Comparative Genomic Analysis of Enterococci across Sectors of the One Health Continuum.

Enterococci are Gram-positive bacteria that can be isolated from a variety of environments including soil, water, plants, and the intestinal tract of humans and animals. Although they are considered commensals in humans, Enterococcus spp. are important opportunistic pathogens. Due to their presence and persistence in diverse environments, Enterococcus spp. are ideal for studying antimicrobial resistance (AMR) from the One Health perspective. We undertook a comparative genomic analysis of the virulome, resistome, mobilome, and the association between the resistome and mobilome of 246 E. faecium and 376 E. faecalis recovered from livestock (swine, beef cattle, poultry, dairy cattle), human clinical samples, municipal wastewater, and environmental sources. Comparative genomics of E. faecium and E. faecalis identified 31 and 34 different antimicrobial resistance genes (ARGs), with 62% and 68% of the isolates having plasmid-associated ARGs, respectively. Across the One Health continuum, tetracycline (tetL and tetM) and macrolide resistance (ermB) were commonly identified in E. faecium and E. faecalis. These ARGs were frequently associated with mobile genetic elements along with other ARGs conferring resistance against aminoglycosides [ant(6)-la, aph(3')-IIIa], lincosamides [lnuG, lsaE], and streptogramins (sat4). Study of the core E. faecium genome identified two main clades, clade 'A' and 'B', with clade A isolates primarily originating from humans and municipal wastewater and carrying more virulence genes and ARGs related to category I antimicrobials. Overall, despite differences in antimicrobial usage across the continuum, tetracycline and macrolide resistance genes persisted in all sectors.

Production systems and important antimicrobial resistant-pathogenic bacteria in poultry: a review.

Economic losses and market constraints caused by bacterial diseases such as colibacillosis due to avian pathogenic Escherichia coli and necrotic enteritis due to Clostridium perfringens remain major problems for poultry producers, despite substantial efforts in prevention and control. Antibiotics have been used not only for the treatment and prevention of such diseases, but also for growth promotion. Consequently, these practices have been linked to the selection and spread of antimicrobial resistant bacteria which constitute a significant global threat to humans, animals, and the environment. To break down the antimicrobial resistance (AMR), poultry producers are restricting the antimicrobial use (AMU) while adopting the antibiotic-free (ABF) and organic production practices to satisfy consumers' demands. However, it is not well understood how ABF and organic poultry production practices influence AMR profiles in the poultry gut microbiome. Various Gram-negative (Salmonella enterica serovars, Campylobacter jejuni/coli, E. coli) and Gram-positive (Enterococcus spp., Staphylococcus spp. and C. perfringens) bacteria harboring multiple AMR determinants have been reported in poultry including organically- and ABF-raised chickens. In this review, we discussed major poultry production systems (conventional, ABF and organic) and their impacts on AMR in some potential pathogenic Gram-negative and Gram-positive bacteria which could allow identifying issues and opportunities to develop efficient and safe production practices in controlling pathogens.

Virulence Genotype and Phenotype of Multiple Antimicrobial-Resistant Escherichia coli Isolates from Broilers Assessed from a "One-Health" Perspective.

ABSTRACT: Extraintestinal pathogenic Escherichia coli (ExPEC) include several serotypes that have been associated with colibacillosis in poultry and with urinary tract infections (UTIs) and newborn meningitis in humans. In this study, 57 antimicrobial-resistant E. coli from apparently healthy broiler chickens were characterized for their health and safety risks. These isolates belonged to 12 serotypes, and isolates of the same serotype were clonal based on single nucleotide variant analysis. Most of the isolates harbored plasmids; IncC and IncFIA were frequently detected. The majority of the resistant isolates harbored plasmid-mediated resistance genes, including aph(3″)-Ib, aph(6)-Id, blaCMY-2, floR, sul1, sul2, tet(A), and tet(B), in agreement with their resistant phenotypes. The class 1 integron was detected in all E. coli serotypes except O124:H25 and O7:H6. Of the 57 broiler E. coli isolates, 27 were avian pathogenic, among which 18 were also uropathogenic E. coli and the remainder were other ExPEC. The two isolates of serotype O161:H4 (ST117) were genetically related to the control avian pathogenic strains and a clinical isolate associated with UTIs. A strain of serotype O159:H45 (ST101) also was closely related to a UTI isolate. The detected virulence factors included adhesins, invasins, siderophores, type III secretion systems, and toxins in combination with other virulence determinants. A broiler isolate of serotype O7:H18 (ST38) carried the ibeA gene encoding a protein involved in invasion of brain endothelium on a 102-kbp genetic island. This isolate moderately adhered and invaded Caco-2 cells and induced mortality (42.5%) in a day-old-chick infection model. The results of this study suggest that multiple antimicrobial-resistant E. coli isolates recovered from apparent healthy broilers can be pathogenic and act as reservoirs for antimicrobial resistance genes, highlighting the necessity of their assessment in a "One-Heath" context.

Antimicrobial Resistance Phenotypes and Genotypes of Escherichia coli Isolates from Broiler Chickens Fed Encapsulated Cinnamaldehyde and Citral.

ABSTRACT: This study was conducted to investigate the effects of in-feed encapsulated cinnamaldehyde (CIN) and citral (CIT) alone or in combination on antimicrobial resistance (AMR) phenotypes and genotypes of Escherichia coli isolates recovered from feces of 6-, 16-, 23-, and 27-day-old broiler chickens. The five dietary treatments including the basal diet (negative control [NC]) and the basal diet supplemented with 55 ppm of bacitracin (BAC), 100 ppm of encapsulated CIN, 100 ppm of encapsulated CIT, or 100 ppm each of encapsulated CIN and encapsulated CIT (CIN+CIT). Antimicrobial susceptibility testing of 240 E. coli isolates revealed that the most common resistance was to ß-lactams, aminoglycosides, sulfonamides, and tetracycline; however, the prevalence of AMR decreased (P < 0.05) as birds aged. The prevalence of resistance to amoxicillin-clavulanic acid, ceftiofur, ceftriaxone, cefoxitin, gentamicin, and sulfonamide was lower (P < 0.05) in isolates from the CIN or CIN+CIT groups than in isolates from the NC or BAC groups. Whole genome sequencing of 227 of the 240 isolates revealed 26 AMR genes and 19 plasmids, but the prevalence of some AMR genes and the number of plasmids were lower (P < 0.05) in E. coli isolated from CIN or CIN+CIT birds than in isolates from NC or BAC birds. The most prevalent resistance genes were tet(A) (108 isolates), aac(3)-VIa (91 isolates), aadA1 (86 isolates), blaCMY-2 (78 isolates), sul1 (77 isolates), aph(3)-Ib (58 isolates), aph(6)-Id (58 isolates), and sul2 (24 isolates). The numbers of most virulence genes carried by isolates increased (P < 0.05) in chickens from 6 to 27 days of age. The prevalence of E. coli O21:H16 isolates was lower (P < 0.05) in CIN and CIN+CIT, and the colibacillosis-associated multilocus sequence type (ST117) was most prevalent in isolates from 23-day-old chickens. A phylogenetic tree of whole genome sequences revealed a close relationship between 25 of the 227 isolates and human or broiler extraintestinal pathogenic E. coli strains. These findings indicate that AMR and virulence genotypes of E. coli could be modulated by providing encapsulated CIN or CIN+CIT feed supplements, but further investigation is needed to determine the mechanisms of the effects of these supplements.

Genome Analysis and Multiplex PCR Method for the Molecular Detection of Coresistance to Cephalosporins and Fosfomycin in Salmonella enterica Serovar Heidelberg.

Heidelberg is among the top three Salmonella enterica serovars associated with human foodborne illness in Canada. Traditional culture and antimicrobial susceptibility testing techniques can be time-consuming to identify Salmonella Heidelberg resistant to cephalosporins and fosfomycin. Rapid and accurate detection of such antibiotic-resistant Salmonella Heidelberg isolates is essential to adopt appropriate control measures. In this study, 15 Salmonella Heidelberg strains isolated from feces of Canadian broiler chickens were characterized by whole genome sequencing. Salmonella Heidelberg genomes had an average coverage of greater than 80-fold, an average of 4,761 protein-coding genes, and all belonged to multilocus sequence type ST15. Genome sequences were compared with genomes in the National Center for Biotechnology Information Pathogen Detection database ( www.ncbi.nlm.nih.gov/pathogens/ ), including human outbreak isolates. The Canadian broiler isolates clustered with chicken isolates from the United States and an equine clinical isolate from Ontario, Canada. In agreement with their antimicrobial resistance phenotypes, several chromosomally encoded specific antimicrobial resistance genes including fosA7 and multidrug resistance efflux pump determinants were detected. An AmpC-like ß-lactamase gene, blaCMY-2, linked with a quaternary ammonium compound resistance gene, sugE, on a replicon type IncI1 plasmid was detected in all 15 broiler Salmonella Heidelberg isolates. Of the 205,031 published Salmonella genomes screened in silico, 4,954 (2.4%) contained blaCMY-2, 8,143 (4.0%) contained fosA7, and 919 (0.4%) contained both resistance genes. The combination of both resistance genes (fosA7 and blaCMY-2) was detected in 64% of the Heidelberg genomes and in a small proportion of various other serovars. A PCR method was developed to detect Salmonella Heidelberg in pure culture and chicken feces based on specific primers targeting genes conferring fosfomycin (fosA7) and third-generation cephalosporin (blaCMY-2) resistance as well as the Salmonella-specific invA gene and the universal 16S rRNA genes. The PCR assay was specific and sensitive for blaCMY-2 and fosA7 harboring Salmonella Heidelberg. However, some other Salmonella serovars containing these two resistance genes could also be detected by the developed PCR method.

Identification of Primary Antimicrobial Resistance Drivers in Agricultural Nontyphoidal Salmonella enterica Serovars by Using Machine Learning.

Nontyphoidal Salmonella (NTS) is a leading global cause of bacterial foodborne morbidity and mortality. Our ability to treat severe NTS infections has been impaired by increasing antimicrobial resistance (AMR). To understand and mitigate the global health crisis AMR represents, we need to link the observed resistance phenotypes with their underlying genomic mechanisms. Broiler chickens represent a key reservoir and vector for NTS infections, but isolates from this setting have been characterized in only very low numbers relative to clinical isolates. In this study, we sequenced and assembled 97 genomes encompassing 7 serotypes isolated from broiler chicken in farms in British Columbia between 2005 and 2008. Through application of machine learning (ML) models to predict the observed AMR phenotype from this genomic data, we were able to generate highly (0.92 to 0.99) precise logistic regression models using known AMR gene annotations as features for 7 antibiotics (amoxicillin-clavulanic acid, ampicillin, cefoxitin, ceftiofur, ceftriaxone, streptomycin, and tetracycline). Similarly, we also trained "reference-free" k-mer-based set-covering machine phenotypic prediction models (0.91 to 1.0 precision) for these antibiotics. By combining the inferred k-mers and logistic regression weights, we identified the primary drivers of AMR for the 7 studied antibiotics in these isolates. With our research representing one of the largest studies of a diverse set of NTS isolates from broiler chicken, we can thus confirm that the AmpC-like CMY-2 ß-lactamase is a primary driver of ß-lactam resistance and that the phosphotransferases APH(6)-Id and APH(3″-Ib) are the principal drivers of streptomycin resistance in this important ecosystem.IMPORTANCE Antimicrobial resistance (AMR) represents an existential threat to the function of modern medicine. Genomics and machine learning methods are being increasingly used to analyze and predict AMR. This type of surveillance is very important to try to reduce the impact of AMR. Machine learning models are typically trained using genomic data, but the aspects of the genomes that they use to make predictions are rarely analyzed. In this work, we showed how, by using different types of machine learning models and performing this analysis, it is possible to identify the key genes underlying AMR in nontyphoidal Salmonella (NTS). NTS is among the leading cause of foodborne illness globally; however, AMR in NTS has not been heavily studied within the food chain itself. Therefore, in this work we performed a broad-scale analysis of the AMR in NTS isolates from commercial chicken farms and identified some priority AMR genes for surveillance.

Growth of Salmonella enterica Serovars Typhimurium and Enteritidis in Iron-Poor Media and in Meat: Role of Catecholate and Hydroxamate Siderophore Transporters.

Enteritidis and Typhimurium are among the top Salmonella enterica serovars implicated in human salmonellosis worldwide. This study examined the individual and combined roles of catecholate-iron and hydroxamate-iron transporters in the survival in meat of Salmonella Enteritidis and Typhimurium. Catecholate-iron-III (Fe3+) and hydroxamate-Fe3+ transporter genes fepA, iroN, and fhuACDB were deleted in isolates of these serovars to generate single, double, and triple mutants. Growth rate in high- and low-iron media was compared among mutants, complements, and their wild-type parents. Susceptibility to 14 antibiotics, the ability to produce and utilize siderophores, and survival on cooked chicken breast were evaluated. In iron-poor liquid media, differences were observed between the growth characteristics of mutant Salmonella Enteritidis and Typhimurium. The double Δ iroNΔ fepA and the triple Δ fhuΔ iroNΔ fepA mutants of Salmonella Enteritidis exhibited prolonged lag phases (λ = 9.72 and 9.53 h) and a slow growth rate (µmax = 0.35 and 0.25 h-1) similar to that of its Δ tonB mutant (λ = 10.12 h and µmax = 0.30 h-1). In Salmonella Typhimurium, double Δ iroNΔ fepA and triple Δ fhuΔ iroNΔ fepA mutations induced a similar growth pattern as its Δ tonB mutant. Double deletions of fepA and iroN reduced the siderophore production and the use of enterobactin as an iron source. In the Δ iroNΔ fepA mutant, but not in Δ fhuΔ iroNΔ fepA, the ferrichrome or deferrioxamine promoted growth for both serovars, confirming the specific role of the FhuACDB system in the uptake and transport of hydroxamate Fe3+. Survival of the mutants was also evaluated in a meat assay, and no difference in survival was observed among the mutants compared with wild type. This study showed differences between serovars in the importance of catecholate-iron and hydroxamate-iron uptake on Salmonella growth in iron-restricted media. Data also confirmed that both Salmonella Enteritidis and Typhimurium are well equipped to survive on cooked chicken meat, offering a rich iron condition.

Antimicrobial-Resistant E. coli from Surface Waters in Southwest Ontario Dairy Farms.

Untreated surface waters can be contaminated with a variety of bacteria, including , some of which can be pathogenic for both humans and animals. Therefore, such waters need to be treated before their use in dairy operations to mitigate risks to dairy cow health and milk safety. To understand the molecular ecology of , this study aimed to assess antimicrobial resistance (AMR) in recovered from untreated surface water sources of dairy farms. Untreated surface water samples ( = 240) from 15 dairy farms were collected and processed to isolate . A total of 234 isolates were obtained and further characterized for their serotypes and antimicrobial susceptibility. Of the 234 isolates, 71.4% were pan-susceptible, 23.5% were resistant to one or two antimicrobial classes, and 5.1% were resistant to three or more antimicrobial classes. Whole genome sequence analysis of 11 selected multidrug-resistant isolates revealed AMR genes including and that confer resistance to the critically important extended-spectrum cephalosporins, as well as a variety of plasmids (mainly of the replicon type) and class 1 integrons. Phylogenetic and comparative genome analysis revealed a genetic relationship between some of the sequenced and Shiga toxin-producing O157:H7 (STEC), which warrants further investigation. This study shows that untreated surface water sources contain antimicrobial-resistant which may serve as a reservoir of AMR that could be disseminated through horizontal gene transfer. This is another reason why effective water treatment before usage should be routinely done on dairy farm operations.

Genomic and Proteomic Analyses of Salmonella enterica Serovar Enteritidis Identifying Mechanisms of Induced de novo Tolerance to Ceftiofur.

With the alarming proliferation of antibiotic resistance, it is important to understand the de novo development of bacterial adaptation to antibiotics in formerly susceptible lineages, in the absence of external genetic input from existing resistance pools. A strain of ceftiofur susceptible Salmonella enterica serovar Enteritidis ABB07-SB3071 (MIC = 1.0 µg/ml) was successively exposed to sub-MIC of ceftiofur to allow its adaptation for tolerance to a concentration of 2.0 µg/ml of this antibiotic. Genomic and proteomic comparative analyses of the parental strain and induced tolerant derived lineages were performed to characterize underlying mechanisms of de novo adaptation (tolerance). Expression and localization of specific drug-, heme-, sugar-, amino acid-, and sulfate-transporters were altered, as was the localization of the cell membrane stabilizing protein OsmY in the tolerant strains adapted to 2.0 µg/ml compared to the parental isolate lines. This redistribution of existing transporters acts to minimize the concentrations of ceftiofur in the periplasm, by decreasing facilitated import and increasing active efflux and cytosolic sequestration as determined by high performance liquid chromatography quantification of residual total and extracellular ceftiofur after growth. Genetic, subcellular localization, and abundance changes of specific regulators of transcription, translation, and post-translational dynamics in the derived ceftiofur tolerant lineages decrease metabolic strain on cell walls and enhance periplasmic envelop stability against stress. This produces slower growing, more tolerant populations, which deplete free ceftiofur concentrations significantly more than susceptible parental populations (P < 0.05), as measured by recoverable levels of ceftiofur from cultures of equivalent cellular density incubated with equal ceftiofur concentrations. Genetic and abundance changes to specific carbon and nitrogen metabolism enzymes, not traditionally associated with beta-lactam metabolism, establish an enzymatic framework with the potential to detoxify/degrade ceftiofur, while mutations and changes in subcellular localization in specific cell surface factors enhance the stability of the Gram-negative cell envelop despite the compromising effect of ceftiofur. The observed changes highlight generalizable mechanisms of de novo tolerance without horizontal gene transfer, and thus can inform policies to combat antibiotic tolerance and minimize induction of de novo tolerance.

Genomic Analysis of Third Generation Cephalosporin Resistant Escherichia coli from Dairy Cow Manure.

The production of extended-spectrum ß-lactamases (ESBLs) conferring resistance to new derivatives of ß-lactams is a major public health threat if present in pathogenic Gram-negative bacteria. The objective of this study was to characterize ceftiofur (TIO)- or cefotaxime (FOX)-resistant Escherichia coli isolated from dairy cow manure. Twenty-four manure samples were collected from four farms and incubated under anaerobic conditions for 20 weeks at 4 °C or at 25 °C. A total of 37 TIO- or FOX-resistant E. coli were isolated from two of the four farms to determine their susceptibility to 14 antibiotics. Among the 37 resistant E. coli, 10 different serotypes were identified, with O8:H1 being the predominant serotype (n = 17). Five isolates belonged to each of serotypes O9:NM and O153:H42, respectively. All 37 cephalosporin resistant isolates were multi-resistant with the most prevalent resistance spectrum being amoxicillin-clavulanic acid-ampicillin-cefoxitin-ceftiofur-ceftriaxone-chloramphenicol-streptomycin-sulfisoxazole-tetracycline-trimethoprim-sulfamethoxazole. The genomes of 18 selected isolates were then sequenced and compared to 14 selected human pathogenic E. coli reference genomes obtained from public repositories using different bioinformatics approaches. As expected, all 18 sequenced isolates carried at least one ß-lactamase bla gene: TEM-1, TEM-81, CTX-M115, CTX-M15, OXA-1, or CMY-2. Several other antibiotic resistance genes (ARGs) and virulence determinants were detected in the sequenced isolates and all of them harbored antimicrobial resistance plasmids belonging to classic Inc groups. Our results confirm the presence of diverse ESBL producing E. coli isolates in dairy cow manure stored for a short period of time. Such manure might constitute a reservoir of resistance and virulence genes for other bacteria that share the same environment.

Complete Genome and Plasmid Sequences of Three Canadian Strains of Salmonella enterica subsp. enterica Serovar Enteritidis Belonging to Phage Types 8, 13, and 13a.

Salmonella enterica subsp. enterica serovar Enteritidis is a prominent cause of human salmonellosis frequently linked to poultry products. In Canada, S. Enteritidis phage types 8, 13, and 13a predominate among both clinical and poultry isolates. Here, we report the complete genome and plasmid sequences of poultry isolates of these three phage types.

Analysis of epigenetic stability and conversions in Saccharomyces cerevisiae reveals a novel role of CAF-I in position-effect variegation.

Position-effect variegation (PEV) phenotypes are characterized by the robust multigenerational repression of a gene located at a certain locus (often called gene silencing) and occasional conversions to fully active state. Consequently, the active state then persists with occasional conversions to the repressed state. These effects are mediated by the establishment and maintenance of heterochromatin or euchromatin structures, respectively. In this study, we have addressed an important but often neglected aspect of PEV: the frequency of conversions at such loci. We have developed a model and have projected various PEV scenarios based on various rates of conversions. We have also enhanced two existing assays for gene silencing in Saccharomyces cerevisiae to measure the rate of switches from repressed to active state and vice versa. We tested the validity of our methodology in Δsir1 cells and in several mutants with defects in gene silencing. The assays have revealed that the histone chaperone Chromatin Assembly Factor I is involved in the control of epigenetic conversions. Together, our model and assays provide a comprehensive methodology for further investigation of epigenetic stability and position effects.

Sub-telomeric core X and Y' elements in S. cerevisiae suppress extreme variations in gene silencing.

Telomere Position Effect (TPE) is governed by strong repression signals emitted by telomeres via the Sir2/3/4 Histone Deacetylase complex. These signals are then relayed by weak proto-silencers residing in the subtelomeric core X and Y' elements. Subtelomeres also contain Sub-Telomeric Anti-silencing Regions (STARs). In this study we have prepared telomeres built of different combinations of core X, Y' and STARs and have analyzed them in strains lacking Histone-Acetyltransferase genes as well as in cdc6-1 and Δrif1 strains. We show that core X and Y' dramatically reduce both positive and negative variations in TPE, that are caused by these mutations. We also show that the deletion of Histone-Acetyltransferase genes reduce the silencing activity of an ACS proto-silencer, but also reduce the anti-silencing activity of a STAR. We postulate that core X and Y' act as epigenetic "cushioning" cis-elements.

GCN5 is a positive regulator of origins of DNA replication in Saccharomyces cerevisiae.

GCN5 encodes one of the non-essential Histone Acetyl Transferases in Saccharomyces cerevisiae. Extensive evidence has indicated that GCN5 is a key regulator of gene expression and could also be involved in transcriptional elongation, DNA repair and centromere maintenance. Here we show that the deletion of GCN5 decreases the stability of mini-chromosomes; that the tethering of Gcn5p to a crippled origin of replication stimulates its activity; that high dosage of GCN5 suppresses conditional phenotypes caused by mutant alleles of bona fide replication factors, orc2-1, orc5-1 and mcm5-461. Furthermore, Gcn5p physically associates with origins of DNA replication, while its deletion leads to localized condensation of chromatin at origins. Finally, Deltagcn5 cells display a deficiency in the assembly of pre-replicative complexes. We propose that GCN5 acts as a positive regulator of DNA replication by counteracting the inhibitory effect of Histone Deacetylases.

The dual role of autonomously replicating sequences as origins of replication and as silencers.

Autonomously replicating sequences (ARSs) in Saccharomyces cerevisiae have been extensively characterized as both origins of DNA replication and as chromatin repressors/silencers. It has been conclusively shown that the origin and the silencer activities of ARS are substantially, but not entirely interchangeable and that they are modulated by position effects and chromatin environment. It remains unclear how these two quite divergent functions of ARS co-exist. This perspective focuses on recent advances, which have shown that slight differences in ARSs can modulate their affinity for origin recognition complex and their activity as silencers or origins.

Subtelomeric ACS-containing proto-silencers act as antisilencers in replication factors mutants in Saccharomyces cerevisiae.

Subtelomeric genes are either fully active or completely repressed and can switch their state about once per 20 generations. This meta-stable telomeric position effect is mediated by strong repression signals emitted by the telomere and relayed/enhanced by weaker repressor elements called proto-silencers. In addition, subtelomeric regions contain sequences with chromatin partitioning and antisilencing activities referred to as subtelomeric antisilencing regions. Using extensive mutational analysis of subtelomeric elements, we show that ARS consensus sequence (ACS)-containing proto-silencers convert to antisilencers in several replication factor mutants. We point out the significance of the B1 auxiliary sequence next to ACS in mediating these effects. In contrast, an origin-derived ACS does not convert to antisilencer in mutants and its B1 element has little bearing on silencing. These results are specific for the analyzed ACS and in addition to the effects of each mutation (relative to wild type) on global silencing. Another line of experiments shows that Mcm5p possesses antisilencing activity and is recruited to telomeres in an ACS-dependent manner. Mcm5p persists at this location at the late stages of S phase. We propose that telomeric ACS are not static proto-silencers but conduct finely tuned silencing and antisilencing activities mediated by ACS-bound factors.

Differential requirement of DNA replication factors for subtelomeric ARS consensus sequence protosilencers in Saccharomyces cerevisiae.

The establishment of silent chromatin requires passage through S-phase, but not DNA replication per se. Nevertheless, many proteins that affect silencing are bona fide DNA replication factors. It is not clear if mutations in these replication factors affect silencing directly or indirectly via deregulation of S-phase or DNA replication. Consequently, the relationship between DNA replication and silencing remains an issue of debate. Here we analyze the effect of mutations in DNA replication factors (mcm5-461, mcm5-1, orc2-1, orc5-1, cdc45-1, cdc6-1, and cdc7-1) on the silencing of a group of reporter constructs, which contain different combinations of "natural" subtelomeric elements. We show that the mcm5-461, mcm5-1, and orc2-1 mutations affect silencing through subtelomeric ARS consensus sequences (ACS), while cdc6-1 affects silencing independently of ACS. orc5-1, cdc45-1, and cdc7-1 affect silencing through ACS, but also show ACS-independent effects. We also demonstrate that isolated nontelomeric ACS do not recapitulate the same effects when inserted in the telomere. We propose a model that defines the modes of action of MCM5 and CDC6 in silencing.