Comparative analysis of a rapid diagnostic test and scoring tools for ESBL detection in Enterobacterales bloodstream infections for optimizing antimicrobial therapy.

IMPORTANCE: Our study addresses a significant issue in the medical and scientific community-the delayed administration of appropriate antimicrobial treatments due to the time-consuming process of phenotypic susceptibility data collection in gram-negative bloodstream infections. Our research indicates that a multiplex PCR rapid diagnostic test (RDT) significantly outperformed two clinical scoring tools in predicting ceftriaxone susceptibility. Multiplex PCR also led to reduced instances of undertreatment with ceftriaxone and minimized overtreatment with carbapenems. Furthermore, multiplex PCR demonstrated high sensitivity and specificity in predicting ceftriaxone susceptibility. The results of our study underscore the potential RDTs to reduce the time to appropriate antimicrobial therapy, leading to improved patient outcomes and reduced healthcare costs.

Resistance mechanisms for Gram-negative bacteria-specific lipopeptides, turnercyclamycins, differ from that of colistin.

IMPORTANCE: Bacterial resistance to antibiotics is a crisis. Acinetobacter baumannii is among the CDC urgent threat pathogens in part for this reason. Lipopeptides known as turnercyclamycins are produced by symbiotic bacteria that normally live in marine mollusks, where they may be involved in shaping their symbiotic niche. Turnercyclamycins killed Gram-negative pathogens including drug-resistant Acinetobacter, but how do the mechanisms of resistance compare to other lipopeptide drugs? Here, we define resistance from a truncation of MlaA, a protein involved in regulating bacterial membrane phospholipids. Intriguingly, this resistance mechanism only affected one turnercyclamycin variant, which differed only in two atoms in the lipid tail of the compounds. We could not obtain significant resistance to the second turnercyclamycin variant, which was also effective in an infection model. This study reveals an unexpected subtlety in resistance to lipopeptide antibiotics, which may be useful in the design and development of antibiotics to combat drug resistance.

Genomic Characterization of 2 Cutibacterium acnes Isolates from a Surgical Site Infection Reveals Large Genomic Inversion.

Background: Cutibacterium acnes is a common commensal of human skin but may also present as an opportunistic pathogen in prosthetic joint and wound infections. Unfortunately, few complete genomes of C. acnes are publicly available, and even fewer are of isolates associated with infection. Here we report the isolation, characterization, and complete genomes of 2 C. acnes isolates from a surgical site infection of an elbow. Methods: We used standard microbiological methods for phenotypic characterization and performed whole genome sequencing on 2 C. acnes isolates using a combination of short-read and long-read sequencing. Results: Antibiotic susceptibility testing showed beta-lactamase negative and low minimal inhibitory concentrations to all antibiotics tested, with the exception of metronidazole. We assembled complete genomes of the 2 isolates, which are approximately 2.5 megabases in length. The isolates belong to the single-locus sequence type (SLST) H1 and the multi-locus sequence type (MLST) IB. Both isolates have similar composition of known virulence genes, and we found no evidence of plasmids but did find phage-associated genes. Notably, the 2 genomes are 99.97% identical but contain a large genomic inversion encompassing approximately half of the genome. Conclusions: This is the first characterization of this large-scale genomic inversion in nearly identical isolates from the same wound. This report adds to the limited numbers of publicly available infection-associated complete genomes of C. acnes.

Enterobacterial common antigen biosynthesis in Yersinia pestis is tied to antimicrobial peptide resistance.

Resistance to antimicrobial peptides (AMPs) plays an important role in allowing Yersinia pestis to maintain a successful infection in the flea vector Xenopsylla cheopis . Mutants that are unable to modify lipid A in their outer membrane with aminoarabinose (Ara4N), showed increased sensitivity to AMPs such as polymyxin B (PB), as well as decreased survival in fleas. A deletion mutant of wecE , a gene involved in biosynthesis of enterobacterial common antigen (ECA), also displayed hypersusceptibility to PB in vitro. Additional mutants in the ECA biosynthetic pathway were generated, some designed to cause accumulation of intermediate products that sequester undecaprenyl phosphate (Und-P), a lipid carrier that is also used in numerous other pathways, including for peptidoglycan, O-antigen, and Ara4N biosynthesis. Mutants that accumulate Und-PP-linked intermediates (ECA-lipid II) showed increased susceptibility to PB, reduced Ara4N-modified lipid A, altered cell morphology, and decreased ability to maintain flea infections. These effects are consistent with a model where Y. pestis has a sufficiently limited free Und-P pool such that sequestration of Und-P as ECA-lipid II prevents adequate Ara4N biosynthesis, ultimately resulting in AMP hypersusceptibility.

Bacterial culture time to detection in platelet components: An evidence synthesis and estimation of detection failures.

BACKGROUND: Non-pathogen reduction platelet bacterial risk control strategies in the US FDA guidance include at least one culture. Almost all of these strategies have a culture hold time of ≥12 h. Studies have reported time to detection (TTD) of bacterial cultures inoculated with bacteria from contaminated platelets, but these data and estimates of risk associated with detection failures have not been synthesized. METHODS: We performed a literature search to identify studies reporting TTD for samples obtained from spiked platelet components. Using extracted data, regression analysis was used to estimate TTD for culture bottles at different inoculum sizes. Detection failures were defined as events in which contaminated components are transfused to a patient. We then used published data on time of transfusion (ToT) to estimate the risk of detection failures in practice. RESULTS: The search identified 1427 studies, of which 16 were included for analysis. TTD data were available for 16 different organisms, including 14 in aerobic cultures and 11 in anaerobic cultures. For inocula of 1 colony forming unit (CFU), the average TTD for aerobic organisms was 19.2 h while it was 24.9 h in anaerobic organisms, but there was substantial overall variation. A hold time of 12 versus 24 h had minimal effect for most organisms. CONCLUSION: TTD variation occurs between bacterial species and within a particular species. Under typical inventory management, the relative contribution of culture detection failures is much smaller than the residual risk from sampling failures. Increasing the hold period beyond 12 h has limited value.

Antibacterial activity of Xenopsylla cheopis attacins against Yersinia pestis.

Antimicrobial peptide resistance has been proposed to play a major role in the flea-borne transmission of Yersinia pestis . However, the antimicrobial peptide response in fleas and their interaction with Y. pestis is largely unknown. Attacins are one of the most abundantly expressed antimicrobial peptides within the first hours after Y. pestis infection of Xenopsylla cheopis , a major vector of plague. In this study, we report the cloning, expression, and purification of two X. cheopis attacin peptides and describe their interactions with and antimicrobial activities against Y. pestis . These flea attacins were shown to bind lipopolysaccharides and have potent activity against Y. pestis , however the mechanism of killing does not involve extensive membrane damage. Treatment with attacins rapidly inhibits Y. pestis colony formation and results in oxidative stress, yet live-cell imaging revealed that bacteria continue to grow and divide for several hours in the presence of attacins before undergoing morphological changes and subsequent lysis. This data provides insights into an early battle between vector and pathogen that may impact transmission of one of the most virulent diseases known to man.

Yersinia pestis Lipopolysaccharide Remodeling Confers Resistance to a Xenopsylla cheopis Cecropin.

Fleas are major vectors of Yersinia pestis, the causative agent of plague. It has been proposed that Y. pestis has developed the ability to overcome the innate immune responses of fleas. Despite the fact that they transmit a number of bacterial infections, very little is known about the immune responses in fleas. In this study, we describe the antimicrobial activities of a cecropin from Xenopsylla cheopis (cheopin), an efficient vector for Y. pestis in the wild. This is the first cecropin-class antimicrobial peptide described from Siphonaptera insects. Cheopin showed potent activity against Gram-negative bacteria but little activity against wild-type Y. pestis KIM6+. Deletion of the aminoarabinose operon, which is responsible for the 4-amino-4-deoxy-l-arabinose (Ara4N) modification of LPS, rendered Y. pestis highly susceptible to cheopin. Confocal microscopy and whole cell binding assays indicated that Ara4N modification reduces the affinity of cheopin for Y. pestis. Further, cheopin only permeabilized bacterial membranes in the absence of Ara4N-modified LPS, which was correlated with bacterial killing. This study provides insights into innate immunity of the flea and evidence for the crucial role of Ara4N modification of Y. pestis LPS in conferring resistance against flea antimicrobial peptides.

Shipworm symbiosis ecology-guided discovery of an antibiotic that kills colistin-resistant Acinetobacter.

Teredinibacter turnerae is an intracellular bacterial symbiont in the gills of wood-eating shipworms, where it is proposed to use antibiotics to defend itself and its animal host. Several biosynthetic gene clusters are conserved in T. turnerae and their host shipworms around the world, implying that they encode defensive compounds. Here, we describe turnercyclamycins, lipopeptide antibiotics encoded in the genomes of all sequenced T. turnerae strains. Turnercyclamycins are bactericidal against challenging Gram-negative pathogens, including colistin-resistant Acinetobacter baumannii. Phenotypic screening identified the outer membrane as the likely target. Turnercyclamycins and colistin operate by similar cellular, although not necessarily molecular, mechanisms, but turnercyclamycins kill colistin-resistant A. baumannii, potentially filling an urgent clinical need. Thus, by exploring environments that select for the properties we require, we harvested the fruits of evolution to discover compounds with potential to target unmet health needs. Investigating the symbionts of shipworms is a powerful example of this principle.

Platelet Component False Positive Detection Rate in Aerobic and Anaerobic Primary Culture: A Systematic Review and Meta-Analysis.

Septic reactions from platelet transfusions are one of the leading causes of transfusion-associated mortality. The FDA guidance for platelet bacterial risk control includes bacterial culture using both aerobic and anaerobic bottles. Several studies have reported false positive rates (FPR) of culture, but these data have not been summarized or influencing factors analyzed. A systematic review and meta-analysis was performed according to published guidelines to assess the false positive rate and influencing factors. Eighteen studies were included for analysis. The combined aerobic/anaerobic FPR was 2.4 events per thousand (EPT) with a prediction interval of 0.5 to 5.7, while the aerobic FPR rate was 1.0 EPT (prediction interval: 0.2-2.2) and the anaerobic rate was 1.8 EPT. Estimates were based on a total of almost 5 million units tested. The rate of false positives due to instrument error was between 0.5-1.7 EPT, while it was between 0.3-1.0 EPT for sampling contamination based on whether only aerobic, anaerobic, or aerobic/anaerobic cultures were performed. The FPR is approximately 2 to 5 times higher than the literature reported true positive rate of 0.5 EPT.

Clinical Laboratory Perspective on Streptococcus halichoeri, an Unusual Nonhemolytic, Lancefield Group B Streptococcus Causing Human Infections.

Streptococcus halichoeri is a relatively newly identified species of pyogenic streptococci that causes zoonotic infection in humans. S. halichoeri was first described in 2004 as indigenous to seals, and only 8 reports of human S. halichoeri infection have been published. S. halichoeri grows as small, white, nonhemolytic colonies and may be strongly catalase-positive on routine blood agar media, which can lead to isolates being misidentified as coagulase-negative staphylococci. S. halichoeri tests positive for Lancefield group B antigen, like S. agalactiae, but can be identified with matrix-assisted laser desorption/ionization time of flight mass spectrometry or partial 16S rRNA sequencing. We describe 3 cases of S. halichoeri bone and joint infections in patients in the United States with underlying health conditions. In addition, we examine the microbiologic characteristics of S. halichoeri and discuss the importance of fully identifying this organism that might otherwise be disregarded as a skin commensal.

The incremental benefit of anaerobic culture for controlling bacterial risk in platelets: a systematic review and meta-analysis.

BACKGROUND AND OBJECTIVES: Septic transfusion reactions are a principal cause of transfusion-related mortality. The frequency of detectable bacterial contamination is greater in platelets compared to other blood components because platelets are stored at room temperature. Most strategies outlined in the September 2019 FDA guidance require both aerobic culture (AC) and anaerobic culture (AnC) testing. We performed a systematic review and meta-analysis in an effort to provide the best available estimate of the effectiveness of AnC. MATERIALS AND METHODS: Our analysis was performed according to published guidelines. Broad and context-specific meta-analyses of bacterial detection rates in platelets by AnC were performed to assess the practical effectiveness of AnC as a risk control measure. RESULTS: Seven studies with a total of 1 767 014 tested platelet components were included for analysis. With exclusion of positives due to Cutibacterium/Propionibacterium species and redundancy due to AC results, AnC detected 0·06 contamination events per thousand (EPT) components tested, twofold lower than the AC (0·12 EPT). CONCLUSION: Excluding Cutibacterium/Propionibacterium species, AnC detects occasional bacterial contamination events that are not detected by AC (~1 in 17 000 platelet components).

The comparative safety of bacterial risk control strategies for platelet components: a simulation study.

BACKGROUND: Bacterial contamination of platelets is a problem that can lead to harmful septic transfusion reactions. The US Food and Drug Administration published a guidance in September 2019 detailing several permissible risk control strategies. Our objective was to compare the safety of each bacterial testing strategy for apheresis platelets. STUDY DESIGN AND METHODS: We used simulation to compare safety of the nine risk control strategies involving apheresis platelet testing. The primary outcome was the risk of exposure. An exposure event occurred if a patient received platelets exceeding a specific contamination threshold (>0, 103 , and 105 colony-forming units (CFU/mL). We generated a range of bacterial contamination scenarios (inoculum size, doubling time, lag time) and compared risk of exposure for each policy in each contamination scenario. We then computed the average risk difference over all scenarios. RESULTS: At the 0 CFU/mL exposure threshold, two-step policies that used secondary culture ranked best (all top three), while single-step 24-hour culture with 3-day expiration ranked last (ninth). This latter policy performed well (median rank of 1) at both the 103 and 105 CFU/mL thresholds, but 48-hour culture with 7-day expiration performed relatively poorly. At these higher thresholds, median ranks of two-step policies that used secondary culture were again top three. Two-step policies that used rapid testing improved at the higher (105 CFU/mL) harm threshold, with median rankings between 1 and 5. CONCLUSION: Two-step policies that used secondary culture were generally safer than single-step policies. Performance of two-step policies that used rapid testing depended on the CFU per milliter threshold of exposure used.

Characterization of six clinical isolates of Chimaeribacter gen. nov., a novel genus related to the Yersiniaceae family and the three species Chimaeribacter arupi sp. nov., Chimaeribacter coloradensis sp. nov, and Chimaeribacter californicus sp. nov.

Eight genetically related, Gram-negative bacterial strains, isolated from clinical specimens between 2012 and 2016, were submitted to arup Laboratories for species identification. The lack of species- or genus-level matches in curated 16S rRNA gene databases prompted us to undertake the polyphasic characterization of these so far undescribed organisms. Six isolates available for additional testing were oxidase negative, catalase positive, pleomorphic, Gram-negative rods displaying temperature-dependent motility and producing yellow-pigmented colonies with three distinct morphotypes: medium-sized shiny, large mucoid and agar-pitting. Biochemical reactions and sugar fermentation patterns were most similar to members of the genus Serratia. Fatty acid profiles were highly similar across all six organisms, with the major components being: C16 : 0; C17 : 0 cyclo; C14 : 0 3-OH/iso-C16 : 1 I; C18 : 1 ω7c; and C16 : 1 ω7c/C16 : 1 ω6c. Whole-genome comparisons and multi locus sequence analysis (using the coding genes atpD, rpoB, gyrB and infB) suggest that the strains here described constitute three individual species within a novel genus related to the family Yersiniaceae. We propose for this novel taxon the name Chimaeribacter gen. nov., referring to the presentation of multiple characteristics typical of distinct Enterobacterales genera within a single organism. Four isolates are representative of a single species: Chimaeribacter arupi sp. nov (2016-Iso1, 2016-Iso2, type strain 2016-Iso3T=DSM 110101T=ATCC TSD-180T and 2013-Iso5). The remaining two isolates constitute the novel species Chimaeribacter coloradensis sp. nov. (type strain 2016-Iso4T=DSM 110102T=ATCC TSD-182T) and Chimaeribacter californicus sp. nov. (type strain 2015-Iso6T=DSM 110100T=ATCC TSD-181T). Our work provides the first formal characterization of the genus Chimaeribacter and forms the basis to study its taxonomic diversity.

Population dynamics of an Escherichia coli ST131 lineage during recurrent urinary tract infection.

Recurrent urinary tract infections (rUTIs) are extremely common, with ~ 25% of all women experiencing a recurrence within 1 year of their original infection. Escherichia coli ST131 is a globally dominant multidrug resistant clone associated with high rates of rUTI. Here, we show the dynamics of an ST131 population over a 5-year period from one elderly woman with rUTI since the 1970s. Using whole genome sequencing, we identify an indigenous clonal lineage (P1A) linked to rUTI and persistence in the fecal flora, providing compelling evidence of an intestinal reservoir of rUTI. We also show that the P1A lineage possesses substantial plasmid diversity, resulting in the coexistence of antibiotic resistant and sensitive intestinal isolates despite frequent treatment. Our longitudinal study provides a unique comprehensive genomic analysis of a clonal lineage within a single individual and suggests a population-wide resistance mechanism enabling rapid adaptation to fluctuating antibiotic exposure.

Multi-state study of Enterobacteriaceae harboring extended-spectrum beta-lactamase and carbapenemase genes in U.S. drinking water.

Community-associated acquisition of extended-spectrum beta-lactamase- (ESBL) and carbapenemase-producing Enterobacteriaceae has significantly increased in recent years, necessitating greater inquiry into potential exposure routes, including food and water sources. In high-income countries, drinking water is often neglected as a possible source of community exposure to antibiotic-resistant organisms. We screened coliform-positive tap water samples (n = 483) from public and private water systems in six states of the United States for blaCTX-M, blaSHV, blaTEM, blaKPC, blaNDM, and blaOXA-48-type genes by multiplex PCR. Positive samples were subcultured to isolate organisms harboring ESBL or carbapenemase genes. Thirty-one samples (6.4%) were positive for blaCTX-M, ESBL-type blaSHV or blaTEM, or blaOXA-48-type carbapenemase genes, including at least one positive sample from each state. ESBL and blaOXA-48-type Enterobacteriaceae isolates included E. coli, Kluyvera, Providencia, Klebsiella, and Citrobacter species. The blaOXA-48-type genes were also found in non-fermenting Gram-negative species, including Shewanella, Pseudomonas and Acinetobacter. Multiple isolates were phenotypically non-susceptible to third-generation cephalosporin or carbapenem antibiotics. These findings suggest that tap water in high income countries could serve as an important source of community exposure to ESBL and carbapenemase genes, and that these genes may be disseminated by non-Enterobacteriaceae that are not detected as part of standard microbiological water quality testing.