Isolation of a lytic bacteriophage against extensively drug-resistant Acinetobacter baumannii infections and its dramatic effect in rat model of burn infection.

OBJECTIVES: Acinetobacter Baumannii is an opportunistic nosocomial pathogen belonging to the Moraxellaceae family. The emergence of multidrug resistant strains of this pathogen caused many problems for hospitals and patients. The aim of the current study was to isolate, identify, and morphologically, physiologically, and in vivo analyze a new lytic bacteriophage targeting extensively drug-resistant (XDR) A. baumannii. MATERIALS AND METHODS: Different wastewater samples were tested for isolation of lytic bacteriophage against 19 A. baumannii isolates obtained from patients hospitalized in a hospital in Arak, Iran, from January 2019 to March 2019. The phenotypic and genotypic characteristics of A. baumannii strains (resistance genes including: adeA, adeB, adeC, adeR, adeS, ISAba1, blaOXA-23, blaOXA-24) were analyzed. The isolated phage characteristics including adsorption time, pH and thermal stability, host range, one-step growth rate, electron microscopy examination, and therapeutic efficacy of the phage were also investigated. Therapeutic efficacy of the phage was evaluated in a rat model with burn infection of XDR A. baumannii. The lesion image was taken on different days after burning and infection induction and was compared with phage untreated lesions. RESULTS: The results showed unique characteristics of the isolated phage (vB-AbauM-Arak1) including high specificity for Acinetobacter baumannii, stability at a relatively wide range of temperatures and pH values, short adsorption time, short latent period, and large burst size. In relation to the therapeutic efficacy of the phage, the lesion area decreased in phage-treated groups over 14 days than in those untreated, significantly (p < 0.05). CONCLUSION: Our findings demonstrated that isolated lytic phage was able to eliminate burn infections caused by XDR A. baumannii in a rat model. So, it may be recommended as alternative options toward to developing a treatment for extensively drug resistant Acinetobacter infections.

Multidrug-Resistant Infections and Outcome of Critically Ill Patients with Coronavirus Disease 2019: A Single Center Experience.

Background: The aim of this study was to assess the drivers of multidrug-resistant (MDR) bacterial infection development in coronavirus disease 2019 (COVID-19) and its impact on patient outcome. Methods: Retrospective analysis on data from 32 consecutive patients with COVID-19, admitted to our intensive care unit (ICU) from March to May 2020. Outcomes considered were MDR infection and ICU mortality. Results: Fifty percent of patients developed an MDR infection during ICU stay after a median time of 8 [4-11] days. Most common MDR pathogens were carbapenem-resistant Klebsiella pneumoniae and Acinetobacter baumannii, causing bloodstream infections and pneumonia. MDR infections were linked to a higher length of ICU stay (p = 0.002), steroid therapy (p = 0.011), and associated with a lower ICU mortality (odds ratio: 0.439, 95% confidence interval: 0.251-0.763; p < 0.001). Low-dose aspirin intake was associated with both MDR infection (p = 0.043) and survival (p = 0.015). Among MDR patients, mortality was related with piperacillin-tazobactam use (p = 0.035) and an earlier onset of MDR infection (p = 0.042). Conclusions: MDR infections were a common complication in critically ill COVID-19 patients at our center. MDR risk was higher among those dwelling longer in the ICU and receiving steroids. However, MDR infections were not associated with a worse outcome.

Acinetobacter baumannii infections in Amazon Region driven by extensively drug resistant international clones, 2016-2018.

BACKGROUND: Acinetobacter baumannii is a leading cause of nosocomial infections. This species is characterised by the presence of pandemic lineages (International Clones) that present a broad antimicrobial resistance profile. OBJECTIVE: To perform the molecular epidemiology of carbapenem-resistant A. baumannii from a clinical setting in the Amazon Basin, and to characterise their antimicrobial resistance determinants. METHODS: The genetic relationship of carbapenem-resistant A. baumannii were assessed by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). Class A, B and D ß-lactamase genes were screened by polymerase chain reaction (PCR) and sequencing. The antimicrobial susceptibility profile was obtained by Disc-diffusion method and minimum inhibitory concentration (MIC) determination. FINDINGS: All carbapenem-resistant A. baumannii strains belonged to three international clones, IC-1, IC-5 and IC-6, the latter recently reported by the first time in Brazil. The major determinant of carbapenem resistance in IC-1 and IC-5 strains was bla OXA-23, associated with ISAba1 and ISAba3, respectively, while IC-6 harboured the bla OXA-72. CONCLUSIONS: The A. baumannii epidemiology in Brazilian Amazon Region was unknown. It was demonstrated that A. baumannii XDR international clones were responsible for nosocomial infections in Boa Vista during 2016-2018, revealing that the epidemiological scenario of A. baumannii infections in Amazon Region resembles those from the cosmopolitan regions worldwide.

Molecular epidemiology of carbapenem-resistant Acinetobacter baumannii isolates from a Korean hospital that carry blaOXA-23.

To elucidate the epidemicity of carbapenem-resistant Acinetobacter baumannii (CRAB), we investigated the antimicrobial susceptibility, the genetic basis of antimicrobial resistance, and the ability to form biofilms of 147 CRAB isolates collected between 2013 and 2015 from a Korean hospital based on sequence types (STs). Six different STs were identified: ST191 (n=47) and ST208 (n=36) were clones that had already been identified in the study hospital, whereas ST229 (n=28), ST369 (n=18), ST357 (n=17), and ST552 (n=1) were previously unknown. All the CRAB isolates exhibited an extensively drug-resistance. Twelve isolates, including ST191 and ST208, were resistant to tigecycline, and two were resistant to colistin. All the isolates carried ISAbaI-blaOXA-23 structures. The presence of the armA gene and/or a combination of aminoglycoside-modifying enzyme genes (aacC1, aadA1, aacA4, aphA1, and aphA6) was responsible for high-level resistance to aminoglycosides (minimal inhibitory concentrations≥256mg/L). All the ST229 isolates carried the blaPER-1 gene, whereas all the ST357 and ST369 isolates carried both aacA4 and aadA1. The ST229 isolates exhibited the greatest tendency to form biofilms, whereas the ST369 isolates exhibited significantly less tendency to form biofilms than other isolates. In conclusion, we discovered clonal diversity in the CRAB isolates from the study hospital. The resistant gene profiles and biofilm formation capabilities of the emerging CRAB STs differed from those of the circulating STs. The potential relationship between these genotypic and phenotypic traits and the epidemic capacity of CRAB STs requires further investigation.

Potential of a lytic bacteriophage to disrupt Acinetobacter baumannii biofilms in vitro.

AIM: The ability of Acinetobacter baumannii to form biofilms and develop antibiotic resistance makes it difficult to control infections caused by this bacterium. In this study, we explored the potential of a lytic bacteriophage to disrupt A. baumannii biofilms. MATERIALS & METHODS: The potential of the lytic bacteriophage to disrupt A. baumannii biofilms was assessed by performing electron microscopy, live/dead bacterial staining, crystal violet staining and by determining adenosine triphosphate release. RESULTS: The bacteriophage inhibited the formation of and disrupted preformed A. baumannii biofilms. Results of disinfection assay showed that the lytic bacteriophage lysed A. baumannii cells suspended in blood or grown on metal surfaces. CONCLUSION: These results suggest the potential of the lytic bacteriophage to disrupt A. baumannii biofilms.

Personalized Therapeutic Cocktail of Wild Environmental Phages Rescues Mice from Acinetobacter baumannii Wound Infections.

Multidrug-resistant bacterial pathogens are an increasing threat to public health, and lytic bacteriophages have reemerged as a potential therapeutic option. In this work, we isolated and assembled a five-member cocktail of wild phages against Acinetobacter baumannii and demonstrated therapeutic efficacy in a mouse full-thickness dorsal infected wound model. The cocktail lowers the bioburden in the wound, prevents the spread of infection and necrosis to surrounding tissue, and decreases infection-associated morbidity. Interestingly, this effective cocktail is composed of four phages that do not kill the parent strain of the infection and one phage that simply delays bacterial growth in vitro via a strong but incomplete selection event. The cocktail here appears to function in a combinatorial manner, as one constituent phage targets capsulated A. baumannii bacteria and selects for loss of receptor, shifting the population to an uncapsulated state that is then sensitized to the remaining four phages in the cocktail. Additionally, capsule is a known virulence factor for A. baumannii, and we demonstrated that the emergent uncapsulated bacteria are avirulent in a Galleria mellonella model. These results highlight the importance of anticipating population changes during phage therapy and designing intelligent cocktails to control emergent strains, as well as the benefits of using phages that target virulence factors. Because of the efficacy of this cocktail isolated from a limited environmental pool, we have established a pipeline for developing new phage therapeutics against additional clinically relevant multidrug-resistant pathogens by using environmental phages sourced from around the globe.

A case report demonstrating the utility of next generation sequencing in analyzing serial samples from the lung following an infection with influenza A (H7N9) virus.

BACKGROUND: Bacterial pneumonia is a well-recognized sequela of patient suffering from influenza, and a key factor, with cytokine dysregulation, that contribute to severe disease and mortality. OBJECTIVES: To obtain a comprehensive assessment of lung microbial community dynamics in a fatal influenza H7N9 case during the whole clinical course, we undertook a longitudinal study. STUDY DESIGN: Serial bronchoalveolar lavage fluid samples were collected from a H7N9 patient after illness onset, and the microbiome was characterized by using next-generation sequencing and microbiological approaches. Furthermore, the kinetics of circulating cytokine storms related to viral and secondary bacterial infection were analyzed. RESULTS: Within complex and dynamic communities, the lung microbiome with H7N9 infection were dominated by gram-negative bacteria, Acinetobacter baumannii after the viral invasion and during the whole clinical course. Sputum and blood culture confirmed the secondary bacterial infection with multidrug-resistant A. baumannii 9 days later. The dynamics of the bacterial infection with carbapenem-resistant A. baumannii correlated with antibiotic therapy. Our observations also indicated that sustained high levels of host inflammatory factors, consisting of a set of distinct cytokines associated with disease stage, may contribute to disease progression and death. CONCLUSIONS: This study demonstrates an initial attempt to explore the dynamic microbiome involved inH7N9 infection and its response to antimicrobial therapy, as well as host cytokine response to infection by using next-generation sequencing. These type of investigations with longitudinal follow-up to understand dynamics of microbial community and cytokines involved in lung infection may provide opportunities for development and optimization of targeted antimicrobial therapy and even new therapeutic strategies.

Ability of bacteriophage in resolving wound infection caused by multidrug-resistant Acinetobacter baumannii in uncontrolled diabetic rats.

Acinetobacter baumannii, a substantial nosocomial pathogen, has developed resistance to almost all available antimicrobial drugs. Bacteriophage therapy is a possible alternative treatment for multidrug-resistant (MDR) bacterial infections. In this study, we have successfully isolated bacteriophage active against clinical strains of A. baumannii by enrichment from hospital sewage sludge using representatives of those strains. The bacteriophage isolated against A. baumannii formed plaques against beta-lactamases producing strains of A. baumannii. The utility of bacteriophage specific for A. baumannii to resolve wound infection in uncontrolled diabetic rats was evaluated. Five groups of uncontrolled diabetic rats were used. Group I was noninfected (Control), Group II was infected with MDR A. baumannii and challenged with bacteriophage, Group III was infected with MDR A. baumannii, Group IV was infected with MDR A. baumannii and challenged with antibiotic colistin, and Group V consisted of noninfected rats and sprayed with phage (Phage control). A significant decrease in infection, period of epithelization, and wound contraction was observed in the phage-challenged group when compared with antibiotic-treated uncontrolled diabetic rats and the control group. To conclude the study, new insights are provided into the biology of the broad host range of A. baumannii phage, demonstrating that A. baumannii phage has prospects for the treatment of infections caused by the MDR A. baumannii.

Acinetobacter baumannii extensively drug resistant lineages in Buenos Aires hospitals differ from the international clones I-III.

As a way to contribute to the assessment of Acinetobacter baumannii clinical population structure, multi-locus sequence typing (MLST) was performed in a collection of 93 isolates from Buenos Aires (1983-2012) and Rosario (2006-2009) hospitals. Sequence types (STs) were achieved by Bartual (B) and Institut Pasteur (P) schemes. PFGE typing, antimicrobial susceptibility assays, and the amplification of the OXA carbapenemase genes most prevalent in our region, were also performed. e-Burst clustered the 25 STs(B) (15 novels) into 5 clonal complexes (CC) and 5 singletons, and grouped the 18 STs(P) (12 novels) into 3 CC and 4 singletons. Bartual scheme divided the CC79(P) into two groups. CC113(B)/CC79(P) prevailed in Buenos Aires at least in 1992-2009, being responsible for epidemic and for endemic infections and acquiring the XDR (extensively drug-resistant) pattern throughout the years. While, CC119(B)/CC79(P) was apparently present before the CC113(B)/CC79(P)domain. CC103(B)/CC15(P) was the second most prevalent CC. Interestingly, CC110(B)/ST25(P) apparently increased over the last years. Conversely, CC109(B)/CC1(P) (international clone I) predominated in Rosario, although the presence of CC113(B)/CC79(P), CC103(B)/CC15(P) and CC110(B)/ST25(P) was observed. Nineteen novel STs clustered in CC79(P), CC15(P), CC113(B), CC109(B) and CC103(B), suggesting their clonal expansion during persistence. PFGE typing proved transmission of strains intra- and inter-hospitals in each city. Except for one, all the recent isolates (2007-2012) harboured the blaOXA-23-like. All isolates were susceptible to colistin. Tigecycline MIC(90) was 1mg/L and the rifampicin MIC>512mg/l was found among isolates in three hospitals. In conclusion, the international clone II (CC92(B)/CC2(P)) was not found among our isolates. CC113(B)/CC79(P), CC103(B)/CC15(P), and ST25(P), suggested also as major components in the A. baumannii population together with the international clone I, were present in Buenos Aires and Rosario with different prevalence rate. Their recent isolates showed high distribution of the blaOXA-23-like as well as the XDR pattern.