Zophobas morio larvae as a novel model for the study of Acinetobacter virulence and antimicrobial resistance.

The use of mammalian models for in vivo testing of bacterial virulence raises ethical concerns and is expensive and time-consuming. As an alternative, non-mammalian models are sought. Galleria mellonella larvae have been used as a model to study several bacterial pathogens. However, their maintenance is challenging, and commercial supply is low. In this study, we aimed to establish the Zophobas morio larvae as an alternative non-mammalian model for the evaluation of the pathogenicity and antimicrobial susceptibility of Acinetobacter baumannii. We infected Z. morio with Acinetobacter strains and determined the optimal temperature and inoculum. To visualize the bacterial distribution within the larvae, hematoxylin and eosin (H&E) staining was performed. Next, a survival model of infected larvae was established, and virulence was compared between strains. The effect of antimicrobial treatment in relation to antibiotic susceptibility was studied. Our results demonstrate that Z. morio can be used as a model system for in vivo studies of A. baumannii.

Carbapenem-resistant Acinetobacter baumannii carrier detection: a simple and efficient protocol.

Timely detection of carbapenem-resistant Acinetobacter baumannii (CRAB) carriers is essential to direct infection control measures. In this work, we aimed to develop a practical protocol to detect CRAB from screening samples. To choose a selective medium that detects CRAB with high sensitivity and specificity, 111 A. baumannii clinical isolates were inoculated on three types of agar: mSuperCARBA (SC), CHROMagar Acinetobacter (CaA), and modified CHROMagar Acinetobacter (mCaA) containing 4.5 mg/mL meropenem. SC was non-selective, CaA was the most sensitive (100%), but only moderately specific (72%), and mCaA was highly specific (97%) and sensitive (98%). Confirmation of the carbapenem-resistant phenotype using PCR-based detection of blaOXA-23, blaOXA-24, and blaOXA-58 genes was specific but not sensitive, detecting only 58% of CRAB isolates. Identification of A. baumannii using either gyrB or blaOXA-51 PCR was excellent. Next, we used the same methodology in routine screening for CRAB carriage. mCaA had the best yield, with high sensitivity but moderate specificity to differentiate between CRAB and other carbapenem-resistant organisms. Skin sampling using sponges and 6 hour enrichment was highly sensitive (98%), while other body sites had poor sensitivity (27%- 41%). Shorter incubation had slightly lower yield, and longer incubation did not improve the detection. Performing PCR for blaOXA-51 and gyrB on colonies growing on modified mCaA differentiated between CRAB and other species with high accuracy (98% and 99%, respectively). Based on our results, we present a procedure for easy and reliable detection of CRAB carriage using skin sampling, short enrichment, selection on mCaA, and PCR-based identification. IMPORTANCE: Carbapenem-resistant Acinetobacter baumannii (CRAB) is a substantial cause of nosocomial infections, classified among the most significant multidrug-resistant pathogens by the World Health Organization and by the US Centers for Disease Control. Limiting the spread of CRAB is an important goal of infection control, but laboratory methods for identification of CRAB carriers are not standardized. In this work, we compared different selective agar plates, tested the efficiency of A. baumannii identification by PCR for species-specific genes, and used PCR-based detection of common resistance genes to confirm the carbapenem-resistant phenotype. During a prospective study, we also determined the optimal sample enrichment time. Based on our results, we propose a simple and efficient protocol for the detection of CRAB carriage using skin sampling, short enrichment, selection on appropriate agar plates, and PCR-based identification, resulting in a turn-around time of 24 hours.

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.

Phenotypic and Genomic Characterization of Nine String-Positive Carbapenem-Resistant Acinetobacter baumannii Isolates from Israel.

A positive "string test" indicates the ability of bacterial colonies grown on agar plates to form viscous strings of >5 mm when stretched. This phenotype is strongly associated with hypervirulence in Klebsiella pneumoniae but has never been described in carbapenem-resistant Acinetobacter baumannii (CRAB), an emerging human pathogen of high clinical significance. In this work, we screened 1,000 CRAB isolates, among which we identified and characterized 9 string-positive CRAB (stCRAB) isolates. Phenotypic and genotypic analyses revealed that the isolates were not phylogenetically related and possessed different antibiotic resistance and virulence profiles. Transmission electron microscopy (TEM) showed the presence of capsule in string-positive isolates. String-positive isolates were more motile but did not form more biofilm than non-string-positive isolates. They were less virulent in a murine thigh fitness model and a Galleria mellonella survival assay. In conclusion, here, we describe string-positive A. baumannii isolates and their phenotypic and molecular characteristics. We found that unlike K. pneumoniae, stCRAB isolates were not associated with increased virulence. IMPORTANCE Acinetobacter baumannii has been considered a major health care threat in recent years. Despite many efforts, the pathogenesis and molecular mechanism of A. baumannii virulence remain poorly understood. Moreover, the plasticity of its genome frequently gives rise to new and more virulent isolates. Our current study is of significant importance as it concerns a previously undescribed A. baumannii phenotype. The string-positive phenotype is strongly associated with increased fitness and virulence in other Gram-negative bacteria such as K. pneumoniae. Although no clear correlation with virulence or fitness was found in our 9 stCRAB isolates, this could have been due to the limited statistical power of our research. We suggest that this phenotype should be taken into consideration as due to its genome plasticity, the next change can give rise to string-positive and hypervirulent strains, as is known for K. pneumoniae. Additional future research is needed regarding its possible consequences.

In vivo fitness of carbapenem-resistant Acinetobacter baumannii strains in murine infection is associated with treatment failure in human infections.

OBJECTIVES: Mortality among patients with carbapenem-resistant Acinetobacter baumannii (CRAB) infections varies between studies. We examined whether in vivo fitness of CRAB strains is associated with clinical outcomes in patients with CRAB infections. METHODS: Isolates were collected from patients enrolled in the AIDA trial with hospital-acquired pneumonia, bloodstream infections and/or urinary tract infections caused by CRAB. The primary outcome was 14-day clinical failure, defined as failure to meet all criteria: alive; haemodynamically stable; improved or stable Sequential Organ Failure Assessment (SOFA) score; improved or stable oxygenation; and microbiological cure of bacteraemia. The secondary outcome was 14-day mortality. We tested in vivo growth using a neutropenic murine thigh infection model. Fitness was defined based on the CFU count 24 hours after injection of an inoculum of 105 CFU. We used mixed-effects logistic regression to test the association between fitness and the two outcomes. RESULTS: The sample included 266 patients; 215 (80.8%) experienced clinical failure. CRAB fitness ranged from 5.23 to 10.08 log CFU/g. The odds of clinical failure increased by 62% for every 1-log CFU/g increase in fitness (OR 1.62, 95% CI 1.04-2.52). After adjusting for age, Charlson score, SOFA score and acquisition in the intensive care unit, fitness remained significant (adjusted OR 1.63, 95% CI 1.03-2.59). CRAB fitness had a similar effect on 14-day mortailty, although the association was not statistically significant (OR 1.56, 95% CI 0.95-2.57). It became significant after adjusting for age, Charlson score, SOFA score and recent surgery (adjusted OR 1.88, 95% CI 1.09-3.25). CONCLUSIONS: In vivo CRAB fitness was associated with clinical failure in patients with CRAB infection.

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.