MALDI-TOF mass spectrometry misidentification of Cutibacterium namnetense and Cutibacterium modestum: Implications for multiplex PCR phylotyping of Cutibacterium acnes.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) can misidentify Cutibacterium namnetense and Cutibacterium modestum as Cutibacterium acnes. We now describe how such MALDI-TOF MS misidentification explains previous reports of C. acnes isolates that could not be characterised using a multiplex PCR phylotyping assay.

Gut microbiome correlates of recurrent urinary tract infection: a longitudinal, multi-center study.

Background: Urinary tract infections (UTI) affect approximately 250 million people annually worldwide. Patients often experience a cycle of antimicrobial treatment and recurrent UTI (rUTI) that is thought to be facilitated by a gut reservoir of uropathogenic Escherichia coli (UPEC). Methods: 125 patients with UTI caused by an antibiotic-resistant organism (ARO) were enrolled from July 2016 to May 2019 in a longitudinal, multi-center cohort study. Multivariate statistical models were used to assess the relationship between uropathogen colonization and recurrent UTI (rUTI), controlling for clinical characteristics. 644 stool samples and 895 UPEC isolates were interrogated for taxonomic composition, antimicrobial resistance genes, and phenotypic resistance. Cohort UTI gut microbiome profiles were compared against published healthy and UTI reference microbiomes, as well as assessed within-cohort for timepoint- and recurrence-specific differences. Findings: Risk of rUTI was not independently associated with clinical characteristics. The UTI gut microbiome was distinct from healthy reference microbiomes in both taxonomic composition and antimicrobial resistance gene (ARG) burden, with 11 differentially abundant taxa at the genus level. rUTI and non-rUTI gut microbiomes in the cohort did not generally differ, but gut microbiomes from urinary tract colonized patients were elevated in E. coli abundance 7-14 days post-antimicrobial treatment. Corresponding UPEC gut isolates from urinary tract colonizing lineages showed elevated phenotypic resistance against 11 of 23 tested drugs compared to non-colonizing lineages. Interpretation: The gut microbiome is implicated in UPEC urinary tract colonization during rUTI, serving as an ARG-enriched reservoir for UPEC. UPEC can asymptomatically colonize the gut and urinary tract, and post-antimicrobial blooms of gut E. coli among urinary tract colonized patients suggest that cross-habitat migration of UPEC is an important mechanism of rUTI. Thus, treatment duration and UPEC populations in both the urinary and gastrointestinal tract should be considered in treating rUTI and developing novel therapeutics. Funding: This work was supported in part by awards from the U.S. Centers for Disease Control and Prevention Epicenter Prevention Program (grant U54CK000482; principal investigator, V.J.F.); to J.H.K. from the Longer Life Foundation (an RGA/Washington University partnership), the National Center for Advancing Translational Sciences (grants KL2TR002346 and UL1TR002345), and the National Institute of Allergy and Infectious Diseases (NIAID) (grant K23A1137321) of the National Institutes of Health (NIH); and to G.D. from NIAID (grant R01AI123394) and the Eunice Kennedy Shriver National Institute of Child Health and Human Development (grant R01HD092414) of NIH. R.T.'s research was funded by the Deutsche Forschungsgemeinschaft (DFG; German Research Foundation; grant 402733540). REDCap is Supported by Clinical and Translational Science Award (CTSA) Grant UL1 TR002345 and Siteman Comprehensive Cancer Center and NCI Cancer Center Support Grant P30 CA091842. The content is solely the responsibility of the authors and does not necessarily represent the official views of the funding agencies.

Genomic surveillance of Clostridioides difficile transmission and virulence in a healthcare setting.

Clostridioides difficile infection (CDI) is a major cause of healthcare-associated diarrhea, despite the widespread implementation of contact precautions for patients with CDI. Here, we investigate strain contamination in a hospital setting and the genomic determinants of disease outcomes. Across two wards over 6 months, we selectively cultured C. difficile from patients (n = 384) and their environments. Whole-genome sequencing (WGS) of 146 isolates revealed that most C. difficile isolates were from clade 1 (131/146, 89.7%), while only one isolate of the hypervirulent ST1 was recovered. Of culture-positive admissions (n = 79), 19 (24%) patients were colonized with toxigenic C. difficile on admission to the hospital. We defined 25 strain networks at ≤2 core gene single nucleotide polymorphisms; two of these networks contain strains from different patients. Strain networks were temporally linked (P < 0.0001). To understand the genomic correlates of the disease, we conducted WGS on an additional cohort of C. difficile (n = 102 isolates) from the same hospital and confirmed that clade 1 isolates are responsible for most CDI cases. We found that while toxigenic C. difficile isolates are associated with the presence of cdtR, nontoxigenic isolates have an increased abundance of prophages. Our pangenomic analysis of clade 1 isolates suggests that while toxin genes (tcdABER and cdtR) were associated with CDI symptoms, they are dispensable for patient colonization. These data indicate that toxigenic and nontoxigenic C. difficile contamination persist in a hospital setting and highlight further investigation into how accessory genomic repertoires contribute to C. difficile colonization and disease. IMPORTANCE: Clostridioides difficile infection remains a leading cause of hospital-associated diarrhea, despite increased antibiotic stewardship and transmission prevention strategies. This suggests a changing genomic landscape of C. difficile. Our study provides insight into the nature of prevalent C. difficile strains in a hospital setting and transmission patterns among carriers. Longitudinal sampling of surfaces and patient stool revealed that both toxigenic and nontoxigenic strains of C. difficile clade 1 dominate these two wards. Moreover, quantification of transmission in carriers of these clade 1 isolates underscores the need to revisit infection prevention measures in this patient group. We identified unique genetic signatures associated with virulence in this clade. Our data highlight the complexities of preventing transmission of this pathogen in a hospital setting and the need to investigate the mechanisms of in vivo persistence and virulence of prevalent lineages in the host gut microbiome.

Secretory leukocyte protease inhibitor protects against severe urinary tract infection in mice.

Millions suffer from urinary tract infections (UTIs) worldwide every year with women accounting for the majority of cases. Uropathogenic Escherichia coli (UPEC) causes most of these primary infections and leads to 25% becoming recurrent or chronic. To repel invading pathogens, the urinary tract mounts a vigorous innate immune response that includes the secretion of antimicrobial peptides (AMPs), rapid recruitment of phagocytes, and exfoliation of superficial umbrella cells. Here, we investigate secretory leukocyte protease inhibitor (SLPI), an AMP with antiprotease, antimicrobial, and immunomodulatory functions, known to play protective roles at other mucosal sites, but not well characterized in UTIs. Using a preclinical model of UPEC-caused UTI, we show that urine SLPI increases in infected mice and that SLPI is localized to bladder epithelial cells. UPEC-infected SLPI-deficient (Slpi-/-) mice suffer from higher urine bacterial burdens, prolonged bladder inflammation, and elevated urine neutrophil elastase (NE) levels compared to wild-type (Slpi+/+) controls. Combined with bulk bladder RNA sequencing, our data indicate that Slpi-/- mice have a dysregulated immune and tissue repair response following UTI. We also measure SLPI in urine samples from a small group of female subjects 18-49 years old and find that SLPI tends to be higher in the presence of a uropathogen, except in patients with a history of recent or recurrent UTI, suggesting a dysregulation of SLPI expression in these women. Taken together, our findings show SLPI promotes clearance of UPEC in mice and provides preliminary evidence that SLPI is likewise regulated in response to uropathogen exposure in women.IMPORTANCEAnnually, millions of people suffer from urinary tract infections (UTIs) and more than $3 billion are spent on work absences and treatment of these patients. While the early response to UTI is known to be important in combating urinary pathogens, knowledge of host factors that help curb infection is still limited. Here, we use a preclinical model of UTI to study secretory leukocyte protease inhibitor (SLPI), an antimicrobial protein, to determine how it protects the bladder against infection. We find that SLPI is increased during UTI, accelerates the clearance of bacteriuria, and upregulates genes and pathways needed to fight an infection while preventing prolonged bladder inflammation. In a small clinical study, we show SLPI is readily detectable in human urine and is associated with the presence of a uropathogen in patients without a previous history of UTI, suggesting SLPI may play an important role in protecting from bacterial cystitis.

Comparison of Clostridioides difficile nucleic acid amplification test (NAAT) results using fresh and frozen stool specimens and rectal swabs.

IMPORTANCE: Nucleic acid amplification tests (NAATs) are frequently used in Clostridioides difficile research and diagnostic testing, but the effect of freezing specimens on C. difficile NAAT performance is not well characterized. This study evaluated the concordance of NAAT results between fresh and frozen specimens (fecal and rectal swabs) and found it to be very good to excellent. The results indicate that frozen fecal and rectal swab specimens may be used for C. difficile NAAT testing in research when fresh specimens are not available.

Impact of diagnostic stewardship interventions in the collection process of fungal blood cultures.

We implemented 2 interventions to improve utilization and contamination at our institution: kits to improve appropriate sample collection and an electronic order alert displaying appropriate indications of fungal blood cultures. An electronic order alert when ordering fungal blood cultures was associated with decreased utilization without decrease in positivity rate.

Comparative genomics reveals the correlations of stress response genes and bacteriophages in developing antibiotic resistance of Staphylococcus saprophyticus.

IMPORTANCE: Staphylococcus saprophyticus is the second most common bacteria associated with urinary tract infections (UTIs) in women. The antimicrobial treatment regimen for uncomplicated UTI is normally nitrofurantoin, trimethoprim-sulfamethoxazole (TMP-SMX), or a fluoroquinolone without routine susceptibility testing of S. saprophyticus recovered from urine specimens. However, TMP-SMX-resistant S. saprophyticus has been detected recently in UTI patients, as well as in our cohort. Herein, we investigated the understudied resistance patterns of this pathogenic species by linking genomic antibiotic resistance gene (ARG) content to susceptibility phenotypes. We describe ARG associations with known and novel SCCmec configurations as well as phage elements in S. saprophyticus, which may serve as intervention or diagnostic targets to limit resistance transmission. Our analyses yielded a comprehensive database of phenotypic data associated with the ARG sequence in clinical S. saprophyticus isolates, which will be crucial for resistance surveillance and prediction to enable precise diagnosis and effective treatment of S. saprophyticus UTIs.

Diagnostic and commensal Staphylococcus pseudintermedius genomes reveal niche adaptation through parallel selection of defense mechanisms.

Staphylococcus pseudintermedius is historically understood as a prevalent commensal and pathogen of dogs, though modern clinical diagnostics reveal an expanded host-range that includes humans. It remains unclear whether differentiation across S. pseudintermedius populations is driven primarily by niche-type or host-species. We sequenced 501 diagnostic and commensal isolates from a hospital, veterinary diagnostic laboratory, and within households in the American Midwest, and performed a comparative genomics investigation contrasting human diagnostic, animal diagnostic, human colonizing, pet colonizing, and household-surface S. pseudintermedius isolates. Though indistinguishable by core and accessory gene architecture, diagnostic isolates harbor more encoded and phenotypic resistance, whereas colonizing and surface isolates harbor similar CRISPR defense systems likely reflective of common household phage exposures. Furthermore, household isolates that persist through anti-staphylococcal decolonization report elevated rates of base-changing mutations in - and parallel evolution of - defense genes, as well as reductions in oxacillin and trimethoprim-sulfamethoxazole susceptibility. Together we report parallel niche-specific bolstering of S. pseudintermedius defense mechanisms through gene acquisition or mutation.

Secretory Leukocyte Protease Inhibitor Protects Against Severe Urinary Tract Infection in Mice.

Millions suffer from urinary tract infections (UTIs) worldwide every year with women accounting for the majority of cases. Uropathogenic Escherichia coli (UPEC) causes most of these primary infections and leads to 25% becoming recurrent or chronic. To repel invading pathogens, the urinary tract mounts a vigorous innate immune response that includes the secretion of antimicrobial peptides (AMPs), rapid recruitment of phagocytes and exfoliation of superficial umbrella cells. Here, we investigate secretory leukocyte protease inhibitor (SLPI), an AMP with antiprotease, antimicrobial and immunomodulatory functions, known to play protective roles at other mucosal sites, but not well characterized in UTIs. Using a mouse model of UPEC-caused UTI, we show that urine SLPI increases in infected mice and that SLPI is localized to bladder epithelial cells. UPEC infected SLPI-deficient (Slpi-/-) mice suffer from higher urine bacterial burdens, prolonged bladder inflammation, and elevated urine neutrophil elastase (NE) levels compared to wild-type (Slpi+/+) controls. Combined with bulk bladder RNA sequencing, our data indicate that Slpi-/- mice have a dysregulated immune and tissue repair response following UTI. We also measure SLPI in urine samples from a small group of female subjects 18-49 years old and find that SLPI tends to be higher in the presence of a uropathogen, except in patients with history of recent or recurrent UTI (rUTI), suggesting a dysregulation of SLPI expression in these women. Taken together, our findings show SLPI protects against acute UTI in mice and provides preliminary evidence that SLPI is likewise regulated in response to uropathogen exposure in women.

Seroprevalence of severe acute respiratory coronavirus virus 2 (SARS-CoV-2) antibodies among healthcare personnel in the Midwestern United States, September 2020-April 2021.

Objective: To determine the prevalence of severe acute respiratory coronavirus virus 2 (SARS-CoV-2) IgG nucleocapsid (N) antibodies among healthcare personnel (HCP) with no prior history of COVID-19 and to identify factors associated with seropositivity. Design: Prospective cohort study. Setting: An academic, tertiary-care hospital in St. Louis, Missouri. Participants: The study included 400 HCP aged ≥18 years who potentially worked with coronavirus disease 2019 (COVID-19) patients and had no known history of COVID-19; 309 of these HCP also completed a follow-up visit 70-160 days after enrollment. Enrollment visits took place between September and December 2020. Follow-up visits took place between December 2020 and April 2021. Methods: At each study visit, participants underwent SARS-CoV-2 IgG N-antibody testing using the Abbott SARS-CoV-2 IgG assay and completed a survey providing information about demographics, job characteristics, comorbidities, symptoms, and potential SARS-CoV-2 exposures. Results: Participants were predominately women (64%) and white (79%), with median age of 34.5 years (interquartile range [IQR], 30-45). Among the 400 HCP, 18 (4.5%) were seropositive for IgG N-antibodies at enrollment. Also, 34 (11.0%) of 309 were seropositive at follow-up. HCP who reported having a household contact with COVID-19 had greater likelihood of seropositivity at both enrollment and at follow-up. Conclusions: In this cohort of HCP during the first wave of the COVID-19 pandemic, ∼1 in 20 had serological evidence of prior, undocumented SARS-CoV-2 infection at enrollment. Having a household contact with COVID-19 was associated with seropositivity.

Molecular epidemiology of Clostridioides difficile isolates in a non-outbreak setting at a comprehensive cancer center.

Ribotyping was performed on Clostridioides difficile isolates from patients with malignancies. Thirty-one (27.9%) isolates from 111 episodes of colitis were recovered representing 14 ribotypes with 25 (80.6%) belonging to 6 ribotypes (014/020, 1/VPI/077/087, 05/015, 015/046, 05/053, 106/174). We identified three novel ribotypes with 1 carrying gene encoding for binary toxin.

A novel immune modulator IM33 mediates a glia-gut-neuronal axis that controls lifespan.

Aging is a complex process involving various systems and behavioral changes. Altered immune regulation, dysbiosis, oxidative stress, and sleep decline are common features of aging, but their interconnection is poorly understood. Using Drosophila, we discover that IM33, a novel immune modulator, and its mammalian homolog, secretory leukocyte protease inhibitor (SLPI), are upregulated in old flies and old mice, respectively. Knockdown of IM33 in glia elevates the gut reactive oxygen species (ROS) level and alters gut microbiota composition, including increased Lactiplantibacillus plantarum abundance, leading to a shortened lifespan. Additionally, dysbiosis induces sleep fragmentation through the activation of insulin-producing cells in the brain, which is mediated by the binding of Lactiplantibacillus plantarum-produced DAP-type peptidoglycan to the peptidoglycan recognition protein LE (PGRP-LE) receptor. Therefore, IM33 plays a role in the glia-microbiota-neuronal axis, connecting neuroinflammation, dysbiosis, and sleep decline during aging. Identifying molecular mediators of these processes could lead to the development of innovative strategies for extending lifespan.

Association between SARS-CoV-2 Symptoms, Ct Values, and Serological Response in Vaccinated and Unvaccinated Healthcare Personnel.

BACKGROUND: SARS-CoV-2 vaccines are effective at reducing symptomatic and asymptomatic COVID-19. Limited studies have compared symptoms, threshold cycle (Ct) values from reverse transcription (RT)-PCR testing, and serological testing results between previously vaccinated vs unvaccinated populations with SARS-CoV-2 infection. METHODS: Healthcare personnel (HCP) with a positive SARS-CoV-2 RT-PCR test within the previous 14 to 28 days completed surveys including questions about demographics, medical conditions, social factors, and symptoms of COVID-19. Ct values were observed, and serological testing was performed for anti-nucleocapsid (anti-N) and anti-Spike (anti-S) antibodies at enrollment and 40 to 90 days later. Serological results were compared to HCP with no known SARS-CoV-2 infection and negative anti-N testing. RESULTS: There were 104 unvaccinated/not fully vaccinated and 77 vaccinated HCP with 2 doses of an mRNA vaccine at time of infection. No differences in type or duration of symptoms were reported (P = 0.45). The median (interquartile range [IQR]) Ct was 21.4 (17.6-24.6) and 21.5 (18.1-24.6) for the unvaccinated and vaccinated HCP, respectively. Higher anti-N IgG was observed in unvaccinated HCP (5.08 S/CO, 3.08-6.92) than vaccinated (3.61 signal to cutoff ratio [S/CO], 2.16-5.05). Anti-S IgG was highest among vaccinated HCP with infection (34 285 aribitrary units [AU]/mL, 17 672-61 775), followed by vaccinated HCP with no prior infection (1452 AU/mL, 791-2943), then unvaccinated HCP with infection (829 AU/mL, 290-1555). Anti-S IgG decreased 1.56% (0.9%-1.79%) per day in unvaccinated and 0.38% (0.03%-0.94%) in vaccinated HCP. CONCLUSIONS: Vaccinated HCP infected with SARS-CoV-2 reported comparable symptoms and had similar Ct values relative to unvaccinated. However, vaccinated HCP had increased and prolonged anti-S and decreased anti-N response relative to unvaccinated.

Intensive care unit sinks are persistently colonized with multidrug resistant bacteria and mobilizable, resistance-conferring plasmids.

Contamination of hospital sinks with microbial pathogens presents a serious potential threat to patients, but our understanding of sink colonization dynamics is largely based on infection outbreaks. Here, we investigate the colonization patterns of multidrug-resistant organisms (MDROs) in intensive care unit sinks and water from two hospitals in the USA and Pakistan collected over 27 months of prospective sampling. Using culture-based methods, we recovered 822 bacterial isolates representing 104 unique species and genomospecies. Genomic analyses revealed long-term colonization by Pseudomonas spp. and Serratia marcescens strains across multiple rooms. Nanopore sequencing uncovered examples of long-term persistence of resistance-conferring plasmids in unrelated hosts. These data indicate that antibiotic resistance (AR) in Pseudomonas spp. is maintained both by strain colonization and horizontal gene transfer (HGT), while HGT maintains AR within Acinetobacter spp. and Enterobacterales, independent of colonization. These results emphasize the importance of proactive, genomic-focused surveillance of built environments to mitigate MDRO spread. IMPORTANCE Hospital sinks are frequently linked to outbreaks of antibiotic-resistant bacteria. Here, we used whole-genome sequencing to track the long-term colonization patterns in intensive care unit (ICU) sinks and water from two hospitals in the USA and Pakistan collected over 27 months of prospective sampling. We analyzed 822 bacterial genomes, representing over 100 different species. We identified long-term contamination by opportunistic pathogens, as well as transient appearance of other common pathogens. We found that bacteria recovered from the ICU had more antibiotic resistance genes (ARGs) in their genomes compared to matched community spaces. We also found that many of these ARGs are harbored on mobilizable plasmids, which were found shared in the genomes of unrelated bacteria. Overall, this study provides an in-depth view of contamination patterns for common nosocomial pathogens and identifies specific targets for surveillance.

Uncharacterized and lineage-specific accessory genes within the Proteus mirabilis pan-genome landscape.

Proteus mirabilis is a Gram-negative bacterium recognized for its unique swarming motility and urease activity. A previous proteomic report on four strains hypothesized that, unlike other Gram-negative bacteria, P. mirabilis may not exhibit significant intraspecies variation in gene content. However, there has not been a comprehensive analysis of large numbers of P. mirabilis genomes from various sources to support or refute this hypothesis. We performed comparative genomic analysis on 2,060 Proteus genomes. We sequenced the genomes of 893 isolates recovered from clinical specimens from three large US academic medical centers, combined with 1,006 genomes from NCBI Assembly and 161 genomes assembled from Illumina reads in the public domain. We used average nucleotide identity (ANI) to delineate species and subspecies, core genome phylogenetic analysis to identify clusters of highly related P. mirabilis genomes, and pan-genome annotation to identify genes of interest not present in the model P. mirabilis strain HI4320. Within our cohort, Proteus is composed of 10 named species and 5 uncharacterized genomospecies. P. mirabilis can be subdivided into three subspecies; subspecies 1 represented 96.7% (1,822/1,883) of all genomes. The P. mirabilis pan-genome includes 15,399 genes outside of HI4320, and 34.3% (5,282/15,399) of these genes have no putative assigned function. Subspecies 1 is composed of several highly related clonal groups. Prophages and gene clusters encoding putatively extracellular-facing proteins are associated with clonal groups. Uncharacterized genes not present in the model strain P. mirabilis HI4320 but with homology to known virulence-associated operons can be identified within the pan-genome. IMPORTANCE Gram-negative bacteria use a variety of extracellular facing factors to interact with eukaryotic hosts. Due to intraspecies genetic variability, these factors may not be present in the model strain for a given organism, potentially providing incomplete understanding of host-microbial interactions. In contrast to previous reports on P. mirabilis, but similar to other Gram-negative bacteria, P. mirabilis has a mosaic genome with a linkage between phylogenetic position and accessory genome content. P. mirabilis encodes a variety of genes that may impact host-microbe dynamics beyond what is represented in the model strain HI4320. The diverse, whole-genome characterized strain bank from this work can be used in conjunction with reverse genetic and infection models to better understand the impact of accessory genome content on bacterial physiology and pathogenesis of infection.

Gut pathogen colonization precedes bloodstream infection in the neonatal intensive care unit.

Bacterial bloodstream infections (BSIs) resulting in late-onset sepsis affect up to half of extremely preterm infants and have substantial morbidity and mortality. Bacterial species associated with BSIs in neonatal intensive care units (NICUs) commonly colonize the preterm infant gut microbiome. Accordingly, we hypothesized that the gut microbiome is a reservoir of BSI-causing pathogenic strains that increase in abundance before BSI onset. We analyzed 550 previously published fecal metagenomes from 115 hospitalized neonates and found that recent ampicillin, gentamicin, or vancomycin exposure was associated with increased abundance of Enterobacteriaceae and Enterococcaceae in infant guts. We then performed shotgun metagenomic sequencing on 462 longitudinal fecal samples from 19 preterm infants (cases) with BSI and 37 non-BSI controls, along with whole-genome sequencing of the BSI isolates. Infants with BSI caused by Enterobacteriaceae were more likely than infants with BSI caused by other organisms to have had ampicillin, gentamicin, or vancomycin exposure in the 10 days before BSI. Relative to controls, gut microbiomes of cases had increased relative abundance of the BSI-causing species and clustered by Bray-Curtis dissimilarity according to BSI pathogen. We demonstrated that 11 of 19 (58%) of gut microbiomes before BSI, and 15 of 19 (79%) of gut microbiomes at any time, harbored the BSI isolate with fewer than 20 genomic substitutions. Last, BSI strains from the Enterobacteriaceae and Enterococcaceae families were detected in multiple infants, indicating BSI-strain transmission. Our findings support future studies to evaluate BSI risk prediction strategies based on gut microbiome abundance in hospitalized preterm infants.

Evaluation of the NG-Test CARBA 5 Lateral Flow Assay with an IMP-27-Producing Morganella morganii and Other Morganellaceae.

An isolate of Morganella morganii (MMOR1) that tested susceptible to 3rd/4th-generation cephalosporins and intermediate to meropenem was characterized as positive for NDM and IMP carbapenemases by NG-Test CARBA 5. Our objective was to further investigate this result, given the inconsistent susceptibility profile and unusual epidemiological profile for our region. The MMOR1 isolate was retested for antimicrobial susceptibilities and characterized for carbapenemase production. MMOR1 tested susceptible to ceftazidime, ceftriaxone, cefepime, aztreonam, and ertapenem, and intermediate to meropenem and imipenem. The isolate tested positive by carbapenem inactivation method (CIM) and CIM+EDTA (eCIM) testing, indicating metallo-ß-lactamase production. The isolate tested negative for all carbapenemase genes on Xpert Carba-R, but positive for IMP on repeat testing of NG-Test CARBA 5. Whole-genome sequencing revealed MMOR1 contained blaIMP-27, but no other carbapenemase genes. Additional testing with NG-Test CARBA 5 revealed a false-positive NDM band when the assay was overloaded with test inoculum. Supplementary isolates were tested with an overloaded inoculum (n = 6 M. morganii; n = 1 P. mirabilis; n = 1 IMP-27-producing P. rettgeri; n = 1 IMP-1-producing E. coli; n = 1 K. pneumoniae), and two non-carbapenemase-producing carbapenem non-susceptible M. morganii also generated a false-positive NDM band; though, this was not universal among this species. A dual IMP+/NDM+ M. morganii is an unusual result that should prompt additional investigation, especially in nonendemic regions and when the susceptibility profile is incompatible. IMP-27 is not detected by Xpert Carba-R but is variably detected by NG-Test CARBA 5. The microorganism inoculum used for NG-Test CARBA 5 must be carefully controlled for accurate results. IMPORTANCE The detection of carbapenemase-producing carbapenem-resistant Enterobacterales (CP-CRE) is an important function of the clinical microbiology laboratory, where positive identifications have immediate implications for infection control and surveillance strategies in the inpatient setting and can inform appropriate selection of therapy among the various novel anti-CP-CRE agents. NG-Test CARBA 5 is a relatively new lateral flow assay used for detection of carbapenemases in CP-CRE. Here, we describe the characterization of a Morganella morganii isolate that generated a false-positive NDM carbapenemase detection by this assay, and perform bacterial test inoculum experiments with additional isolates to further investigate a cause of false-positive results using the NG-Test CARBA 5. While a lateral flow assay like the NG-Test CARBA 5 is a very desirable test format for clinical laboratories, there are pitfalls to avoid when performing this test and interpreting results, including recognizing an overloaded test assay, which could lead to false-positive results.

Genomic Analyses of Longitudinal Mycobacterium abscessus Isolates in a Multicenter Cohort Reveal Parallel Signatures of In-Host Adaptation.

BACKGROUND: Nontuberculous mycobacteria (NTM) are ubiquitous in the environment and an increasingly frequent cause of opportunistic infections. Mycobacterium abscessus complex (MABC) is one of the major NTM lung pathogens that disproportionately colonize and infect the lungs of individuals with cystic fibrosis (CF). MABC infection can persist for years, and antimicrobial treatment is frequently ineffective. METHODS: We sequenced the genomes of 175 isolates longitudinally collected from 30 patients with MABC lung infection. We contextualized our cohort amidst the broader MABC phylogeny and investigated genes undergoing parallel adaptation across patients. Finally, we tested the phenotypic consequences of parallel mutations by conducting antimicrobial resistance and mercury-resistance assays. RESULTS: We identified highly related isolate pairs across hospital centers with low likelihood of transmission. We further annotated nonrandom parallel mutations in 22 genes and demonstrated altered macrolide susceptibility co-occurring with a nonsynonymous whiB1 mutation. Finally, we highlighted a 23-kb mercury-resistance plasmid whose loss during chronic infection conferred phenotypic susceptibility to organic and nonorganic mercury compounds. CONCLUSIONS: We characterized parallel genomic processes through which MABC is adapting to promote survival within the host. The within-lineage polymorphisms we observed have phenotypic effects, potentially benefiting fitness in the host at the putative detriment of environmental survival.

Comparative Evaluation of Current Biochemical-, Sequencing-, and Proteomic-Based Identification Methods for the Streptococcus bovis Group.

The Streptococcus bovis group (previously group D streptococci) consists of seven distinct species and subspecies. Definitive identification within the group is important, as certain organisms have been associated with gastrointestinal carcinoma, bacteremia, infective endocarditis, meningitis, biliary tract disease, and carcinoma, among others. Definitive identification, however, remains elusive due to limitations and inconsistencies across commonly used identification platforms in the United States. Here, we compared the performance of standard biochemical (Trek Gram-positive identification [GPID] plate, Vitek 2 GPID), sequencing (16S rDNA, sodA) databases (NCBI, RDP, CDC MicrobeNet), and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) platforms (Vitek MS, Bruker Biotyper MS) using a set of eight type strains representing all seven strains within the S. bovis group. Despite the evaluation of contemporary methods, no single platform was able to definitively identify all type strains within the S. bovis group. Vitek MS (85.7%, 7/8) provided the most accurate definitive identifications, followed by sodA sequencing (75%, 6/8). Vitek 2 and Bruker Biotyper RUO platforms performed the next best (62.5%, 5/8). All remaining platforms failed to adequately differentiate type strains within the S. bovis group (range, 0 to 37.5%). Laboratorians and clinicians should be aware of the identification limitations of routine testing algorithms and incorporate reflex testing, when appropriate, to platforms such as Vitek MS and/or sodA sequencing that are more able to definitively identify S. bovis group organisms. Further clinical evaluation was conducted using 65 clinical isolates from three geographically distinct U.S. institutions. Future improvements in identification platforms may reveal new clinical and epidemiological trends for members of the S. bovis group.