Induction of Efflux-Mediated Macrolide Resistance in Streptococcus pneumoniae

ABSTRACTThe antimicrobial efflux system encoded by the operonmef(E)-melon the mobile genetic element MEGA inStreptococcus pneumoniaeand other Gram-positive bacteria is inducible by macrolide antibiotics and antimicrobial peptides. Induction may affect the clinical response to the use of macrolides. We developedmef(E)reporter constructs and a disk diffusion induction and resistance assay to determine the kinetics and basis ofmef(E)-melinduction. Induction occurred rapidly, with a >15-fold increase in transcription within 1 h of exposure to subinhibitory concentrations of erythromycin. A spectrum of environmental conditions, including competence and nonmacrolide antibiotics with distinct cellular targets, did not inducemef(E).Using 16 different structurally defined macrolides, induction was correlated with the amino sugar attached to C-5 of the macrolide lactone ring, not with the size (e.g., 14-, 15- or 16-member) of the ring or with the presence of the neutral sugar cladinose at C-3. Macrolides with a monosaccharide attached to C-5, known to block exit of the nascent peptide from the ribosome after the incorporation of up to eight amino acids, inducedmef(E)expression. Macrolides with a C-5 disaccharide, which extends the macrolide into the ribosomal exit tunnel, disrupting peptidyl transferase activity, did not induce it. The induction ofmef(E)did not require macrolide efflux, but the affinity of macrolides for the ribosome determined the availability for efflux and pneumococcal susceptibility. The induction ofmef(E)-melexpression by inducing macrolides appears to be based on specific interactions of the macrolide C-5 saccharide with the ribosome that alleviate transcriptional attenuation ofmef(E)-mel.

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Recognition of Streptococcus pneumoniae and Muramyl Dipeptide by NOD2 Results in Potent Induction of MMP-9, Which Can Be Controlled by Lipopolysaccharide Stimulation

Infection and Immunity ◽

10.1128/iai.02150-14 ◽

2014 ◽

Vol 82 (12) ◽

pp. 4952-4958 ◽

Cited By ~ 10

Author(s):

Marloes Vissers ◽

Yvonne Hartman ◽

Laszlo Groh ◽

Dirk J. de Jong ◽

Marien I. de Jonge ◽

...

Keyword(s):

Streptococcus Pneumoniae ◽

Mononuclear Cells ◽

Muramyl Dipeptide ◽

Tissue Inhibitor Of Metalloproteinase ◽

Gram Negative Bacteria ◽

Gram Positive ◽

Potent Inducer ◽

Gram Negative ◽

Content Type ◽

Gram Positive Bacteria

ABSTRACTMatrix metallopeptidase 9 (MMP-9) is a protease involved in the degradation of extracellular matrix collagen. Evidence suggests that MMP-9 is involved in pathogenesis duringStreptococcus pneumoniaeinfection. However, not much is known about the induction of MMP-9 and the regulatory processes involved. We show here that the Gram-positive bacteria used in this study induced large amounts of MMP-9, in contrast to the Gram-negative bacteria that were used. An important pathogen-associated molecular pattern (PAMP) for Gram-positive bacteria is muramyl dipeptide (MDP). MDP is a very potent inducer of MMP-9 and showed a dose-dependent MMP-9 induction. Experiments using peripheral blood mononuclear cells (PBMCs) from Crohn's disease patients with nonfunctional NOD2 showed that MMP-9 induction byStreptococcus pneumoniaeand MDP is NOD2 dependent. Increasing amounts of lipopolysaccharide (LPS), an important PAMP for Gram-negative bacteria, resulted in decreasing amounts of MMP-9. Moreover, the induction of MMP-9 by MDP could be counteracted by simultaneously adding LPS. The inhibition of MMP-9 expression by LPS was found to be regulated posttranscriptionally, independently of tissue inhibitor of metalloproteinase 1 (TIMP-1), an endogenous inhibitor of MMP-9. Collectively, these data show thatStreptococcus pneumoniaeis able to induce large amounts of MMP-9. These high MMP-9 levels are potentially involved inStreptococcus pneumoniaepathogenesis.

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Postantibiotic Suppression of Growth of Erythromycin A-Susceptible and -Resistant Gram-Positive Bacteria by the Ketolides Telithromycin (HMR 3647) and HMR 3004

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.44.6.1749-1753.2000 ◽

2000 ◽

Vol 44 (6) ◽

pp. 1749-1753 ◽

Cited By ~ 17

Author(s):

Wendy J. Munckhof ◽

Glenn Borlace ◽

John D. Turnidge

Keyword(s):

Staphylococcus Aureus ◽

Streptococcus Pneumoniae ◽

Streptococcus Pyogenes ◽

Gram Positive ◽

Gram Positive Bacteria ◽

Resistant Strains ◽

Erythromycin A

ABSTRACT We investigated the in vitro postantibiotic effects (PAEs) of the ketolides telithromycin (HMR 3647) and HMR 3004 and analyzed the results using the sigmoid E max model. Mean maximum telithromycin PAEs against erythromycin A-susceptible strains of Staphylococcus aureus, Streptococcus pyogenes, and Streptococcus pneumoniae were 3.7, 8.9, and 9.7 h, respectively, while maximum PAEs for erythromycin A-resistant strains were much shorter. Mean maximum HMR 3004 PAEs were 3.2 to 4.4 h for all species.

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Coping with an Essential Poison: a Genetic Suppressor Analysis Corroborates a Key Function of c-di-AMP in Controlling Potassium Ion Homeostasis in Gram-Positive Bacteria

Journal of Bacteriology ◽

10.1128/jb.00166-18 ◽

2018 ◽

Vol 200 (12) ◽

Cited By ~ 11

Author(s):

Fabian M. Commichau ◽

Jörg Stülke

Keyword(s):

Streptococcus Pneumoniae ◽

Ion Homeostasis ◽

Second Messenger ◽

Potassium Ion ◽

Complex Media ◽

Content Type ◽

Potassium Homeostasis ◽

Intracellular Potassium ◽

Gram Positive Bacteria ◽

Suppressor Analysis

ABSTRACT Cyclic di-AMP (c-di-AMP) is an important second messenger in bacteria. In most Firmicutes , the molecule is required for growth in complex media but also toxic upon accumulation. In an article on their current study, Zarrella and coworkers present a suppressor analysis of a Streptococcus pneumoniae strain that is unable to degrade c-di-AMP (T. M. Zarrella, D. W. Metzger, and G. Bai, J Bacteriol 200:e00045-18, 2018, https://doi.org/10.1128/JB.00045-18 ). Their study identifies new links between c-di-AMP and potassium homeostasis and supports the hypothesis that c-di-AMP serves as a second messenger to report about the intracellular potassium concentrations.

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The Pneumococcal Type 1 Pilus Genes Are Thermoregulated and Are Repressed by a Member of the Snf2 Protein Family

Journal of Bacteriology ◽

10.1128/jb.00078-17 ◽

2017 ◽

Vol 199 (15) ◽

Cited By ~ 3

Author(s):

Alan Basset ◽

Muriel Herd ◽

Raecliffe Daly ◽

Simon L. Dove ◽

Richard Malley

Keyword(s):

Streptococcus Pneumoniae ◽

Feedback Loop ◽

Respiratory Infections ◽

Protein Family ◽

Negative Regulator ◽

Negative Regulators ◽

Nasopharyngeal Colonization ◽

Content Type ◽

Gram Positive Bacteria

ABSTRACT In Streptococcus pneumoniae, the type 1 pilus is involved in many steps of pathogenesis, including adherence to epithelial cells, mediation of inflammation, escape from macrophages, and the formation of biofilms. The type 1 pilus genes are expressed in a bistable fashion with cells switching between “on” and “off” expression states. Bistable expression of these genes is due to their control by RlrA, a positive regulator subject to control by a positive-feedback loop. The type 1 pilus genes are also thought to be negatively regulated by a large number of repressors. Here we show that expression of the type 1 pilus genes is thermosensitive and switched off at growth temperatures below 31°C. We also report that the on expression state of the type 1 pilus genes is highly stable, a phenomenon which we show likely contributed to the erroneous identification of many proteins as negative regulators of these genes. Finally, we exploited the effect of low temperature on pilus gene expression to help identify SP_1523, an Snf2-type protein, as a novel negative regulator of the pilus genes. Our findings establish that the type 1 pilus genes are thermoregulated and are repressed by a member of the Snf2 protein family. They also refute the notion that these genes are controlled by 8 previously described negative regulators. IMPORTANCE Streptococcus pneumoniae is the leading cause of death from respiratory infections in children. Many bacterial factors contribute to pneumococcal virulence and nasopharyngeal colonization. The type 1 pneumococcal pilus plays an important role in mouse models and in epithelial adherence and is expressed in a bistable fashion. Here we show that the “on” state is highly stable, which may explain the prior misidentification of negative regulators of pilus expression. We also show that expression of pilus genes is thermosensitive: virtually no expression can be detected at temperatures found in the anterior nares of humans. We took advantage of this property to identify a negative regulator of pilus expression, a member of a family of proteins widely conserved across Gram-positive bacteria.

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Dynamic Distribution of the SecA and SecY Translocase Subunits and Septal Localization of the HtrA Surface Chaperone/Protease during Streptococcus pneumoniae D39 Cell Division

mBio ◽

10.1128/mbio.00202-11 ◽

2011 ◽

Vol 2 (5) ◽

Cited By ~ 36

Author(s):

Ho-Ching Tiffany Tsui ◽

Susan K. Keen ◽

Lok-To Sham ◽

Kyle J. Wayne ◽

Malcolm E. Winkler

Keyword(s):

Cell Division ◽

Streptococcus Pneumoniae ◽

Stationary Phase ◽

Streptococcus Pyogenes ◽

Phospholipid Content ◽

Gram Positive ◽

General Role ◽

Content Type ◽

Gram Positive Bacteria ◽

Dividing Cells

ABSTRACT The Sec translocase pathway is the major route for protein transport across and into the cytoplasmic membrane of bacteria. Previous studies reported that the SecA translocase ATP-binding subunit and the cell surface HtrA protease/chaperone formed a single microdomain, termed “ExPortal,” in some species of ellipsoidal (ovococcus) Gram-positive bacteria, including Streptococcus pyogenes. To investigate the generality of microdomain formation, we determined the distribution of SecA and SecY by immunofluorescent microscopy in Streptococcus pneumoniae (pneumococcus), which is an ovococcus species evolutionarily distant from S. pyogenes. In the majority (≥75%) of exponentially growing cells, S. pneumoniae SecA (SecA Spn ) and SecY Spn located dynamically in cells at different stages of division. In early divisional cells, both Sec subunits concentrated at equators, which are future sites of constriction. Further along in division, SecA Spn and SecY Spn remained localized at mid-cell septa. In late divisional cells, both Sec subunits were hemispherically distributed in the regions between septa and the future equators of dividing cells. In contrast, the HtrA Spn homologue localized to the equators and septa of most (>90%) dividing cells, whereas the SrtA Spn sortase located over the surface of cells in no discernable pattern. This dynamic pattern of Sec distribution was not perturbed by the absence of flotillin family proteins, but was largely absent in most cells in early stationary phase and in ∆cls mutants lacking cardiolipin synthase. These results do not support the existence of an ExPortal microdomain in S. pneumoniae. Instead, the localization of the pneumococcal Sec translocase depends on the stage of cell division and anionic phospholipid content. IMPORTANCE Two patterns of Sec translocase distribution, an ExPortal microdomain in certain ovococcus-shaped species like Streptococcus pyogenes and a spiral pattern in rod-shaped species like Bacillus subtilis, have been reported for Gram-positive bacteria. This study provides evidence for a third pattern of Sec localization in the ovococcus human pathogen Streptococcus pneumoniae. The SecA motor and SecY channel subunits of the Sec translocase localize dynamically to different places in the mid-cell region during the division cycle of exponentially growing, but not stationary-phase, S. pneumoniae. Unexpectedly, the S. pneumoniae HtrA (HtrA Spn ) protease/chaperone principally localizes to cell equators and division septa. The coincident localization of SecA Spn , SecY Spn , and HtrA Spn to regions of peptidoglycan (PG) biosynthesis in unstressed, growing cells suggests that the pneumococcal Sec translocase directs assembly of the PG biosynthesis apparatus to regions where it is needed during division and that HtrA Spn may play a general role in quality control of proteins exported by the Sec translocase.

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Allosteric Activation of Escherichia coli Glucosamine-6-Phosphate Deaminase (NagB)In VivoJustified by Intracellular Amino Sugar Metabolite Concentrations

Journal of Bacteriology ◽

10.1128/jb.00870-15 ◽

2016 ◽

Vol 198 (11) ◽

pp. 1610-1620 ◽

Cited By ~ 8

Author(s):

Laura I. Álvarez-Añorve ◽

Isabelle Gaugué ◽

Hannes Link ◽

Jorge Marcos-Viquez ◽

Dana M. Díaz-Jiménez ◽

...

Keyword(s):

Escherichia Coli ◽

Sugar Metabolism ◽

Fold Increase ◽

Amino Sugar ◽

Amino Sugars ◽

High Concentration ◽

Futile Cycle ◽

Content Type ◽

E Coli ◽

The Impact

ABSTRACTWe have investigated the impact of growth on glucosamine (GlcN) andN-acetylglucosamine (GlcNAc) on cellular metabolism by quantifying glycolytic metabolites inEscherichia coli. Growth on GlcNAc increased intracellular pools of both GlcNAc6P and GlcN6P 10- to 20-fold compared to growth on glucose. Growth on GlcN produced a 100-fold increase in GlcN6P but only a slight increase in GlcNAc6P. Changes to the amounts of downstream glycolytic intermediates were minor compared to growth on glucose. The enzyme glucosamine-6P deaminase (NagB) is required for growth on both GlcN and GlcNAc. It is an allosteric enzyme inE. coli, displaying sigmoid kinetics with respect to its substrate, GlcN6P, and is allosterically activated by GlcNAc6P. The high concentration of GlcN6P, accompanied by the small increase in GlcNAc6P, drivesE. coliNagB (NagBEc) into its high activity state, as observed during growth on GlcN (L. I. Álvarez-Añorve, I. Bustos-Jaimes, M. L. Calcagno, and J. Plumbridge, J Bacteriol 191:6401–6407, 2009,http://dx.doi.org/10.1128/JB.00633-09). The slight increase in GlcNAc6P during growth on GlcN is insufficient to displace NagC, the GlcNAc6P-responsive repressor of thenaggenes, from its binding sites, so there is only a small increase innagBexpression. We replaced the gene for the allosteric NagBEcenzyme with that of the nonallosteric,B. subtilishomologue, NagBBs. We detected no effects on growth rates or competitive fitness on glucose or the amino sugars, nor did we detect any effect on the concentrations of central metabolites, thus demonstrating the robustness of amino sugar metabolism and leaving open the question of the role of allostery in the regulation of NagB.IMPORTANCEChitin, the polymer ofN-acetylglucosamine, is an abundant biomaterial, and both glucosamine andN-acetylglucosamine are valuable nutrients for bacteria. The amino sugars are components of numerous essential macromolecules, including bacterial peptidoglycan and mammalian glycosaminoglycans. Controlling the biosynthetic and degradative pathways of amino sugar metabolism is important in all organisms to avoid loss of nitrogen and energy via a futile cycle of synthesis and breakdown. The enzyme glucosamine-6P deaminase (NagB) is central to this control, andN-acetylglucosamine-6P is the key signaling molecule regulating amino sugar utilization inEscherichia coli. Here, we investigate how the metabolic status of the bacteria impacts on the activity of NagBEcand theN-acetylglucosamine-6P-sensitive transcriptional repressor, NagC.

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Reactive Oxygen Species Contribute to the Bactericidal Effects of the Fluoroquinolone Moxifloxacin in Streptococcus pneumoniae

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02299-15 ◽

2015 ◽

Vol 60 (1) ◽

pp. 409-417 ◽

Cited By ~ 19

Author(s):

M. J. Ferrándiz ◽

A. J. Martín-Galiano ◽

C. Arnanz ◽

T. Zimmerman ◽

A. G. de la Campa

Keyword(s):

Reactive Oxygen Species ◽

Streptococcus Pneumoniae ◽

Hydroxyl Radical ◽

Fenton Reaction ◽

Reactive Oxygen ◽

Fold Increase ◽

Oxygen Species ◽

Acetyl Coa ◽

Transcriptomic Response ◽

Content Type

ABSTRACTWe studied the transcriptomic response ofStreptococcus pneumoniaeto the fluoroquinolone moxifloxacin at a concentration that inhibits DNA gyrase. Treatment of the wild-type strain R6, at a concentration of 10× the MIC, triggered a response involving 132 genes after 30 min of treatment. Genes from several metabolic pathways involved in the production of pyruvate were upregulated. These included 3 glycolytic enzymes, which ultimately convert fructose 6-phosphate to pyruvate, and 2 enzymes that funnel phosphate sugars into the glycolytic pathway. In addition, acetyl coenzyme A (acetyl-CoA) carboxylase was downregulated, likely leading to an increase in acetyl-CoA. When coupled with an upregulation in formate acetyltransferase, an increase in acetyl-CoA would raise the production of pyruvate. Since pyruvate is converted by pyruvate oxidase (SpxB) into hydrogen peroxide (H2O2), an increase in pyruvate would augment intracellular H2O2. Here, we confirm a 21-fold increase in the production of H2O2and a 55-fold increase in the amount of hydroxyl radical in cultures treated during 4 h with moxifloxacin. This increase in hydroxyl radical through the Fenton reaction would damage DNA, lipids, and proteins. These reactive oxygen species contributed to the lethality of the drug, a conclusion supported by the observed protective effects of an SpxB deletion. These results support the model whereby fluoroquinolones cause redox alterations. The transcriptional response ofS. pneumoniaeto moxifloxacin is compared with the response to levofloxacin, an inhibitor of topoisomerase IV. Levofloxacin triggers the transcriptional activation of iron transport genes and also enhances the Fenton reaction.

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Role of Phase II Enzymes in the Bioactivation of 1,4-Dichlorobenzene and 1,4-Dibromobenzene

Alternatives to Laboratory Animals ◽

10.1177/026119299602400422 ◽

1996 ◽

Vol 24 (4) ◽

pp. 603-608

Author(s):

Moreno Paolini ◽

Laura Pozzetti ◽

Renata Mesirca ◽

Andrea Sapone ◽

Paola Silingardi ◽

...

Keyword(s):

Dna Binding ◽

Phase I ◽

Phase Ii ◽

Covalent Binding ◽

Fold Increase ◽

Test System ◽

Oxidative Enzymes ◽

Transferase Activity ◽

Genetic Toxicology

The use of sodium phenobarbital (PB, CYP2B1 inducer) combined with β-naphthoflavone (β-NF, 1A1) to induce certain Phase I reactions in S9 liver fractions is a standard method for conducting short-term bioassays for genotoxicity. However, because post-oxidative enzymes are also able to activate many precarcinogens, we tested the possibility of adapting S9 liver fractions derived from Phase II-induced rodents to the field of genetic toxicology. In this study, S9 liver fractions derived from Swiss albino CD1 mice fed 7.5g/kg 2-(3)-tert-butyl-4-hydroxyanisole (BHA; a monofunctional Phase II-inducer) for 3 weeks, show a clear pattern of induction with an approximately 3.5–9.5-fold increase in glutathione S-transferase activity. In vitro DNA binding of the promutagenic agents, [14C]-l,4-dichlorobenzene (DCB) and [14C]-1,4-dibromobenzene (DBB), is mediated by such metabolic liver preparations and showed a significant increase in covalent binding capability. In some instances, enzyme activity was more elevated when compared to that obtained with traditional (Phase I-induced) S9. Together with DNA binding, the genetic response of these chemicals in the diploid D7 strain of Saccharomyces cerevisiae used as a biological test system, revealed the ability of the BHA-derived preparations to activate the promutagenic agents, as exemplified by the significant enhancement of mitotic gene-conversion (up to 5.2-fold for DCB and 3.4-fold for DBB) and reverse point mutation (up to 3.6-fold for DCB and 2.5-fold for DBB) at a 4mM concentration. This novel metabolising biosystem, with enhanced Phase II activity, is recommended together with a traditional S9, for detecting unknown promutagens in genotoxicity studies. The routine use of either oxidative or post-oxidative S9 increases the responsiveness of the test and can contribute to the identification of promutagens not detected when using traditional protocols.

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Antibodies to Streptococcus pneumoniae Capsular Polysaccharide Enhance Pneumococcal Quorum Sensing

mBio ◽

10.1128/mbio.00176-11 ◽

2011 ◽

Vol 2 (5) ◽

Cited By ~ 22

Author(s):

Masahide Yano ◽

Shruti Gohil ◽

J. Robert Coleman ◽

Catherine Manix ◽

Liise-anne Pirofski

Keyword(s):

Monoclonal Antibodies ◽

Streptococcus Pneumoniae ◽

Quorum Sensing ◽

Direct Effect ◽

Pneumococcal Disease ◽

Effector Cell ◽

Capsular Polysaccharide ◽

Genetic Exchange ◽

Content Type ◽

Specific Antibodies

ABSTRACTThe use of pneumococcal capsular polysaccharide (PPS)-based vaccines has resulted in a substantial reduction in invasive pneumococcal disease. However, much remains to be learned about vaccine-mediated immunity, as seven-valent PPS-protein conjugate vaccine use in children has been associated with nonvaccine serotype replacement and 23-valent vaccine use in adults has not prevented pneumococcal pneumonia. In this report, we demonstrate that certain PPS-specific monoclonal antibodies (MAbs) enhance the transformation frequency of two differentStreptococcus pneumoniaeserotypes. This phenomenon was mediated by PPS-specific MAbs that agglutinate but do not promote opsonic effector cell killing of the homologous serotypeinvitro. Compared to the autoinducer, competence-stimulating peptide (CSP) alone, transcriptional profiling of pneumococcal gene expression after incubation with CSP and one such MAb to the PPS of serotype 3 revealed changes in the expression of competence (com)-related and bacteriocin-like peptide (blp) genes involved in pneumococcal quorum sensing. This MAb was also found to induce a nearly 2-fold increase in CSP2-mediated bacterial killing or fratricide. These observations reveal a novel, direct effect of PPS-binding MAbs on pneumococcal biology that has important implications for antibody immunity to pneumococcus in the pneumococcal vaccine era. Taken together, our data suggest heretofore unsuspected mechanisms by which PPS-specific antibodies could affect genetic exchange and bacterial viability in the absence of host cells.IMPORTANCECurrent thought holds that pneumococcal capsular polysaccharide (PPS)-binding antibodies protect against pneumococcus by inducing effector cell opsonic killing of the homologous serotype. While such antibodies are an important part of how pneumococcal vaccines protect against pneumococcal disease, PPS-specific antibodies that do not exhibit this activity but are highly protective against pneumococcus in mice have been identified. This article examines the effect of nonopsonic PPS-specific monoclonal antibodies (MAbs) on the biology ofStreptococcus pneumoniae. The results showed that in the presence of a competence-stimulating peptide (CSP), such MAbs increase the frequency of pneumococcal transformation. Further studies with one such MAb showed that it altered the expression of genes involved in quorum sensing and increased competence-induced killing or fratricide. These findings reveal a novel, previously unsuspected mechanism by which certain PPS-specific antibodies exert a direct effect on pneumococcal biology that has broad implications for bacterial clearance, genetic exchange, and antibody immunity to pneumococcus.

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Lachnospiraceae and Bacteroidales Alternative Fecal Indicators Reveal Chronic Human Sewage Contamination in an Urban Harbor

Applied and Environmental Microbiology ◽

10.1128/aem.05480-11 ◽

2011 ◽

Vol 77 (19) ◽

pp. 6972-6981 ◽

Cited By ~ 79

Author(s):

Ryan J. Newton ◽

Jessica L. VandeWalle ◽

Mark A. Borchardt ◽

Marc H. Gorelick ◽

Sandra L. McLellan

Keyword(s):

Sequence Analysis ◽

Genetic Marker ◽

Microbial Community Composition ◽

Fold Increase ◽

Fecal Pollution ◽

Plate Count ◽

Rrna Gene ◽

Fecal Indicators ◽

Fecal Indicator ◽

Content Type

ABSTRACTThe complexity of fecal microbial communities and overlap among human and other animal sources have made it difficult to identify source-specific fecal indicator bacteria. However, the advent of next-generation sequencing technologies now provides increased sequencing power to resolve microbial community composition within and among environments. These data can be mined for information on source-specific phylotypes and/or assemblages of phylotypes (i.e., microbial signatures). We report the development of a new genetic marker for human fecal contamination identified through microbial pyrotag sequence analysis of the V6 region of the 16S rRNA gene. Sequence analysis of 37 sewage samples and comparison with database sequences revealed a human-associated phylotype within theLachnospiraceaefamily, which was closely related to the genusBlautia. This phylotype, termed Lachno2, was on average the second most abundant fecal bacterial phylotype in sewage influent samples from Milwaukee, WI. We developed a quantitative PCR (qPCR) assay for Lachno2 and used it along with the qPCR-based assays for humanBacteroidales(based on the HF183 genetic marker), totalBacteroidalesspp., and enterococci and the conventionalEscherichia coliand enterococci plate count assays to examine the prevalence of fecal and human fecal pollution in Milwaukee's harbor. Both the conventional fecal indicators and the human-associated indicators revealed chronic fecal pollution in the harbor, with significant increases following heavy rain events and combined sewer overflows. The two human-associated genetic marker abundances were tightly correlated in the harbor, a strong indication they target the same source (i.e., human sewage). Human adenoviruses were routinely detected under all conditions in the harbor, and the probability of their occurrence increased by 154% for every 10-fold increase in the human indicator concentration. Both Lachno2 and humanBacteroidalesincreased specificity to detect sewage compared to general indicators, and the relationship to a human pathogen group suggests that the use of these alternative indicators will improve assessments for human health risks in urban waters.

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