Laboratory approaches to determining blood culture contamination rates: an ASM Laboratory Practices Subcommittee report.

Blood culture contamination (BCC) is the presence of specific commensal and environmental organisms cultivated from a single blood culture set out of a blood culture series and that do not represent true bacteremia. BCC can impact quality of care and lead to negative outcomes, unnecessary antibiotic exposure, prolonged hospital stays, and substantial costs. As part of the laboratory's quality management plan, microbiology laboratory personnel are tasked with monitoring BCC rates, preparing BCC rate reports, and providing feedback to the appropriate committees within their healthcare system. The BCC rate is calculated by the laboratory using pre-set criteria. However, pre-set criteria are not universally defined and depend on the individual institution's patient population and practices. This mini-review provides practical recommendations on elaborating BCC rate reports, the parameters to define for the pre-set criteria, how to collect and interpret the data, and additional analysis to include in a BCC report.

A diagnostic stewardship approach to prevent unnecessary testing of an enteric bacterial molecular panel.

IMPORTANCE: Testing for enteric bacterial pathogens in patients hospitalized for more than 3 days is almost always inappropriate. Our study validates the utility of the 3-day rule and the use of clinical decision support tools to decrease unnecessary testing of enteropathogenic bacteria other than C. difficile. Overriding the restriction was very low yield. Our study highlights the importance of diagnostic stewardship and further refines the criteria for allowing providers to override the restriction while monitoring the impact of the interventions.

Clinical significance of Nosocomiicoccus ampullae isolated from blood cultures.

IMPORTANCE: Nosocomiicoccus species are recently described as members of the Staphylococcaceae family. With their inclusion in commercial matrix-assisted laser desorption/ionization-time of flight mass spectrometry databases, Nosocomiicoccus species can now be identified when Gram-positive cocci in clusters are detected in positive blood cultures. However, their clinical significance is not known, making it difficult for the clinical microbiology laboratory to decide the extent of work-up. Based on our study, Nosocomiicoccus species demonstrate low pathogenicity and opportunistic potential. If isolated from a single blood culture set, limited work-up should be performed to an extent similar to other possible blood culture contaminants.

Dissemination and genome characterization of a human adenovirus F41 in a patient with B-Cell lymphoma.

Adenovirus (HAdV) F41 is a common cause of gastroenteritis and has rarely been reported associated with disseminated disease. In this report, an adult patient with a history of ulcerative colitis, cryptogenic cirrhosis, stage III adenocarcinoma, high-grade diffuse large B-cell lymphoma on chemotherapy was diagnosed with disseminated adenovirus infection. HAdV DNA was quantified in stool, plasma, and urine with viral loads of 7, 4, and 3 log10 copies/mL, respectively. The patient's course was rapidly progressive and he passed away 2 days after initiation of antiviral therapy. The patient's infecting virus was characterized as HAdV-F41 by whole genome sequencing.

Human Colon Cancer-Derived Clostridioides difficile Strains Drive Colonic Tumorigenesis in Mice.

Defining the complex role of the microbiome in colorectal cancer and the discovery of novel, protumorigenic microbes are areas of active investigation. In the present study, culturing and reassociation experiments revealed that toxigenic strains of Clostridioides difficile drove the tumorigenic phenotype of a subset of colorectal cancer patient-derived mucosal slurries in germ-free ApcMin/+ mice. Tumorigenesis was dependent on the C. difficile toxin TcdB and was associated with induction of Wnt signaling, reactive oxygen species, and protumorigenic mucosal immune responses marked by the infiltration of activated myeloid cells and IL17-producing lymphoid and innate lymphoid cell subsets. These findings suggest that chronic colonization with toxigenic C. difficile is a potential driver of colorectal cancer in patients. SIGNIFICANCE: Colorectal cancer is a leading cause of cancer and cancer-related deaths worldwide, with a multifactorial etiology that likely includes procarcinogenic bacteria. Using human colon cancer specimens, culturing, and murine models, we demonstrate that chronic infection with the enteric pathogen C. difficile is a previously unrecognized contributor to colonic tumorigenesis. See related commentary by Jain and Dudeja, p. 1838. This article is highlighted in the In This Issue feature, p. 1825.

Antibiotic Treatment Shapes the Antigenic Environment During Chronic TB Infection, Offering Novel Targets for Therapeutic Vaccination.

The lengthy and complicated current regimen required to treat drug-susceptible tuberculosis (TB) reflects the ability of Mycobacterium tuberculosis (Mtb) to persist in host tissues. The stringent response pathway, governed by the dual (p)ppGpp synthetase/hydrolase, Rel Mtb , is a major mechanism underlying Mtb persistence and antibiotic tolerance. In the current study, we addressed the hypothesis that Rel Mtb is a "persistence antigen" presented during TB chemotherapy and that enhanced immunity to Rel Mtb can enhance the tuberculocidal activity of the first-line anti-TB drug, isoniazid, which has reduced efficacy against Mtb persisters. C57BL/6 mice and Hartley guinea pigs were aerosol-infected with M. tuberculosis (Mtb) and, 4 weeks later, received either human-equivalent daily doses of isoniazid alone, or isoniazid in combination with a DNA vaccine targeting relMtb . After isoniazid treatment, there was a significant reduction in dominant antigen ESAT6-reactive CD4+ or TB10.4-reactive CD8+ T cells in the lungs and spleens of mice. However, the total number of Rel Mtb -reactive CD4+ T cells remained stable in mouse lungs and spleens, as did the number of Rel Mtb -reactive CD8+T cells. Therapeutic vaccination with relMtb DNA vaccine enhanced the activity of isoniazid in Mtb-infected C57BL/6 mice and guinea pigs. When treatment with isoniazid was discontinued, mice immunized with the relMtb DNA vaccine showed a lower mean lung bacterial burden at relapse compared to the control group. Our work shows that antitubercular treatment shapes the antigenic environment, and that therapeutic vaccination targeting the Mtb stringent response may represent a novel approach to enhance immunity against Mtb persisters, with the ultimate goal of shortening curative TB treatment.

Altered Mycobacterium tuberculosis Cell Wall Metabolism and Physiology Associated With RpoB Mutation H526D.

Background:Mycobacterium tuberculosis (Mtb) rpoB mutations are associated with global metabolic remodeling. However, the net effects of rpoB mutations on Mtb physiology, metabolism and function are not completely understood. Based on previous work, we hypothesized that changes in the expression of cell wall molecules in Mtb mutant RpoB 526D lead to changes in cell wall permeability and to altered resistance to environmental stresses and drugs. Methods: The phenotypes of a fully drug-susceptible clinical strain of Mtb and its paired rifampin-monoresistant, RpoB H526D mutant progeny strain were compared. Results: The rpoB mutant showed altered colony morphology, bacillary length and cell wall thickness, which were associated with increased cell wall permeability and susceptibility to the cell wall detergent sodium dodecyl sulfate (SDS) after exposure to nutrient starvation. Relative to the isogenic rifampin-susceptible strain, the RpoB H526D mutant showed altered bacterial cellular metabolic activity and an eightfold increase in susceptibility to the cell-wall acting drug vancomycin. Conclusion: Our data suggest that RpoB mutation H526D is associated with altered cell wall physiology and resistance to cell wall-related stress. These findings are expected to contribute to an improved understanding of the pathogenesis of drug-resistant M. tuberculosis infections.

In vitro and in vivo fitness costs associated with Mycobacterium tuberculosis RpoB mutation H526D.

AIM: There is controversy regarding the potential fitness costs of rifampicin (RIF) resistance-conferring mutations in the Mycobacterium tuberculosis (Mtb) rpoB gene. We characterized the pathogenicity of an Mtb RpoB H526D mutant. MATERIALS & METHODS: A mutant containing the RpoB H526D mutation was isolated from wild-type Mtb grown on RIF-containing plates and complemented for determination of in vitro and in vivo fitness costs. RESULTS: The RpoB H526D mutant showed reduced survival relative to control strains during progressive hypoxia and delayed growth following resuscitation from nutrient starvation (p < 0.05), which was associated with reduced expression of the resuscitation-promoting factor genes rpfB, rpfC and rpfE. Relative to the isogenic wild-type strain, the mutant showed significantly attenuated growth and long-term survival as well as reduced inflammation in mouse lungs. Conclusion & future perspective: Our data suggest that RpoB H526D mutation confers a fitness cost during growth-limiting conditions in vitro and in mouse lungs.

Efficacy of a conjugate vaccine containing polymannuronic acid and flagellin against experimental Pseudomonas aeruginosa lung infection in mice.

Vaccines that could effectively prevent Pseudomonas aeruginosa pulmonary infections in the settings of cystic fibrosis (CF) and nosocomial pneumonia could be exceedingly useful, but to date no effective immunotherapy targeting this pathogen has been successfully developed for routine use in humans. Evaluations using animals and limited human trials of vaccines and their associated immune effectors against different P. aeruginosa antigens have suggested that antibody to the conserved surface polysaccharide alginate, as well as the flagellar proteins, often give high levels of protection. However, alginate itself does not elicit protective antibody in humans, and flagellar vaccines containing the two predominant serotypes of this antigen may not provide sufficient coverage against variant flagellar types. To evaluate if combining these antigens in a conjugate vaccine would be potentially efficacious, we conjugated polymannuronic acid (PMA), containing the blocks of mannuronic acid conserved in all P. aeruginosa alginates, to type a flagellin (FLA) and evaluated immunogenicity, opsonic killing activity, and passive protective efficacy in mice. The PMA-FLA conjugate was highly immunogenic in mice and rabbits and elicited opsonic antibodies against mucoid but not nonmucoid P. aeruginosa, but nonetheless rabbit antibody to PMA-FLA showed evidence of protective efficacy against both types of this organism in a mouse lung infection model. Importantly, the PMA-FLA conjugate vaccine did not elicit antibodies that neutralized the Toll-like receptor 5 (TLR5)-activating activity of flagellin, an important part of innate immunity to flagellated microbial pathogens. Conjugation of PMA to FLA appears to be a promising path for developing a broadly protective vaccine against P. aeruginosa.

Evaluation of flagella and flagellin of Pseudomonas aeruginosa as vaccines.

Pseudomonas aeruginosa is a serious pathogen in hospitalized, immunocompromised, and cystic fibrosis (CF) patients. P. aeruginosa is motile via a single polar flagellum made of polymerized flagellin proteins differentiated into two major serotypes: a and b. Antibodies to flagella delay onset of infection in CF patients, but whether immunity to polymeric flagella and that to monomeric flagellin are comparable has not been addressed, nor has the question of whether such antibodies might negatively impact Toll-like receptor 5 (TLR5) activation, an important component of innate immunity to P. aeruginosa. We compared immunization with flagella and that with flagellin for in vitro effects on motility, opsonic killing, and protective efficacy using a mouse pneumonia model. Antibodies to flagella were superior to antibodies to flagellin at inhibiting motility, promoting opsonic killing, and mediating protection against P. aeruginosa pneumonia in mice. Protection against the flagellar type strains PAK and PA01 was maximal, but it was only marginal against motile clinical isolates from flagellum-immunized CF patients who nonetheless became colonized with P. aeruginosa. Purified flagellin was a more potent activator of TLR5 than were flagella and also elicited higher TLR5-neutralizing antibodies than did immunization with flagella. Antibody to type a but not type b flagella or flagellin inhibited TLR5 activation by whole bacterial cells. Overall, intact flagella appear to be superior for generating immunity to P. aeruginosa, and flagellin monomers might induce antibodies capable of neutralizing innate immunity due to TLR5 activation, but solid immunity to P. aeruginosa based on flagellar antigens may require additional components beyond type a and type b proteins from prototype strains.

Airway epithelial control of Pseudomonas aeruginosa infection in cystic fibrosis.

Defective expression or function of the cystic fibrosis transmembrane conductance regulator (CFTR) underlies the hypersusceptibility of cystic fibrosis (CF) patients to chronic airway infections, particularly with Pseudomonas aeruginosa. CFTR is involved in the specific recognition of P. aeruginosa, thereby contributing to effective innate immunity and proper hydration of the airway surface layer (ASL). In CF, the airway epithelium fails to initiate an appropriate innate immune response, allowing the microbe to bind to mucus plugs that are then not properly cleared because of the dehydrated ASL. Recent studies have identified numerous CFTR-dependent factors that are recruited to the epithelial plasma membrane in response to infection and that are needed for bacterial clearance, a process that is defective in CF patients hypersusceptible to infection with this organism.