be taken to prevent the AMR spread.

Comparison of Antimicrobial Resistance and Pan-Genome of Clinical and Non-Clinical Enterococcus cecorum from Poultry Using Whole-Genome Sequencing.

Enterococcus cecorum is an emerging avian pathogen, particularly in chickens, but can be found in both diseased (clinical) and healthy (non-clinical) poultry. To better define differences between E. cecorum from the two groups, whole-genome sequencing (WGS) was used to identify and compare antimicrobial resistance genes as well as the pan-genome among the isolates. Eighteen strains selected from our previous study were subjected to WGS using Illumina MiSeq and comparatively analyzed. Assembled contigs were analyzed for resistance genes using ARG-ANNOT. Resistance to erythromycin was mediated by ermB, ermG, and mefA, in clinical isolates and ermB and mefA, in non-clinical isolates. Lincomycin resistance genes were identified as linB, lnuB, lnuC, and lnuD with lnuD found only in non-clinical E. cecorum; however, lnuB and linB were found in only one clinical isolate. For both groups of isolates, kanamycin resistance was mediated by aph3-III, while tetracycline resistance was conferred by tetM, tetO, and tetL. No mutations or known resistance genes were found for isolates resistant to either linezolid or chloramphenicol, suggesting possible new mechanisms of resistance to these drugs. A comparison of WGS results confirmed that non-clinical isolates contained more resistance genes than clinical isolates. The pan-genome of clinical and non-clinical isolates resulted in 3651 and 4950 gene families, respectively, whereas the core gene sets were comprised of 1559 and 1534 gene families in clinical and non-clinical isolates, respectively. Unique genes were found more frequently in non-clinical isolates than clinical. Phylogenetic analysis of the isolates and all the available complete and draft genomes showed no correlation between healthy and diseased poultry. Additional genomic comparison is required to elucidate genetic factors in E. cecorum that contribute to disease in poultry.

MOLECULAR CHARACTERIZATION OF CLOSTRIDIUM PERFRINGENS ISOLATED FROM TURKEYS

A total of 82 C. perfringensisolates (41%) were recovered from 200 samples collected from 100 turkeys (20 apparently healthy, 40 clinically diseased and 40 freshly dead), 4-6 weeks old. The clinical signs of diseased birds were sudden mortality, depression, ruffled feathers, diarrhea, dehydration, emaciation and decrease in feed consumption with increase in water consumption. While, postmortem findings were consistent with necrotic enteritis (NE), the small intestine was severely affected. Small and demarcated lesions were found in the duodenal loop, hepatitis and cholecystitis were also observed. Unopened intestines were gas-filled and enlarged with thin wall. The intestinal contents were dark due to necrotic material. The opened intestines showed necrotic lesions of varying severity of the mucosa. Isolation and biochemical identification of C.perfringenswere done.Five C.Perfringens field isolates were analyzed by PCR assay to determine the presence of some toxin genes.In all tested isolates, the Cpa gene (alpha toxin) was detected (100%) confirming the isolates were C.perfringens. While, Cpb (beta toxin) gene was detected in two samples (40%). But the Etx gene (epsilon toxin) was not detected in any isolate (0%). NetB gene was detected in two isolates (40%). Antimicrobial susceptibility of 40 C.perfringens field isolates recovered from turkeys revealed that C.perfringens isolates were sensitive to Ampicillin (85%), Amoxicillin (85%), Penicillin (82.5%), Florfenicol (72.5%), Enrofloxacin (67.5%), Vancomycin (65%), Bacitracin (60%), Oxytetracyclin (32.5%), Lincomycin (22.5%) and Clindamycin (15%).These five C. perfringens isolates were also screened by PCR which detected the presence of tetracycline resistance gene tet(K) in three isolates (60%) and Lincomycin resistance gene lin(B) in three isolates (60%).

Dosage effects of lincomycin mycelial residues on lincomycin resistance genes and soil microbial communities

Lincomycin mycelial residues (LMRs) are one kind of byproduct of the pharmaceutical industry. Hydrothermal treatment has been used to dispose of them and land application is an attractive way to reuse the treated LMRs. However, the safe dose for soil amendment remains unclear. In this study, a lab-scale incubation experiment was conducted to investigate the influence of the amendment dosage on lincomycin resistance genes and soil bacterial communities via quantitative PCR and 16S rRNA sequencing. The results showed that introduced lincomycin degraded quickly in soil and became undetectable after 50 days. Degradation rate of the high amendment amount (100 mg kg−1) was almost 4 times faster than that of low amendment amount (10 mg kg−1). Moreover, the introduced LMRs induced the increase of lincomycin resistance genes after incubation for 8 days, and two genes (lmrA and lnuB) showed a dosage-related increase. For example, the abundance of gene lmrA was 17.78, 74.13 and 128.82 copies g−1 soil for lincomycin concentration of 10, 50 and 100 mg kg−1, respectively. However, the abundance of lincomycin resistance genes recovered to the control level as the incubation period extended to 50 days, indicating a low persistence in soil. In addition, LMRs application markedly shifted the bacterial composition and significant difference was found between control soil, 10 mg kg−1 and 50 mg kg−1 lincomycin amended soil. Actually, several genera bacteria were significantly related to the elevation of lincomycin resistance genes. These results provided a comprehensive understanding of the effects of lincomycin dosage on the fate of resistance genes and microbial communities in LMRs applied soil.

New genetic context of lnu(B) composed of two multi-resistance gene clusters in clinical Streptococcus agalactiae ST-19 strains

BackgroundClindamycin is a lincosamide antibiotic used to treat staphylococcal and streptococcal infections. Reports of clinical Streptococcus agalactiae isolates with the rare lincosamide resistance/macrolide susceptibility (LR/MS) phenotype are increasing worldwide. In this study, we characterised three clinical S. agalactiae strains with the unusual L phenotype from China.MethodsThree clinical S. agalactiae strains, Sag3, Sag27 and Sag4104, with the L phenotype were identified from 186 isolates collected from 2016 to 2018 in Shanghai, China. The MICs of clindamycin, erythromycin, tetracycline, levofloxacin, and penicillin were determined using Etest. PCR for the lnu(B) gene was conducted. Whole genome sequencing and sequence analysis were carried out to investigate the genetic context of lnu(B). Efforts to transfer lincomycin resistance by conjugation and to identify the circular form by inverse PCR were made.ResultsSag3, Sag27, and Sag4104 were susceptible to erythromycin (MIC ≤0.25 mg/L) but resistant to clindamycin (MIC ≥1 mg/L). lnu(B) was found to be responsible for the L phenotype. lnu(B) in Sag3 and Sag27 were chromosomally located in an aadE-spw-lsa(E)-lnu(B) resistance gene cluster adjacent to an upstream 7-kb tet(L)-cat resistance gene cluster. Two resistance gene clusters were flanked by the IS6-like element, IS1216. Sag4104 only contained partial genes of aadE-spw-lsa(E)-lnu(B) resistance gene cluster and was also flanked by IS1216.ConclusionThese results established the presence of the L phenotype associated with lnu(B) in clinical S. agalactiae isolates in China. The lnu(B)-containing multi-resistance gene cluster possibly acts as a composite transposon flanked by IS1216 and as a vehicle for the dissemination of multidrug resistance among S. agalactiae.

Phenotypic, molecular and genomic characterization of Actinobaculum suis isolated from swine in Brazil

Urinary tract infections (UTI) are considered one of the most important diseases of sows due to its close relationship with reproductive problems such as reduced litter size, increase in the rate of return to estrous, vulvar discharge, abortion, mastitis and anestrus. Actinobaculum suis is one of the main agents involved in porcine urinary tract infection and is responsible for the most severe and fatal cases in sows. In the present report, 23 A. suis strains isolated from a sow and boars in Brazil were identified by PCR and further characterized by broth microdilution, molecular typing by pulsed-field gel electrophoresis (PFGE), single-enzyme amplified fragment length polymorphism (SE-AFLP), and whole-genome sequencing. All strains were sensitive to ceftiofur, linezolid, nitrofurantoin, quinupristin-dalfopristin and vancomycin. Ciprofloxacin, daptomycin, lincomycin, erythromycin and tylosin resistance was observed in 100% of tested strains. Tetracycline and tigecycline also presented high resistance rates (87% and 30.4%, respectively). PFGE with eight different restriction enzymes and three programs did not enable strain characterization; however, all strains were typed by SE-AFLP that clustered strains according to their origin, thus proving an effective tool for A. suis genotyping. Whole-genome sequencing and comparative analysis enabled species differentiation from closely related genus. This is the first report of genomic characterization of A. suis.

TetR-Type Regulator SLCG_2919 Is a Negative Regulator of Lincomycin Biosynthesis in Streptomyces lincolnensis.

Lincomycin A (Lin-A) is a widely used antibacterial antibiotic fermented by Streptomyces lincolnensis However, the transcriptional regulatory mechanisms underlying lincomycin biosynthesis have seldom been investigated. Here, we first identified a TetR family transcriptional regulator (TFR), SLCG_2919, which negatively modulates lincomycin biosynthesis in S. lincolnensis LCGL. SLCG_2919 was found to specifically bind to promoter regions of the lincomycin biosynthetic gene cluster (lin cluster), including 25 structural genes, three resistance genes, and one regulatory gene, and to inhibit the transcription of these genes, demonstrating a directly regulatory role in lincomycin biosynthesis. Furthermore, we found that SLCG_2919 was not autoregulated, but directly repressed its adjacent gene, SLCG_2920, which encodes an ATP/GTP binding protein whose overexpression increased resistance against lincomycin and Lin-A yields in S. lincolnensis The precise SLCG_2919 binding site within the promoter region of SLCG_2920 was determined by a DNase I footprinting assay and by electrophoretic mobility shift assays (EMSAs) based on base substitution mutagenesis, with the internal 10-nucleotide (nt) AT-rich sequence (AAATTATTTA) shown to be essential for SLCG_2919 binding. Our findings indicate that SLCG_2919 is a negative regulator for controlling lincomycin biosynthesis in S. lincolnensis The present study improves our understanding of molecular regulation for lincomycin biosynthesis.IMPORTANCE TetR family transcriptional regulators (TFRs) are generally found to regulate diverse cellular processes in bacteria, especially antibiotic biosynthesis in Streptomyces species. However, knowledge of their function in lincomycin biosynthesis in S. lincolnensis remains unknown. The present study provides a new insight into the regulation of lincomycin biosynthesis through a TFR, SLCG_2919, that directly modulates lincomycin production and resistance. Intriguingly, SLCG_2919 and its adjoining gene, SLCG_2920, which encodes an ATP/GTP binding protein, were extensively distributed in diverse Streptomyces species. In addition, we revealed a new TFR binding motif, in which SLCG_2919 binds to the promoter region of SLCG_2920, dependent on the intervening AT-rich sequence rather than on the flanking inverted repeats found in the binding sites of other TFRs. These insights into transcriptional regulation of lincomycin biosynthesis by SLCG_2919 will be valuable in paving the way for genetic engineering of regulatory elements in Streptomyces species to improve antibiotic production.

Whole-Genome Sequence Analysis of Multidrug-Resistant Campylobacter Isolates: a Focus on Aminoglycoside Resistance Determinants.

A whole-genome sequencing (WGS) approach was conducted in order to identify the molecular determinants associated with antimicrobial resistance in 12 multidrug-resistant Campylobacter jejuni and Campylobacter coli isolates, with a focus on aminoglycoside resistance determinants. Two variants of a new aminoglycoside phosphotransferase gene [aph(2″)-Ii1 and aph(2″)-Ii2 ] putatively associated with gentamicin resistance were found. In addition, the following new genes were identified for the first time in Campylobacter: a lincosamide nucleotidyltransferase gene [lnu(G)], likely associated with lincomycin resistance, and two resistance enzyme genes (spw and apmA) similar to those found in Staphylococcus aureus, which may confer spectinomycin and gentamicin resistance, respectively. A C1192T mutation of the 16S rRNA gene that may be involved in spectinomycin resistance was also found in a C. coli isolate. Genes identified in the present study were located either on the bacterial chromosome or on plasmids that could be transferred naturally. Their role in aminoglycoside resistance remains to be supported by genetic studies. Regarding the other antimicrobial agents studied, i.e., ampicillin, ciprofloxacin, erythromycin, and tetracycline, a perfect correlation between antimicrobial phenotypes and genotypes was found. Overall, our data suggest that WGS analysis is a powerful tool for identifying resistance determinants in Campylobacter and can disclose the full genetic elements associated with resistance, including antimicrobial compounds not tested routinely in antimicrobial susceptibility testing.

A possible mechanism for lincomycin induction of secondary metabolism in Streptomyces coelicolor A3(2).

Lincomycin forms cross-links within the peptidyl transferase loop region of the 23S ribosomal RNA (rRNA) of the 50S subunit of the bacterial ribosome, which is the site of peptide bond formation, thereby inhibiting protein synthesis. We have previously reported that lincomycin at concentrations below the minimum inhibitory concentration potentiates the production of secondary metabolites in actinomycete strains, suggesting that activation of these strains by utilizing the dose-dependent response of lincomycin could be used to effectively induce the production of cryptic secondary metabolites. Here, we aimed to elucidate the fundamental mechanisms underlying lincomycin induction of secondary metabolism in actinomycetes. In the present study, the dose-dependent response of lincomycin on gene expression of the model actinomycete Streptomyces coelicolor A3(2) and possible relationships to secondary metabolism were investigated. RNA sequencing analysis indicated that lincomycin produced enormous changes in gene expression profiles. Moreover, reverse transcription PCR and/or comparative proteome analysis revealed that in S. coelicolor A3(2), lincomycin, which was used at concentrations for markedly increased blue-pigmented antibiotic actinorhodin production, rapidly enhanced expression of the gene encoding the lincomycin-efflux ABC transporter, the 23S rRNA methyltransferase, and the ribosome-splitting factor to boost the intrinsic lincomycin resistance mechanisms and to reconstruct the probably stalled 70S ribosomes with lincomycin; and in contrast temporarily but dramatically reduced mRNA levels of housekeeping genes, such as those encoding FoF1 ATP synthase, RNA polymerase, ribosomal proteins, and transcription and translation factors, with an increase in intracellular NTPs. A possible mechanism for lincomycin induction of secondary metabolism in S. coelicolor A3(2) is discussed on the basis of these results.