Use of Fourier-transform infrared spectroscopy for real-time outbreak investigation of OXA-48-producing Escherichia coli.

BACKGROUND: Efficient infection control during carbapenem-resistant Enterobacterales outbreaks demands rapid and simple techniques for outbreak investigations. WGS, the current gold standard for outbreak identification, is expensive, time-consuming and requires a high level of expertise. Fourier-transform infrared (FTIR) spectroscopy (IR Biotyper) is a rapid typing method based on infrared radiation applied to samples, which provides a highly specific absorption spectrum. OBJECTIVES: To investigate an outbreak of OXA-48-producing Escherichia coli in real-time using FTIR and subsequently compare the results with WGS. METHODS: Twenty-one isolates were collected during a nosocomial outbreak, and identification and antibiotic susceptibilities were confirmed by VITEK®2. FTIR was conducted for all isolates, and nine representative isolates were sequenced. RESULTS: FTIR was able to correctly determine the clonal relatedness of the isolates and to identify the outbreak cluster, as confirmed by WGS. By WGS, isolates in the main FTIR cluster belonged to the same MLST type and core-genome MLST type, and they harboured similar plasmids and resistance genes, whereas the singletons external to the FTIR cluster had different genetic content. CONCLUSIONS: FTIR can operate as a rapid, efficient and reliable first-line tool for outbreak investigations during a real-time ongoing E. coli outbreak, which can contribute to limiting the spread of pathogens.

Occurrence, Typing, and Resistance Genes of ESBL/AmpC-Producing Enterobacterales in Fresh Vegetables Purchased in Central Israel.

Beta-lactam resistance can lead to increased mortality, higher healthcare expenses, and limited therapeutic options. The primary mechanism of beta-lactam resistance is the production of extended-spectrum beta-lactamases (ESBL) and AmpC beta-lactamases. The spread of beta-lactamase-producing Enterobacterales via the food chain may create a resistance reservoir. The aims of this study were to determine the prevalence of ESBL/AmpC-producing Enterobacterales in vegetables, to examine the association between EBSL/AmpC-producing bacteria and types of vegetables, packaging, and markets, and to investigate the genetic features of ESBL-producing isolates. The antibiotic susceptibilities were determined using VITEK. Phenotypic ESBL/AmpC production was confirmed using disk diffusion. ESBL-producing isolates were subjected to Fourier-transform infrared (FT-IR) spectroscopy and to whole genome sequencing using Oxford Nanopore sequencing technology. Of the 301 vegetable samples, 20 (6.6%) were positive for ESBL producers (16 Klebsiella pneumoniae and 4 Escherichia coli), and 63 (20.9%) were positive for AmpC producers (56 Enterobacter cloacae complex, 4 Enterobacter aerogenes/cancerogenus, and 3 Pantoea spp., Aeromonas hydrophila, and Citrobacter braakii). The blaCTX-M and blaSHV genes were most common among ESBL-producing isolates. The beta-lactamase genes of the ESBL producers were mainly carried on plasmids. Multilocus sequence typing and FT-IR typing revealed high diversity among the ESBL producers. AmpC producers were significantly more common in leafy greens and ESBL producers were significantly less common in climbing vegetables. The presence of ESBL/AmpC-producing Enterobacterales in raw vegetables may contribute to the dissemination of resistance genes in the community.

Prevalence and Clinical Consequences of Colistin Heteroresistance and Evolution into Full Resistance in Carbapenem-Resistant Acinetobacter baumannii.

Colistin heteroresistance (HR) refers to a bacterial population comprised of several subpopulations with different levels of resistance to colistin. In this study, we discuss the classic form of HR, in which a resistant subpopulation exists within a predominantly susceptible population. We investigated the prevalence of colistin HR and its evolution into full resistance among 173 clinical carbapenem-resistant Acinetobacter baumannii isolates and examined the effect of HR on clinical outcomes. To determine HR, we performed population analysis profiling. Our results showed a high prevalence of HR (67.1%). To examine evolution of HR strains into full resistance, the HR strains were grown in colistin-containing broth, transferred onto colistin-containing plates, and colonies on these plates were transferred into colistin-free broth. Many of the HR strains (80.2%) evolved into full resistance, 17.2% reverted to HR, and 2.6% were borderline. We used logistic regression to compare 14-day clinical failure and 14-day mortality between patients infected by HR versus susceptible non-HR carbapenem-resistant A. baumannii. In the subgroup of patients with bacteremia, HR was significantly associated with 14-day mortality. IMPORTANCE To our knowledge, this is the first large-scale study to report on HR in Gram-negative bacteria. We described the prevalence of colistin HR in a large sample of carbapenem-resistant A. baumannii isolates, the evolution of many colistin HR isolates to a resistant phenotype following colistin exposure and withdrawal, and the clinical consequences of colistin HR. We found a high prevalence of HR among clinical carbapenem-resistant A. baumannii isolates; most evolved into a resistant phenotype following colistin exposure and withdrawal. In patients treated with colistin, evolution of HR A. baumannii into full resistance could lead to higher rates of treatment failure and contribute to the reservoir of colistin-resistant pathogens in health care settings.

Analysis of four carbapenem-resistant Acinetobacter baumannii outbreaks using Fourier-transform infrared spectroscopy.

We used Fourier-transform infrared (FTIR) spectroscopy to analyze 4 carbapenem-resistant Acinetobacter baumannii outbreaks. FTIR distinguished between isolates from different hospitals and uncovered the relatedness between isolates from acute-care hospitals and a post-acute-care hospital. Using higher cutoffs reveals more distant relationships and lower cutoffs support analyses of recent events.

OXA-900, a Novel OXA Sub-Family Carbapenemase Identified in Citrobacter freundii, Evades Detection by Commercial Molecular Diagnostics Tests.

Using whole-genome sequencing and cloning of the target gene, we identified blaOXA-900 carbapenemase, a novel blaOXA belonging to a distant and distinct sub-family of blaOXA-48-like. The plasmid-mediated gene was identified in a C. freundii isolate with elevated carbapenem MICs that evaded detection by commercial DNA-based methods. The novel gene, an OXA-48 family carbapenem-hydrolyzing class D ß-lactamase, OXA-900, likely originates from marine environmental Shewanella. Since this plasmid-mediated gene has entered a member of the Enterobacterales and evades detection by commonly used tests, it may gain wide dissemination among Enterobacterales.

Multiplex lateral flow immunochromatographic assay is an effective method to detect carbapenemases without risk of OXA-48-like cross reactivity.

BACKGROUND: It is essential to detect carriers of carbapenemase-producing Enterobacterales in order to implement infection control measures. The objectives of this study was to evaluate the NG-Test® CARBA 5 (CARBA 5) assay for detection of five carbapenemases and to assess the cross reactivity of other OXA-type carbapenemases with the OXA-48-like specific antibodies. METHODS: A total of 197 Enterobacterales isolates were tested. To evaluate the cross reactivity, 73 carbapenem-resistant A. baumannii, harboring OXA-type variants, were tested. Polymerase chain reaction (PCR) served as gold standard for carbapenemase identification. RESULTS: Excellent agreement was found between PCR and CARBA 5, for all but one isolate. The single false positive result (a blaSME positive S. marcescens isolate) was incorrectly positive for blaOXA-48 by CARBA 5. No cross reactivity was observed. The sensitivity and specificity were 100.0% and 98.0%, respectively. CONCLUSIONS: The CARBA 5 assay is highly sensitive and specific and is recommended as a tool for the detection of the main carbapenemases of interest in clinical microbiology laboratories.

Increased Capsule Thickness and Hypermotility Are Traits of Carbapenem-Resistant Acinetobacter baumannii ST3 Strains Causing Fulminant Infection.

BACKGROUND: Acinetobacter baumannii is a successful nosocomial pathogen, causing severe, life-threatening infections in hospitalized patients, including pneumonia and bloodstream infections. The spread of carbapenem-resistant Acinetobacter baumannii (CRAB) strains is a major health threat worldwide. The successful spread of CRAB is mostly due to its highly plastic genome. Although some virulence factors associated with CRAB have been uncovered, many mechanisms contributing to its success are not fully understood. METHODS: Here we describe strains of CRAB that were isolated from fulminant cases in 2 hospitals in Israel. These isolates show a rare hypermucoid (HM) phenotype and were investigated using phenotypic assays, comparative genomics, and an in vivo Galleria mellonella model. RESULTS: The 3 isolates belonged to the ST3 international clonal type and were closely related to each other, as shown by Fourier-transform infrared spectroscopy and phylogenetic analyses. These isolates possessed thickened capsules and a dense filamentous extracellular polysaccharides matrix as shown by transmission electron microscopy (TEM), and overexpressed the capsule polysaccharide synthesis pathway-related wzc gene. CONCLUSIONS: The HM isolates possessed a unique combination of virulence genes involved in iron metabolism, protein secretion, adherence, and membrane glycosylation. HM strains were more virulent than control strains in 2 G. mellonella infection models. In conclusion, our findings demonstrated several virulence factors, all present in 3 CRAB isolates with rare hypermucoid phenotypes.

Evaluation of the MICRONAUT MIC-strip colistin assay for colistin susceptibility testing of carbapenem-resistant Acinetobacter baumannii and Enterobacterales.

We sought to evaluate the MICRONAUT MIC-Strip colistin (MMS) assay, a commercial broth microdilution (BMD) panel, using 273 carbapenem-resistant Acinetobacter baumannii and Enterobacterales isolates. BMD was used as gold standard. MMS had 98.5% sensitivity and 99.5% specificity. All 37 isolates with MICs close to the breakpoint exhibited complete agreement.

Colistin Dependency among Colistin-Heteroresistant Acinetobacter baumannii Isolates.

Colistin dependent (CD) isolates are dependent on colistin for optimal growth. Here we aimed to systematically determine the emergence of CD among colistin-heteroresistant carbapenem-resistant Acinetobacter baumannii (CRAB) isolates. We also examined the phenotypic characteristics of CD and the evolution of CD strains to overt resistance. Additionally, we examined whether detection of growth in blood cultures was impaired by CD. Heteroresistant isolates, as determined by population analysis profiling, were exposed to colistin; when the colony count with colistin was significantly higher than without, isolates were suspected to be CD. CD was confirmed by Etest and growth curves. CD strains with colistin minimum inhibitory concentrations > 2 mg/L after growth in colistin-free media were considered colistin-resistant. Of the 65 heteroresistant strains tested, eight became CD after colistin exposure. These strains attained higher colony counts and growth rates with colistin vs. without, and grew adjacent to the colistin Etest strip. CD strains exhibited increased susceptibilities to multiple antibiotics compared to their parent heteroresistant strains. All CD strains tested became colistin-resistant following growth without colistin. CD strains were detected in blood culture bottles, but time to detection was significantly prolonged compared with parent strains, suggesting that CD may lead to delay in detection of CRAB bacteremia.

Rapid identification of capsulated Acinetobacter baumannii using a density-dependent gradient test.

BACKGROUND: Gram-negative bacterial capsules are associated with production of carbohydrates, frequently resulting in a mucoid phenotype. Infections caused by capsulated or mucoid A. baumannii are associated with increased clinical severity. Therefore, it is clinically and epidemiologically important to identify capsulated A. baumannii. Here, we describe a density-dependent gradient test to distinguish between capsulated and thin/non-capsulated A. baumannii. RESULTS: Thirty-one of 57 A. baumannii isolates displayed a mucoid phenotype. The density-dependent gradient test was comprised of two phases, with silica concentrations of 30% (top phase) and 50% (bottom phase). Twenty-three isolates migrated to the bottom phase, indicating thin or non-capsulated strains, and 34 migrated to the top phase, suggesting strains suspected to be capsulated. There was agreement between the mucoid and the non-mucoid phenotypes and the density-dependent gradient test for all but three isolates. Total carbohydrates extracted from strains suspected to be capsulated were significantly higher. Transmission electron microscopy confirmed the presence of a capsule in the six representative strains suspected to be capsulated. CONCLUSIONS: The density-dependent gradient test can be used to verify capsule presence in mucoid-appearing A. baumannii strains. Identifying capsulated strains can be useful for directing infection control measures to reduce the spread of hypervirulent strains.

Fourier Transform Infrared Spectroscopy Is a New Option for Outbreak Investigation: a Retrospective Analysis of an Extended-Spectrum-Beta-Lactamase-Producing Klebsiella pneumoniae Outbreak in a Neonatal Intensive Care Unit.

The IR Biotyper is a new automated typing system based on Fourier-transform infrared (FT-IR) spectroscopy that gives results within 4 h. We aimed (i) to use the IR Biotyper to retrospectively analyze an outbreak of extended-spectrum beta-lactamase-producing Klebsiella pneumoniae (ESBL-KP) in a neonatal intensive care unit and to compare results to BOX-PCR and whole-genome sequencing (WGS) results as the gold standard and (ii) to assess how the cutoff values used to define clusters affect the discriminatory power of the IR Biotyper. The sample consisted of 18 isolates from 14 patients. Specimens were analyzed in the IR Biotyper using the default analysis settings, and spectra were analyzed using OPUS 7.5 software. The software contains a feature that automatically proposes a cutoff value to define clusters; the cutoff value defines up to which distance the spectra are considered to be in the same cluster. Based on FT-IR, the outbreak represented 1 dominant clone, 1 secondary clone, and several unrelated clones. FT-IR results, using the cutoff value generated by the accompanying software after 4 replicates, were concordant with WGS for all but 1 isolate. BOX-PCR was underdiscriminatory compared to the other two methods. Using the cutoff value generated after 12 replicates, the results of FT-IR and WGS were completely concordant. The IR Biotyper can achieve the same typeability and discriminatory power as genome-based methods. However, to attain this high performance requires either previous, strain-dependent knowledge about the optimal technical parameters to be used or validation by a second method.

Performance of Rapid Polymyxin™ NP and Rapid Polymyxin™ Acinetobacter for the detection of polymyxin resistance in carbapenem-resistant Acinetobacter baumannii and Enterobacterales.

BACKGROUND: The global spread of carbapenem-resistant Enterobacterales (CRE) and Acinetobacter baumannii (CRAB) has prompted the reintroduction of colistin as a last-resort treatment. Although the recommended method for colistin susceptibility testing is broth microdilution (BMD), methods that are more rapid and easy to use are needed. OBJECTIVES: To evaluate the performance of two commercial kits for colistin susceptibility testing: Rapid Polymyxin™ NP (RP-NP) for CRE and Rapid Polymyxin™ Acinetobacter (RP-AB) for CRAB. METHODS: A total of 76 CRE and 87 CRAB isolates were collected from hospitalized patients in Europe and Israel. The isolates were subcultured twice on 5% sheep blood in tryptic soy agar. We tested colistin susceptibility using the RP-NP and RP-AB kits and compared the results with those from BMD. RESULTS: Of the CRE isolates, 25% (19/76) were resistant to colistin using BMD. Categorical agreement between RP-NP and BMD was 93.4% (71/76), major errors 1.8% (1/57) and very major errors 21.1% (4/19). Sensitivity was 78.9% and specificity was 98.2%. Of the CRAB isolates, 58.6% (51/87) were resistant to colistin by BMD. Categorical agreement between RP-AB and BMD was 59.8% (52/87), major errors 13.9% (5/36) and very major errors 58.8% (30/51). Sensitivity of RP-AB was 41.2% and specificity was 86.1%. CONCLUSIONS: In many of the tested isolates, weak or inconclusive colour changes in the test wells caused difficulty in interpretation, resulting in an unacceptable rate of very major errors.