Assessment of the Urinary Microbiota of MSM Using Urine Culturomics Reveals a Diverse Microbial Environment.

BACKGROUND: The urinary tract is not sterile and is populated by microbial communities that influence urinary health. Men who have sex with men (MSM) are understudied yet have increased risk factors for genitourinary infections. Our objective was to interrogate the composition of MSM urinary microbiota. METHODS: Midstream urine specimens (n = 129) were collected from MSM (n = 63) and men seen for routine care (clinical cohort, n = 30). Demographics and sexual/medical history were documented. Specimens underwent culture using standard-of-care and enhanced methods designed to isolate fastidious and anaerobic microorganisms. Isolates were identified by MALDI-TOF mass spectrometry or 16S rRNA gene sequencing. RESULTS: The MSM cohort was younger (mean (SD), 35.4 (11.26) years) compared to the clinical cohort (62.7 (15.95) years). Organism recovery was significantly increased using enhanced vs standard culture for the MSM (mean of 9.1 vs 0.6 species/sample [P < 0.001]) and clinical (7.8 vs 0.9 species/sample [P < 0.001]) cohorts. The microbial composition of MSM urine specimens was dominated by Gram-positive and anaerobic microbes and clustered distinctly from that of clinical urine specimens. Composition of microbial species recovered within the same subject was dynamic between urine specimens but more similar relative to inter-individual comparisons. Principal coordinate analysis showed no correlation between urinary microbiota composition and age, recent antibiotic use, sexually transmitted infection/HIV status, or sexual practices. CONCLUSIONS: Enhanced culture recovered a large diversity of microbial species from MSM urine specimens, especially taxa typically associated with mucosal surfaces. These findings may increase understanding of urologic disease in MSM and improve diagnostic methods for detection of genitourinary infections.

The Sensor Histidine Kinase ArlS Is Necessary for Staphylococcus aureus To Activate ArlR in Response to Nutrient Availability.

Staphylococcus aureus is a versatile opportunistic pathogen whose success is driven by its ability to adapt to diverse environments and host-imposed stresses. Two-component signal transduction systems, such as ArlRS, often mediate these adaptations. Loss of ArlRS or the response regulator ArlR alone impairs the ability of S. aureus to respond to host-imposed manganese starvation and glucose limitation. As sensor histidine kinases and response regulators frequently work as pairs, it has been assumed that ArlS senses and activates ArlR in response to these stimuli. However, recent work suggests that the sensor histidine kinase GraS can also activate ArlR, calling the contribution of ArlS in responding to manganese and glucose availability into question. The results of current studies reveal that ArlS is necessary to activate ArlR in response to manganese sequestration by the host immune effector calprotectin and glucose limitation. Although the loss of ArlS does not completely eliminate ArlR activity, this response regulator is no longer responsive to manganese or glucose availability in the absence of its cognate histidine kinase. Despite the residual activity of ArlR in the absence of ArlS, ArlR phosphorylation by ArlS is required for S. aureus to resist calprotectin-imposed metal starvation. Cumulatively, these findings contribute to the understanding of S. aureus signal transduction in response to nutritional immunity and support the previous observation indicating that ArlRS is activated by a common signal derived from host-imposed manganese and glucose limitation. IMPORTANCE The ability of pathogens, including Staphylococcus aureus, to sense and adapt to diverse environments partially relies on two-component systems, such as ArlRS. Recent work revealed that the response regulator ArlR can be cross-activated by the sensor histidine kinase GraS, rendering the role of its cognate partner, ArlS, in response to manganese and glucose limitation uncertain. The results of this study reveal that ArlS is necessary for the activation of ArlR in response to calprotectin and glucose limitation. Although a low level of ArlR activity remains in the absence of ArlS, ArlS phosphotransfer to ArlR is required for S. aureus to overcome calprotectin-induced nutritional stress. Collectively, this study provides fundamental information to understand how ArlRS mediates staphylococcal adaptation during infection.

Spatiotemporal dynamics of multidrug resistant bacteria on intensive care unit surfaces.

Bacterial pathogens that infect patients also contaminate hospital surfaces. These contaminants impact hospital infection control and epidemiology, prompting quantitative examination of their transmission dynamics. Here we investigate spatiotemporal and phylogenetic relationships of multidrug resistant (MDR) bacteria on intensive care unit surfaces from two hospitals in the United States (US) and Pakistan collected over one year. MDR bacteria isolated from 3.3% and 86.7% of US and Pakistani surfaces, respectively, include common nosocomial pathogens, rare opportunistic pathogens, and novel taxa. Common nosocomial isolates are dominated by single lineages of different clones, are phenotypically MDR, and have high resistance gene burdens. Many resistance genes (e.g., blaNDM, blaOXA carbapenamases), are shared by multiple species and flanked by mobilization elements. We identify Acinetobacter baumannii and Enterococcus faecium co-association on multiple surfaces, and demonstrate these species establish synergistic biofilms in vitro. Our results highlight substantial MDR pathogen burdens in hospital built-environments, provide evidence for spatiotemporal-dependent transmission, and demonstrate potential mechanisms for multi-species surface persistence.

Clinical Effect of Expedited Pathogen Identification and Susceptibility Testing for Gram-Negative Bacteremia and Candidemia by Use of the Accelerate PhenoTM System.

BACKGROUND: Fast diagnostic tests providing earlier identification (ID) of pathogens, and antimicrobial susceptibility testing (AST) may reduce time to appropriate antimicrobial therapy (AAT), decrease mortality, and facilitate antimicrobial deescalation (ADE). Our objective was to determine the theoretical reduction in time to AAT and opportunities for ADE with Accelerate PhenoTM System (AXDX). METHODS: The prospective cohort (April 14, 2016 through June 1, 2017) was from the Barnes-Jewish Hospital, a 1250-bed academic center. Emergency department (ED) or intensive care unit (ICU) blood cultures Gram-stain positive for gram-negative bacilli (GNB) or yeast. AXDX was used in parallel with standard-of-care (SOC) diagnostics to determine differences in time to pathogen ID and AST. Theoretical opportunities for ADE from AXDX results were determined. RESULTS: In total, 429 blood cultures were screened, 153 meeting inclusion criteria: 110 on-panel GNB, 10 Candida glabrata, and 5 Candida albicans. For GNB SOC, median time from blood culture positivity to ID and AST were 28.2 and 52.1 h. Median time to ID and AST after AXDX initiation was 1.37 and 6.7 h for on-panel organisms. For on-panel Candida, time to ID was approximately 21 h faster with AXDX. ADE or AAT was theoretically possible with AXDX in 48.4%. Of on-panel organisms, 24.0% did not receive initial AAT. In-hospital mortality was 46.7% without initial AAT, and 11.6% with AAT. Coverage of AXDX was 75.3%, specificity 99.7%, positive predictive value (PPV) 96.0%, and negative predictive value (NPV) 97.6%. On-panel sensitivity was 91.5%, specificity 99.6%, PPV 96.0%, and NPV 99.0%. CONCLUSIONS: AXDX provides more rapid ID and AST for GNB and ID for yeast than SOC. AXDX could potentially reduce time to AAT and facilitate ADE.

Evaluation of telavancin susceptibility in isolates of Staphylococcus aureus with reduced susceptibility to vancomycin.

Historically, vancomycin has been considered a primary therapeutic option for treating infections with Staphylococcus aureus, but isolates with reduced vancomycin susceptibility (SA-RVS) (MIC ≥ 4 µg/mL) have emerged. Telavancin, a semisynthetic lipoglycopeptide, is an alternative treatment option for S. aureus, but data examining telavancin activity against SA-RVS are limited. In the present study, we characterize 300 isolates of S. aureus isolates (50 vancomycin-susceptible (VSSA) isolates and 250 SA-RVS isolates) from a large tertiary care, academic medical center, 51.8% of which were methicillin resistant (MRSA). Sixteen (6.4%) SA-RVS isolates were non-susceptible to telavancin, whereas all VSSA isolates were susceptible. Additionally, 3.6% of SA-RVS isolates were non-susceptible to daptomycin, with three (1.2%) isolates testing non-susceptible to both telavancin and daptomycin. When tested against other classes of antimicrobials, there were no statistical differences in susceptibility of VSSA and SA-RVS isolates, except for the fluoroquinolones (ciprofloxacin and moxifloxacin). Molecular characterization of the isolates showed that SCCmec types II and IV together represented over half of the SA-RVS isolates; 12.0% of the VSSA isolates were SCCmec type II. Using RepPCR, we detected 16 distinct strain types in this isolate collection, and tst-1 (gene encoding the Staphylococcus toxic shock syndrome super-antigen) carriage was low (5.4%). Overall, we show that in addition to reduced vancomycin susceptibility, a small, but clinically significant, proportion of SA-RVS isolates also demonstrate reduced susceptibility to both telavancin and daptomycin.

Superficieibacter electus gen. nov., sp. nov., an Extended-Spectrum ß-Lactamase Possessing Member of the Enterobacteriaceae Family, Isolated From Intensive Care Unit Surfaces.

Two Gram-negative bacilli strains, designated BP-1(T) and BP-2, were recovered from two different Intensive Care Unit surfaces during a longitudinal survey in Pakistan. Both strains were unidentified using the bioMerieux VITEK MS IVD v2.3.3 and Bruker BioTyper MALDI-TOF mass spectrometry platforms. To more precisely determine the taxonomic identity of BP-1(T) and BP-2, we employed a biochemical and phylogenomic approach. The 16S rRNA gene sequence of strain BP-1(T) had the highest identity to Citrobacter farmeri CDC 2991-81(T) (98.63%) Citrobacter amalonaticus CECT 863(T) (98.56%), Citrobacter sedlakii NBRC 105722(T) (97.74%) and Citrobacter rodentium NBRC 105723(T) (97.74%). The biochemical utilization scheme of BP-1(T) using the Analytic Profile Index for Enterobacteriaceae (API20E) indicated its enzymatic functions are unique within the Enterobacteriaceae but most closely resemble Kluyvera spp., Enterobacter cloacae and Citrobacter koseri/farmeri. Phylogenomic analysis of the shared genes between BP-1(T), BP-2 and type strains from Kluyvera, Citrobacter, Escherichia, Salmonella, Kosakonia, Siccibacter and Shigella indicate that BP-1(T) and BP-2 isolates form a distinct branch from these genera. Average Nucleotide Identity analysis indicates that BP-1(T) and BP-2 are the same species. The biochemical and phylogenomic analysis indicate strains BP-1(T) and BP-2 represent a novel species from a new genus within the Enterobacteriaceae family, for which the name Superficieibacter electus gen. nov., sp. nov., is proposed. The type strain is BP-1(T) (= ATCC BAA-2937, = NBRC 113412).

Frequency of Instrument, Environment, and Laboratory Technologist Contamination during Routine Diagnostic Testing of Infectious Specimens.

Laboratory testing to support the care of patients with highly infectious diseases may pose a risk for laboratory workers. However, data on the risk of virus transmission during routine laboratory testing conducted using standard personal protective equipment (PPE) are sparse. Our objective was to measure laboratory contamination during routine analysis of patient specimens. Remnant specimens were spiked with the nonpathogenic bacteriophage MS2 at 1.0 × 107 PFU/ml, and contamination was assessed using reverse transcriptase PCR (RT-PCR) for MS2. Specimen containers were exteriorly coated with a fluorescent powder to enable the visualization of gross contamination using UV light. Testing was performed by two experienced laboratory technologists using standard laboratory PPE and sample-to-answer instrumentation. Fluorescence was noted on the gloves, bare hands, and laboratory coat cuffs of the laboratory technologist in 36/36 (100%), 13/36 (36%), and 4/36 (11%) tests performed, respectively. Fluorescence was observed in the biosafety cabinet (BSC) in 8/36 (22%) tests, on test cartridges/devices in 14/32 (44%) tests, and on testing accessory items in 29/32 (91%) tests. Fluorescence was not observed on or in laboratory instrumentation or adjacent surfaces. In contrast to fluorescence detection, MS2 detection was infrequent (3/286 instances [1%]) and occurred during test setup for the FilmArray instrument and on FilmArray accessory equipment. The information from this study may provide opportunities for the improvement of clinical laboratory safety practices so as to reduce the risk of pathogen transmission to laboratory workers.

When Good Bugs Go Bad: Epidemiology and Antimicrobial Resistance Profiles of Corynebacterium striatum, an Emerging Multidrug-Resistant, Opportunistic Pathogen.

Infections with Corynebacterium striatum have been described in the literature over the last 2 decades, with the majority being bacteremia, central line infections, and occasionally, endocarditis. In recent years, the frequency of C. striatum infections appears to be increasing; a factor likely contributing to this is the increased ease and accuracy of the identification of Corynebacterium spp., including C. striatum, from clinical cultures. The objective of this study was to retrospectively characterize C. striatum isolates recovered from specimens submitted as part of routine patient care at a 1,250-bed, tertiary-care academic medical center. Multiple strain types were recovered, as demonstrated by repetitive-sequence-based PCR. Most of the strains of C. striatum characterized were resistant to antimicrobials commonly used to treat Gram-positive organisms, such as penicillin, ceftriaxone, meropenem, clindamycin, and tetracycline. The MIC50 for ceftaroline was >32 µg/ml. Although there are no interpretive criteria for susceptibility with telavancin, it appeared to have potent in vitro efficacy against this species, with MIC50 and MIC90 values of 0.064 and 0.125 µg/ml, respectively. Finally, as previously reported in case studies, we demonstrated rapid in vitro development of daptomycin resistance in 100% of the isolates tested (n = 50), indicating that caution should be exhibited when using daptomycin for the treatment of C. striatum infections. C. striatum is an emerging, multidrug-resistant pathogen that can be associated with a variety of infection types.

Draft Genome Sequence of the blaOXA-436- and blaNDM-1-Harboring Shewanella putrefaciens SA70 Isolate.

We sequenced a carbapenem-resistant Shewanella putrefaciens isolate cultured from the sink handle of a Pakistan hospital room. Assembly annotation indicates that the isolate has a chromosomal blaOXA-436 carbapenemase and a plasmid-borne blaNDM-1 gene. To our knowledge, this is the first report of a Shewanella species harboring blaNDM.

Assessment of Healthcare Worker Protocol Deviations and Self-Contamination During Personal Protective Equipment Donning and Doffing.

OBJECTIVE To evaluate healthcare worker (HCW) risk of self-contamination when donning and doffing personal protective equipment (PPE) using fluorescence and MS2 bacteriophage. DESIGN Prospective pilot study. SETTING Tertiary-care hospital. PARTICIPANTS A total of 36 HCWs were included in this study: 18 donned/doffed contact precaution (CP) PPE and 18 donned/doffed Ebola virus disease (EVD) PPE. INTERVENTIONS HCWs donned PPE according to standard protocols. Fluorescent liquid and MS2 bacteriophage were applied to HCWs. HCWs then doffed their PPE. After doffing, HCWs were scanned for fluorescence and swabbed for MS2. MS2 detection was performed using reverse transcriptase PCR. The donning and doffing processes were videotaped, and protocol deviations were recorded. RESULTS Overall, 27% of EVD PPE HCWs and 50% of CP PPE HCWs made ≥1 protocol deviation while donning, and 100% of EVD PPE HCWs and 67% of CP PPE HCWs made ≥1 protocol deviation while doffing (P=.02). The median number of doffing protocol deviations among EVD PPE HCWs was 4, versus 1 among CP PPE HCWs. Also, 15 EVD PPE protocol deviations were committed by doffing assistants and/or trained observers. Fluorescence was detected on 8 EVD PPE HCWs (44%) and 5 CP PPE HCWs (28%), most commonly on hands. MS2 was recovered from 2 EVD PPE HCWs (11%) and 3 CP PPE HCWs (17%). CONCLUSIONS Protocol deviations were common during both EVD and CP PPE doffing, and some deviations during EVD PPE doffing were committed by the HCW doffing assistant and/or the trained observer. Self-contamination was common. PPE donning/doffing are complex and deserve additional study. Infect Control Hosp Epidemiol 2017;38:1077-1083.

Evaluation of Genotypic and Phenotypic Methods to Detect Carbapenemase Production in Gram-Negative Bacilli.

BACKGROUND: Carbapenemase-producing gram-negative bacteria (CP-GNB) are an urgent and expanding public health threat. Rapid and accurate identification of these organisms facilitates infection prevention efforts in healthcare facilities. The objective of our study was to evaluate methods to detect and identify CP-GNB. METHODS: We examined 189 carbapenem-resistant GNB(CR-GNB), including Enterobacteriaceae, Pseudomonas aeruginosa, and Acinetobacter baumannii complex, using 3 different methods: 2 methods to screen isolates of GNB for carbapenemase production [the carbapenem inactivation method (CIM) and 2 chromogenic agars] and a molecular method (Cepheid GeneXpert Carba-R) to identify the mechanism of carbapenem resistance and the associated resistance genes (blaKPC, blaNDM, blaIMP, blaOXA-48-like, and blaVIM). RESULTS: The CIM was a simple and inexpensive phenotypic screen to differentiate between CR-GNB and CP-GNB, with improved analytical performance characteristics and inter-reader correlation compared to the modified Hodge test. Both chromogenic agars evaluated (HardyCHROM CRE and chromID CARBA) were able to support growth of most of the organisms tested, including isolates possessing the blaOXA-48-like gene. However, these media had a low analytical specificity for carbapenemase production, with breakthrough of CR-GNB that did not produce a carbapenemase. The Xpert Carba-R assay was rapid and easy to perform, and demonstrated 100% positive and negative agreement for characterization of genetic determinants of carbapenem resistance. CONCLUSIONS: Screening by CIM followed by the Xpert Carba-R PCR is an accurate method for detecting and characterizing CP-GNB, including Enterobacteriaceae, P. aeruginosa, and A. baumannii complex.