RESUMEN
Antimicrobial resistance (AMR) is a public health threat where efficient surveillance is needed to prevent outbreaks. Existing methods for detection of gastrointestinal colonization of multidrug-resistant organisms (MDRO) are limited to specific organisms or resistance mechanisms. Metagenomic next-generation sequencing (mNGS) is a more rapid and agnostic diagnostic approach for microbiome and resistome investigations. We determined if mNGS can detect MDRO from rectal swabs in concordance with standard microbiology results. We performed and compared mNGS performance on short-read Illumina MiSeq (N = 10) and long-read Nanopore MinION (N = 5) platforms directly from rectal swabs to detect vancomycin-resistant enterococci (VRE) and carbapenem-resistant Gram-negative organisms (CRO). We detected Enterococcus faecium (N = 8) and Enterococcus faecalis (N = 2) with associated van genes (9/10) in concordance with VRE culture-based results. We studied the microbiome and identified CRO, Pseudomonas aeruginosa (N = 1), Enterobacter cloacae (N = 1), and KPC-producing Klebsiella pneumoniae (N = 1). Nanopore real-time analysis detected the blaKPC gene in 2.3 min and provided genetic context (blaKPC harbored on pKPC_Kp46 IncF plasmid). Illumina sequencing provided accurate allelic variant determination (i.e., blaKPC-2) and strain typing of the K. pneumoniae (ST-15). We demonstrated an agnostic approach for surveillance of MDRO, examining advantages of both short- and long-read mNGS methods for AMR detection.
Asunto(s)
Carbapenémicos , Farmacorresistencia Bacteriana , Bacterias Gramnegativas/genética , Infecciones por Bacterias Gramnegativas/diagnóstico , Enterococos Resistentes a la Vancomicina/genética , Genoma Bacteriano , Infecciones por Bacterias Gramnegativas/microbiología , Humanos , Metagenómica , Recto/microbiologíaRESUMEN
Sepsis remains one of the most lethal and costly conditions treated in U.S. hospitals, with approximately 50% of cases caused by Gram-negative bacterial infections. Septic shock is induced when lipopolysaccharide (LPS), the main component of Gram-negative outer bacterial membrane, signals through the Toll-like receptor 4 (TLR4) complex. Lethal endotoxemia, a model for septic shock, was induced in WT C57BL6 and TLR4-/- mice by administration of Escherichia coli LPS. WT LPS treated mice showed high morbidity, while PBS treated LPS and treated TLR4-/- mice did not. ANOVA analysis of label-free quantification of longitudinal serum proteome revealed 182 out of 324 proteins in LPS injected WT mice that were significantly changed across four time points (0, 6, 12, and 18 h). No significant changes were identified in the two control groups. From the 182 identified proteins, examples of known sepsis biomarkers were validated by ELISA, which showed similar trends as MS proteomics data. Longitudinal analysis within individual mice produced 3-fold more significantly changed proteins than pair-wise comparison. A subsequent global analysis of WT and TLR4-/- mice identified pathways activated independent of TLR4. These pathways represent possible compensatory mechanisms that allow for control of Gram-negative bacterial infection regardless of host immune status.
Asunto(s)
Sepsis , Choque Séptico , Animales , Lipopolisacáridos/toxicidad , Ratones , Proteómica , Sepsis/genética , Receptor Toll-Like 4/genéticaRESUMEN
Infectious diseases have a substantial global health impact. Clinicians need rapid and accurate diagnoses of infections to direct patient treatment and improve antibiotic stewardship. Current technologies employed in routine diagnostics are based on bacterial culture followed by morphological trait differentiation and biochemical testing, which can be time-consuming and labor-intensive. With advances in mass spectrometry (MS) for clinical diagnostics, the U.S. Food and Drug Administration has approved two microbial identification platforms based on matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS analysis of microbial proteins. We recently reported a novel and complementary approach by comparing MALDI-TOF mass spectra of microbial membrane lipid fingerprints to identify ESKAPE pathogens. However, this lipid-based approach used a sample preparation method that required more than a working day from sample collection to identification. Here, we report a new method that extracts lipids efficiently and rapidly from microbial membranes using an aqueous sodium acetate (SA) buffer that can be used to identify clinically relevant Gram-positive and -negative pathogens and fungal species in less than an hour. The SA method also has the ability to differentiate antibiotic-susceptible and antibiotic-resistant strains, directly identify microbes from biological specimens, and detect multiple pathogens in a mixed sample. These results should have positive implications for the manner in which bacteria and fungi are identified in general hospital settings and intensive care units.
Asunto(s)
Bacterias/aislamiento & purificación , Candida albicans/aislamiento & purificación , Farmacorresistencia Microbiana , Lípidos de la Membrana/orina , Microextracción en Fase Sólida/métodos , Bacterias/química , Candida albicans/química , Humanos , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodosRESUMEN
The optimal method to screen for gastrointestinal colonization with carbapenem-resistant organisms (CRO) has yet to be established. The direct MacConkey (direct MAC) plate method demonstrates high sensitivity for CRO detection, but established zone diameter (ZD) criteria for ertapenem (≤27 mm) and meropenem (≤32 mm) result in high rates of false positives upon confirmatory testing. To increase specificity, we screened for CRO in two high-risk wards using the direct MAC plate method, recorded ZDs for each sample, and generated receiver operating characteristic (ROC) curves to evaluate the optimal ZD cutoff criteria. Of 6,868 swabs obtained over an 18-month period, 4,766 (69%) had growth on MAC plates, and 2,500 (36%) met criteria for further evaluation based on previously established ZDs around the carbapenem disks. A total of 812 (12%) swabs were confirmed positive for at least one CRO and included 213 (3%) carbapenemase-producing organisms (CPO), resulting in a specificity of 78% for the direct MAC plate method. Reducing the ertapenem and meropenem ZDs to ≤25 mm improved specificity to 83%, decreasing the confirmatory testing workload by 32%. The sensitivities with the lower ZD criteria were 89% for CRO and 94% for CPO, respectively. The direct MAC plate method criteria for CRO testing can be modified to balance the sensitivity and specificity of CRO while reducing the burden on clinical microbiology laboratories. These modifications can be particularly helpful in regions with a low CRO prevalence.
Asunto(s)
Antibacterianos/farmacología , Carbapenémicos/farmacología , Pruebas Antimicrobianas de Difusión por Disco/métodos , Bacterias Gramnegativas/efectos de los fármacos , Recto/microbiología , Resistencia betalactámica , Ertapenem/farmacología , Bacterias Gramnegativas/aislamiento & purificación , Infecciones por Bacterias Gramnegativas/microbiología , Humanos , Meropenem/farmacología , Curva ROC , Sensibilidad y EspecificidadRESUMEN
Acinetobacter baumannii is a prevalent nosocomial pathogen with a high incidence of multidrug resistance. Treatment of infections due to this organism with colistin, a last-resort antibiotic of the polymyxin class, can result in the emergence of colistin-resistant strains. Colistin resistance primarily occurs via modifications of the terminal phosphate moieties of lipopolysaccharide-derived lipid A, which reduces overall membrane electronegativity. These modifications are readily identified by mass spectrometry (MS). In this study, we prospectively collected Acinetobacter baumannii complex clinical isolates from a hospital system in Pennsylvania over a 3-year period. All isolates were evaluated for colistin resistance using standard MIC testing by both agar dilution and broth microdilution, as well as genospecies identification and lipid A profiling using MS analyses. Overall, an excellent correlation between colistin susceptibility and resistance, determined by MIC testing, and the presence of a lipid A modification, determined by MS, was observed with a sensitivity of 92.9% and a specificity of 94.0%. Additionally, glycolipid profiling was able to differentiate A. baumannii complex organisms based on their membrane lipids. With the growth of MS use in clinical laboratories, a reliable MS-based glycolipid phenotyping method that identifies colistin resistance in A. baumannii complex clinical isolates, as well as other Gram-negative organisms, represents an alternative or complementary approach to existing diagnostics.
Asunto(s)
Acinetobacter baumannii/efectos de los fármacos , Membrana Celular/química , Colistina/farmacología , Glucolípidos/química , Espectrometría de Masas , Antibacterianos/farmacología , Farmacorresistencia Bacteriana , Pruebas de Sensibilidad Microbiana , Estudios ProspectivosRESUMEN
Infectious diseases propagated by arthropod vectors, such as tularemia, are commonly initiated via dermal infection of the skin. However, due to the technical difficulties in achieving accurate and reproducible dermal deposition, intradermal models are less commonly used. To overcome these limitations, we used microneedle arrays (MNAs), which are micron-scale polymeric structures, to temporarily disrupt the barrier function of the skin and deliver a bacterial inoculum directly to the dermis of an animal. MNAs increase reliability by eliminating leakage of the inoculum or blood from the injection site, thereby providing a biologically relevant model for arthropod-initiated disease. Here, we validate the use of MNAs as a means to induce intradermal infection using a murine model of tularemia initiated by Francisella novicida We demonstrate targeted delivery of the MNA bolus to the dermal layer of the skin, which subsequently led to innate immune cell infiltration. Additionally, F. novicida-coated MNAs were used to achieve lethality in a dose-dependent manner in C57BL/6 mice. The immune profile of infected mice mirrored that of established F. novicida infection models, consisting of markedly increased serum levels of interleukin-6 and keratinocyte chemoattractant, splenic T-cell depletion, and an increase in splenic granulocytes, together confirming that MNAs can be used to reproducibly induce tularemia-like pathogenesis in mice. When MNAs were used to immunize mice using an attenuated F. novicida mutant (F. novicida ΔlpxD1), all immunized mice survived a lethal subcutaneous challenge. Thus, MNAs can be used to effectively deliver viable bacteria in vivo and provide a novel avenue to study intradermally induced microbial diseases in animal models.
Asunto(s)
Francisella/patogenicidad , Inyecciones Intradérmicas/instrumentación , Agujas , Piel/microbiología , Tularemia/prevención & control , Animales , Anticuerpos Antibacterianos/sangre , Vacunas Bacterianas/administración & dosificación , Vacunas Bacterianas/inmunología , Dermis/inmunología , Dermis/microbiología , Modelos Animales de Enfermedad , Femenino , Inmunización/instrumentación , Inmunización/métodos , Interleucina-6/sangre , Ratones , Ratones Endogámicos C57BL , Mutación , Reproducibilidad de los Resultados , Piel/inmunología , Bazo/inmunología , Tularemia/inmunología , Vacunas Atenuadas/administración & dosificación , Vacunas Atenuadas/inmunologíaRESUMEN
Objectives: In this study, we characterize a concurrent disseminated infection with a virulent hypermucoviscous (HMV) Klebsiella pneumoniae and an OXA-181-producing XDR K. pneumoniae from a patient with recent hospitalization in India. During exposure to meropenem therapy, the highly susceptible HMV K. pneumoniae became resistant to carbapenems, consistent with the acquisition of blaOXA-181. Methods: Twelve K. pneumoniae isolates were recovered from the patient and the hospital room environment over a 3 month hospitalization. Phenotypic and molecular studies were completed to characterize the isolates. Oxford Nanopore and Illumina MiSeq WGS were performed to study phylogeny (MLST and SNPs), plasmids and virulence genes and demonstrate changes in the organism's resistome that occurred over time. Results: WGS revealed that the HMV K. pneumoniae belonged to ST23 and harboured an IncH1B virulence plasmid, while the XDR K. pneumoniae belonged to ST147 and possessed two MDR plasmids (IncR and IncFII), the blaOXA-181-bearing ColKP3 plasmid and chromosomal mutations conferring the XDR phenotype. Sequential isolates demonstrated plasmid diversification (fusion of the IncR and IncFII plasmids), mobilization of resistance elements (ompK35 inactivation by ISEcp1-blaCTX-M-15 mobilization, varying numbers of resistance genes on plasmid scaffolds) and chromosomal mutations (mutations in mgrB) leading to further antibiotic resistance that coincided with antibiotic pressure. Importantly, the HMV strain in this study was unable to preserve the carbapenem-resistant phenotype without the selective pressure of meropenem. Conclusions: To the best of our knowledge, we are the first to report a carbapenem-resistant HMV K. pneumoniae strain in the USA. Ultimately, this case demonstrates the role of antibiotic pressure in the acquisition and loss of important genetic elements.
Asunto(s)
Antibacterianos/farmacología , Proteínas Bacterianas/genética , Farmacorresistencia Bacteriana Múltiple/genética , Klebsiella pneumoniae/efectos de los fármacos , Klebsiella pneumoniae/genética , Adulto , Técnicas de Tipificación Bacteriana , Genoma Bacteriano , Hospitalización , Humanos , India , Infecciones por Klebsiella/tratamiento farmacológico , Klebsiella pneumoniae/patogenicidad , Masculino , Meropenem/farmacología , Pruebas de Sensibilidad Microbiana , Tipificación de Secuencias Multilocus , Plásmidos , Viaje , Resultado del Tratamiento , Estados Unidos , Virulencia , beta-Lactamasas/genéticaRESUMEN
Accurate detection of carbapenemase-producing glucose-nonfermenting Gram-negative bacilli (CPNFs), including Pseudomonas aeruginosa and Acinetobacter baumannii, is necessary to prevent their dissemination within health care settings. We performed a method comparison study of 11 phenotypic carbapenemase detection assays to evaluate their accuracy for the detection of CPNFs. A total of 96 carbapenem-resistant glucose-nonfermenting isolates were included, of which 29% produced carbapenemases. All CPNFs were molecularly characterized to identify ß-lactamase genes. A total of 86% of the carbapenemase-producing P. aeruginosa isolates produced class B carbapenemases. Several assays performed with a sensitivity of >90% for the detection of carbapenemase-producing P. aeruginosa, including all rapid chromogenic assays and the modified carbapenem inactivation method. Most included assays, with the exception of the Manual Blue Carba assay, the Modified Carba NP assay, the boronic acid synergy test, and the metallo-ß-lactamase Etest, had specificities of >90% for detecting carbapenemase-producing P. aeruginosa Class D carbapenemases were the most prevalent carbapenemases among the carbapenemase-producing A. baumannii strains, with 60% of the carbapenemase-producing A. baumannii isolates producing acquired OXA-type carbapenemases. Although several assays achieved >90% specificity in identifying carbapenemase-producing A. baumannii, no assays achieved a sensitivity of greater than 90%. Our findings suggest that the available phenotypic tests generally appear to have excellent sensitivity and specificity for detecting carbapenemase-producing P. aeruginosa isolates. However, further modifications to existing assays or novel assays may be necessary to accurately detect carbapenemase-producing A. baumannii.
Asunto(s)
Acinetobacter baumannii/enzimología , Proteínas Bacterianas/análisis , Pseudomonas aeruginosa/enzimología , beta-Lactamasas/análisis , beta-Lactamasas/genética , Acinetobacter baumannii/efectos de los fármacos , Acinetobacter baumannii/genética , Acinetobacter baumannii/aislamiento & purificación , Proteínas Bacterianas/genética , Glucosa/metabolismo , Humanos , Pseudomonas aeruginosa/efectos de los fármacos , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/aislamiento & purificación , Resistencia betalactámica/genéticaRESUMEN
Early identification of carbapenemase-producing Enterobacteriaceae (CPE) is essential to prevent their dissemination within health care settings. Our objective was to evaluate the accuracy of 11 phenotypic assays for the detection of CPE. Two collections of carbapenem-resistant Enterobacteriaceae (CRE) isolates were evaluated, including 191 retrospective isolates (122 CP-CRE and 69 non-CP isolates) as well as 45 prospective clinical isolates (15 CP-CRE and 30 non-CP-CRE) obtained over a 3-month period. The sensitivity and specificity of each test was determined, with molecular genotype serving as the gold standard. Among the retrospective cohort, sensitivities ranged from 72% for the boronic acid synergy test for the detection of KPC producers to ≥98% for the modified Carba NP, the Rapidec Carba NP, the manual Blue Carba, and the modified carbapenem inactivation method for the detection of any CPE. Sensitivity differed among tests across enzyme classes. All assays had excellent specificity exceeding 95%, with the exception of the boronic acid synergy test (88%) and modified Hodge test (91%). Prospectively, 45 CRE isolates were encountered over a 3-month period, including 15 CPE (33%) and 30 non-CP-CRE (67%). Results from the prospective cohort were similar. However, a decrease in specificity was observed across most tests, likely due to restricted inclusion of non-CP-CRE to assess the specificity of the assays. Overall, accuracy of CPE detection varied across phenotypic tests. Local epidemiology of CP genotypes, turnaround time, and ease of incorporation into the laboratory workflow should be considered when selecting a phenotypic assay for clinical use.
Asunto(s)
Proteínas Bacterianas/análisis , Técnicas Bacteriológicas/métodos , Enterobacteriaceae/enzimología , Enterobacteriaceae/aislamiento & purificación , beta-Lactamasas/análisis , Humanos , Estudios Prospectivos , Estudios Retrospectivos , Sensibilidad y EspecificidadRESUMEN
Carbapenemase-producing (CP) Enterobacteriaceae are largely responsible for the rapid spread of carbapenem-resistant Enterobacteriaceae (CRE). Distinguishing CP-CRE from non-CP-CRE has important infection control implications. In a cohort of 198 CRE isolates, for isolates that remained susceptible or intermediate to some carbapenem antibiotics, an ertapenem MIC of 0.5 µg/ml and meropenem, imipenem, and doripenem MICs of 2 µg/ml were best able to distinguish CP-CRE from non-CP-CRE isolates.
Asunto(s)
Proteínas Bacterianas/metabolismo , Carbapenémicos/farmacología , Enterobacteriaceae/efectos de los fármacos , Pruebas de Sensibilidad Microbiana/métodos , beta-Lactamasas/metabolismo , Farmacorresistencia Bacteriana/efectos de los fármacos , Enterobacteriaceae/aislamiento & purificación , Enterobacteriaceae/metabolismo , Humanos , Curva ROCRESUMEN
Rectal swabs from high-risk patients were screened for carbapenem-resistant organisms (CROs) using several methods. The direct MacConkey plate method was the most sensitive for CROs (95%), while chromID CARBA and the Check-Direct CPE screen assay were the most sensitive for the detection of carbapenemase-producing organisms (CPOs) (100%; all blaKPC). All methods had a specificity of >90% for CROs, and for CPOs, the specificity ranged from 85 to 98%. Broth enrichment methods performed poorly compared to direct inoculation methods, negating the need for the broth enrichment step.
Asunto(s)
Técnicas Bacteriológicas/métodos , Carbapenémicos/farmacología , Bacterias Gramnegativas/efectos de los fármacos , Bacterias Gramnegativas/aislamiento & purificación , Infecciones por Bacterias Gramnegativas/diagnóstico , Recto/microbiología , Resistencia betalactámica , Proteínas Bacterianas/análisis , Infecciones por Bacterias Gramnegativas/microbiología , Humanos , Sensibilidad y Especificidad , beta-Lactamasas/análisisRESUMEN
The activity of antimicrobial peptides (AMPs) has significant bacterial species bias, the mechanisms of which are not fully understood. We employed single-molecule tracking to measure the affinity of three different AMPs to hybrid supported bilayers composed of lipid A extracted from four different Gram negative bacteria and observed a strong empirical anticorrelation between the affinity of a particular AMP to a given lipid A layer and the activity of that AMP towards the bacterium from which that lipid A was extracted. This suggested that the species bias of AMP activity is directly related to AMP interactions with bacterial outer membranes, despite the fact that the mechanism of antimicrobial activity occurs at the inner membrane. The trend also suggested that the interactions between AMPs and the outer membrane lipid A (even in the absence of other components, such as lipopolysaccharides) capture effects that are relevant to the minimum inhibitory concentration.