High-resolution microbiome analysis reveals exclusionary Klebsiella species competition in preterm infants at risk for necrotizing enterocolitis.

Intestinal colonization with Klebsiella has been linked to necrotizing enterocolitis (NEC), but methods of analysis usually failed to discriminate Klebsiella species or strains. A novel ~ 2500-base amplicon (StrainID) that spans the 16S and 23S rRNA genes was used to generate amplicon sequence variant (ASV) fingerprints for Klebsiella oxytoca and Klebsiella pneumoniae species complexes (KoSC and KpSC, respectively) and co-occurring fecal bacterial strains from 10 preterm infants with NEC and 20 matched controls. Complementary approaches were used to identify cytotoxin-producing isolates of KoSC. Klebsiella species colonized most preterm infants, were more prevalent in NEC subjects versus controls, and replaced Escherichia in NEC subjects. Single KoSC or KpSC ASV fingerprinted strains dominated the gut microbiota, suggesting exclusionary Klebsiella competition for luminal resources. Enterococcus faecalis was co-dominant with KoSC but present infrequently with KpSC. Cytotoxin-producing KoSC members were identified in most NEC subjects and were less frequent in controls. Few Klebsiella strains were shared between subjects. We conclude that inter-species Klebsiella competition, within an environment of KoSC and E. faecalis cooperation, appears to be an important factor for the development of NEC. Preterm infants seem to acquire Klebsiella primarily through routes other than patient-to-patient transmission.

Bacterial Indole as a Multifunctional Regulator of Klebsiella oxytoca Complex Enterotoxicity.

Gastrointestinal microbes respond to biochemical metabolites that coordinate their behaviors. Here, we demonstrate that bacterial indole functions as a multifactorial mitigator of Klebsiella grimontii and Klebsiella oxytoca pathogenicity. These closely related microbes produce the enterotoxins tilimycin and tilivalline; cytotoxin-producing strains are the causative agent of antibiotic-associated hemorrhagic colitis and have been associated with necrotizing enterocolitis of premature infants. We demonstrate that carbohydrates induce cytotoxin synthesis while concurrently repressing indole biosynthesis. Conversely, indole represses cytotoxin production. In both cases, the alterations stemmed from differential transcription of npsA and npsB, key genes involved in tilimycin biosynthesis. Indole also enhances conversion of tilimycin to tilivalline, an indole analog with reduced cytotoxicity. In this context, we established that tilivalline, but not tilimycin, is a strong agonist of pregnane X receptor (PXR), a master regulator of xenobiotic detoxification and intestinal inflammation. Tilivalline binding upregulated PXR-responsive detoxifying genes and inhibited tubulin-directed toxicity. Bacterial indole, therefore, acts in a multifunctional manner to mitigate cytotoxicity by Klebsiella spp.: suppression of toxin production, enhanced conversion of tilimycin to tilivalline, and activation of PXR. IMPORTANCE The human gut harbors a complex community of microbes, including several species and strains that could be commensals or pathogens depending on context. The specific environmental conditions under which a resident microbe changes its relationship with a host and adopts pathogenic behaviors, in many cases, remain poorly understood. Here, we describe a novel communication network involving the regulation of K. grimontii and K. oxytoca enterotoxicity. Bacterial indole was identified as a central modulator of these colitogenic microbes by suppressing bacterial toxin (tilimycin) synthesis and converting tilimycin to tilivalline while simultaneously activating a host receptor, PXR, as a means of mitigating tissue cytotoxicity. On the other hand, fermentable carbohydrates were found to inhibit indole biosynthesis and enhance toxin production. This integrated network involving microbial, host, and metabolic factors provides a contextual framework to better understand K. oxytoca complex pathogenicity.

Cytotoxin-producing Klebsiella oxytoca in the preterm gut and its association with necrotizing enterocolitis.

Necrotizing enterocolitis (NEC) is a devastating intestinal inflammatory disease of premature infants associated with gut bacterial dysbiosis. Using 16S rRNA-based methods, our laboratory identified an unclassified Enterobacteriaceae sequence (NEC_unk_OTU) with high abundance in NEC fecal samples. We aimed to identify this bacterium and determine its potential role in the disease. NCBI database searches for the 16S sequence, selective culture systems, biotyping and polymerase chain reaction were employed to refine classification of NEC_unk_OTU and identify toxin-encoding genes from the index NEC case. Bacterial cytotoxin production was confirmed by mass spectrometry and apoptosis assays. Additional fecal samples from 9 NEC and 5 non-NEC controls were analyzed using similar methods and multi-locus sequence typing (MLST) was performed to investigate clonal relationships and define sequence types of the isolates. NEC_unk_OTU was identified as Klebsiella oxytoca, a pathobiont known to cause antibiotic-associated hemorrhagic colitis, but not previously linked to NEC. Including the index case, cytotoxin-producing strains of K. oxytoca were isolated from 6 of 10 subjects with NEC; in these, the K. oxytoca 16S sequence predominated the fecal microbiota. Cytotoxin-producing strains of K. oxytoca also were isolated from 4 of 5 controls; in these, however, the abundance of the corresponding 16S sequence was very low. MLST analysis of the toxin-positive isolates demonstrated no clonal relationships and similar genetic clustering between cases and controls. These results suggest cytotoxin-producing strains of K. oxytoca colonize a substantial proportion of premature infants. Some, perhaps many, cases of NEC may be precipitated by outgrowth of this opportunistic pathogen.

Influence of iron and aeration on Staphylococcus aureus growth, metabolism, and transcription.

Staphylococcus aureus is a prominent nosocomial pathogen and a major cause of biomaterial-associated infections. The success of S. aureus as a pathogen is due in part to its ability to adapt to stressful environments. As an example, the transition from residing in the nares to residing in the blood or deeper tissues is accompanied by changes in the availability of nutrients and elements such as oxygen and iron. As such, nutrients, oxygen, and iron are important determinants of virulence factor synthesis in S. aureus. In addition to influencing virulence factor synthesis, oxygen and iron are critical cofactors in enzymatic and electron transfer reactions; thus, a change in iron or oxygen availability alters the bacterial metabolome. Changes in metabolism create intracellular signals that alter the activity of metabolite-responsive regulators such as CodY, RpiRc, and CcpA. To assess the extent of metabolomic changes associated with oxygen and iron limitation, S. aureus cells were cultivated in iron-limited medium and/or with decreasing aeration, and the metabolomes were examined by nuclear magnetic resonance (NMR) spectroscopy. As expected, oxygen and iron limitation dramatically decreased tricarboxylic acid (TCA) cycle activity, creating a metabolic block and significantly altering the metabolome. These changes were most prominent during post-exponential-phase growth, when TCA cycle activity was maximal. Importantly, many of the effects of iron limitation were obscured by aeration limitation. Aeration limitation not only obscured the metabolic effects of iron limitation but also overrode the transcription of iron-regulated genes. Finally, in contrast to previous speculation, we confirmed that acidification of the culture medium occurs independent of the availability of iron.

Staphylococcal response to oxidative stress.

Staphylococci are a versatile genus of bacteria that are capable of causing acute and chronic infections in diverse host species. The success of staphylococci as pathogens is due in part to their ability to mitigate endogenous and exogenous oxidative and nitrosative stress. Endogenous oxidative stress is a consequence of life in an aerobic environment; whereas, exogenous oxidative and nitrosative stress are often due to the bacteria's interaction with host immune systems. To overcome the deleterious effects of oxidative and nitrosative stress, staphylococci have evolved protection, detoxification, and repair mechanisms that are controlled by a network of regulators. In this review, we summarize the cellular targets of oxidative stress, the mechanisms by which staphylococci sense oxidative stress and damage, oxidative stress protection and repair mechanisms, and regulation of the oxidative stress response. When possible, special attention is given to how the oxidative stress defense mechanisms help staphylococci control oxidative stress in the host.

The Staphylococcus aureus two-component regulatory system, GraRS, senses and confers resistance to selected cationic antimicrobial peptides.

The two-component regulatory system, GraRS, appears to be involved in staphylococcal responses to cationic antimicrobial peptides (CAPs). However, the mechanism(s) by which GraRS is induced, regulated, and modulated remain undefined. In this study, we used two well-characterized MRSA strains (Mu50 and COL) and their respective mutants of graR and vraG (encoding the ABC transporter-dependent efflux pump immediately downstream of graRS), and show that (i) the expression of two key determinants of net positive surface charge (mprF and dlt) is dependent on the cotranscription of both graR and vraG, (ii) reduced expression of mprF and dlt in graR mutants was phenotypically associated with reduced surface-positive charge, (iii) this net reduction in surface-positive charge in graR and vraG mutants, in turn, correlated with enhanced killing by a range of CAPs of diverse structure and origin, including those from mammalian platelets (tPMPs) and neutrophils (hNP-1) and from bacteria (polymyxin B), and (iv) the synthesis and translocation of membrane lysyl-phosphatidylglycerol (an mprF-dependent function) was substantially lower in graR and vraG mutants than in parental strains. Importantly, the inducibility of mprF and dlt transcription via the graRS-vraFG pathway was selective, with induction by sublethal exposure to the CAPs, RP-1 (platelets), and polymyxin B, but not by other cationic molecules (hNP-1, vancomycin, gentamicin, or calcium-daptomycin). Although graR regulates expression of vraG, the expression of graR was codependent on an intact downstream vraG locus. Collectively, these data support an important role of the graRS and vraFG loci in the sensing of and response to specific CAPs involved in innate host defenses.

CcpA coordinates central metabolism and biofilm formation in Staphylococcus epidermidis.

Staphylococcus epidermidis is an opportunistic bacterium whose infections often involve the formation of a biofilm on implanted biomaterials. In S. epidermidis, the exopolysaccharide facilitating bacterial adherence in a biofilm is polysaccharide intercellular adhesin (PIA), whose synthesis requires the enzymes encoded within the intercellular adhesin operon (icaADBC). In vitro, the formation of S. epidermidis biofilms is enhanced by conditions that repress tricarboxylic acid (TCA) cycle activity, such as growth in a medium containing glucose. In many Gram-positive bacteria, repression of TCA cycle genes in response to glucose is accomplished by catabolite control protein A (CcpA). CcpA is a member of the GalR-LacI repressor family that mediates carbon catabolite repression, leading us to hypothesize that catabolite control of S. epidermidis biofilm formation is indirectly regulated by CcpA-dependent repression of the TCA cycle. To test this hypothesis, ccpA deletion mutants were constructed in strain 1457 and 1457-acnA and the effects on TCA cycle activity, biofilm formation and virulence were assessed. As anticipated, deletion of ccpA derepressed TCA cycle activity and inhibited biofilm formation; however, ccpA deletion had only a modest effect on icaADBC transcription. Surprisingly, deletion of ccpA in strain 1457-acnA, a strain whose TCA cycle is inactive and where icaADBC transcription is derepressed, strongly inhibited icaADBC transcription. These observations demonstrate that CcpA is a positive effector of biofilm formation and icaADBC transcription and a repressor of TCA cycle activity.

Fur is required for the activation of virulence gene expression through the induction of the sae regulatory system in Staphylococcus aureus.

Our previous studies showed that both Sae and Fur are required for the induction of eap and emp expression in low iron. In this study, we show that expression of sae is also iron-regulated, as sae expression is activated by Fur in low iron. We also demonstrate that both Fur and Sae are required for full induction of the oxidative stress response and expression of non-covalently bound surface proteins in low-iron growth conditions. In addition, Sae is required for the induced expression of the important virulence factors isdA and isdB in low iron. Our studies also indicate that Fur is required for the induced expression of the global regulators Agr and Rot in low iron and a number of extracellular virulence factors such as the haemolysins which are also Sae- and Agr-regulated. Hence, we show that Fur is central to a complex regulatory network that is required for the induced expression of a number of important S. aureus virulence determinants in low iron.

Transcriptomic response of Listeria monocytogenes to iron limitation and Fur mutation.

Iron is required by almost all bacteria, but concentrations above physiological levels are toxic. In bacteria, intracellular iron is regulated mostly by the ferric uptake regulator, Fur, or a similar functional protein. Iron limitation results in the regulation of a number of genes, especially those involved in iron uptake. A subset of these genes is the Fur regulon under the control of Fur. In the present study, we have identified Fur- and iron-regulated genes in Listeria monocytogenes by DNA microarray analysis using a fur mutant and its isogenic parent. To identify genes regulated exclusively in response to iron limitation, the whole-genome transcriptional responses to the iron limitation of a fur mutant and its isogenic parent were compared. Fur-regulated genes were identified by comparing the transcriptional profile of the parent with the transcriptional profile of the isogenic fur mutant. Our studies have identified genes regulated exclusively in response to iron and those that are negatively regulated by Fur. We have identified at least 14 genes that were negatively regulated directly by Fur. Under iron-limited conditions, these genes were upregulated, while the expression of fur was found to be downregulated. To further investigate the regulation of fur in response to iron, an ectopic fur promoter-lacZ transcriptional fusion strain was constructed, and its isogenic fur and perR mutant derivatives were generated in L. monocytogenes 10403S. Analysis of the iron limitation of the perR mutant indicated that the regulation of genes under the negative control of Fur was significantly inhibited. Our results indicate that Fur and PerR proteins negatively regulate fur and that under iron-limited conditions, PerR is required for the negative regulation of genes controlled by Fur.

Staphylococcus aureus cell wall stress stimulon gene-lacZ fusion strains: potential for use in screening for cell wall-active antimicrobials.

lacZ fusion strains were constructed using the promoters of five cell wall stress stimulon genes: pbp2, tcaA, vraSR, sgtB, and lytR. All fusion strains were induced only in the presence of cell wall-active antibiotics, suggesting the potential of these strains for use in high-throughput screening for new cell wall-active agents.

Molecular characterization of the Fur protein of Listeria monocytogenes.

Iron is essential for the survival of almost all organisms, although excess iron can result in the generation of free radicals which are toxic to cells. To avoid the toxic effects of free radicals, the concentration of intracellular iron is generally regulated by the ferric uptake regulator Fur in bacteria. The 150 aa fur ORF from Listeria monocytogenes was cloned into pRSETa, and the His-tagged fusion protein was purified by nickel affinity column chromatography. DNA binding activity of this protein was studied by an electrophoretic mobility shift assay using the end-labelled promoters P(fhuDC) and P(fur). The results showed a decrease in migration for both promoter DNAs in the presence of the Fur protein, and the change in migration was competitively inhibited with an excess of the same unlabelled promoters. No shift in migration was observed when a similar assay was performed using non-specific end-labelled DNA. The assay showed that binding of Fur to P(fur) or P(fhuDC) was independent of iron or manganese ions, and was not inhibited in the presence of 2 mM EDTA. Inductively coupled plasma MS of the Fur protein showed no iron or manganese, but 0.48 mole zinc per mole protein was detected. A DNase I protection assay revealed that Fur specifically bound to and protected a 19 bp consensus Fur box sequence located in the promoters of fur and fhuDC. There was no requirement for iron or manganese in this assay also. However, Northern blot analysis showed an increase in fur transcription under iron-restricted compared to high-level conditions. Thus, the study suggests that under in vitro conditions, the affinity of the Fur protein for the 19 bp Fur box sequence does not require iron, but iron availability regulates fur transcription in vivo. Thus, the regulation by Fur in this intracellular pathogen may be dependent on either the structure of the DNA binding domain or other intracellular factors yet to be identified.

Effects of oxacillin and tetracycline on autolysis, autolysin processing and atl transcription in Staphylococcus aureus.

Autolysins are peptidoglycan hydrolases involved in cell growth and cell lysis. Atl is an important autolysin of Staphylococcus aureus and is essential for penicillin-induced autolysis. The objective of our study was to examine the effect of oxacillin, chloramphenicol and tetracycline on autolysis, peptidoglycan hydrolase profiles and transcription of atl encoding the major S. aureus autolysin on cells grown in the presence of minimum inhibitory concentrations of the antibiotics. Growth of methicillin-susceptible strains in the presence of oxacillin led to increased autolysis, a loss of low molecular weight and a gain of high molecular weight peptidoglycan hydrolase bands suggesting altered proteolytic processing of peptidoglycan hydrolases, and a decrease in atl transcription. In contrast, growth in the presence of tetracycline led to a decrease in autolysis, an increase in atl transcription, and a drastic decrease in the protein concentration of freeze-thaw extracts obtained for peptidoglycan hydrolase analysis. Growth of methicillin-resistant strains in the presence of oxacillin had only moderate effects on autolysis and peptidoglycan hydrolase profiles.

Regulation of the expression of cell wall stress stimulon member gene msrA1 in methicillin-susceptible or -resistant Staphylococcus aureus.

Genome-wide transcriptional profiling studies of the response of Staphylococcus aureus to cell wall-active antibiotics have led to the discovery of a cell wall stress stimulon of genes induced by these agents. msrA1, encoding methionine sulfoxide reductase, is a highly induced member gene of this stimulon. In the present study we show that msrA1 induction by oxacillin is common to all methicillin-susceptible strains studied but did not occur in two homogeneous and two heterogeneous methicillin-resistant strains. However, msrA1 was induced by vancomycin and/or D-cycloserine in methicillin-resistant strains. Lysozyme and lysostaphin treatment did not induce msrA1 expression. Oxacillin-induced msrA1 expression was enhanced by ca. 30% in a SigB+ derivative (SH1000) of the SigB-defective RN450 (NCTC 8325-4) strain. msrA1 expression was not affected in mutants in the global regulatory systems agr and sar. Glycerol monolaurate, an inhibitor of signal transduction, inhibited the oxacillin-induced transcription of msrA1 and other cell wall stress stimulon member genes, vraS and dnaK. These observations suggest that the cell wall stress stimulon is induced by inhibition of the process of peptidoglycan biosynthesis, and the inhibitory effects of glycerol monolaurate indicate that gene expression is dependent on a signal transduction pathway.