New design strategies for ultra-specific CRISPR-Cas13a-based RNA detection with single-nucleotide mismatch sensitivity.

An increasingly pressing need for clinical diagnostics has required the development of novel nucleic acid-based detection technologies that are sensitive, fast, and inexpensive, and that can be deployed at point-of-care. Recently, the RNA-guided ribonuclease CRISPR-Cas13 has been successfully harnessed for such purposes. However, developing assays for detection of genetic variability, for example single-nucleotide polymorphisms, is still challenging and previously described design strategies are not always generalizable. Here, we expanded our characterization of LbuCas13a RNA-detection specificity by performing a combination of experimental RNA mismatch tolerance profiling, molecular dynamics simulations, protein, and crRNA engineering. We found certain positions in the crRNA-target-RNA duplex that are particularly sensitive to mismatches and establish the effect of RNA concentration in mismatch tolerance. Additionally, we determined that shortening the crRNA spacer or modifying the direct repeat of the crRNA leads to stricter specificities. Furthermore, we harnessed our understanding of LbuCas13a allosteric activation pathways through molecular dynamics and structure-guided engineering to develop novel Cas13a variants that display increased sensitivities to single-nucleotide mismatches. We deployed these Cas13a variants and crRNA design strategies to achieve superior discrimination of SARS-CoV-2 strains compared to wild-type LbuCas13a. Together, our work provides new design criteria and Cas13a variants to use in future easier-to-implement Cas13-based RNA detection applications.

Species Delineation and Comparative Genomics within the Campylobacter ureolyticus Complex.

Campylobacter ureolyticus is an emerging pathogen increasingly appreciated as a common cause of gastroenteritis and extra-intestinal infections in humans. Outside the setting of gastroenteritis, little work has been done to describe the genomic content and relatedness of the species, especially regarding clinical isolates. We reviewed the epidemiology of clinical C. ureolyticus cultured by our institution over the past 10 years. Fifty-one unique C. ureolyticus isolates were identified between January 2010 and August 2022, mostly originating from abscesses and blood cultures. To clarify the taxonomic relationships between isolates and to attribute specific genes with different clinical manifestations, we sequenced 19 available isolates from a variety of clinical specimen types and conducted a pangenomic analysis with publicly available C. ureolyticus genomes. Digital DNA:DNA hybridization suggested that these C. ureolyticus comprised a species complex of 10 species clusters (SCs) and several subspecies clusters. Although some orthologous genes or gene functions were enriched in isolates found in different SCs and clinical specimens, no association was significant. Nearly a third of the isolates possessed antimicrobial resistance genes, including the ermA resistance gene, potentially conferring resistance to macrolides, the treatment of choice for severe human campylobacteriosis. This work effectively doubles the number of publicly available C. ureolyticus genomes, provides further clarification of taxonomic relationships within this bacterial complex, and identifies target SCs for future analysis.

Genomic Analysis of Clostridioides difficile in 2 Regions of the United States Reveals a Diversity of Strains and Limited Transmission.

BACKGROUND: The distribution of Clostridioides difficile strains and transmission dynamics in the United States are not well defined. Whole-genome sequencing across 2 Centers for Disease Control and Prevention Emerging Infections Program C. difficile infection (CDI) surveillance regions (Minnesota and New York) was performed to identify predominant multilocus sequence types (MLSTs) in community-associated (CA) and healthcare-associated (HCA) disease and assess transmission. METHODS: Whole-genome sequencing was performed on C. difficile isolates from patients with CDI over 3 months between 2016 and 2017. Patients were residents of the catchment area without a positive C. difficile test in the preceding 8 weeks. CDI cases were epidemiologically classified as HCA or CA. RESULTS: Of 422 isolates, 212 (50.2%) were HCA and 203 (48.1%) were CA. Predominant MLSTs were sequence type (ST) 42 (9.3%), ST8 (7.8%), and ST2 (8.1%). MLSTs associated with HCA-CDI included ST1 (76%), ST53 (83.3%), and ST43 (80.0%), while those associated with CA-CDI included ST3 (76.9%) and ST41 (77.8%). ST1 was more frequent in New York than in Minnesota (10.8% vs 3.1%). Thirty-three pairs were closely related genomically, 14 of which had potential patient-to-patient transmission supported by record review. CONCLUSIONS: The genomic epidemiology of C. difficile across 2 regions of the United States indicates the presence of a diverse strain profile and limited direct transmission.

Multicenter Evaluation of the Unyvero Platform for Testing Bronchoalveolar Lavage Fluid.

Bronchoalveolar lavage (BAL) culture is a standard, though time-consuming, approach for identifying microorganisms in patients with severe lower respiratory tract (LRT) infections. The sensitivity of BAL culture is relatively low, and prior antimicrobial therapy decreases the sensitivity further, leading to overuse of empirical antibiotics. The Unyvero LRT BAL Application (Curetis GmbH, Germany) is a multiplex molecular panel that detects 19 bacteria, 10 antibiotic resistance markers, and a fungus, Pneumocystis jirovecii, in BAL fluid in ∼4.5 h. Its performance was evaluated using 1,016 prospectively collected and 392 archived specimens from 11 clinical trial sites in the United States. Overall positive and negative percent agreements with culture results for identification of bacteria that grow in routine cultures were 93.4% and 98.3%, respectively, with additional potential pathogens identified by Unyvero in 21.7% of prospectively collected specimens. For detection of P. jirovecii, the positive percent agreement with standard testing was 87.5%. Antibiotic resistance marker results were compared to standard antibiotic susceptibility test results to determine positive predictive values (PPVs). PPVs ranged from 80 to 100%, based on the microorganism and specific resistance marker(s). The Unyvero LRT BAL Application provides accurate detection of common agents of bacterial pneumonia and of P. jirovecii The sensitivity and rapidity of this panel suggest significant clinical value for choosing appropriate antibiotics and for antibiotic stewardship.

A Multiplex Microsphere IgG Assay for SARS-CoV-2 Using ACE2-Mediated Inhibition as a Surrogate for Neutralization.

The coronavirus disease 2019 (COVID-19) pandemic has highlighted the challenges inherent to the serological detection of a novel pathogen such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Serological tests can be used diagnostically and for surveillance, but their usefulness depends on their throughput, sensitivity, and specificity. Here, we describe a multiplex fluorescent microsphere-based assay, 3Flex, that can detect antibodies to three major SARS-CoV-2 antigens-spike (S) protein, the spike ACE2 receptor-binding domain (RBD), and nucleocapsid (NP). Specificity was assessed using 213 prepandemic samples. Sensitivity was measured and compared to that of the Abbott Architect SARS-CoV-2 IgG assay using serum samples from 125 unique patients equally binned (n = 25) into 5 time intervals (≤5, 6 to 10, 11 to 15, 16 to 20, and ≥21 days from symptom onset). With samples obtained at ≤5 days from symptom onset, the 3Flex assay was more sensitive (48.0% versus 32.0%), but the two assays performed comparably using serum obtained ≥21 days from symptom onset. A larger collection (n = 534) of discarded sera was profiled from patients (n = 140) whose COVID-19 course was characterized through chart review. This revealed the relative rise, peak (S, 23.8; RBD, 23.6; NP, 16.7 [in days from symptom onset]), and decline of the antibody response. Considerable interperson variation was observed with a subset of extensively sampled intensive care unit (ICU) patients. Using soluble ACE2, inhibition of antibody binding was demonstrated for S and RBD, and not for NP. Taking the data together, this study described the performance of an assay built on a flexible and high-throughput serological platform that proved adaptable to the emergence of a novel infectious agent.

Trends in Incidence of Methicillin-resistant Staphylococcus aureus Bloodstream Infections Differ by Strain Type and Healthcare Exposure, United States, 2005-2013.

BACKGROUND: Previous reports suggested that US methicillin-resistant Staphylococcus aureus (MRSA) strain epidemiology has changed since the rise of USA300 MRSA. We describe invasive MRSA trends by strain type. METHODS: Data came from 5 Centers for Disease Control and Prevention Emerging Infections Program sites conducting population-based surveillance and collecting isolates for invasive MRSA (ie, from normally sterile body sites), 2005-2013. MRSA bloodstream infection (BSI) incidence per 100 000 population was stratified by strain type and epidemiologic classification of healthcare exposures. Invasive USA100 vs USA300 case characteristics from 2013 were compared through logistic regression. RESULTS: From 2005 to 2013, USA100 incidence decreased most notably for hospital-onset (6.1 vs 0.9/100 000 persons, P < .0001) and healthcare-associated, community-onset (10.7 vs 4.9/100 000 persons, P < .0001) BSIs. USA300 incidence for hospital-onset BSIs also decreased (1.5 vs 0.6/100 000 persons, P < .0001). However, USA300 incidence did not significantly change for healthcare-associated, community-onset (3.9 vs 3.3/100 000 persons, P = .05) or community-associated BSIs (2.5 vs 2.4/100 000 persons, P = .19). Invasive MRSA was less likely to be USA300 in patients who were older (adjusted odds ratio [aOR], 0.97 per year [95% confidence interval {CI}, .96-.98]), previously hospitalized (aOR, 0.36 [95% CI, .24-.54]), or had central lines (aOR, 0.44 [95% CI, .27-.74]), and associated with USA300 in people who inject drugs (aOR, 4.58 [95% CI, 1.16-17.95]). CONCLUSIONS: Most of the decline in MRSA BSIs was from decreases in USA100 BSI incidence. Prevention of USA300 MRSA BSIs in the community will be needed to further reduce burden from MRSA BSIs.

Multicenter Evaluation of the New Etest Gradient Diffusion Method for Piperacillin-Tazobactam Susceptibility Testing of Enterobacterales, Pseudomonas aeruginosa, and Acinetobacter baumannii Complex.

Piperacillin-tazobactam (P/T) is a ß-lactam-ß-lactamase inhibitor combination frequently used in the hospital setting. Etest is a gradient diffusion method that represents an alternative to broth microdilution (BMD) for performing antimicrobial susceptibility testing. We conducted a multicenter evaluation of the performance of the new P/T Etest compared to that of BMD following U.S. Food and Drug Administration (FDA) and International Standards Organization (ISO) standard ISO 20776-2 criteria using Clinical and Laboratory Standards Institute (CLSI)-FDA and European Committee on Antimicrobial Susceptibility Testing (EUCAST) interpretive breakpoints, respectively. A total of 977 isolates (775 Enterobacterales isolates, 119 Pseudomonas aeruginosa isolates, and 83 Acinetobacter baumannii complex isolates) were tested. Overall essential agreement (EA) was 96.4% and 96.6% for Enterobacterales when FDA and ISO 20776-2 criteria, respectively, were followed. EA was 98.3% for P. aeruginosa and 91.6% for the A. baumannii complex when both the FDA and ISO criteria were followed. Applying CLSI-FDA breakpoints, categorical agreement (CA) reached 93.0%, 93.3%, and 89.2% for the Enterobacterales, P. aeruginosa, and the A. baumannii complex, respectively. Two very major errors (VMEs; 1.1%) were found among the Enterobacterales (for 2 Klebsiella pneumoniae isolates). No additional major errors (MEs) or VMEs were found. Applying EUCAST breakpoints, CA was 94.8% and 95.8% for Enterobacterales and P. aeruginosa, respectively (no breakpoints are currently available for the A. baumannii complex). No VMEs were observed among the Enterobacterales, but 2 (0.4%) MEs were found. Among the P. aeruginosa isolates, 2 (6.9%) VMEs and 3 (3.3%) MEs were observed. These errors resulted when P/T Etest MICs were 1 doubling dilution apart from the BMD MICs. In conclusion, the new P/T Etest represents an accurate tool for performing antimicrobial susceptibility testing of Enterobacterales, P. aeruginosa, and A. baumannii complex isolates with limited category errors.

Carbapenem-Resistant Pseudomonas aeruginosa at US Emerging Infections Program Sites, 2015.

Pseudomonas aeruginosa is intrinsically resistant to many antimicrobial drugs, making carbapenems crucial in clinical management. During July-October 2015 in the United States, we piloted laboratory-based surveillance for carbapenem-resistant P. aeruginosa (CRPA) at sentinel facilities in Georgia, New Mexico, Oregon, and Tennessee, and population-based surveillance in Monroe County, NY. An incident case was the first P. aeruginosa isolate resistant to antipseudomonal carbapenems from a patient in a 30-day period from any source except the nares, rectum or perirectal area, or feces. We found 294 incident cases among 274 patients. Cases were most commonly identified from respiratory sites (120/294; 40.8%) and urine (111/294; 37.8%); most (223/280; 79.6%) occurred in patients with healthcare facility inpatient stays in the prior year. Genes encoding carbapenemases were identified in 3 (2.3%) of 129 isolates tested. The burden of CRPA was high at facilities under surveillance, but carbapenemase-producing CRPA were rare.

Next-Generation-Sequencing-Based Hospital Outbreak Investigation Yields Insight into Klebsiella aerogenes Population Structure and Determinants of Carbapenem Resistance and Pathogenicity.

Klebsiella aerogenes is a nosocomial pathogen associated with drug resistance and outbreaks in intensive care units. In a 5-month period in 2017, we experienced an increased incidence of cultures for carbapenem-resistant K. aerogenes (CR-KA) from an adult cardiothoracic intensive care unit (CICU) involving 15 patients. Phylogenomic analysis following whole-genome sequencing (WGS) identified the outbreak CR-KA isolates to group together as a tight monoclonal cluster (with no more than six single nucleotide polymorphisms [SNPs]), suggestive of a protracted intraward transmission event. No clonal relationships were identified between the CICU CR-KA strains and additional hospital CR-KA patient isolates from different wards and/or previous years. Carbapenemase-encoding genes and drug-resistant plasmids were absent in the outbreak strains, and carbapenem resistance was attributed to mutations impacting AmpD activity and membrane permeability. The CICU outbreak strains harbored an integrative conjugative element (ICE) which has been associated with pathogenic Klebsiella pneumoniae lineages (ICEKp10). Comparative genomics with global K. aerogenes genomes showed our outbreak strains to group closely with global sequence type 4 (ST4) strains, which, along with ST93, likely represent dominant K. aerogenes lineages associated with human infections. For poorly characterized pathogens, scaling analyses to include sequenced genomes from public databases offer the opportunity to identify emerging trends and dominant clones associated with specific attributes, syndromes, and geographical locations.

A Nationwide Screen of Carbapenem-Resistant Klebsiella pneumoniae Reveals an Isolate with Enhanced Virulence and Clinically Undetected Colistin Heteroresistance.

The convergence of hypervirulence and multidrug resistance in Klebsiella pneumoniae is a significant concern. Here, we report the first screen for hypermucoviscosity, a trait associated with increased virulence, using a U.S. surveillance collection of carbapenem-resistant (CR) K. pneumoniae isolates. We identified one hypermucoviscous isolate, which carried a gene encoding the KPC-3 carbapenemase, among numerous resistance genes. The strain further exhibited colistin heteroresistance undetected by diagnostics. This convergence of diverse resistance mechanisms and increased virulence underscores the need for enhanced K. pneumoniae surveillance.

MRSA nares swab is a more accurate predictor of MRSA wound infection compared with clinical risk factors in emergency department patients with skin and soft tissue infections.

OBJECTIVES: Skin and soft tissue infections (SSTI) caused by methicillin-resistant Staphylococcus aureus (MRSA) are prevalent in the emergency department (ED). We determined whether MRSA nasal carriage better identifies patients with MRSA wound infection than clinical risk factors or emergency medicine (EM) provider's choice of discharge prescriptions. METHODS: Adult patients presenting to a large academic medical centre ED in the USA with SSTI between May 2010 and November 2011 were screened. Research assistants administered a questionnaire regarding MRSA risk factors, and MRSA nares swab PCR testing, wound culture results and information on antibiotics prescribed at discharge were collected. Measures of classification accuracy for nares swab, individual risk factors and physician's prescription for MRSA coverage were compared with gold standard wound culture. RESULTS: During the study period, 116 patients with SSTI had both wound cultures and nares swabs for MRSA. S. aureus was isolated in 59.5%, most often MRSA (75.4%). Thirty patients (25.9%) had a positive MRSA nares swab and culture for a sensitivity of 57.7% and specificity of 92.2%. Positive predictive value (PPV) for MRSA nares swab was 85.7% and positive likelihood ratio was 7.4, while negative predictive value was 72.8% and negative likelihood ratio 0.5. None of the individual risk factors nor EM provider's prescription for MRSA coverage had a PPV or positive likelihood ratio higher than nares swabs. CONCLUSIONS: MRSA nares swab is a more accurate predictor of MRSA wound infection compared with clinical risk factors or EM provider's choice of antibiotics. MRSA nares swab may be a useful tool in the ED.

In Vitro Activity of Solithromycin against Bordetella pertussis, an Emerging Respiratory Pathogen.

There has been an increase in the number of pertussis cases reported since the introduction of the acellular pertussis vaccine. While children that present with pertussis have a characteristic whooping cough, adults can simply have a persistent, nonspecific cough and remain undiagnosed. Macrolide antibiotics, such as azithromycin, are the currently recommended treatment for pertussis. Solithromycin is a new macrolide and the first fluoroketolide with broad activity against a wide spectrum of bacterial pathogens and has completed clinical development for community-acquired bacterial pneumonia. This study reports the potent in vitro activity of solithromycin against a collection of recent isolates of Bordetella pertussis.

Semen-derived enhancer of viral infection (SEVI) binds bacteria, enhances bacterial phagocytosis by macrophages, and can protect against vaginal infection by a sexually transmitted bacterial pathogen.

The semen-derived enhancer of viral infection (SEVI) is a positively charged amyloid fibril that is derived from a self-assembling proteolytic cleavage fragment of prostatic acid phosphatase (PAP(248-286)). SEVI efficiently facilitates HIV-1 infection in vitro, but its normal physiologic function remains unknown. In light of the fact that other amyloidogenic peptides have been shown to possess direct antibacterial activity, we investigated whether SEVI could inhibit bacterial growth. Neither SEVI fibrils nor the unassembled PAP(248-286) peptide had significant direct antibacterial activity in vitro. However, SEVI fibrils bound to both Gram-positive (Staphylococcus aureus) and Gram-negative (Escherichia coli and Neisseria gonorrhoeae) bacteria, in a charge-dependent fashion. Furthermore, SEVI fibrils but not the monomeric PAP(248-286) peptide promoted bacterial aggregation and enhanced the phagocytosis of bacteria by primary human macrophages. SEVI also enhanced binding of bacteria to macrophages and the subsequent release of bacterially induced proinflammatory cytokines (tumor necrosis factor alpha [TNF-α], interleukin-6 [IL-6], and IL-1ß). Finally, SEVI fibrils inhibited murine vaginal colonization with Neisseria gonorrhoeae. These findings demonstrate that SEVI has indirect antimicrobial activity and that this activity is dependent on both the cationic charge and the fibrillar nature of SEVI.

A single-blinded randomized clinical trial comparing polymyxin B-trimethoprim and moxifloxacin for treatment of acute conjunctivitis in children.

OBJECTIVE: To perform a randomized controlled trial comparing moxifloxacin hydrochloride with polymyxin B-trimethoprim for the treatment of acute conjunctivitis. STUDY DESIGN: Patients ages 1-18 years old with acute conjunctivitis had cultures performed and were randomized to receive either moxifloxacin hydrochloride or polymyxin B-trimethoprim ophthalmic solution for 7 days. Response to treatment was determined by phone query on day 4-6 and by examination with post-treatment conjunctival culture on day 7-10. RESULTS: One hundred and twenty-four patients were enrolled. Eighty patients (65%) had recognized pathogens (55 Haemophilus influenzae, 22 Streptococcus pneumoniae, 4 Moraxella catarrhalis) isolated from their conjunctiva. One hundred fourteen (56/62 moxifloxacin and 58/62 polymyxin B-trimethoprim) completed the 4-6 day evaluation, with 43/56 (77%) of the moxifloxacin group and 42/58 (72%) of the polymyxin B-trimethoprim group clinically cured according to parents (noninferiority test P = .04). Eighty-nine (39/56 moxifloxacin and 50/58 polymyxin B-trimethoprim) patients completed the 7-10 day evaluation. Clinical cure was observed in 37/39 (95%) of the moxifloxacin and 49/51 (96%) of the polymyxin B-trimethoprim treated groups (noninferiority test P ≤ .01). Clinical cure rates for culture positive and negative conjunctivitis were not different. There was no statistically significant difference in bacteriologic cure rates between the 2 groups. CONCLUSIONS: Polymyxin B-trimethoprim continues to be an effective treatment for acute conjunctivitis with a clinical response rate that does not differ from moxifloxacin. Use of polymyxin B-trimethoprim for the treatment of conjunctivitis would result in significant cost savings compared with fluoroquinolones.

Complete genome sequence of a human bacteremia isolate of Kalamiella piersonii.

The complete genome of Kalamiella piersonii clinical isolate URMC-2103A041 from human bacteremia was determined using the hybrid assembly of short- and long-read sequencing chemistry. The genome contains a 3.93 Mb chromosome, three circular plasmids, and one linear plasmid.

Trends and Non-Clinical Predictors of Respiratory Syncytial Virus (RSV) and Influenza Diagnosis in an Urban Pediatric Population.

Objective: To evaluate the demographic, maternal, and community-level predictors of pediatric respiratory syncytial virus (RSV) and influenza diagnosis among an urban population of children residing in Rochester, NY. Study design: A test-negative case-control design was used to investigate various non-clinical determinants of RSV and influenza diagnosis among 1,808 children aged 0-14 years who presented to the University of Rochester Medical Center (URMC) or an affiliated health clinic in Rochester, NY between 2012-2019. These children were all tested for RSV and influenza via polymerase-chain-reaction (PCR) method, including RSV and influenza diagnosis of all severity types. Test results were linked to medical records, birth certificates, questionnaires administered through the Statewide Perinatal Data System, and the US census by census tracts to obtain information on child, maternal, demographic, and socio-economic characteristics. Results: Overall the strongest predictor of RSV and influenza diagnosis was child's age, with every year increase in child's age, risk for RSV decreased (OR: 0.75; 95% CI: 0.71, 0.79) and risk for influenza increased (OR: 1.20; 95%: 1.16, 1.24). In addition to age, non-private insurance type was positively associated with influenza diagnosis. When considering the proportion of positive cases for RSV and influenza over all PCR tests by respiratory season, a spike in influenza cases was observed in 2018-2019. Conclusions: Age was a strong predictor of RSV and influenza diagnosis among this urban sample of children.

Invasive fungal infections following liver transplantation: incidence, risk factors, survival, and impact of fluconazole-resistant Candida parapsilosis (2003-2007).

Invasive fungal infections (IFIs) are associated with a high mortality rate for liver transplantation (LT) recipients. To study the incidence of and risk factors for IFIs in LT recipients and the associated mortality rates, we retrospectively reviewed the records of first-time deceased donor LT recipients (January 2003 to December 2007). The incidence of IFIs was 12%. Non-albicans Candida species accounted for 55% of IFIs; 50% of these IFIs were Candida parapsilosis. Only 43% of Candida isolates were fluconazole-susceptible (minimum inhibitory concentration ≤ 8 µ/mL). All C. parapsilosis isolates were fluconazole-resistant, and this coincided with a surge of these isolates during a peak period of LT. Factors associated with IFIs included a creatinine level > 2 mg/mL [hazard ratio (OR) = 2.4, 95% confidence interval (CI) = 1.2-5.0, P = 0.01], a Model for End-Stage Liver Disease score > 25 (OR = 2.4, 95% CI = 1.2-4.9, P = 0.02), pretransplant fungal colonization (OR = 7.0, 95% CI = 3.2-15.3, P < 0.001), and a daily prophylactic fluconazole dosage < 200 mg (OR = 2.8, 95% CI = 1.1-7.4, P = 0.03). According to a multivariate analysis, only pretransplant fungal colonization was associated with IFIs (OR = 7.8, 95% CI = 3.9-16.2, P < 0.001). The 1-year patient survival rates with and without IFIs were 41% and 80%, respectively, and the survival rates with C. parapsilosis, other non-albicans Candida, and Candida albicans IFIs were 28%, 50%, and 75%, respectively. In conclusion, IFIs after LT (especially non-albicans Candida species and fluconazole-resistant C. parapsilosis) were associated with reduced survival. The risk factors highlight the importance of pretransplant risk assessments. The identification of pretransplant fungal colonization may allow for risk modifications before or at the time of LT. Additionally, the number of LT procedures and prophylactic strategies may affect institutional outbreaks of resistant Candida strains.

Whole-Genome Sequences of Human Monkeypox Virus Strains from Two 2022 Global Outbreak Cases in Western New York State.

The genomes of two human monkeypox virus strains from recently reported cases in our local region that were associated with the 2022 global outbreak were sequenced. Genomes from clinical isolates provide valuable information for epidemiological tracking and analysis of strain evolution and can be especially important during the early phases of outbreaks.

Predictors of agr dysfunction in methicillin-resistant Staphylococcus aureus (MRSA) isolates among patients with MRSA bloodstream infections.

Despite emerging evidence that dysfunction in the accessory gene regulator (agr) locus is associated with deleterious outcomes among patients treated with vancomycin for methicillin-resistant Staphylococcus aureus (MRSA) infections, factors predictive of agr dysfunction have not been evaluated. This study describes the epidemiology of agr dysfunction, identifies predictors of agr dysfunction in MRSA isolates among those with MRSA bloodstream infections, and describes the relationship between agr dysfunction and other microbiologic phenotypes. A cross-sectional study of patients with MRSA bloodstream infections at two institutions in upstate New York was performed. Clinical data on demographics, comorbidities, disease severity, hospitalization history, and antibiotic history were collected. Microbiologic phenotypes, including agr dysfunction, MIC values by broth microdilution (BMD) and Etest, and vancomycin heteroresistance (hVISA) were tested. Multivariable analyses were performed to identify factors predictive of agr dysfunction. Among 200 patients with an MRSA bloodstream infection, the proportion of strains with agr dysfunction was 31.5%. The distribution of MICs determined by both BMD and Etest were equivalent across agr groups, and there was no association between agr dysfunction and the presence of hVISA. Severity of illness, comorbidities, and hospitalization history were comparable between agr groups. In the multivariate analysis, prior antibiotic exposure was the only factor of variables studied found to be predictive of agr dysfunction. This relationship was predominantly driven by prior beta-lactam and fluoroquinolone administration in the bivariate analysis. Identifying these institution-specific risk factors can be used to develop a process to assess the risk of agr dysfunction and guide empirical antibiotic therapy decisions.

Colistin Heteroresistance Is Largely Undetected among Carbapenem-Resistant Enterobacterales in the United States.

Heteroresistance is a form of antibiotic resistance where a bacterial strain is comprised of a minor resistant subpopulation and a majority susceptible subpopulation. We showed previously that colistin heteroresistance can mediate the failure of colistin therapy in an in vivo infection model, even for isolates designated susceptible by clinical diagnostics. We sought to characterize the extent of colistin heteroresistance among the highly drug-resistant carbapenem-resistant Enterobacterales (CRE). We screened 408 isolates for colistin heteroresistance. These isolates were collected between 2012 and 2015 in eight U.S. states as part of active surveillance for CRE. Colistin heteroresistance was detected in 10.1% (41/408) of isolates, and it was more common than conventional homogenous resistance (7.1%, 29/408). Most (93.2%, 38/41) of these heteroresistant isolates were classified as colistin susceptible by standard clinical diagnostic testing. The frequency of colistin heteroresistance was greatest in 2015, the last year of the study. This was especially true among Enterobacter isolates, of which specific species had the highest rates of heteroresistance. Among Klebsiella pneumoniae isolates, which were the majority of isolates tested, there was a closely related cluster of colistin-heteroresistant ST-258 isolates found mostly in Georgia. However, cladistic analysis revealed that, overall, there was significant diversity in the genetic backgrounds of heteroresistant K. pneumoniae isolates. These findings suggest that due to being largely undetected in the clinic, colistin heteroresistance among CRE is underappreciated in the United States.IMPORTANCE Heteroresistance is an underappreciated phenomenon that may be the cause of some unexplained antibiotic treatment failures. Misclassification of heteroresistant isolates as susceptible may lead to inappropriate therapy. Heteroresistance to colistin was more common than conventional resistance and was overwhelmingly misclassified as susceptibility by clinical diagnostic testing. Higher proportions of colistin heteroresistance observed in certain Enterobacter species and clustering among heteroresistant Klebsiella pneumoniae strains may inform colistin treatment recommendations. Overall, the rate of colistin nonsusceptibility was more than double the level detected by clinical diagnostics, suggesting that the prevalence of colistin nonsusceptibility among CRE may be higher than currently appreciated in the United States.