Antibiotic Resistance Genes and Antibiotic Susceptibility of Oral Enterococcus faecalis Isolates Compared to Isolates from Hospitalized Patients and Food

The presence of Salmonella spp. in marine animals is a consequence of contamination from terrestrial sources (human activities and animals). Bacteria present in marine environments, including Salmonella spp., can be antibiotic resistant or harbor resistance genes. In this study, Salmonella spp. detection was performed on 176 marine animals stranded in the Sicilian coasts (south Italy). Antibiotic susceptibility, by disk diffusion method and MIC determination, and antibiotic resistance genes, by molecular methods (PCR) of the Salmonella spp. strains, were evaluated. We isolated Salmonella spp. in three animals, though no pathological signs were detected. Our results showed a low prevalence of Salmonella spp. (1.7%) and a low incidence of phenotypic resistance in three Salmonella spp. strains isolated. Indeed, of the three strains, only Salmonella subsp. enterica serovar Typhimurium from S. coeruleoalba and M. mobular showed phenotypic resistance: the first to ampicillin, tetracycline, and sulphamethoxazole, while the latter only to sulphamethoxazole. However, all strains harbored resistance genes (blaTEM, blaOXA, tet(A), tet(D), tet(E), sulI, and sulII). Although the low prevalence of Salmonella spp. found in this study does not represent a relevant health issue, our data contribute to the collection of information on the spread of ARGs, elements involved in antibiotic resistance, now considered a zoonosis in a One Health approach.

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CRISPR-Cas and Restriction-Modification Act Additively against Conjugative Antibiotic Resistance Plasmid Transfer in Enterococcus faecalis

mSphere ◽

10.1128/msphere.00064-16 ◽

2016 ◽

Vol 1 (3) ◽

Cited By ~ 43

Author(s):

Valerie J. Price ◽

Wenwen Huo ◽

Ardalan Sharifi ◽

Kelli L. Palmer

Keyword(s):

Antibiotic Resistance ◽

High Risk ◽

Enterococcus Faecalis ◽

Resistance Genes ◽

Treatment Options ◽

Antibiotic Resistance Genes ◽

Multidrug Resistant ◽

Content Type ◽

New Genes ◽

Restriction Modification

ABSTRACT Enterococcus faecalis is a bacterium that normally inhabits the gastrointestinal tracts of humans and other animals. Although these bacteria are members of our native gut flora, they can cause life-threatening infections in hospitalized patients. Antibiotic resistance genes appear to be readily shared among high-risk E. faecalis strains, and multidrug resistance in these bacteria limits treatment options for infections. Here, we find that CRISPR-Cas and restriction-modification systems, which function as adaptive and innate immune systems in bacteria, significantly impact the spread of antibiotic resistance genes in E. faecalis populations. The loss of these systems in high-risk E. faecalis suggests that they are immunocompromised, a tradeoff that allows them to readily acquire new genes and adapt to new antibiotics. Enterococcus faecalis is an opportunistic pathogen and a leading cause of nosocomial infections. Conjugative pheromone-responsive plasmids are narrow-host-range mobile genetic elements (MGEs) that are rapid disseminators of antibiotic resistance in the faecalis species. Clustered regularly interspaced short palindromic repeat (CRISPR)-Cas and restriction-modification confer acquired and innate immunity, respectively, against MGE acquisition in bacteria. Most multidrug-resistant E. faecalis isolates lack CRISPR-Cas and possess an orphan locus lacking cas genes, CRISPR2, that is of unknown function. Little is known about restriction-modification defense in E. faecalis. Here, we explore the hypothesis that multidrug-resistant E. faecalis strains are immunocompromised. We assessed MGE acquisition by E. faecalis T11, a strain closely related to the multidrug-resistant hospital isolate V583 but which lacks the ~620 kb of horizontally acquired genome content that characterizes V583. T11 possesses the E. faecalis CRISPR3-cas locus and a predicted restriction-modification system, neither of which occurs in V583. We demonstrate that CRISPR-Cas and restriction-modification together confer a 4-log reduction in acquisition of the pheromone-responsive plasmid pAM714 in biofilm matings. Additionally, we show that the orphan CRISPR2 locus is functional for genome defense against another pheromone-responsive plasmid, pCF10, only in the presence of cas9 derived from the E. faecalis CRISPR1-cas locus, which most multidrug-resistant E. faecalis isolates lack. Overall, our work demonstrated that the loss of only two loci led to a dramatic reduction in genome defense against a clinically relevant MGE, highlighting the critical importance of the E. faecalis accessory genome in modulating horizontal gene transfer. Our results rationalize the development of antimicrobial strategies that capitalize upon the immunocompromised status of multidrug-resistant E. faecalis. IMPORTANCE Enterococcus faecalis is a bacterium that normally inhabits the gastrointestinal tracts of humans and other animals. Although these bacteria are members of our native gut flora, they can cause life-threatening infections in hospitalized patients. Antibiotic resistance genes appear to be readily shared among high-risk E. faecalis strains, and multidrug resistance in these bacteria limits treatment options for infections. Here, we find that CRISPR-Cas and restriction-modification systems, which function as adaptive and innate immune systems in bacteria, significantly impact the spread of antibiotic resistance genes in E. faecalis populations. The loss of these systems in high-risk E. faecalis suggests that they are immunocompromised, a tradeoff that allows them to readily acquire new genes and adapt to new antibiotics.

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Effects of Biofilm Growth on Plasmid Copy Number and Expression of Antibiotic Resistance Genes in Enterococcus faecalis

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02010-12 ◽

2013 ◽

Vol 57 (4) ◽

pp. 1850-1856 ◽

Cited By ~ 17

Author(s):

L. C. Cook ◽

G. M. Dunny

Keyword(s):

Antibiotic Resistance ◽

Enterococcus Faecalis ◽

Resistance Genes ◽

Copy Number ◽

Antibiotic Resistance Genes ◽

Plasmid Copy Number ◽

Plasmid Copy ◽

Rapid Reduction ◽

Biofilm Growth ◽

Content Type

ABSTRACTBiofilm growth causes increased average plasmid copy number as well as increased copy number heterogeneity inEnterococcus faecaliscells carrying plasmid pCF10. In this study, we examined whether biofilm growth affected the copy number and expression of antibiotic resistance determinants for several plasmids with diverse replication systems. Four differentE. faecalisplasmids, unrelated to pCF10, demonstrated increased copy number in biofilm cells. In biofilm cells, we also observed increased transcription of antibiotic resistance genes present on these plasmids. The increase in plasmid copy number correlated with increased plating efficiency on high concentrations of antibiotics. Single-cell analysis of strains carrying two different plasmids suggested that the increase in plasmid copy number associated with biofilm growth was restricted to a subpopulation of biofilm cells. Regrowth of harvested biofilm cells in liquid culture resulted in a rapid reduction of plasmid copy number to that observed in the planktonic state. These results suggest a possible mechanism by which biofilm growth could reduce susceptibility to antibiotics in clinical settings.

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Antibiotic susceptibility profiles and frequency of resistance genes in clinical Shiga toxin-producing Escherichia coli isolates from Michigan over a 14-year period

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01189-21 ◽

2021 ◽

Author(s):

Sanjana Mukherjee ◽

Heather M. Blankenship ◽

Jose A. Rodrigues ◽

Rebekah E. Mosci ◽

James T. Rudrik ◽

...

Keyword(s):

Escherichia Coli ◽

Antibiotic Resistance ◽

Resistance Genes ◽

Shiga Toxin ◽

Antibiotic Susceptibility ◽

Genetic Relatedness ◽

Antibiotic Resistance Genes ◽

Genomic Analysis ◽

Mechanisms Of Resistance ◽

Susceptibility Profiles

Background: Shiga toxin-producing Escherichia coli (STEC) is an important foodborne pathogen that contributes to over 250,000 infections in the US each year. Because antibiotics are not recommended for STEC infections, resistance in STEC has not been widely researched despite an increased likelihood for the transfer of resistance gene from STEC to opportunistic pathogens residing within the same microbial community. Methods: Between 2001 and 2014, 969 STEC isolates were collected from Michigan patients. Serotyping and antibiotic susceptibility profiles to clinically relevant antibiotics were determined using disc diffusion, while epidemiological data was used to identify factors associated with resistance. Whole genome sequencing was used to examine genetic relatedness and identify genetic determinants and mechanisms of resistance in the non-O157 isolates. Results: Increasing frequencies of resistance to at least one antibiotic was observed over the 14 years (p=0.01). While the non-O157 serogroups were more commonly resistant than O157 (Odds Ratio: 2.4; 95% Confidence Interval:1.43-4.05), the frequency of ampicillin resistance among O157 isolates was significantly higher in Michigan compared to the national average (p=0.03). Genomic analysis of 321 non-O157 isolates uncovered 32 distinct antibiotic resistance genes (ARGs). Although mutations in genes encoding resistance to ciprofloxacin and ampicillin were detected in four isolates, most of the horizontally acquired ARGs conferred resistance to aminoglycosides, β-lactams, sulfonamides and/or tetracycline. Conclusions: This study provides insight into the mechanisms of resistance in a large collection of clinical non-O157 STEC isolates and demonstrates that antibiotic resistance among all STEC serogroups has increased over time, prompting the need for enhanced surveillance.

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Conjugative delivery of CRISPR-Cas9 for the selective depletion of antibiotic-resistant enterococci

10.1101/678573 ◽

2019 ◽

Cited By ~ 1

Author(s):

Marinelle Rodrigues ◽

Sara W. McBride ◽

Karthik Hullahalli ◽

Kelli L. Palmer ◽

Breck A. Duerkop

Keyword(s):

Antibiotic Resistance ◽

Enterococcus Faecalis ◽

Resistance Genes ◽

Bacterial Infections ◽

Antibiotic Resistance Genes ◽

Multidrug Resistant ◽

Conjugative Plasmid ◽

Antibiotic Resistant ◽

Resistance Determinants

AbstractThe innovation of new therapies to combat multidrug-resistant (MDR) bacteria is being outpaced by the continued rise of MDR bacterial infections. Of particular concern are hospital-acquired infections (HAIs) recalcitrant to antibiotic therapies. The Gram-positive intestinal pathobiontEnterococcus faecalisis associated with HAIs and some strains are MDR. Therefore, novel strategies to controlE. faecalispopulations are needed. We previously characterized anE. faecalisType II CRISPR-Cas system and demonstrated its utility in the sequence-specific removal of antibiotic resistance determinants. Here we present work describing the adaption of this CRISPR-Cas system into a constitutively expressed module encoded on a pheromone-responsive conjugative plasmid that efficiently transfers toE. faecalisfor the selective removal of antibiotic resistance genes. Usingin vitrocompetition assays, we show that these CRISPR-Cas-encoding delivery plasmids, or CRISPR-Cas antimicrobials, can reduce the occurrence of antibiotic resistance in enterococcal populations in a sequence-specific manner. Furthermore, we demonstrate that deployment of CRISPR-Cas antimicrobials in the murine intestine reduces the occurrence of antibiotic-resistantE. faecalisby several orders of magnitude. Finally, we show thatE. faecalisdonor strains harboring CRISPR-Cas antimicrobials are immune to uptake of antibiotic resistance determinantsin vivo. Our results demonstrate that conjugative delivery of CRISPR-Cas antimicrobials may be adaptable for future deployment from probiotic bacteria for exact targeting of defined MDR bacteria or for precision engineering of polymicrobial communities in the mammalian intestine.ImportanceCRISPR-Cas nucleic acid targeting systems hold promise for the amelioration of multidrug-resistant enterococci, yet the utility of such tools in the context of the intestinal environment where enterococci reside is understudied. We describe the development of a CRISPR-Cas antimicrobial, deployed on a conjugative plasmid, for the targeted removal of antibiotic resistance genes from intestinalEnterococcus faecalis. We demonstrate that CRISPR-Cas targeting reduces antibiotic resistance ofE. faecalisby several orders of magnitude in the intestine. Although barriers exist that influence the penetrance of the conjugative CRISPR-Cas antimicrobial among target recipientE. faecaliscells, the removal of antibiotic resistance genes inE. faecalisupon uptake of the CRISPR-Cas antimicrobial is absolute. In addition, cells that obtain the CRISPR-Cas antimicrobial are immunized against the acquisition of new antibiotic resistance genes. This study suggests a potential path toward plasmid based CRISPR-Cas therapies in the intestine.

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Enterococcus faecalisCRISPR-Cas is a robust barrier to conjugative antibiotic resistance dissemination in the murine intestine

10.1101/312751 ◽

2018 ◽

Cited By ~ 2

Author(s):

Valerie J. Price ◽

Sara W. McBride ◽

Karthik Hullahalli ◽

Anushila Chatterjee ◽

Breck A. Duerkop ◽

...

Keyword(s):

Antibiotic Resistance ◽

Enterococcus Faecalis ◽

Resistance Genes ◽

Antibiotic Resistance Genes ◽

Conjugative Plasmids ◽

Resistance Plasmids ◽

The Impact ◽

Mammalian Intestine

AbstractCRISPR-Cas systems are barriers to horizontal gene transfer (HGT) in bacteria. Little is known about CRISPR-Cas interactions with conjugative plasmids, and studies investigating CRISPR-Cas/plasmid interactions inin vivomodels relevant to infectious disease are lacking. These are significant gaps in knowledge because conjugative plasmids disseminate antibiotic resistance genes among pathogensin vivo, and it is essential to identify strategies to reduce the spread of these elements. We use enterococci as models to understand the interactions of CRISPR-Cas with conjugative plasmids.Enterococcus faecalisis a native colonizer of the mammalian intestine and harbors pheromone-responsive plasmids (PRPs). PRPs mediate inter- and intraspecies transfer of antibiotic resistance genes. We assessedE. faecalisCRISPR-Cas anti-PRP activity in the mouse intestine and under varyingin vitroconditions. We observed striking differences in CRISPR-Cas efficiencyin vitroversusin vivo. With few exceptions, CRISPR-Cas blocked intestinal PRP dissemination, whilein vitro, the PRP frequently escaped CRISPR-Cas defense. Our results further the understanding of CRISPR-Cas biology by demonstrating that standardin vitroexperiments do not adequately model thein vivoanti-plasmid activity of CRISPR-Cas. Additionally, our work identifies several variables that impact the apparentin vitroanti-plasmid activity of CRISPR-Cas, including planktonic versus biofilm settings, different donor/recipient ratios, production of a plasmid-encoded bacteriocin, and the time point at which matings are sampled. Our results are clinically significant because they demonstrate that barriers to HGT encoded by normal human microbiota can have significant impacts onin vivoantibiotic resistance dissemination.ImportanceCRISPR-Cas is a type of immune system encoded by bacteria that is hypothesized to be a natural impediment to the spread of antibiotic resistance genes. In this study, we directly assessed the impact of CRISPR-Cas on antibiotic resistance dissemination in the mammalian intestine and under varyingin vitroconditions. We observed a robust effect of CRISPR-Cas onin vivobut notin vitrodissemination of antibiotic resistance plasmids in the native mammalian intestinal colonizerEnterococcus faecalis. We conclude that standard laboratory experiments currently do not appropriately model thein vivoconditions where antibiotic resistance dissemination occurs betweenE. faecalisstrains. Moreover, our results demonstrate that CRISPR-Cas encoded by native members of the mammalian intestinal microbiota can block the spread of antibiotic resistance plasmids.

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Enterococcus faecalis CRISPR-Cas Is a Robust Barrier to Conjugative Antibiotic Resistance Dissemination in the Murine Intestine

mSphere ◽

10.1128/msphere.00464-19 ◽

2019 ◽

Vol 4 (4) ◽

Cited By ~ 7

Author(s):

Valerie J. Price ◽

Sara W. McBride ◽

Karthik Hullahalli ◽

Anushila Chatterjee ◽

Breck A. Duerkop ◽

...

Keyword(s):

Antibiotic Resistance ◽

Enterococcus Faecalis ◽

Resistance Genes ◽

Antibiotic Resistance Genes ◽

Content Type ◽

Conjugative Plasmids ◽

Resistance Plasmids ◽

Mammalian Intestine

ABSTRACT CRISPR-Cas systems are barriers to horizontal gene transfer (HGT) in bacteria. Little is known about CRISPR-Cas interactions with conjugative plasmids, and studies investigating CRISPR-Cas/plasmid interactions in in vivo models relevant to infectious disease are lacking. These are significant gaps in knowledge because conjugative plasmids disseminate antibiotic resistance genes among pathogens in vivo, and it is essential to identify strategies to reduce the spread of these elements. We use enterococci as models to understand the interactions of CRISPR-Cas with conjugative plasmids. Enterococcus faecalis is a native colonizer of the mammalian intestine and harbors pheromone-responsive plasmids (PRPs). PRPs mediate inter- and intraspecies transfer of antibiotic resistance genes. We assessed E. faecalis CRISPR-Cas anti-PRP activity in the mouse intestine and under different in vitro conditions. We observed striking differences in CRISPR-Cas efficiency in vitro versus in vivo. With few exceptions, CRISPR-Cas blocked intestinal PRP dissemination, while in vitro, the PRP frequently escaped CRISPR-Cas defense. Our results further the understanding of CRISPR-Cas biology by demonstrating that standard in vitro experiments do not adequately model the in vivo antiplasmid activity of CRISPR-Cas. Additionally, our work identifies several variables that impact the apparent in vitro antiplasmid activity of CRISPR-Cas, including planktonic versus biofilm settings, different donor-to-recipient ratios, production of a plasmid-encoded bacteriocin, and the time point at which matings are sampled. Our results are clinically significant because they demonstrate that barriers to HGT encoded by normal (healthy) human microbiota can have significant impacts on in vivo antibiotic resistance dissemination. IMPORTANCE CRISPR-Cas is a type of immune system in bacteria that is hypothesized to be a natural impediment to the spread of antibiotic resistance genes. In this study, we directly assessed the impact of CRISPR-Cas on antibiotic resistance dissemination in the mammalian intestine and under different in vitro conditions. We observed a robust effect of CRISPR-Cas on in vivo but not in vitro dissemination of antibiotic resistance plasmids in the native mammalian intestinal colonizer Enterococcus faecalis. We conclude that standard in vitro experiments currently do not appropriately model the in vivo conditions where antibiotic resistance dissemination occurs between E. faecalis strains in the intestine. Moreover, our results demonstrate that CRISPR-Cas present in native members of the mammalian intestinal microbiota can block the spread of antibiotic resistance plasmids.

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Genomic Based Characterization of Enterococcus Spp-An Emerging Pathogen Isolated from Human Gut

10.21203/rs.3.rs-214831/v1 ◽

2021 ◽

Author(s):

Zumara younus ◽

Sagar M. Goyal ◽

Vikash Singh ◽

Aamer Ikram ◽

Muhammad Imran

Keyword(s):

Antibiotic Resistance ◽

Enterococcus Faecalis ◽

Resistance Genes ◽

Antibiotic Resistance Genes ◽

Ecological Niches ◽

Opportunistic Pathogens ◽

Human Gut ◽

Enterococcus Spp ◽

Enterococcus Casseliflavus ◽

Enterococcus Gallinarum

Abstract Background Enterococci are ubiquitous microorganisms having diverse ecological niches but mostly prominently in gastrointestinal tract of humans and animals. Production of enterocins make them used as probiotics, but in last few years their role as probiotic become ambiguous. This ambiguity in their probiotic role is related to presence of virulence factors and antibiotic resistance genes. Moreover, these virulence traits are also known to be transfer genetically which make them opportunistic pathogens in gastrointestinal track. These reports suggest serious concerns related to enterococcus before using them as probiotics. In present study Whole-genome sequencing (WGS) of Enterococcus spp was done for checking presence of resistance and virulence genes, isolated from human gut.Methods and resultsFour human origin Enterococcus spp including Enterococcus faecalis, Enterococcus casseliflavus, and two Enterococcus gallinarum were isolated from human fecal samples, further cultured on blood and MacConkey agar. Sanger sequencing was done using Applied Biosystems 3730xl DNA Analyzer. These strains were further subjected to WGS using oxford nano pore technology MinION. Raw data was analyzed using free online tool epi2me. The Comprehensive Antibiotic Resistance Database (CARD) and RAST software’s were used to look for presence of antibiotic resistance genes in these strains. Resistance determinants for clinically important antibiotics (vancomycin) and functional virulence factor genes were detected. G-view server was used for comparative genomics of all strains.Conclusion:The draft genomic sequencing of enterococcus suggested that Enterococcus faecalis, Enterococcus casseliflavus and Enterococcus gallinarum strains are opportunistic pathogens, having antibiotic resistance genes. All isolates have vancomycin resistance genes which they also expressed phenotypically. Some genes related to bacteriocin resistance were also present in E. casseliflavus and E. gallinarum.

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Antibiotic susceptibility profiles of Pediococcus pentosaceus from various origins and their implications for the safety assessment of strains with food-technology applications

Journal of Food Protection ◽

10.4315/jfp-20-363 ◽

2020 ◽

Author(s):

Noam Shani ◽

Simone Oberhaensli ◽

Emmanuelle Arias-Roth

Keyword(s):

Antibiotic Resistance ◽

Resistance Genes ◽

Antibiotic Susceptibility ◽

False Negative ◽

Antibiotic Resistance Genes ◽

Fermented Foods ◽

Food Technology ◽

Pediococcus Pentosaceus ◽

Minimal Inhibitory Concentrations ◽

Technology Applications

In the fight against the spread of antibiotic resistance (ABR), authorities usually require that strains “intentionally added into the food chain” be tested for their antibiotic susceptibility. This applies to strains used in starter or adjunct cultures for the production of fermented foods, such as many strains of Pediococcus pentosaceus . The European Food Safety Authority (EFSA) recommends testing strains for their antibiotic susceptibility based on both genomic and phenotypic approaches. Furthermore, it proposes a set of antibiotics to assess, as well as a list of microbiological cutoffs (MCs) allowing classifying lactic acid bacteria as susceptible or resistant. Accurate MCs are essential, on the one hand, to avoid false negative strains, which may carry ABR genes and remain unnoticed, and on the other, to avoid false positive strains, which may be discarded while screening potential candidates for food-technology applications. Due to relatively scarce data, MCs have been defined for the whole Pediococcus genus, although differences between different species should be expected. In this study, we investigated the antibiotic susceptibility of thirty-five strains of P. pentosaceus isolated from various matrices in the last seventy years. Minimal inhibitory concentrations (MICs) were determined using a standard protocol, and MIC distributions were established. Phenotypic analyses were complemented with genome sequencing and by seeking known antibiotic resistance genes. The genomes of all the strains were free of known antibiotic resistance genes, but most displayed MICs above the currently defined MCs for chloramphenicol, and all showed excessive MICs for tetracycline. Based on the distributions, we calculated and proposed new MCs for chloramphenicol (16 instead of 4 mg/L) and tetracycline (256 instead of 8 mg/L).

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