Exoenzyme Y Contributes to End-Organ Dysfunction Caused by Pseudomonas aeruginosa Pneumonia in Critically Ill Patients: An Exploratory Study.

Pseudomonas aeruginosa is an opportunistic pathogen that causes pneumonia in immunocompromised and intensive care unit (ICU) patients. During host infection, P. aeruginosa upregulates the type III secretion system (T3SS), which is used to intoxicate host cells with exoenzyme (Exo) virulence factors. Of the four known Exo virulence factors (U, S, T and Y), ExoU has been shown in prior studies to associate with high mortality rates. Preclinical studies have shown that ExoY is an important edema factor in lung infection caused by P. aeruginosa, although its importance in clinical isolates of P. aeruginosa is unknown. We hypothesized that expression of ExoY would be highly prevalent in clinical isolates and would significantly contribute to patient morbidity secondary to P. aeruginosa pneumonia. A single-center, prospective observational study was conducted at the University of Alabama at Birmingham Hospital. Mechanically ventilated ICU patients with a bronchoalveolar lavage fluid culture positive for P. aeruginosa were included. Enrolled patients were followed from ICU admission to discharge and clinical P. aeruginosa isolates were genotyped for the presence of exoenzyme genes. Ninety-nine patients were enrolled in the study. ExoY was present in 93% of P. aeruginosa clinical isolates. Moreover, ExoY alone (ExoY+/ExoU-) was present in 75% of P. aeruginosa isolates, compared to 2% ExoU alone (ExoY-/ExoU+). We found that bacteria isolated from human samples expressed active ExoY and ExoU, and the presence of ExoY in clinical isolates was associated with end-organ dysfunction. This is the first study we are aware of that demonstrates that ExoY is important in clinical outcomes secondary to nosocomial pneumonia.

Virulent Pseudomonas aeruginosa infection converts antimicrobial amyloids into cytotoxic prions.

Pseudomonas aeruginosa infection elicits the production of cytotoxic amyloids from lung endothelium, yet molecular mechanisms of host-pathogen interaction that underlie the amyloid production are not well understood. We examined the importance of type III secretion system (T3SS) effectors in the production of cytotoxic amyloids. P aeruginosa possessing a functional T3SS and effectors induced the production and release of cytotoxic amyloids from lung endothelium, including beta amyloid, and tau. T3SS effector intoxication was sufficient to generate cytotoxic amyloid release, yet intoxication with exoenzyme Y (ExoY) alone or together with exoenzymes S and T (ExoS/T/Y) generated the most virulent amyloids. Infection with lab and clinical strains engendered cytotoxic amyloids that were capable of being propagated in endothelial cell culture and passed to naïve cells, indicative of a prion strain. Conversely, T3SS-incompetent P aeruginosa infection produced non-cytotoxic amyloids with antimicrobial properties. These findings provide evidence that (1) endothelial intoxication with ExoY is sufficient to elicit self-propagating amyloid cytotoxins during infection, (2) pulmonary endothelium contributes to innate immunity by generating antimicrobial amyloids in response to bacterial infection, and (3) ExoY contributes to the virulence arsenal of P aeruginosa through the subversion of endothelial amyloid host-defense to promote a lung endothelial-derived cytotoxic proteinopathy.

Factors Associated With Ventriculoperitoneal Shunt Placement in Patients With Cryptococcal Meningitis.

OBJECTIVE: Increased intracranial pressure (ICP) is an important complication of cryptococcal meningitis (CM) and impacts morbidity and mortality. Factors associated with permanent ventriculoperitoneal (VP) shunt placement are poorly characterized. METHOD: We conducted a retrospective cohort study of patients with CM at the University of Alabama at Birmingham from 1996 through 2015. Characteristics of patients at time of CM diagnosis who did and did not receive a VP shunt were compared with use of the 2-group chi-square test or Fisher exact test for categorical variables and the 2-group t test for continuous variables. Stepwise logistic regression analysis was used to determine predictors of shunt placement. RESULTS: Of 422 patients with cryptococcosis, 257 (60.9%) had CM. Mean age was 47.7 years, 71.6% were male, and 44.4% were African American. The most common underlying conditions were HIV (42.4%), solid organ transplantation (29.6%), and corticosteroid use (34.2%). Forty-four (17.1%) received a VP shunt a median of 17 days (range, 1-320 days) post-diagnosis. By multivariable analysis, baseline opening pressure >30 cm H2O (OR, 9.4; 95% CI, 3.0, 28.8; P < .0001), being a normal host (OR, 6.3; 95% CI, 1.5, 26.1; P = .011) and hydrocephalus (OR, 4.9, 95% CI, 1.3, 17.9); P = .017) were associated with increased odds of shunting (Table 2). In contrast, age (OR, 0.96; 95% CI, 0.92, 0.99; P = .037) and male gender (OR, 0.18; 95% CI, 0.06, 0.55; P = .023) were associated with decreased odds of shunting. CONCLUSIONS: Identification of factors at time of CM diagnosis associated with need for permanent VP shunt placement may allow for earlier, more aggressive treatment and potentially improve outcomes associated with increased ICP from cryptococcal meningitis.

Infection-induced endothelial amyloids impair memory.

Patients with nosocomial pneumonia exhibit elevated levels of neurotoxic amyloid and tau proteins in the cerebrospinal fluid (CSF). In vitro studies indicate that pulmonary endothelium infected with clinical isolates of either Pseudomonas aeruginosa, Klebsiella pneumoniae, or Staphylococcus aureus produces and releases cytotoxic amyloid and tau proteins. However, the effects of the pulmonary endothelium-derived amyloid and tau proteins on brain function have not been elucidated. Here, we show that P. aeruginosa infection elicits accumulation of detergent insoluble tau protein in the mouse brain and inhibits synaptic plasticity. Mice receiving endothelium-derived amyloid and tau proteins via intracerebroventricular injection exhibit a learning and memory deficit in object recognition, fear conditioning, and Morris water maze studies. We compared endothelial supernatants obtained after the endothelia were infected with P. aeruginosa possessing an intact [P. aeruginosa isolated from patient 103 (PA103) supernatant] or defective [mutant strain of P. aeruginosa lacking a functional type 3 secretion system needle tip complex (ΔPcrV) supernatant] type 3 secretion system. Whereas the PA103 supernatant impaired working memory, the ΔPcrV supernatant had no effect. Immunodepleting amyloid or tau proteins from the PA103 supernatant with the A11 or T22 antibodies, respectively, overtly rescued working memory. Recordings from hippocampal slices treated with endothelial supernatants or CSF from patients with or without nosocomial pneumonia indicated that endothelium-derived neurotoxins disrupted the postsynaptic synaptic response. Taken together, these results establish a plausible mechanism for the neurologic sequelae consequent to nosocomial bacterial pneumonia.-Balczon, R., Pittet, J.-F., Wagener, B. M., Moser, S. A., Voth, S., Vorhees, C. V., Williams, M. T., Bridges, J. P., Alvarez, D. F., Koloteva, A., Xu, Y., Zha, X.-M., Audia, J. P., Stevens, T., Lin, M. T. Infection-induced endothelial amyloids impair memory.

Streptococcus mutans serotypes and collagen-binding proteins Cnm/Cbm in children with caries analysed by PCR.

Streptococcus mutans, a primary bacterium associated with dental caries, has four known clinical serotypes (c, e, fand k). Certain serotypes, the presence of multiple serotypes and strains with collagen-binding proteins (CBP, Cnm and Cbm) have been linked with systemic disease. Evaluation of S mutans serotype distribution and caries association is needed in the United States. The purpose of this study was to evaluate the prevalence of S mutans serotypes from two cohorts of African-American children in rural Alabama using three sample types (saliva, plaque and individual S mutans isolates) by PCR detection for association with caries. Detection of CBP was also performed by PCR. In total, 129 children were evaluated and overall prevalence of serotypes were: serotype c(98%), e(26%), f(7%) and k(52%). Serotype c was statistically associated with higher caries scores in older children (P < 0.001) and serotype k was statistically more likely in females (P = 0.004). Fourteen per cent of children had CBP. Thirteen S mutans isolates from five children tested positive for both CBP. This study is the first to report on the prevalence of S mutans serotypes in a US population using the PCR-based approach. The frequency of serotype k in this study is the highest reported in any population, illustrating the need for further study to determine the prevalence of this clinically relevant serotype in the US. This is the first study to report S mutans isolates with both Cnm and Cbm in the same strain, and further analysis is needed to determine the clinical significance of these strains.

Improvement of gram-negative susceptibility to fluoroquinolones after implementation of a pre-authorization policy for fluoroquinolone use: A decade-long experience.

OBJECTIVE: Due to concerns over increasing fluoroquinolone (FQ) resistance among gram-negative organisms, our stewardship program implemented a preauthorization use policy. The goal of this study was to assess the relationship between hospital FQ use and antibiotic resistance. DESIGN: Retrospective cohort. SETTING: Large academic medical center. METHODS: We performed a retrospective analysis of FQ susceptibility of hospital isolates for 5 common gram-negative bacteria: Acinetobacter spp., Enterobacter cloacae, Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa. Primary endpoint was the change of FQ susceptibility. A Poisson regression model was used to calculate the rate of change between the preintervention period (1998-2005) and the postimplementation period (2006-2016). RESULTS: Large rates of decline of FQ susceptibility began in 1998, particularly among P. aeruginosa, Acinetobacter spp., and E. cloacae. Our FQ restriction policy improved FQ use from 173 days of therapy (DOT) per 1,000 patient days to <60 DOT per 1,000 patient days. Fluoroquinolone susceptibility increased for Acinetobacter spp. (rate ratio [RR], 1.038; 95% confidence interval [CI], 1.005-1.072), E. cloacae (RR, 1.028; 95% CI, 1.013-1.044), and P. aeruginosa (RR, 1.013; 95% CI, 1.006-1.020). No significant change in susceptibility was detected for K. pneumoniae (RR, 1.002; 95% CI, 0.996-1.008), and the susceptibility for E. coli continued to decline, although the decline was not as steep (RR, 0.981; 95% CI, 0.975-0.987). CONCLUSIONS: A stewardship-driven FQ restriction program stopped overall declining FQ susceptibility rates for all species except E. coli. For 3 species (ie, Acinetobacter spp, E. cloacae, and P. aeruginosa), susceptibility rates improved after implementation, and this improvement has been sustained over a 10-year period.

Echinocandin resistance among Candida isolates at an academic medical centre 2005-15: analysis of trends and outcomes.

Objectives: To identify the frequency of micafungin resistance among clinically significant isolates of Candida stored at our institution from 2005 to 2015. Chart review of patients with resistant isolates then informed the clinical setting and outcomes associated with these infections. Methods: Clinical Candida isolates had been stored at -80°C in Brucella broth with 20% glycerol from 2005. Isolates were tested using broth microdilution to determine micafungin MICs. All Candida glabrata isolates and all isolates demonstrating decreased susceptibility to micafungin were screened for FKS mutations using a Luminex assay. Results: In total, 3876 Candida isolates were tested for micafungin resistance, including 832 C. glabrata isolates. Of those, 33 isolates from 31 patients were found to have either decreased susceptibility to micafungin and/or an FKS mutation. C. glabrata accounted for the majority of these isolates. While bloodstream infections were found to have a very high mortality rate, isolates from other sites were uncommonly associated with 30-day mortality. Overall resistance rates were very low. Conclusions: Echinocandin resistance in C. glabrata has been increasingly reported but rates at our institution remain very low. We hypothesize that a focus on antifungal stewardship may have led to these observations. Knowledge of local resistance patterns is key to appropriate empirical treatment strategies.

Transmission patterns of Streptococcus mutans demonstrated by a combined rep-PCR and MLST approach.

OBJECTIVE: Clinical typing methods of the oral pathogen Streptococcus mutans with molecular analysis can be very specific, but expensive. Repetitive extragenic palindromic PCR (rep-PCR) is a relatively inexpensive pre-screening alternative for isolate selection for additional analyses. This study evaluated the prediction accuracy of using rep-PCR to identify S. mutans multilocus sequence typing (MLST) sequence types (ST) among children and their family members. Potential S. mutans strain sources were evaluated for evidence of transmission. MATERIAL AND METHODS: Ten dendrograms (rep-PCR), with 20 isolates each of the 10 most common S. mutans genotypes, were generated from different subjects. Using a cut-off of 98% similarity, 7-11 isolates of each genotype were selected for MLST analysis to determine ST match/no-match. RESULTS: Overall, rep-PCR was 75% effective at determining MLST ST match/no-match and 90% effective when applied to related individuals. Most genotypes were further differentiated by MLST. MLST ST diversity was greatest for one genotype (genotype 12, G12) and evidence of transmission among children and their family members was identified by rep-PCR and MLST. Younger children (6 months to 4 years old) shared ST with their mothers but 50% of older children (5-9 years old) had ST not identified in their mother. Six ST were shared between different families and probable source members were identified. CONCLUSION: This study confirms that rep-PCR offers an affordable option to predict diverse isolates for downstream applications. CLINICAL RELEVANCE: Using a combined rep-PCR and MLST approach, it is possible to track probable transmission and strain sources for S. mutans genotypes.

Association Between Early Childhood Caries and Colonization with Streptococcus mutans Genotypes From Mothers.

PURPOSE: The purpose of this study was to evaluate Streptococcus mutans genotypes (GT) between mother and child (M-C) in a high caries risk cohort to explore the association with early childhood caries (ECC). METHODS: Sixty-nine infants (each approximately one year old) had periodic oral examinations (dmfs) and microbial samples collected from dental plaque, saliva, and other oral surfaces. Their mothers had an examination and plaque collected. S mutans isolates were genotyped using repetitive extragenic palindromic-PCR (rep-PCR). Statistical analyses were conducted for associations of S mutans in M-C dyads with caries outcomes. RESULTS: Twenty-seven S mutans genotypes (GT) from 3,414 isolates were identified. M-C were categorized as GT match (n equals 40) or no-match (n equals 29). When modeling the severity of ECC at 36 months (approximately four years old), the estimated dmfs in the match group was 2.61 times that of the no-match group (P=.014). CONCLUSIONS: Colonization of children with Streptococcus mutans genotypes that matched with mothers was shown to be highly associated with early childhood caries. Although the data suggest vertical transmission of S mutans in 40 of 69 children that shared GT with their mother, it is possible that other individuals transmitted the S mutans. Nonetheless, these findings support the importance of the mother's oral microbial status as a contributing influence to their children's oral health.

Pseudo-outbreak of Brevundimonas diminuta Attributed to Contamination of Culture Medium Supplement.

We report an epidemiological investigation of a cluster of Brevundimonas diminuta isolates cultured from sterile sites. Inoculation of supplement medium yielded growth of B. diminuta. Molecular typing indicated likely contamination of the lot. No B. diminuta was further isolated after replacement of the supplement with a new lot number. Infect Control Hosp Epidemiol 2017;38:598-601.

Pseudomonas aeruginosa infection liberates transmissible, cytotoxic prion amyloids.

Patients who recover from pneumonia subsequently have elevated rates of death after hospital discharge as a result of secondary organ damage, the causes of which are unknown. We used the bacterium Pseudomonas aeruginosa, a common cause of hospital-acquired pneumonia, as a model for investigating this phenomenon. We show that infection of pulmonary endothelial cells by P. aeruginosa induces production and release of a cytotoxic amyloid molecule with prion characteristics, including resistance to various nucleases and proteases. This cytotoxin was self-propagating, was neutralized by anti-amyloid Abs, and induced death of endothelial cells and neurons. Moreover, the cytotoxin induced edema in isolated lungs. Endothelial cells and isolated lungs were protected from cytotoxin-induced death by stimulation of signal transduction pathways that are linked to prion protein. Analysis of bronchoalveolar lavage fluid collected from human patients with P. aeruginosa pneumonia demonstrated cytotoxic activity, and lavage fluid contained amyloid molecules, including oligomeric τ and Aß. Demonstration of long-lived cytotoxic agents after Pseudomonas infection may establish a molecular link to the high rates of death as a result of end-organ damage in the months after recovery from pneumonia, and modulation of signal transduction pathways that have been linked to prion protein may provide a mechanism for intervention.-Balczon, R., Morrow, K. A., Zhou, C., Edmonds, B., Alexeyev, M., Pittet, J.-F., Wagener, B. M., Moser, S. A., Leavesley, S., Zha, X., Frank, D. W., Stevens, T. Pseudomonas aeruginosa infection liberates transmissible, cytotoxic prion amyloids.

Genetic Diversity and Evidence for Transmission of Streptococcus mutans by DiversiLab rep-PCR.

This two-part study investigated the genetic diversity and transmission of Streptococcus mutans using the DiversiLab repetitive extragenic palindromic PCR (rep-PCR) approach. For children with S. mutans and participating household members, analysis for evidence of unrelated child-to-child as well as intra-familial transmission was evaluated based on commonality of genotypes. A total of 169 index children and 425 household family members from Uniontown, Alabama were evaluated for genetic diversity using rep-PCR. Thirty-four unique rep-PCR genotypes were observed for 13,906 S. mutans isolates. For transmission, 117 child and household isolates were evaluated for shared genotype (by child and by genotype cases, multiple matches possible for each child). Overall, children had 1-9 genotypes and those with multiple genotypes were 2.3 times more likely to have caries experience (decayed, missing and filled teeth/surfaces>0). Only 28% of children shared all genotypes within the household, while 72% had at least 1 genotype not shared with anyone in the household. Children had genotype(s) not shared with any household members in 157 cases. In 158 cases children and household members shared a genotype in which 55% (87/158 cases) were shared with more than one family member. Children most frequently shared genotypes with their mothers (54%; 85/158), siblings (46%; 72/158) and cousins (23%; 37/158). A reference library for S. mutans for epidemiological surveillance using the DiversiLab rep-PCR approach is detailed. The genetic diversity of S. mutans in this population demonstrated frequent commonality of genotypes. Evidence for both child-to-child and intra-familial transmission of S. mutans was observed by rep-PCR.

Assessment of clonality and serotypes of Streptococcus mutans among children by multilocus sequence typing.

Studies using multilocus sequence typing (MLST) have demonstrated that Streptococcus mutans isolates are genetically diverse. Our laboratory previously demonstrated clonality of S. mutans using MLST but could not discount the possibility of sampling bias. In this study, the clonality of randomly selected S. mutans plaque isolates from African-American children was examined using MLST. Serotype and the presence of collagen-binding proteins (CBPs) encoded by cnm/cbm were also assessed. One-hundred S. mutans isolates were randomly selected for MLST analysis. Sequence analysis was performed and phylogenetic trees were generated using start2 and mega. Thirty-four sequence types were identified, of which 27 were unique to this population. Seventy-five per cent of the isolates clustered into 16 clonal groups. The serotypes observed were c (n = 84), e (n = 3), and k (n = 11). The prevalence of S. mutans isolates of serotype k was notably high, at 17.5%. All isolates were cnm/cbm negative. The clonality of S. mutans demonstrated in this study illustrates the importance of localized population studies and are consistent with transmission. The prevalence of serotype k, a recently proposed systemic pathogen, observed in this study, is higher than reported in most populations and is the first report of S. mutans serotype k in a United States population.

Assessment of two multilocus sequence typing (MLST) schemes available for Streptococcus mutans.

OBJECTIVE: Two multilocus sequencing typing (MLST) schemes are currently available for Streptococcus mutans. The first, introduced by Nakano et al. in 2007, consists of 8 conserved housekeeping genes. The second, introduced in 2010 by Do et al., includes 6 housekeeping genes and 2 putative virulence genes. The purpose of the current study was to compare the two MLST schemes for use in validating repetitive extragenic palindromic polymerase chain reaction (rep-PCR) genotypes. DESIGN: Thirty-three S. mutans isolates, representing the 11 most commonly occurring rep-PCR genotype groups, were selected for MLST. MLST was performed with SYBR Green™ PCR with published primers for both MLST schemes. Amplicons were purified, sequenced, and data checked against the www.PubMLST.org database for allelic and sequence type (ST) assignment. Discriminatory power, congruence, and convenience criteria were evaluated. Concatenated sequences for each scheme were analyzed using MEGA to generate phylogenetic trees using minimum evolution with bootstrap. RESULTS: No significant difference in discriminatory power was observed between the two MLST schemes for S. mutans. Clonal clusters were consistent for both schemes. Overall, MLST demonstrated marginally greater discriminatory power than rep-PCR; however all methods were found to be congruent. New alleles and ST are reported for each scheme and added to the PubMLST database. CONCLUSIONS: Clonality, supported by both methods and rep-PCR, indicates S. mutans genotypes are shared between unrelated subjects. Both Nakano and Do schemes demonstrates similar genotype discrimination for S. mutans isolates suggesting each are well designed and may be used to verify rep-PCR genotypes.

Mutans streptococci enumeration and genotype selection using different bacitracin-containing media.

The primary etiological agents associated with dental caries include the mutans streptococci (MS) comprised of Streptococcus mutans and Streptococcus sobrinus. The effective cultivation and isolation of MS are necessary for the study of MS, including their proper clinical assessment in the epidemiological study of dental caries. Several selective media have been developed for the isolation, enumeration, and characterization of MS. However, inhibition of MS may occur, reducing counts and perhaps limiting selection of some strains. The purpose of this study was to compare five culture media containing bacitracin recommended for the isolation of MS. Five commonly used bacitracin-containing media (MSB, MSKB, GTSB, TYS20B, and TYCSB) used for MS isolation were quantitatively evaluated. Standard plate counts were performed in duplicate for 2 prototype MS strains (S. mutans UA159 and S. sobrinus 6715) and for MS isolates from clinical saliva samples obtained from 16 children (approximate age 5years) to determine total plate counts, and total S. mutans counts. Selected isolates (n=249) from all five media for 5 saliva samples were further confirmed as S. mutans with real-time PCR then subsequently evaluated qualitatively with rep-PCR for genotype determination. All media resulted in variable enumeration with no significant difference in MS counts. MS prototype strains grew well on all five media; clinical isolates demonstrated more variability in counts but no overall significant differences were found. MSB demonstrated comparable ability to grow S. mutans but allowed for more non-S. mutans growth. All 5 media identified a consistent predominant genotype by rep-PCR. Recovery of minor genotypes was not inhibited by media type.

Withdrawn Biofeature: Henry D. Isenberg, Ph.D.

Withdrawn.

Characteristics of Streptococcus mutans genotypes and dental caries in children.

This longitudinal cohort study evaluated the diversity, commonality, and stability of Streptococcus mutans genotypes associated with dental caries history. Sixty-seven 5- and 6-yr-old children, considered as being at high caries risk, had plaque collected from baseline through 36 months for S. mutans isolation and genotyping using repetitive extragenic palindromic-PCR (4,392 total isolates). Decayed, missing, or filled surfaces (dmfs (primary teeth)/DMFS (secondary teeth)) for each child were recorded at baseline. At baseline, 18 distinct genotypes were found among 911 S. mutans isolates from 67 children (diversity), and 13 genotypes were shared by at least two children (commonality). The number of genotypes per individual was positively associated with the proportion of decayed surfaces (p-ds) at baseline. Twenty-four of the 39 children who were available at follow-up visits maintained a predominant genotype for the follow-up periods (stability) and this was negatively associated with the p-ds. The observed diversity, commonality, and stability of S. mutans genotypes represent a pattern of dental caries epidemiology in this high-caries-risk community, which suggests that fewer decayed surfaces are significantly associated with lower diversity and higher stability of S. mutans genotypes.

Association between vancomycin-resistant Enterococci bacteremia and ceftriaxone usage.

OBJECTIVE: Vancomycin-resistant enterococci (VRE) have become a public health concern with implications for patient mortality and costs. Hospital antibiotic usage may impact VRE incidence, but the relationship is poorly understood. Animal investigations suggest that ceftriaxone may be associated with VRE proliferation. We measured antimicrobial usage and VRE bloodstream infection (VRE-BSI) incidence to test our hypothesis that increased ceftriaxone usage would be associated with a higher incidence of VRE-BSI. DESIGN: Retrospective cohort study. SETTING: University of Alabama at Birmingham Medical Center, a 900-bed urban tertiary care hospital. PARTICIPANTS: All patients admitted during the study period contributed data. METHODS: We conducted a retrospective analysis of antimicrobial usage and VRE-BSI from 2005 to 2008 (43 months). Antimicrobial usage was quantified as days of therapy (DOTs) per 1,000 patient-days. VRE-BSI incidence was calculated as cases per 1,000 patient-days. Negative binomial regression with adjustment for correlation between consecutive observations was used to measure the association between antimicrobial usage and VRE-BSI incidence at the hospital- and care-unit levels. RESULTS: VRE-BSI incidence increased from 0.06 to 0.17 infections per 1,000 patient-days. Hospital VRE-BSI incidence was associated with prior-month ceftriaxone DOTs (incidence rate ratio, 1.38 per 10 DOTs; P = .005). After controlling for ceftriaxone, prior-month cephalosporin usage (class) was not predictive of VRE-BSI (P = .70). Similarly, prior-month usage of piperacillin-tazobactam, ceftazidime, cefepime, cefazolin, or vancomycin was not predictive of VRE-BSI when considered individually (P≥ .4 for all comparisons). The final model suggests that type of intensive care unit was related to VRE-BSI incidence. CONCLUSIONS: Ceftriaxone usage in the prior month, but not cephalosporin (class) or vancomycin usage, was related to VRE-BSI incidence. These findings suggest that an antimicrobial stewardship program that limits ceftriaxone may reduce nosocomial VRE-BSI incidence.