Bdellovibrio svalbardensis sp. nov., a newly described predator isolated from Svalbard, Norway.

Identified as a newly described species from a biocrust in Svalbard, Norway (78° 54' 8.27″ N 12° 01' 20.34″ E), isolate PAP01T has different characteristics from any known predatory bacteria. The isolate was vibrio-shaped strain that employed flagellar motility. Phylogenetic analysis based on 16S rRNA gene sequences revealed that the isolate clustered within the genus Bdellovibrio in the family Bdellovibrionaceae. 16S rRNA gene sequence similarities between strain PAP01T and the type strain (Bdellovibrio bacteriovorus HD100) was 95.7 %. The PAP01T genome has a size of 3.898 Mbp and possesses 3732 genes and a G+C content of 45.7 mol%. The results of genetic and physiological tests indicated the phenotypic differentiation of strain PAP01T from the two other Bdellovibrio species with validly published names. Based on the physiological and phylogenetic data, as well as the prey range spectrum and osmolality sensitivities, isolate PAP01T represents a novel species within the genus Bdellovibrio, for which the name Bdellovibrio svalbardensis sp. nov. is proposed. The type strain is PAP01T (=KCTC 92583T=DSM 115080T).

Predatory bacteria as potential biofilm control and eradication agents in the food industry.

Biofilms are a major concern within the food industry since they have the potential to reduce productivity in situ (within the field), impact food stability and storage, and cause downstream food poisoning. Within this review, predatory bacteria as potential biofilm control and eradication agents are discussed, with a particular emphasis on the intraperiplasmic Bdellovibrio-and-like organism (BALO) grouping. After providing a brief overview of predatory bacteria and their activities, focus is given to how BALOs fulfill four attributes that are essential for biocontrol agents to be successful in the food industry: (1) Broad spectrum activity against pathogens, both plant and human; (2) Activity against biofilms; (3) Safety towards humans and animals; and (4) Compatibility with food. As predatory bacteria possess all of these characteristics, they represent a novel form of biofilm biocontrol that is ripe for use within the food industry.

Staphylococcus aureus Sensitivity to Membrane Disrupting Antibacterials Is Increased under Microgravity.

In a survey of the International Space Station (ISS), the most common pathogenic bacterium identified in samples from the air, water and surfaces was Staphylococcus aureus. While growth under microgravity is known to cause physiological changes in microbial pathogens, including shifts in antibacterial sensitivity, its impact on S. aureus is not well understood. Using high-aspect ratio vessels (HARVs) to generate simulated microgravity (SMG) conditions in the lab, we found S. aureus lipid profiles are altered significantly, with a higher presence of branch-chained fatty acids (BCFAs) (14.8% to 35.4%) with a concomitant reduction (41.3% to 31.4%) in straight-chain fatty acids (SCFAs) under SMG. This shift significantly increased the sensitivity of this pathogen to daptomycin, a membrane-acting antibiotic, leading to 12.1-fold better killing under SMG. Comparative assays with two additional compounds, i.e., SDS and violacein, confirmed S. aureus is more susceptible to membrane-disrupting agents, with 0.04% SDS and 0.6 mg/L violacein resulting in 22.9- and 12.8-fold better killing in SMG than normal gravity, respectively. As humankind seeks to establish permanent colonies in space, these results demonstrate the increased potency of membrane-active antibacterials to control the presence and spread of S. aureus, and potentially other pathogens.

Use of Resazurin To Rapidly Enumerate Bdellovibrio and Like Organisms and Evaluate Their Activities.

A method to rapidly quantify predatory bacterial cell populations using resazurin reduction to resorufin and its resulting fluorescence kinetics (dF/dt) are described. The reliability of this method to measure the predatory populations was demonstrated with the type strain, Bdellovibrio bacteriovorus HD100, as well as B. bacteriovorus 109J and two natural isolates, Halobacteriovorax strains JA-1 and JA-3, with clear correlation when densities were between 107 and 109 PFU/ml. Resazurin was also used to evaluate how B. bacteriovorus HD100 and Halobacteriovorax strain JA-1 respond to harmful conditions, i.e., exposure to sodium dodecyl sulfate (SDS), with both the dF/dt and PFU/ml indicating Halobacteriovorax strain JA-1 is more sensitive to this surfactant. Tests were also performed using media of different osmolalities, with the dF/dt values matching the 24-h predatory activities reasonably well. Finally, this method was successfully applied in near real-time analyses of predator-prey dynamics and, when coupled with SDS, was capable of differentiating between the predatory and prey populations. All of these tests serve to prove this method is (i) very rapid, needing only 15 min from start to finish; (ii) very reliable with different predatory bacterial species; and (iii) very versatile as it can be easily adapted to measure predatory numbers and activities in a range of experiments. IMPORTANCEBdellovibrio and like organisms are predatory bacteria that are capable of attacking, killing, and consuming many bacterial pathogens, including multidrug-resistant strains. These qualities have led to them being labeled as "living antibiotics." Research work with these remarkable strains, however, has been hampered by long growth times needed to quantify the predatory populations through plaque assays, which typically take 4 days to develop. Here, we describe a fluorescence-based method using the conversion of resazurin (low fluorescence) to resorufin (high fluorescence) after it is reduced by the predators' NADH. Not only do we show that the fluorescence correlates strongly with the predatory concentration and that we can use it to evaluate if the predators are viable, but the entire procedure from start to finish takes only 15 min, drastically reducing the time researchers need to quantify the predatory numbers. Employing this technique will greatly advance research related to predatory bacteria and their potential applications.

The Kiss of Death: Serratia marcescens Antibacterial Activities against Staphylococcus aureus Requires Both de novo Prodigiosin Synthesis and Direct Contact.

Prodigiosin possesses antibacterial activities, but as a highly hydrophobic compound, it raised the question about how Serratia marcescens introduce this compound to other microbes. Here, we demonstrate that the production of prodigiosin by newly isolated S. marcescens RH10 correlates with its antibacterial activity against a multidrug-resistant strain of S. aureus, with this pathogen's viability decreasing 6-log over 24 h. While S. marcescens RH10 does secrete membrane vesicles that carry prodigiosin, this antibiotic was not active in this form, with 5 mg/L prodigiosin leading to only a 1.22-fold reduction in the S. aureus viability while the same quantity of purified prodigiosin led to a 2800-fold reduction. Contact assays, however, showed increased activity, with a 3-log loss in the S. aureus viabilities in only 6 h as long as de novo production of prodigiosin occurred. The role of prodigiosin was confirmed further by generating an isogenic ΔpigA mutant in S. marcescens RH10, based on the draft genome sequence reported here, to inhibit the synthesis of prodigiosin. In all experiments performed, this mutant was unable to kill S. aureus. Finally, the possibility that the type VI secretion system present in S. marcescens may also be important was also explored as it is known to be used by this strain to kill other microbes. The results here, however, found no obvious activity against S. aureus. In conclusion, the results presented here show prodigiosin requires both cell-to-cell contact and de novo synthesis for it to be effective as an antibiotic for its native host. IMPORTANCE The antibacterial activities of prodigiosin are well-established but, as a hydrophobic molecule, the mechanisms used to introduce it to susceptible microbes has never been studied. We found here, in contrast to violacein, another hydrophobic antibiotic that can be transferred using membrane vesicles (MVs), prodigiosin is also carried from Serratia marcescens in MVs released but its resulting activities were severely mitigated compared to the freely added compound, suggesting it is more tightly bound to the MVs than violacein. This led us to hypothesize that cell-to-cell contact is needed, which we demonstrate here. As well, we show de novo synthesis of prodigiosin is needed for it to be effective. As violacein- and prodigiosin-producing bacterial strains are both beneficial to amphibians, where they help protect the skin against pathogens, the findings presented here provide an important ecological perspective as they show the mechanisms used differ according to the antibacterial produced.

Predation of colistin- and carbapenem-resistant bacterial pathogenic populations and their antibiotic resistance genes in simulated microgravity.

As mankind evaluates moving toward permanently inhabiting outer space and other planetary bodies, alternatives to antibiotic that can effectively control drug-resistant pathogens are needed. The activity of one such alternative, Bdellovibrio bacteriovorus HD100, was explored here, and was found to be as active or better in simulated microgravity (SMG) conditions as in flask and normal gravity (NG) cultures, with the prey viabilities decreasing by 3- to 7-log CFU/mL in 24 h. The activity of B. bacteriovorus HD100 under SMG was also appraised with three different carbapenem- and colistin-resistant pathogenic bacterial strains. In addition to being more efficient at killing two of these pathogens under SMG conditions (with losses of 5- to 6-log CFU/mL), we also explored the ability of B. bacteriovorus HD100 to hydrolyze the carbapenem- and colistin-resistant gene pools, i.e., mcr-1, blaKPC-2 and blaOXA-51, present in these clinical isolates. We found removal efficiencies of 97.4 ± 0.9 %, 97.8 ± 0.4 % and 99.3 ± 0.1 %, respectively, in SMG cultures, while similar reductions were also seen in the flask and NG cultures. These results illustrate the potential applicability of B. bacteriovorus HD100 as an antibiotic to combat the ever-growing threat of multidrug-resistant (MDR) pathogens during spaceflight, such as in the International Space Station (ISS).

Biotechnological Activities and Applications of Bacterial Pigments Violacein and Prodigiosin.

In this review, we discuss violacein and prodigiosin, two chromogenic bacterial secondary metabolites that have diverse biological activities. Although both compounds were "discovered" more than seven decades ago, interest into their biological applications has grown in the last two decades, particularly driven by their antimicrobial and anticancer properties. These topics will be discussed in the first half of this review. The latter half delves into the current efforts of groups to produce these two compounds. This includes in both their native bacterial hosts and heterogeneously in other bacterial hosts, including discussing some of the caveats related to the yields reported in the literature, and some of the synthetic biology techniques employed in this pursuit.

Concentric liquid reactors for chemical synthesis and separation.

Recent years have witnessed increased interest in systems that are capable of supporting multistep chemical processes without the need for manual handling of intermediates. These systems have been based either on collections of batch reactors1 or on flow-chemistry designs2-4, both of which require considerable engineering effort to set up and control. Here we develop an out-of-equilibrium system in which different reaction zones self-organize into a geometry that can dictate the progress of an entire process sequence. Multiple (routinely around 10, and in some cases more than 20) immiscible or pairwise-immiscible liquids of different densities are placed into a rotating container, in which they experience a centrifugal force that dominates over surface tension. As a result, the liquids organize into concentric layers, with thicknesses as low as 150 micrometres and theoretically reaching tens of micrometres. The layers are robust, yet can be internally mixed by accelerating or decelerating the rotation, and each layer can be individually addressed, enabling the addition, sampling or even withdrawal of entire layers during rotation. These features are combined in proof-of-concept experiments that demonstrate, for example, multistep syntheses of small molecules of medicinal interest, simultaneous acid-base extractions, and selective separations from complex mixtures mediated by chemical shuttles. We propose that 'wall-less' concentric liquid reactors could become a useful addition to the toolbox of process chemistry at small to medium scales and, in a broader context, illustrate the advantages of transplanting material and/or chemical systems from traditional, static settings into a rotating frame of reference.

Consumption of Oleic Acid During Matriphagy in Free-Living Nematodes Alleviates the Toxic Effects of the Bacterial Metabolite Violacein.

Maternal behaviors benefit the survival of young, contributing directly to the mother's reproductive fitness. An extreme form of this is seen in matriphagy, when a mother performs the ultimate sacrifice and offers her body as a meal for her young. Whether matriphagy offers only a single energy-rich meal or another possible benefit to the young is unknown. Here, we characterized the toxicity of a bacterial secondary metabolite, namely, violacein, in Caenorhabditis elegans and found it is not only toxic towards adults, but also arrests growth and development of C. elegans larvae. To counteract this, C. elegans resorted to matriphagy, with the mothers holding their eggs within their bodies and hatching the young larvae internally, which eventually led to the mothers' death. This violacein-induced matriphagy alleviated some of the toxic effects of violacein, allowing a portion of the internally-hatched young to bypass developmental arrest. Using genetic and pharmacological experiments, we found the consumption of oleate, a monounsaturated fatty acid produced by the mother, during matriphagy is partially responsible. As such, our study provides experimental evidence of why such a drastic and peculiar maternal behavior may have arisen in nematode natural habitats.

Compounds affecting predation by and viability of predatory bacteria.

Bdellovibrio-and-like organisms (BALOs) are a small group of bacteria that actively predate on other Gram-negative bacterial species. Although viewed mostly in a positive light, such as their potential use as living antibiotics to reduce pathogenic strain populations, several studies have also highlighted the need to control their activities, such as in the production of biodiesel. Consequently, this mini-review discusses research being conducted to characterize compounds and environmental settings that influence predation rates and the mechanisms by which they accomplish this, with a heavy emphasis on studies published within the last decade.Key points• This review discusses bacterial predators and factors impacting their activities. • Emphasis is on recent articles, particularly those discussing prey metabolites. • The implications on possible applications of bacterial predators are discussed.

Chromobacterium violaceum delivers violacein, a hydrophobic antibiotic, to other microbes in membrane vesicles.

This study describes Chromobacterium violaceum's use of extracellular membrane vesicles (MVs) to both solubilize and transport violacein to other microorganisms. Violacein is a hydrophobic bisindole with known antibiotic activities against other microorganisms. Characterization of the MVs found they carried more violacein than protein (1.37 ± 0.19-fold), suggesting they may act as a reservoir for this compound. However, MVs are not produced in response to violacein - a ΔvioA isogenic mutant, which is incapable of making violacein, actually produced significantly more MVs (3.2-fold) than the wild-type strain. Although violacein is insoluble in water (Log Poctanol:water = 3.34), 79.5% remained in the aqueous phase when it was present within the C. violaceum MVs, an increase in solubility of 1740-fold. Moreover, tests with a strain of Staphylococcus aureus showed MV-associated violacein is bactericidal, with 3.1 mg/l killing 90% of S. aureus in 6 h. Tests with the ΔvioA MVs found no loss in the S. aureus viability, even when its MVs were added at much higher concentrations, demonstrating violacein is the active component within the wild-type MVs. In conclusion, our study clearly demonstrates C. violaceum produces MVs and uses them as vehicles to solubilize violacein and transport this hydrophobic antibiotic to other microbes.

Viscosity has dichotomous effects on Bdellovibrio bacteriovorus HD100 predation.

Bdellovibrio bacteriovorus HD100 is a highly motile predatory bacterium that consumes other Gram-negative bacteria for its sustenance. Here, we describe the impacts the media viscosity has both on the motility of predator and its attack rates. Experiments performed in polyethylene glycol (PEG) solutions, a linear polymer, found a viscosity of 10 mPa s (5% PEG) negatively impacted predation over a 24-h period. When the viscosity was increased to 27 mPa s (10% PEG), predation was nearly abolished. Tests with three other B. bacteriovorus strains, i.e., 109J and two natural isolates, found identical results. Short-term (2-h) experiments, however, found attack rates were improved in 1% PEG, which had a viscosity of 5.4 mPa s, using bioluminescent prey and their viabilities. In contrast, when experiments were performed in dextran, a branched polymer, no increase in predation was seen even though the viscosity was a comparable 5.1 mPa s. The enhanced attack rates in this solution coincided with a 31% increase in B. bacteriovorus HD100 swimming speeds (62 µm s-1 in 1% PEG vs. 47.5 µm s-1 in HEPES-salt).

Cyanide Production by Chromobacterium piscinae Shields It from Bdellovibrio bacteriovorus HD100 Predation.

Predation of Chromobacterium piscinae by Bdellovibrio bacteriovorus HD100 was inhibited in dilute nutrient broth (DNB) but not in HEPES. Experiments showed that the effector responsible was present in the medium, as cell-free supernatants retained the ability to inhibit predation, and that the effector was not toxic to B. bacteriovorus Violacein, a bisindole secondary metabolite produced by C. piscinae, was not responsible. Further characterization of C. piscinae found that this species produces sufficient concentrations of cyanide (202 µM) when grown in DNB to inhibit the predatory activity of B. bacteriovorus, but that in HEPES, the cyanide concentrations were negligible (19 µM). The antagonistic role of cyanide was further confirmed, as the addition of hydroxocobalamin, which chelates cyanide, allowed predation to proceed. The activity of cyanide against B. bacteriovorus was found to be twofold, depending on the life cycle stage of this predator. For the attack-phase predatory cells, cyanide caused the cells to lose motility and tumble, while for intraperiplasmic predators, development and lysis of the prey cell were halted. These findings suggest that cyanogenesis in nature may be employed by the bacterial strains that produce this compound to prevent and reduce their predation by B. bacteriovorusIMPORTANCE Bacterial predators actively attack, kill, and enter the periplasm of susceptible Gram-negative bacteria, where they consume the prey cell components. To date, the activity of B. bacteriovorus HD100 has been demonstrated against more than 100 human pathogens. As such, this strain and others are being considered as potential alternatives or supplements to conventional antibiotics. However, the production of secondary metabolites by prey bacteria is known to mitigate, and even abolish, predation by bacterivorous nematodes and protists. With the exception of indole, which was shown to inhibit predation, the effects of bacterial secondary metabolites on B. bacteriovorus and its activities have not been considered. Consequently, we undertook this study to better understand the mechanisms that bacterial strains employ to inhibit predation by B. bacteriovorus HD100. We report here that cyanogenic bacterial strains can inhibit predation and show that cyanide affects both attack-phase predators and those within prey, i.e., in the bdelloplast.

Combined Application of Bacterial Predation and Violacein to Kill Polymicrobial Pathogenic Communities.

Violacein is a bisindole antibiotic that is effective against Gram-positive bacteria while the bacterial predator, Bdellovibrio bacteriovorus HD100, predates on Gram-negative strains. In this study, we evaluated the use of both together against multidrug resistant pathogens. The two antibacterial agents did not antagonize the activity of the other. For example, treatment of Staphylococcus aureus with violacein reduced its viability by more than 2,000-fold with or without B. bacteriovorus addition. Likewise, predation of Acinetobacter baumannii reduced the viability of this pathogen by more than 13,000-fold, regardless if violacein was present or not. When used individually against mixed bacterial cultures containing both Gram-positive and Gram-negative strains, violacein and B. bacteriovorus HD100 were effective against only their respective strains. The combined application of both violacein and B. bacteriovorus HD100, however, reduced the total pathogen numbers by as much as 84,500-fold. Their combined effectiveness was also demonstrated using a 4-species culture containing S. aureus, A. baumannii, Bacillus cereus and Klebsiella pneumoniae. When used alone, violacein and bacterial predation reduced the total population by only 19% and 68%, respectively. In conjunction with each other, the pathogen viability was reduced by 2,965-fold (99.98%), illustrating the prospective use of these two antimicrobials together against mixed species populations.

Investigating the Responses of Human Epithelial Cells to Predatory Bacteria.

One beguiling alternative to antibiotics for treating multi-drug resistant infections are Bdellovibrio-and-like-organisms (BALOs), predatory bacteria known to attack human pathogens. Consequently, in this study, the responses from four cell lines (three human and one mouse) were characterized during an exposure to different predatory bacteria, Bdellovibrio bacteriovorus HD100, Bacteriovorus BY1 and Bacteriovorax stolpii EB1. TNF-α levels were induced in Raw 264.7 mouse macrophage cultures with each predator, but paled in comparison to those obtained with E. coli. This was true even though the latter strain was added at an 11.1-fold lower concentration (p < 0.01). Likewise, E. coli led to a significant (54%) loss in the Raw 264.7 murine macrophage viability while the predatory strains had no impact. Tests with various epithelial cells, including NuLi-1 airway, Caco2, HT29 and T84 colorectal cells, gave similar results, with E. coli inducing IL-8 production. The viabilities of the NuLi-1 and Caco-2 cells were slightly reduced (8%) when exposed to the predators, while T84 viability remained steady. In no cases did the predatory bacteria induce actin rearrangement. These results clearly demonstrate the gentle natures of predatory bacteria and their impacts on human cells.

High-level production of violacein by the newly isolated Duganella violaceinigra str. NI28 and its impact on Staphylococcus aureus.

A violacein-producing bacterial strain was isolated and identified as a relative of Duganella violaceinigra YIM 31327 based upon phylogenetic analyses using the 16S rRNA, gyrB and vioA gene sequences and a fatty acid methyl ester (FAME) analysis. This new strain was designated D. violaceinigra str. NI28. Although these two strains appear related based upon these analyses, the new isolate was phenotypically different from the type strain as it grew 25% faster on nutrient media and produced 45-fold more violacein. When compared with several other violacein producing strains, including Janthinobacterium lividum, D. violaceinigra str. NI28 was the best violacein producer. For instance, the crude violacein yield with D. violaceinigra str. NI28 was 6.0 mg/OD at 24 hours, a value that was more than two-fold higher than all the other strains. Finally, the antibacterial activity of D. violaceinigra str. NI28 crude violacein was assayed using several multidrug resistant Staphylococcus aureus. Addition of 30 µM crude violacein led to a 96% loss in the initial S. aureus population while the minimum inhibitory concentration was 1.8 µM. Consequently, this novel isolate represents a phenotypic variant of D. violaceinigra capable of producing much greater quantities of crude violacein, an antibiotic effective against multidrug resistant S. aureus.

Violacein: Properties and Production of a Versatile Bacterial Pigment.

Violacein-producing bacteria, with their striking purple hues, have undoubtedly piqued the curiosity of scientists since their first discovery. The bisindole violacein is formed by the condensation of two tryptophan molecules through the action of five proteins. The genes required for its production, vioABCDE, and the regulatory mechanisms employed have been studied within a small number of violacein-producing strains. As a compound, violacein is known to have diverse biological activities, including being an anticancer agent and being an antibiotic against Staphylococcus aureus and other Gram-positive pathogens. Identifying the biological roles of this pigmented molecule is of particular interest, and understanding violacein's function and mechanism of action has relevance to those unmasking any of its commercial or therapeutic benefits. Unfortunately, the production of violacein and its related derivatives is not easy and so various groups are also seeking to improve the fermentative yields of violacein through genetic engineering and synthetic biology. This review discusses the recent trends in the research and production of violacein by both natural and genetically modified bacterial strains.

Changes in pneumococcal nasopharyngeal colonization among children with respiratory tract infections before and after use of the two new extended-valency pneumococcal conjugated vaccines.

BACKGROUND: The 10-valent pneumococcal non-typeable Haemophilus influenzae protein D conjugate vaccine (PHiD-CV) and the 13-valent pneumococcal conjugate vaccine (PCV13) replaced the 7-valent PCV (PCV7) in May 2010 in Korea. We investigated the dynamics of pneumococcal nasopharyngeal (NP) colonization in children with a respiratory illness before and after use of PHiD-CV and PCV13. METHODS: From March 2009 to December 2012 NP secretions were obtained from 2176 children aged < 5 years with respiratory diseases. We used the multiplex polymerase chain reaction (PCR) technique to determine pneumococcal serotypes. RESULTS: Among the samples, 468 (21.5%) specimens were positive by multiplex PCR. The overall pneumococcal colonization rate remained stable during the 2009-2012 periods. The serotypes present in PCV7 and serotype 19A decreased in frequency from 36.8% and 26.4% in 2009 to 10.1% and 11.4% in 2012, respectively (χ(2) for trend, P < 0.001 and P = 0.007, respectively). The frequency of non-PCV13 serotypes increased from 36.8% in 2009 to 78.5% in 2012 (χ(2) for trend, P < 0.001). There was no significant difference in carriage rates of each serotype between groups of children that received PCV7, PHiD-CV, or PCV13. CONCLUSIONS: Compared with the period of PCV7 vaccination, overall carriage rate was not affected by the introduction of new PCVs. However, serotype distribution now consists mostly of non-vaccine serotypes. PCVs affect mucosal immunity against Streptococcus pneumoniae (SP) in NP carriage; but, global SP colonization seems to be maintained by replacement.

Limitation of the delta neutrophil index for assessing bacteraemia in immunocompromised children.

BACKGROUND: Delta neutrophil index (DNI) has been reported to be useful in assessing the prognosis of suspected bacteraemia in adults. However, data in children are limited. The purpose of this study was to investigate the correlation between the DNI and bacteraemia in children and to compare DNI values between immunocompetent and immunocompromised hosts. METHODS: We retrospectively collected data on 8593 children who underwent simultaneous blood culture and DNI testing. DNI was determined with a blood cell analyser (ADVIA 120, Siemens, Inc.). The children were divided into immunocompromised (n=664) and immunocompetent groups (n=7929). RESULTS: DNI was higher in the bacteraemia group than in the non-bacteraemia group (P<0.001). According to the group, DNI was higher in immunocompromised than immunocompetent patients (P<0.001). Within the immunocompromised group, there was no significant difference between the bacteraemia and non-bacteraemia subgroups due to a higher DNI as compared to the immunocompetent group. CONCLUSIONS: DNI could be an additional method for the early diagnosis of bacteraemia in children. However, the use of DNI for the prediction of bacteraemia in immunocompromised cases has limitations. Further studies on the usefulness of DNI according to specific diseases are needed.