Carbapenem-resistant Acinetobacter Baumannii Isolates Research Articles

Induced Heteroresistance in Carbapenem-Resistant Acinetobacter baumannii (CRAB) via Exposure to Human Pleural Fluid (HPF) and Its Impact on Cefiderocol Susceptibility.

Infections caused by Carbapenem-resistant Acinetobacter baumannii (CRAB) isolates, such as hospital-acquired pneumonia (HAP), bacteremia, and skin and soft tissue infections, among others, are particularly challenging to treat. Cefiderocol, a chlorocatechol-substituted siderophore antibiotic, was approved by the U.S. Food and Drug Administration (FDA) in 2019 and prescribed for the treatment of CRAB infections. Despite the initial positive treatment outcomes with this antimicrobial, recent studies reported a higher-than-average all-cause mortality rate in patients treated with cefiderocol compared to the best available therapy. The cause(s) behind these outcomes remains unconfirmed. A plausible hypothesis is heteroresistance, a phenotype characterized by the survival of a small proportion of cells in a population that is seemingly isogenic. Recent results have demonstrated that the addition of human fluids to CRAB cultures leads to cefiderocol heteroresistance. Here, we describe the molecular and phenotypic analyses of CRAB heteroresistant bacterial subpopulations to better understand the nature of the less-than-expected successful outcomes after cefiderocol treatment. Isolation of heteroresistant variants of the CRAB strain AMA40 was carried out in cultures supplemented with cefiderocol and human pleural fluid (HPF). Two AMA40 variants, AMA40 IHC1 and IHC2, were resistant to cefiderocol. To identify mutations and gene expression changes associated with cefiderocol heteroresistance, we subjected these variants to whole genome sequencing and global transcriptional analysis. We then assessed the impact of these mutations on the pharmacodynamic activity of cefiderocol via susceptibility testing, EDTA and boronic acid inhibition analysis, biofilm formation, and static time-kill assays. Heteroresistant variants AMA40 IHC1 and AMA40 IHC2 have 53 chromosomal mutations, of which 40 are common to both strains. None of the mutations occurred in genes associated with high affinity iron-uptake systems or β-lactam resistance. However, transcriptional analyses demonstrated significant modifications in levels of expression of genes associated with iron-uptake systems or β-lactam resistance. The blaNDM-1 and blaADC-2, as well as various iron-uptake system genes, were expressed at higher levels than the parental strain. On the other hand, the carO and ompA genes' expression was reduced. One of the mutations common to both heteroresistant strains was mapped within ppiA, a gene associated with iron homeostasis in other species. Static time-kill assays demonstrated that supplementing cation-adjusted Mueller-Hinton broth with human serum albumin (HAS), the main protein component of HPF, considerably reduced cefiderocol killing activity for all three strains tested. Notably, collateral resistance to amikacin was observed in both variants. We conclude that exposing CRAB to fluids with high HSA concentrations facilitates the rise of heteroresistance associated with point mutations and transcriptional upregulation of genes coding for β-lactamases and biofilm formation. The findings from this study hold significant implications for understanding the emergence of CRAB resistance mechanisms against cefiderocol treatment. This understanding is vital for the development of treatment guidelines that can effectively address the challenges posed by CRAB infections.

Prevalence of Carbapenemases in Carbapenem-Resistant Acinetobacter baumannii Isolates from the Kingdom of Bahrain.

Acinetobacter baumannii is regarded as a significant cause of death in hospitals. The WHO recently added carbapenem-resistant Acinetobacter baumannii (CRAB) to its global pathogen priority list. There is a dearth of information on CRAB from our region. Fifty CRAB isolates were collected from four main hospitals in Bahrain for this study. Bacterial identification and antibiotic susceptibility tests were carried out using the BD PhoenixTM and VITEK-2 compact, respectively. Using conventional PCR, these isolates were further screened for carbapenem resistance markers (blaOXA-51, blaOXA-23, blaOXA-24, blaOXA-40, blaIMP, blaNDM, blaVIM, and blaKPC). All of the isolates were resistant to imipenem (100%), meropenem (98%), and cephalosporins (96-98%), followed by other commonly used antibiotics. All these isolates were least resistant to gentamicin (64%). The detection of resistance determinants showed that the majority harbored blaOXA-51 (100%) and blaIMP (94%), followed by blaOXA-23 (82%), blaOXA-24 (46%), blaOXA-40 (14%), blaNDM (6%), blaVIM (2%), and blaKPC (2%). The study isolates showed a high level of antibiotic resistance. Class D carbapenemases were more prevalent in our CRAB isolate collection. The resistance genes were found in various combinations. This study emphasizes the importance of strengthening surveillance and stringent infection control measures in clinical settings to prevent the emergence and further spread of such isolates.

In Vitro Activity of Cefiderocol against a Global Collection of Carbapenem-Resistant Acinetobacter baumannii Isolates.

Background: Cefiderocol is a novel siderophore cephalosporin with potent activity against multi-drug-resistant Gram-negative pathogens including carbapenem-resistant Acinetobacter baumannii (CRAB). Methods: The susceptibility of 313 non-duplicate CRAB isolates with defined carbapenem resistance mechanisms from a global collection to cefiderocol, ceftazidime, ceftazidime/avibactam, ceftolozane/tazobactam, ciprofloxacin, colistin, imipenem/relebactam, meropenem, meropenem/vaborbactam, minocycline, and piperacillin/tazobactam was determined using the broth microdilution method. Isolates were obtained from various body sites from patients in 47 countries in five world regions between 2012 and 2016. The identification of carbapenem resistance mechanisms and assignment to A. baumannii international clonal lineages were based on whole genome sequencing. Results: Cefiderocol showed greater activity than comparator antimicrobials of the β-lactam class, including novel β-lactams combined with β-lactamase inhibitors, ciprofloxacin, and minocycline. Cefiderocol MIC50 and MIC90 values were 0.5 mg/L and 4 mg/L, respectively, while colistin had comparable activity with a higher MIC50 at 1 mg/L and a lower MIC90 value of 2 mg/L. Many isolates with elevated cefiderocol MICs ≥ 4 mg/L represented A. baumannii international clone (IC) 1 and harbored a metallo-β-lactamase. Conclusions: While cefiderocol is a useful addition to the limited armamentarium of drugs targeting this problematic pathogen, a considerable part of CRAB isolates had elevated MIC values in a range of 4 -> 32 mg/L, including all isolates with a metallo-β-lactamase.

Functional characterization and clinical implication of a novel epidemic carbapenem-resistant Acinetobacter baumannii genetic marker

ObjectivesThe aim of the study was to evaluate the distribution and function of contact-dependent growth inhibition (CDI) systems associated with carbapenem-resistant Acinetobacter baumannii (CRAB) isolates. MethodsIsolates were examined by multilocus sequence typing (MLST) and polymerase chain reaction (PCR) for the presence of CDI genes in CRAB and carbapenem-susceptible A. baumannii (CSAB) from patients with invasive disease in a medical center in Taiwan. Inter-bacterial competition assays were performed to characterize the in vitro function of the CDI system. ResultsA total of 89 (61.0%) CSAB and 57 (39.0%) CRAB isolates were collected and examined. ST787 (20/57; 35.1%) was the predominant sequence type among CRAB, followed by ST455 (10/57; 17.5%). More than half the CRAB (56.1%, 32/57) belonged to CC455 and more than one third (38.6%, 22/57) to CC92. A novel CDI system, cdiTYTH1, was found in 87.7% (50/57) of the CRAB but in only 1.1% (1/89) of the CSAB isolates (P<0.00001). The cdiTYTH1 was also identified in 94.4% (17/18) of previously genome-sequenced CRAB isolates and only one CSAB isolate from Taiwan. Two other previously reported CDI (cdi19606-1 and cdi19606-2) were not found in these isolates, except both were found in one CSAB. All six CRAB without cdiTYTH1 showed growth inhibition by a CSAB carrying cdiTYTH1 in vitro. All clinical CRAB isolates belonging to the predominant CC455 carried the newly identified cdiTYTH1. ConclusionsThis CDI system was widespread in CRAB clinical isolates and appeared to be an epidemic genetic marker for CRAB in Taiwan. The cdiTYTH1 was functional in vitro in bacterial competition assay.

Carbapenem Resistant Acinetobacter baumannii: Current Status of Problem in a Tertiary Care Hospital, Jaipur

ABSTRACT Background: Carbapenem-resistant Acinetobacter baumannii (CRAB) is a serious global health challenge. It is increasingly associated with nosocomial infections and outbreaks in healthcare facilities. This study was conducted in a tertiary care teaching hospital in Jaipur to quantitate the burden of CRAB in our setup, examine the clinico-epidemiological profile of patients infected with this bug, and determine their antibiotic susceptibility pattern. Methods: Clinical specimens collected from patients admitted in various wards and ICUs were processed. Bacterial identification and susceptibility testing were done using standard laboratory techniques and VITEK 2 automated system. Only one isolate per patient was included for study purposes. The data on the sociodemographic and epidemiological profile was collected and analyzed statistically. Results: A total of 4897 clinical samples were received in the department of microbiology during the study period, out of which 1517 exhibited significant growth, sample positivity rate being 31%. Acinetobacter baumannii was the third most common gram-negative isolate (228/1067; 21.3%). Out of the total 228 A. baumannii isolates, 221 (97%) were carbapenem resistant. The maximum number of CRAB isolates were obtained from endotracheal secretions (103), followed by blood (59). All patients were critically ill and needed intensive care. The antibiotic susceptibility testing of these clinical isolates revealed a high level of resistance (99%) against ceftazidime, meropenem, and imipenem with the least resistance against colistin (2%) and tigecycline (9%). Conclusions: CRAB is an important global pathogen contributing to increased morbidity and mortality in hospitalized patients. Viable treatment options that may be used with success against bugs include minocycline, tigecycline, and polymyxins.

Patient and facility characteristics of an NDM-producing Acinetobacter baumannii outbreak in California, 2020–2022

Background: Carbapenem-resistant Acinetobacter baumannii (CRAB) are bacteria that cause healthcare-associated infections and outbreaks. Most produce carbapenemases like New Delhi metallo-β-lactamase (NDM), which are more commonly found in carbapenem-resistant Enterobacterales but rarely in CRAB. In 2018, selected laboratories began participating in a public health sentinel surveillance program by routinely submitting CRAB and other antimicrobial-resistant isolates to the AR Laboratory Network for specialized testing. In May 2020, the Antimicrobial Resistance Laboratory Network detected the first NDM-CRAB case in California, triggering an investigation. Initial whole-genome sequencing of subsequent isolates indicated high relatedness. Methods: We defined confirmed cases as patients with NDM detected in CRAB isolates and probable cases as NDM detected in a screening swab from a patient epidemiologically linked to a known case(s) with specimens collected during May 2020–September 2022. We defined outbreak facilities as having (1) 1 or more newly identified cases during a point-prevalence survey in response to a known case or (2) at least 2 cases identified within 4 weeks of each other that were epidemiologically linked. We analyzed demographic and specimen characteristics, as well as healthcare exposure history using R Studio version 1.3.959 software. Results: Of 230 total patients, 176 (77%) were confirmed and 54 (23%) were probable cases; 150 (65%) were identified through colonization screening. Among 176 NDM-CRAB isolates, the most common specimen sources were respiratory (n = 29), wound (n = 28), and urine (n = 24), and 87 (49%) of 176 isolates were nonsusceptible to all antimicrobials tested. Among patients, median age was 65 years (range, 24–97), 127 (55%) were male, 37 (15%) were Hispanic or Latino, and 100 (43%) were White. We identified 37 outbreak facilities across 13 counties, including 25 acute-care hospitals (ACHs), 6 skilled nursing facilities (SNFs), 5 ventilator-equipped SNFs (vSNFs), and 1 long-term ACH. We identified 125 cases (54%) in SNFs and vSNFs and 93 cases (40%) in ACHs; among ACH patients, 43 (46%) had been SNF or vSNF residents within the prior year. No patients reported international exposure. Conclusions: The first known case of NDM-CRAB in California was detected by sentinel surveillance. In this extensive regional outbreak, most cases were identified by screening at public health and clinical laboratories. Transmission occurred across healthcare settings connected by patient sharing, underscoring the importance of communication, active surveillance, and implementation of infection prevention and control practices to mitigate spread within and between facilities. Expanding these efforts, with support and resources from public health departments, is key to detecting, characterizing, and containing future outbreaks of antimicrobial-resistant pathogens.Disclosure: None

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.

Characterisation of pellicle-forming ability in clinical carbapenem-resistant Acinetobacter baumannii.

Acinetobacter baumannii was reported to have resistance towards carbapenems and the ability to form an air-liquid biofilm (pellicle) which contributes to their virulence. The GacSA two-component system has been previously shown to play a role in pellicle formation. Therefore, this study aims to detect the presence of gacA and gacS genes in carbapenem-resistant Acinetobacter baumannii (CRAB) isolates recovered from patients in intensive care units and to investigate their pellicle forming ability. The gacS and gacA genes were screened in 96 clinical CRAB isolates using PCR assay. Pellicle formation assay was performed in Mueller Hinton medium and Luria Bertani medium using borosilicate glass tubes and polypropylene plastic tubes. The biomass of the pellicle was quantitated using the crystal violet staining assay. The selected isolates were further assessed for their motility using semi-solid agar and monitored in real-time using real-time cell analyser (RTCA). All 96 clinical CRAB isolates carried the gacS and gacA genes, however, only four isolates (AB21, AB34, AB69 and AB97) displayed the ability of pellicle-formation phenotypically. These four pellicle-forming isolates produced robust pellicles in Mueller Hinton medium with better performance in borosilicate glass tubes in which biomass with OD570 ranging from 1.984±0.383 to 2.272±0.376 was recorded. The decrease in cell index starting from 13 hours obtained from the impedance-based RTCA showed that pellicle-forming isolates had entered the growth stage of pellicle development. These four pellicle-forming clinical CRAB isolates could be potentially more virulent, therefore further investigation is warranted to provide insights into their pathogenic mechanisms.

Clinical Outcomes and Associated Factors for Mortality among Pediatric Patients with Carbapenem-Resistant Acinetobacter baumannii

Background: Acinetobacter baumannii bacteremia is a hospital-acquired infection with a high mortality rate. Up to 80% of hospital-acquired A. baumannii infections are caused by carbapenem-resistant Acinetobacter baumannii (CRAB) strains. Objective: To determine the overall 30-day mortality rate, factors associated with mortality, and antibiotic drug susceptibility patterns of CRAB isolates among children with hospital-acquired CRAB bloodstream infections. Materials and Methods: A retrospective review was conducted among hospitalized pediatric patients between January 2017 and September 2022 at King Chulalongkorn Memorial Hospital, Bangkok. The inclusion criteria were CRAB bacteremia in children under 18 years of age. Thirty-day mortality after CRAB bacteremia was analyzed using Kaplan-Meier estimates. Associated factors were analyzed by Poisson regression. Antibiotic susceptibility patterns of nine antimicrobial agents were summarized. Results: Fifty-eight patients with 66 episodes of CRAB bacteremia were identified. The median age was 7.5 months (IQR 0.8 to 60.0), and 86.4% of the patients were admitted to the intensive care unit. Central line-associated bloodstream infections (CLABSI) were identified in 90.9% of cases. Most patients (74.2%) received colistin combination with sulbactam regimen. The 30-day mortality rate was 19.7% (95% CI 10.9 to 31.3). Associated factors for mortality rate were septic shock (aRR 7.6, 95% CI 2.3 to 25.0) and underlying congenital heart disease (aRR 3.4, 95% CI 1.0 to 11.7). Drug susceptibility of colistin and tigecycline were 93% and 48%, respectively. Sulbactam was not susceptible. Conclusion: One-fifth of children with CRAB bacteremia died within 30 days. Associated factors with mortality were septic shock and congenital heart disease. Colistin had the highest in vitro drug susceptibility rate. The common regimen used in the present study was colistin combination with sulbactam therapy. Keywords: Acinetobacter baumannii; Bacteremia; Carbapenem resistance; Susceptibility; Mortality; Pediatrics

Endemicity and diversification of carbapenem-resistant Acinetobacter baumannii in an intensive care unit

Carbapenem-resistant Acinetobacter baumannii (CRAB) is a major public health concern globally. Often studied in the context of hospital outbreaks, little is known about the persistence and evolutionary dynamics of endemic CRAB populations. A three-month cross-sectional observational study was conducted in a 28-bed intensive care unit (ICU) in Hangzhou, China. A total of 5068 samples were collected from the hospital environment (n=3985), patients (n=964) and staff (n=119). CRAB isolates were obtained from 10.5% of these samples (n=532). All of these isolates, plus an additional 19 from clinical infections, were characterised through whole-genome sequencing. The ICU CRAB population was dominated by OXA-23-producing global clone 2 isolates (99.3% of all isolates) that could be divided into 20 distinct clusters, defined through genome sequencing. CRAB was persistently present in the ICU, driven by regular introductions of distinct clusters. The hospital environment was heavily contaminated, with CRAB isolated from bed units on 183/335 (54.6%) sampling occasions but from patients on only 72/299 (24.1%) occasions. CRAB was spread to adjacent bed units and rooms, and following re-location of patients within the ICU. We also observed three horizontal gene transfer events between CRAB strains in the ICU, involving three different plasmids. The epidemiology of CRAB in this setting contrasted with previously described clonal outbreaks in high-income countries, highlighting the importance of environmental CRAB reservoirs in ICU epidemiology and the unique challenges in containing the spread of CRAB in ICUs where this important multidrug-resistant pathogen is endemic. This work was undertaken as part of the DETECTIVE research project funded by the Medical Research Council (MR/S013660/1), National Natural Science Foundation of China (81861138054, 32011530116, 31970128, 31770142), Zhejiang Province Medical Platform Backbone Talent Plan (2020RC075), and the National Key Research and Development Program of China grant (2018YFE0102100). W.v.S was also supported by a Wolfson Research Merit Award (WM160092).

Salvage therapy with sulbactam/durlobactam against cefiderocol-resistant Acinetobacter baumannii in a critically ill burn patient: clinical challenges and molecular characterization.

Carbapenem-resistant Acinetobacter baumannii (CRAB) infections are associated with high mortality rates. The optimal treatment regimen for CRAB has not been defined. Cefiderocol has been recently introduced in the armamentarium against CRAB but there is concern about treatment-emergent resistance. Since mortality rates in CRAB infections remain high, further antibiotic options are needed. We report a case of severe infection by CRAB resistant to both colistin and cefiderocol treated with sulbactam/durlobactam and describe the molecular features of the strain. Susceptibility to cefiderocol was detected by disc diffusion according to EUCAST breakpoints. Susceptibility to sulbactam/durlobactam was determined by Etest according to preliminary breakpoints provided by Entasis Therapeutics. Whole Genome Sequencing (WGS) of the CRAB isolate was performed. A burn patient with ventilator-associated pneumonia by CRAB resistant to colistin and cefiderocol received sulbactam/durlobactam as compassionate use. She was alive after 30 days from the end of therapy. Complete microbiological eradication of CRAB was achieved. The isolate harboured blaADC-30, blaOXA-23 and blaOXA-66. A missense mutation in PBP3 was detected. The isolate harboured a mutation in the TonB-dependent siderophore receptor gene piuA that showed a frameshift mutation causing a premature stop codon (K384fs). Moreover, the fepA gene, which is orthologous to pirA, was interrupted by a transposon insertion P635-ISAba125 (IS30 family). Further treatment options for severe infections by CRAB resistant to all available antibiotics are urgently needed. Sulbactam/durlobactam may be a future option against MDR A. baumannii.

Molecular Epidemiological Investigation and Management of Outbreak Caused by Carbapenem-Resistant Acinetobacter baumannii in a Neonatal Intensive Care Unit.

The present study describes an epidemiological investigation into a carbapenem-resistant Acinetobacter baumannii (CRAB) outbreak, which had occurred in a neonatal intensive care unit (NICU), and the subsequent strengthening of infection control interventions. Upon the onset of the outbreak, existing infection control interventions were reviewed, and a set of containment measures were instituted. All CRAB isolates were characterized in terms of antimicrobial susceptibility testing and their genetic relatedness. The investigation process identified gaps within the NICU's existing infection control measures, which had likely resulted in the outbreak. CRAB was isolated from nine preterm infants: five colonized and four infected. All five colonized patients were discharged well. However, three out of four of the infected infants died. Outbreak investigation and genomic subtyping of environmental swabs revealed that mini syringe drivers shared between patients and a sink in the milk preparation room had served as CRAB reservoirs with possible transmission via the hands of healthcare workers. Implementation of immediate actions such as reinforcement of hand hygiene practices, intensified environmental cleaning, geographical cohorting, reviewing of milk handling practices and sink management protocol had resulted in no further CRAB isolation. The CRAB outbreak in the NICU underlines the importance of consistent compliance with infection-control interventions. Integration of epidemiological and microbiological data, together with comprehensive preventive measures, successfully brought the outbreak to a halt.

Phenotypic and Molecular Characteristics of Carbapenem-Resistant Acinetobacter baumannii Isolates from Bulgarian Intensive Care Unit Patients.

Carbapenem-resistant Acinetobacter baumannii (CRAB) is designated as an urgent public health threat, both due to its remarkable multidrug resistance and propensity for clonal spread. This study aimed to explore the phenotypic and molecular characteristics of antimicrobial resistance in CRAB isolates (n = 73) from intensive care unit (ICU) patients in two university hospitals in Bulgaria (2018-2019). The methodology included antimicrobial susceptibility testing, PCR, whole-genome sequencing (WGS), and phylogenomic analysis. The resistance rates were as follows: imipenem, 100%; meropenem, 100%; amikacin, 98.6%; gentamicin, 89%; tobramycin, 86.3%; levofloxacin, 100%; trimethoprim-sulfamethoxazole, 75.3%; tigecycline, 86.3%; colistin, 0%; and ampicillin-sulbactam, 13.7%. All isolates harbored blaOXA-51-like genes. The frequencies of distribution of other antimicrobial resistance genes (ARGs) were: blaOXA-23-like, 98.6%; blaOXA-24/40-like, 2.7%; armA, 86.3%; and sul1, 75.3%. The WGS of selected extensively drug-resistant A. baumannii (XDR-AB) isolates (n = 3) revealed the presence of OXA-23 and OXA-66 carbapenem-hydrolyzing class D β-lactamases in all isolates, and OXA-72 carbapenemase in one of them. Various insertion sequencies, such as ISAba24, ISAba31, ISAba125, ISVsa3, IS17, and IS6100, were also detected, providing increased ability for horizontal transfer of ARGs. The isolates belonged to the widespread high-risk sequence types ST2 (n = 2) and ST636 (n = 1) (Pasteur scheme). Our results show the presence of XDR-AB isolates, carrying a variety of ARGs, in Bulgarian ICU settings, which highlights the crucial need for nationwide surveillance, especially in the conditions of extensive antibiotic usage during COVID-19.

Rifampicin Enhanced Carbapenem Activity with Improved Antibacterial Effects and Eradicates Established Acinetobacter baumannii Biofilms.

Biofilm-mediated infections are critical to public health and a leading cause of resistance among pathogens, amounting to a prolonged hospital stay and increased mortality rate in the intensive care unit. In this study, the antibacterial and antibiofilm activities of rifampicin or carbapenem monotherapies were compared with rifampicin and carbapenem combination therapies against rifampicin-resistant and carbapenem-resistant Acinetobacter baumannii isolates. Among 29 CRAB isolates, 24/29 (83%) were resistant to rifampicin, with MIC values between 2-256 µg/mL. Checkerboard assays disclosed that combination therapies at FICIs between 1/8 and 1/4 improved the activity of carbapenems at subinhibitory concentrations. Time-kill kinetics indicated a 2- to 4-log reduction at 1/2 MIC rifampicin + 1/4 MIC carbapenem and 1/4 MIC rifampicin + 1/4 MIC carbapenem against the isolates, with the MIC values ranging from 2-8 µg/mL. The MTT assay revealed a dose-dependent decrease of the cell viability of established bacterial biofilm at 4 MIC rifampicin + 2 MIC carbapenems, with a percentage reduction of 44-75%, compared with monotherapies at 16 MIC. Scanning electron microscopy further confirmed bacterial cell membrane disruption, suggesting a synergism between carbapenem and rifampicin against a representative isolate. The findings demonstrated that the combination of rifampicin with carbapenems could improve antibacterial activities and eradicate established Acinetobacter baumannii biofilm.

Enhanced bacterial killing with a combination of sulbactam/minocycline against dual carbapenemase-producing Acinetobacter baumannii

Carbapenem-resistant Acinetobacter baumannii (CRAB) is often difficult to treat. Considering the current circumstances, there is an unquestionable need for new therapeutic options to treat CRAB infections. In the present study, the synergistic activity of sulbactam-based combination was determined against genetically characterized CRAB isolates. Non-duplicate CRAB isolates (n = 150) recovered from blood culture and endotracheal aspirates were included in this study. The minimum inhibitory concentrations (MICs) of tetracyclines (minocycline, tigecycline, eravacycline) and their comparators (meropenem, sulbactam, cefoperazone/sulbactam, ceftazidime/avibactam, and colistin) were determined using the microbroth dilution method. Six isolates were tested for the synergistic activity of various sulbactam-based combinations using time-kill experiments. Tigecycline and minocycline showed a wide spread of MICs with most isolates in the range of1 to 16mg/L. The MIC90 of eravacycline (0.5mg/L) was four dilutions lower than that of tigecycline (8mg/L). Minocycline with sulbactam was the most active dual combination against OXA-23 like (n = 2) and NDM with OXA-23 like producers (n = 1), which resulted in ≥ 2 log10 kill. The combination of ceftazidime-avibactam with sulbactam showed ≥ 3 log10 kill against all the three tested OXA-23 like producing CRAB isolates, but showed no activity against dual carbapenemase producers. Sulbactam with meropenem showed ≥ 2 log10 kill against one OXA-23 like producing CRAB isolate. The findings suggest that sulbactam-based combination may confer therapeutic benefits against CRAB infections.

Molecular characterisation of colistin and carbapenem-resistant clinical isolates of Acinetobacter baumannii from Southeast Europe

ObjectivesTo characterise 11 colistin- and carbapenem-resistant Acinetobacter baumannii isolates recently emerging in hospital settings. MethodsA. baumannii isolates were collected from hospitalised patients under colistin treatment in three countries of Southeast Europe: Turkey, Croatia, and Bosnia and Herzegovina. Isolates were identified using molecular methods. ResultsIsolates from Turkey and Croatia belong to the sequence types ST195 or ST281 of the clone lineage 2, while the single isolate from Bosnia and Herzegovina belongs to the ST231 of clone lineage 1. All isolates turned out to be highly resistant to colistin (MIC ≥ 16 mg/L) and have point mutations in pmrCAB operon genes. The colistin-resistant isolate from Bosnia and Herzegovina had a unique P170L point mutation in the pmrB gene and the R125H point mutation in the pmrC gene. The L20S mutation in the pmrA gene was detected only in isolates from Croatia and has never been reported before in isolates from this country. ConclusionColistin resistance in A. baumannii in hospitalised patients receiving colistin treatment is a result of chromosomal mutations. The pattern of point mutations in pmrCAB genes suggests a spread of specific colistin-resistant isolates within the hospital.

Early initiation of three-drug combinations for the treatment of carbapenem-resistant A. baumannii among COVID-19 patients.

We evaluated the clinical characteristics and outcomes of patients with COVID-19 who received three-drug combination regimens for treatment of carbapenem-resistant Acinetobacter baumannii (CRAB) infections during a single-centre outbreak. Our objective was to describe the clinical outcomes and molecular characteristics and in vitro synergy of antibiotics against CRAB isolates. Patients with severe COVID-19 admitted between April and July 2020 with CRAB infections were retrospectively evaluated. Clinical success was defined as resolution of signs/symptoms of infection without need for additional antibiotics. Representative isolates underwent whole-genome sequencing (WGS) and in vitro synergy of two- or three-drug combinations was assessed by checkerboard and time-kill assays, respectively. Eighteen patients with CRAB pneumonia or bacteraemia were included. Treatment regimens included high-dose ampicillin-sulbactam, meropenem, plus polymyxin B (SUL/MEM/PMB; 72%), SUL/PMB plus minocycline (MIN; 17%) or other combinations (12%). Clinical resolution was achieved in 50% of patients and 30-day mortality was 22% (4/18). Seven patients had recurrent infections, during which further antimicrobial resistance to SUL or PMB was not evident. PMB/SUL was the most active two-drug combination by checkerboard. Paired isolates collected before and after treatment with SUL/MEM/PMB did not demonstrate new gene mutations or differences in the activity of two- or three-drug combinations. Use of three-drug regimens for severe CRAB infections among COVID-19 resulted in high rates of clinical response and low mortality relative to previous studies. The emergence of further antibiotic resistance was not detected phenotypically or through WGS analysis. Additional studies are needed to elucidate preferred antibiotic combinations linked to the molecular characteristics of infecting strains.

Emergence and Evolution of OXA-23-Producing ST46Pas-ST462Oxf-KL28-OCL1 Carbapenem-Resistant Acinetobacter baumannii Mediated by a Novel ISAba1-Based Tn7534 Transposon

Carbapenem-resistant Acinetobacter baumannii (CRAB) isolates of global clone 1 (GC1) and global clone 2 (GC2) have been widely reported. Nevertheless, non-GC1 and non-GC2 CRAB strains have been studied less. In particular, no reports concerning sequence type 46 (ST46Pas) CRAB strains have been described thus far. In this work, the genomic features and possible evolution mechanism of ST46Pas OXA-23-producing CRAB isolates from clinical specimens are reported for the first time. Antimicrobial susceptibility testing of three ST46Pas strains revealed identical resistance profiles (resistance to imipenem, meropenem, ciprofloxacin and the combination of cefoperazone/sulbactam at a 2:1 ratio). They were found to belong to ST46Pas and ST462Oxf with capsular polysaccharide 28 (KL28) and lipooligosaccharide 1 (OCL1), respectively. Whole-genome sequencing (WGS) revealed that all contained one copy of chromosomal blaOXA-23, which was located in a novel ISAba1-based Tn7534 composite transposon. In particular, another copy of the Tn7534 composite transposon was identified in an Hgz_103-type plasmid with 9 bp target site duplications (TSDs, ACAACATGC) in the A. baumannii ZHOU strain. As the strains originated from two neighboring intensive care units (ICUs), ST46Pas OXA-23-producing CRAB strains may have evolved via transposition events or a pdif module. Based on the GenBank database, ST46Pas strains were collected from various sources; however, most were collected in Hangzhou (China) from 2014 to 2021. Pan-genome analysis revealed 3276 core genes, 0 soft-core genes, 768 shell genes and 443 cloud genes shared among all ST46Pas strains. In conclusion, the emergence of ST46Pas CRAB strains might present a new threat to healthcare settings; therefore, effective surveillance is required to prevent further dissemination.

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.

Carbapenem-Resistant Acinetobacter baumannii: Biofilm-Associated Genes, Biofilm-Eradication Potential of Disinfectants, and Biofilm-Inhibitory Effects of Selenium Nanoparticles.

This study aimed to investigate the biofilm-production ability of carbapenem-resistant Acinetobacter baumannii (CRAB), the biofilm-eradication potential of 70% ethanol and 0.5% sodium hypochlorite, the effects of selenium nanoparticles (SeNPs) against planktonic and biofilm-embedded CRAB, and the relationship between biofilm production and bacterial genotypes. A total of 111 CRAB isolates were tested for antimicrobial susceptibility, biofilm formation, presence of the genes encoding carbapenemases, and biofilm-associated virulence factors. The antibiofilm effects of disinfectants and SeNPs against CRAB isolates were also tested. The vast majority of the tested isolates were biofilm producers (91.9%). The bap, ompA, and csuE genes were found in 57%, 70%, and 76% of the CRAB isolates, with the csuE being significantly more common among biofilm producers (78.6%) compared to non-biofilm-producing CRAB (25%). The tested disinfectants showed a better antibiofilm effect on moderate and strong biofilm producers than on weak producers (p &lt; 0.01). The SeNPs showed an inhibitory effect against all tested planktonic (MIC range: 0.00015 to &gt;1.25 mg/mL) and biofilm-embedded CRAB, with a minimum biofilm inhibitory concentration of less than 0.15 mg/mL for 90% of biofilm producers. In conclusion, SeNPs might be used as promising therapeutic and medical device coating agents, thus serving as an alternative approach for the prevention of biofilm-related infections.