Association Between Environmental Factors and Toxigenic Clostridioides difficile Carriage at Hospital Admission.

Importance: Clostridioides difficile infection is the most frequent health care-associated infection in the United States. However, exposure to this organism might occur outside the health care setting. Objective: To examine whether exposure to environmental factors, such as livestock farms, is associated with a higher probability of being colonized with C difficile at hospital admission. Design, Setting, and Participants: This retrospective cohort study was conducted from May 1, 2017, to June 30, 2018, at a teaching-affiliated hospital in Milwaukee, Wisconsin. All consecutive patients underwent C difficile screening using a nucleic acid amplification test at hospital admission. Data analyses were performed from July 2018 to October 2019. Exposures: The distances from patient residence to the nearest livestock farms, meat processing plants, raw materials services, and sewage facilities were measured in addition to risk factors previously evaluated in other studies. Main Outcomes and Measures: The main outcome was a positive result on C difficile screening tests performed within 72 hours of hospital admission. Results: A total of 3043 patients admitted to the hospital were included in the final analysis. Of those, 1564 (51.4%) were women and 2074 (68.9%) were white, with a mean (SD) age of 62.0 (15.9) years; 978 patients (32.1%) were admitted to hematology-oncology units. At first admission, 318 patients (10.4%) were detected through testing as C difficile carriers. Multivariable logistic regression analyses were performed on a stratified sample of patients based on hematology-oncology admission status. These analyses indicated that although patients admitted to hematology-oncology units were 35% more likely to be colonized with C difficile, no significant association existed between their sociodemographic and economic characteristics or health care and environmental exposures and the likelihood of a positive C difficile test result. In contrast, among patients admitted to non-hematology-oncology units, comorbidities increased the likelihood for colonization by more than 4 times; women had 60% greater colonization than men, and a history of recent hospitalization (ie, within the preceding 6 months) increased the likelihood of colonization by 70%. Residential proximity to livestock farms were all significantly associated with a higher likelihood of a positive C difficile test result. Residential proximity to livestock farms more than doubled the probability of C difficile colonization in patients admitted to non-hematology-oncology units. Conclusions and Relevance: A shorter distance between residence and livestock farms was associated with C difficile colonization. Knowledge of the epidemiology of C difficile in the community surrounding the hospital is important, as it has potential implications for the incidence of hospital-onset C difficile infection.

Clinical Evaluation and Cost Analysis of Great Basin Shiga Toxin Direct Molecular Assay for Detection of Shiga Toxin-Producing Escherichia coli in Diarrheal Stool Specimens.

The Shiga Toxin Direct molecular assay (ST Direct) relies on nucleic acid amplification and solid array-based amplicon detection to identify Shiga toxin-producing Escherichia coli (STEC) in preserved stool specimens. Genes encoding Shiga toxin (stx1 and stx2), as well as the E. coli serotype O:157-specific marker rfbE, are simultaneously detected within 2 h. ST Direct was evaluated using 1,084 prospectively collected preserved stool specimens across five clinical centers. An additional 55 retrospectively collected, frozen specimens were included to increase the number of positive specimens evaluated. Results were compared to results from routine culture and an enzyme immunoassay (EIA) specific for the recovery and identification of STEC. ST Direct was found to be 93.2% sensitive and 99.3% specific for detection of stx1 and stx2 and 95.7% sensitive and 99.3% specific for detection of E. coli serotype O:157. All specimens with false-positive results were found to contain stx1 or stx2 or were found to be positive for serotype O:157 when analyzed using alternative molecular methods. All 4 false-negative stx1 or stx2 results were reported for frozen, retrospectively tested specimens. In all cases, the specimens tested positive for stx by an alternative FDA-cleared nucleic acid amplification test (NAAT) but were negative for stx1 and stx2 following nucleic acid sequence analysis. Based on these data, culture and EIA-based methods for detection of STEC are only 33% sensitive compared to molecular tests. A retrospective cost analysis demonstrated 59% of the cost of routine stool culture to be attributable to the identification of STEC. Taken together, these data suggest that ST Direct may provide a cost-effective, rapid molecular alternative to routine culture for the identification of STEC in preserved stool specimens.

Automatic Digital Analysis of Chromogenic Media for Vancomycin-Resistant-Enterococcus Screens Using Copan WASPLab.

Vancomycin-resistant enterococci (VRE) are an important cause of health care-acquired infections (HAIs). Studies have shown that active surveillance of high-risk patients for VRE colonization can aid in reducing HAIs; however, these screens generate a significant cost to the laboratory and health care system. Digital imaging capable of differentiating negative and "nonnegative" chromogenic agar can reduce the labor cost of these screens and potentially improve patient care. In this study, we evaluated the performance of the WASPLab Chromogenic Detection Module (CDM) (Copan, Brescia, Italy) software to analyze VRE chromogenic agar and compared the results to technologist plate reading. Specimens collected at 3 laboratories were cultured using the WASPLab CDM and plated to each site's standard-of-care chromogenic media, which included Colorex VRE (BioMed Diagnostics, White City, OR) or Oxoid VRE (Oxoid, Basingstoke, United Kingdom). Digital images were scored using the CDM software after 24 or 40 h of growth, and all manual reading was performed using digital images on a high-definition (HD) monitor. In total, 104,730 specimens were enrolled and automation agreed with manual analysis for 90.1% of all specimens tested, with sensitivity and specificity of 100% and 89.5%, respectively. Automation results were discordant for 10,348 specimens, and all discordant images were reviewed by a laboratory supervisor or director. After a second review, 499 specimens were identified as representing missed positive cultures falsely called negative by the technologist, 1,616 were identified as containing borderline color results (negative result but with no package insert color visible), and 8,234 specimens were identified as containing colorimetric pigmentation due to residual matrix from the specimen or yeast (Candida). Overall, the CDM was accurate at identifying negative VRE plates, which comprised 84% (87,973) of the specimens in this study.

Resistance Mechanisms, Epidemiology, and Approaches to Screening for Vancomycin-Resistant Enterococcus in the Health Care Setting.

Infections attributable to vancomycin-resistant Enterococcus (VRE) strains have become increasingly prevalent over the past decade. Prompt identification of colonized patients combined with effective multifaceted infection control practices can reduce the transmission of VRE and aid in the prevention of hospital-acquired infections (HAIs). Increasingly, the clinical microbiology laboratory is being asked to support infection control efforts through the early identification of potential patient or environmental reservoirs. This review discusses the factors that contribute to the rise of VRE as an important health care-associated pathogen, the utility of laboratory screening and various infection control strategies, and the available laboratory methods to identify VRE in clinical specimens.

Is Staphylococcal Screening and Suppression an Effective Interventional Strategy for Reduction of Surgical Site Infection?

BACKGROUND: Staphylococcus aureus has been recognized as a major microbial pathogen for over 100 y, having the capacity to produce a variety of suppurative and toxigenic disease processes. Many of these infections are life-threatening, with particularly enhanced virulence in hospitalized patients with selective risk factors. Strains of methicillin-resistant Staphylococcus aureus (MRSA) have rapidly spread throughout the healthcare environment such that approximately 20% of S. aureus isolates recovered from surgical site infections are methicillin-resistant, (although this is now reducing following national screening and suppression programs and high impact interventions). METHODS: Widespread nasal screening to identify MRSA colonization in surgical patients prior to admission are controversial, but selective, evidence-based studies have documented a reduction of surgical site infection (SSI) after screening and suppression. RESULTS: Culture methods used to identify MRSA colonization involve selective, differential, or chromogenic media. These methods are the least expensive, but turnaround time is 24-48 h. Although real-time polymerase chain reaction (RT-PCR) technology provides rapid turnaround (1-2 h) with exceptional testing accuracy, the costs can range from three to 10 times more than conventional culture methodology. Topical mupirocin, with or without pre-operative chlorhexidine showers or skin wipes, is the current "gold-standard" for nasal decolonization, but inappropriate use of mupirocin is associated with increasing staphylococcal resistance. CONCLUSIONS: Selection of an effective active universal or targeted surveillance strategy should be based upon the relative risk of MSSA or MRSA surgical site infection in patients undergoing orthopedic or cardiothoracic device related surgical procedures.

Detection of Group A Streptococcus in Pharyngeal Swab Specimens by Use of the AmpliVue GAS Isothermal Helicase-Dependent Amplification Assay.

We evaluated the clinical performance (sensitivity and specificity) of the AmpliVue group A Streptococcus (GAS) isothermal helicase-dependent amplification assay using 1,192 pharyngeal swab specimens. AmpliVue GAS assay results were compared to the results of routine throat cultures on selective streptococcal blood agar plates. The sensitivity and specificity of the AmpliVue GAS assay were 98.3% (95% confidence interval [CI], 95 to 100%) and 93.2% (95% CI, 91 to 95%), respectively.

Francisella tularensis live vaccine strain folate metabolism and pseudouridine synthase gene mutants modulate macrophage caspase-1 activation.

Francisella tularensis is a Gram-negative bacterium and the causative agent of the disease tularemia. Escape of F. tularensis from the phagosome into the cytosol of the macrophage triggers the activation of the AIM2 inflammasome through a mechanism that is not well understood. Activation of the AIM2 inflammasome results in autocatalytic cleavage of caspase-1, resulting in the processing and secretion of interleukin-1ß (IL-1ß) and IL-18, which play a crucial role in innate immune responses to F. tularensis. We have identified the 5-formyltetrahydrofolate cycloligase gene (FTL_0724) as being important for F. tularensis live vaccine strain (LVS) virulence. Infection of mice in vivo with a F. tularensis LVS FTL_0724 mutant resulted in diminished mortality compared to infection of mice with wild-type LVS. The FTL_0724 mutant also induced increased inflammasome-dependent IL-1ß and IL-18 secretion and cytotoxicity in macrophages in vitro. In contrast, infection of macrophages with a F. tularensis LVS rluD pseudouridine synthase (FTL_0699) mutant resulted in diminished IL-1ß and IL-18 secretion from macrophages in vitro compared to infection of macrophages with wild-type LVS. In addition, the FTL_0699 mutant was not attenuated in vivo. These findings further illustrate that F. tularensis LVS possesses numerous genes that influence its ability to activate the inflammasome, which is a key host strategy to control infection with this pathogen in vivo.

Cutting edge: mutation of Francisella tularensis mviN leads to increased macrophage absent in melanoma 2 inflammasome activation and a loss of virulence.

The mechanisms by which the intracellular pathogen Francisella tularensis evades innate immunity are not well defined. We have identified a gene with homology to Escherichia coli mviN, a putative lipid II flippase, which F. tularensis uses to evade activation of innate immune pathways. Infection of mice with a F. tularensis mviN mutant resulted in improved survival and decreased bacterial burdens compared to infection with wild-type F. tularensis. The mviN mutant also induced increased absent in melanoma 2 inflammasome-dependent IL-1beta secretion and cytotoxicity in macrophages. The compromised in vivo virulence of the mviN mutant depended upon inflammasome activation, as caspase 1- and apoptosis-associated speck-like protein containing a caspase recruitment domain-deficient mice did not exhibit preferential survival following infection. This study demonstrates that mviN limits F. tularensis-induced absent in melanoma 2 inflammasome activation, which is critical for its virulence in vivo.

Identification of migR, a regulatory element of the Francisella tularensis live vaccine strain iglABCD virulence operon required for normal replication and trafficking in macrophages.

Francisella tularensis, the etiological agent of tularemia, is capable of infecting a wide range of animals and causes a severe, lethal disease in humans. The pathogen evades killing by cells of the innate immune system utilizing genes encoding a pathogenicity island, including iglABCD, and instead utilizes these cells as a niche for replication and dissemination to other organs within the host. Regulators of the igl genes (e.g., MglA, SspA, FevR and PmrA) have been identified, but environmental stimuli and mechanisms of regulation are as yet unknown and are likely to involve additional gene products. In this work, we more closely examine the roles that environmental iron and the ferric uptake repressor protein (Fur) play in the regulation of the iglABCD operon. We also used a genetic approach to identify and characterize a new regulator of the igl operon, designated migR (macrophage intracellular growth regulator; FTL_1542). Quantitative real-time reverse transcription-PCR in a site-directed migR mutant confirmed the reduction in the number of iglC transcripts in this strain and also demonstrated reduced expression of fevR. Comparison of the migR and fevR mutants in monocyte-derived macrophages (MDMs) and epithelial cell lines revealed a reduced ability for each mutant to grow in MDMs, yet only the fevR mutant exhibited impaired replication in epithelial cell lines. Confocal analysis of infected MDMs revealed that although neither mutant reached the MDM cytosol, the fevR mutant was trapped in lamp-1-positive phagosomes, whereas the migR mutant resided in mature phagolysosomes enriched with both lamp-1 and cathepsin D. Disruption of migR and fevR also impaired the ability of F. tularensis to prevent neutrophil oxidant production. Thus, we have identified migR, a gene that regulates expression of the iglABCD operon and is essential for bacterial growth in MDMs and also contributes to the blockade of neutrophil NADPH oxidase activity.

Francisella tularensis genes required for inhibition of the neutrophil respiratory burst and intramacrophage growth identified by random transposon mutagenesis of strain LVS.

Francisella tularensis is a facultative intracellular pathogen and the causative agent of tularemia. We have shown that F. tularensis subspecies holarctica strain LVS prevents NADPH oxidase assembly and activation in human neutrophils, but how this is achieved is unclear. Herein, we used random transposon mutagenesis to identify LVS genes that affect neutrophil activation. Our initial screen identified carA, carB, and pyrB, which encode the small and large subunits of carbamoylphosphate synthase and aspartate carbamoyl transferase, respectively. These strains are uracil auxotrophs, and their growth was attenuated on cysteine heart agar augmented with sheep blood (CHAB) or in modified Mueller-Hinton broth. Phagocytosis of the uracil auxotrophic mutants triggered a respiratory burst in neutrophils, and ingested bacteria were killed and fragmented in phagosomes that contained superoxide. Conversely, phagocytosis did not trigger a respiratory burst in blood monocytes or monocyte-derived macrophages (MDM), and phagosomes containing wild-type or mutant bacteria lacked NADPH oxidase subunits. Nevertheless, the viability of mutant bacteria declined in MDM, and ultrastructural analysis revealed that phagosome egress was significantly inhibited despite synthesis of the virulence factor IglC. Other aspects of infection, such as interleukin-1beta (IL-1beta) and IL-8 secretion, were unaffected. The cultivation of carA, carB, or pyrB on uracil-supplemented CHAB was sufficient to prevent neutrophil activation and intramacrophage killing and supported escape from MDM phagosomes, but intracellular growth was not restored unless uracil was added to the tissue culture medium. Finally, all mutants tested grew normally in both HepG2 and J774A.1 cells. Collectively, our data demonstrate that uracil auxotrophy has cell type-specific effects on the fate of Francisella bacteria.