Staphylococcus aureus α-Toxin Effect on Acinetobacter baumannii Behavior.

Polymicrobial infections are more challenging to treat and are recognized as responsible for significant morbidity and mortality. It has been demonstrated that multiple Gram-negative organisms take advantage of the effects of Staphylococcus aureus α-toxin on mucosal host defense, resulting in proliferation and dissemination of the co-infecting pathogens. Through phenotypic approaches, we observed a decrease in the motility of A. baumannii A118 after exposure to cell-free conditioned media (CFCM) of S. aureus strains, USA300 and LS1. However, the motility of A. baumannii A118 was increased after exposure to the CFCM of S. aureus strains USA300 Δhla and S. aureus LSI ΔagrA. Hemolytic activity was seen in A118, in the presence of CFCM of S. aureus LS1. Further, A. baumannii A118 showed an increase in biofilm formation and antibiotic resistance to tetracycline, in the presence of CFCM of S. aureus USA300. Transcriptomic analysis of A. baumannii A118, with the addition of CFCM from S. aureus USA300, was carried out to study A. baumannii response to S. aureus' released molecules. The RNA-seq data analysis showed a total of 463 differentially expressed genes, associated with a wide variety of functions, such as biofilm formation, virulence, and antibiotic susceptibility, among others. The present results showed that A. baumannii can sense and respond to molecules secreted by S. aureus. These findings demonstrate that A. baumannii may perceive and respond to changes in its environment; specifically, when in the presence of CFCM from S. aureus.

Genomic Analysis of two NDM-1 Providencia stuartii Strains Recovered from a Single Patient.

In the last years, an increasing number of untreatable infections caused by drug-resistant microbes have impacted the health care system. Worldwide, infections caused by carbapenem-resistant (CR) Gram-negative bacilli have dramatically increased. Among the CR-Gram-negative bacilli, those producing carbapenemases, such as NDM-1, are the main concern. Different Enterobacterales harboring NDM-1 have been reported lately. Providencia stuartii, a member of the Morganellaceae family, is ubiquitous in the environment, but is also known to cause nosocomial infections. Here we describe the genomic analysis of two NDM-1- producing P. stuartii strains recovered from the same patient as well as other carbapenem resistant strains recovered from the same hospital. As a result of the genomic analysis thirteen resistance genes, including three to ß-lactams (blaOXA-1, blaTEM-1, blaNDM-1), four to aminoglycosides (aphA6, aac(3)-IId, aac(2')-Ia, aac(6')-Ib-cr5), one to sulfonamides (sul1), two to chloramphenicol (catB3, catA3), one to rifampicin, one to bleomycin (ble), and one to tetracycline (tet(B)) were found. Moreover, a variety of mobile genetic elements, such as insertion sequences, plasmids and phage- related sequences, were found within P. stuartii genomes. The spread of carbapenem-resistant isolates remains a significant clinical and public health concern. Therefore, we considered that the detection of CR isolates is an essential step in addressing this problem.

Genomic analysis of two Acinetobacter baumannii strains belonging to two different sequence types (ST172 and ST25).

OBJECTIVES: Acinetobacter baumannii is an opportunistic nosocomial pathogen that is the main focus of attention in clinical settings owing to its intrinsic ability to persist in the hospital environment and its capacity to acquire determinants of resistance and virulence. Here we present the genomic sequencing, molecular characterisation and genomic comparison of two A. baumannii strains belonging to two different sequence types (STs), one sporadic and one widely distributed in our region. METHODS: Whole-genome sequencing (WGS) of Ab42 and Ab376 was performed using Illumina MiSeq-I and the genomes were assembled with SPAdes. ARG-ANNOT, CARD-RGI, ISfinder, PHAST, PlasmidFinder, plasmidSPAdes and IslandViewer were used to analyse both genomes. RESULTS: Genome analysis revealed that Ab42 belongs to ST172, an uncommon ST, whilst Ab376 belongs to ST25, a widely distributed ST. Molecular characterisation showed the presence of two antibiotic resistance genes in Ab42 and nine in Ab376. No insertion sequences were detected in Ab42, however 22 were detected in Ab376. Moreover, two prophages were found in Ab42 and three in Ab376. In addition, a CRISPR-cas type I-Fb and two plasmids, one of which harboured an AbGRI1-like island, were found in Ab376. CONCLUSIONS: We present WGS analysis of twoA. baumannii strains belonging to two different STs. These findings allowed us to characterise a previously undescribed ST (ST172) and provide new insights to the widely studied ST25.

Distinct Mechanisms of Dissemination of NDM-1 Metallo-ß-Lactamase in Acinetobacter Species in Argentina.

A 4-year surveillance of carbapenem-resistant Acinetobacter spp. isolates in Argentina identified 40 strains carrying blaNDM-1 Genome sequencing revealed that most were Acinetobacter baumannii, whereas seven represented other Acinetobacter spp. The A. baumannii genomes were closely related, suggesting recent spread. blaNDM-1 was located in the chromosome of A. baumannii strains and on a plasmid in non-A. baumannii strains. A resistance gene island carrying blaPER-7 and other resistance determinants was found on a plasmid in some A. baumannii strains.

Human pleural fluid triggers global changes in the transcriptional landscape of Acinetobacter baumannii as an adaptive response to stress.

Acinetobacter baumannii is a feared, drug-resistant pathogen, characterized by its ability to resist extreme environmental and nutrient-deprived conditions. Previously, we showed that human serum albumin (HSA) can increase foreign DNA acquisition specifically and alter the expression of genes associated with pathogenicity. Moreover, in a recent genome-wide transcriptomic study, we observed that pleural fluid (PF), an HSA-containing fluid, increases DNA acquisition, can modulate cytotoxicity, and control immune responses by eliciting changes in the A. baumannii metabolic profile. In the present work, using more stringent criteria and focusing on the analysis of genes related to pathogenicity and response to stress, we analyzed our previous RNA-seq data and performed phenotypic assays to further explore the impact of PF on A. baumannii's microbial behavior and the strategies used to overcome environmental stress. We observed that PF triggered differential expression of genes associated with motility, efflux pumps, antimicrobial resistance, biofilm formation, two-component systems (TCSs), capsule synthesis, osmotic stress, and DNA-damage response, among other categories. Phenotypic assays of A. baumannii A118 and two other clinical A. baumannii strains, revealed differences in their responses to PF in motility, biofilm formation, antibiotic susceptibility, osmotic stress, and outer membrane vesicle (OMV) production, suggesting that these changes are strain specific. We conclude that A. baumannii's pathoadaptive responses is induced by HSA-containing fluids and must be part of this bacterium armamentarium to persist in hostile environments.

Discrepancies in susceptibility testing to colistin in Acinetobacter baumannii: The influence of slow growth and heteroresistance.

The increasing use of polymyxins as last-resort drugs for managing infections by Acinetobacter baumannii has led to the emergence of resistance. This study aimed to determine the resistance mechanisms in Acinetobacter baumannii isolates with colistin MIC ≥ 4 mg/L and to relate the mechanisms of resistance with the difficulties in detecting them. Absolute agreement among the different methodologies (Phoenix automatized system, broth and agar dilution, and a rapid colorimetric test) in the 140 colistin-susceptible isolates was observed; whereas in the 25 resistant isolates, the performance varied according to the colistin MIC value. Most of the discrepancies (irrespective of the methodology that was used) were observed in isolates with an MIC value close to the breakpoint. The number of errors in each method in the resistant isolates was as follows: rapid test, four of 25 (16%); agar dilution, eight of 25 (32%); Phoenix system, 13 of 25 (52%) and its manual reading at 24 h, eight of 25 (32%). Categorical errors were detected in 13 isolates: slow growth was the main reason in five isolates, whereas in the remaining eight isolates, slow growth was detected together with a low proportion of colistin-resistant subpopulations and the colistin MIC value was close to the breakpoint value. To understand the probable reason for the observed MIC values, sequencing of genes associated with colistin resistance was performed. Mutations at lpxA, lpxC, and pmrB genes were detected and it was observed that isolates carrying mutations in lpxC presented slow growth at killing curves.

Human Pleural Fluid Elicits Pyruvate and Phenylalanine Metabolism in Acinetobacter baumannii to Enhance Cytotoxicity and Immune Evasion.

Acinetobacter baumannii (Ab) is one of the most treacherous pathogens among those causing hospital-acquired pneumonia (HAP). A. baumannii possesses an adaptable physiology, seen not only in its antibiotic resistance and virulence phenotypes but also in its metabolic versatility. In this study, we observed that A. baumannii undergoes global transcriptional changes in response to human pleural fluid (PF), a key host-derived environmental signal. Differential gene expression analyses combined with experimental approaches revealed changes in A. baumannii metabolism, affecting cytotoxicity, persistence, bacterial killing, and chemotaxis. Over 1,220 genes representing 55% of the differentially expressed transcriptomic data corresponded to metabolic processes, including the upregulation of glutamate, short chain fatty acid, and styrene metabolism. We observed an upregulation by 1.83- and 2.61-fold of the pyruvate dehydrogenase complex subunits E3 and E2, respectively. We also found that pyruvate (PYR), in conjunction with PF, triggers an A. baumannii pathogenic behavior that adversely impacts human epithelial cell viability. Interestingly, PF also amplified A. baumannii cytotoxicity against murine macrophages, suggesting an immune evasion strategy implemented by A. baumannii. Moreover, we uncovered opposing metabolic strategies dependent on the degree of pathogenicity of the strains, where less pathogenic strains demonstrated greater utilization of PYR to promote persister formation in the presence of PF. Additionally, our transcriptomic analysis and growth studies of A. baumannii suggest the existence of an alternative phenylalanine (PA) catabolic route independent of the phenylacetic acid pathway, which converts PA to phenylpyruvate (PP) and shuttles intermediates into styrene metabolism. This alternative route promoted a neutrophil-evasive state, as PF-induced degradation of PP significantly reduced overall human neutrophil chemotaxis in ex vivo chemotactic assays. Taken together, these data highlight A. baumannii pathoadaptabililty in response to host signals and provide further insight into the role of bacterial metabolism in virulence traits, antibiotic persistence strategies, and host innate immune evasion.

Genetic and Phenotypic Features of a Novel Acinetobacter Species, Strain A47, Isolated From the Clinical Setting.

In 2014, a novel species of Acinetobacter, strain A47, determined to be hospital-acquired was recovered from a single patient soft tissue sample following a traumatic accident. The complexity of the Acinetobacter genus has been established, and every year novel species are identified. However, specific features and virulence factors that allow members of this genus to be successful pathogens are not well understood. Utilizing both genomic and phenotypic approaches, we identified distinct features and potential virulence factors of the A47 strain to understand its pathobiology. In silico analyses confirmed the uniqueness of this strain and other comparative and sequence analyses were used to study the evolution of relevant features identified in this isolate. The A47 genome was further analyzed for genes associated with virulence and genes involved in type IV pili (T4P) biogenesis, hemolysis, type VI secretion system (T6SS), and novel antibiotic resistance determinants were identified. A47 exhibited natural transformation with both genomic and plasmid DNA. It was able to form biofilms on different surfaces, to cause hemolysis of sheep and rabbit erythrocytes, and to kill competitor bacteria. Additionally, surface structures with non-uniform length were visualized with scanning electron microscopy and proposed as pili-like structures. Furthermore, the A47 genome revealed the presence of two putative BLUF type photoreceptors, and phenotypic assays confirmed the modulation by light of different virulence traits. Taken together, these results provide insight into the pathobiology of A47, which exhibits multiple virulence factors, natural transformation, and the ability to sense and respond to light, which may contribute to the success of an A47 as a hospital dwelling pathogen.

A Study on Acinetobacter baumannii and Staphylococcus aureus Strains Recovered from the Same Infection Site of a Diabetic Patient.

Diabetic foot ulcer infections are frequently polymicrobial in nature and exhibit increased morbidity and mortality, as well as, treatment failures. Interactions between Acinetobacter baumannii and Staphylococcus aureus were studied, which showed strain-dependent changes in growth and antibiotic susceptibility. This study examined the interactions between two clinical strains of A. baumannii (1929) and S. aureus (1928) that were recovered from skin and soft tissues of a diabetic patient. When S. aureus 1928 and A. baumannii 1929 were co-cultured together, there was no significant decrease in growth in either clinical strains, indicating that both strains can co-exist in the same site of infection. Additionally, neither strains experienced statistically significant changes in susceptibility. These findings highlight that these two pathogens can be found in the same niche of infection, which may lead to more aggressive outcome of the infection.

Whole Genome Sequence Analysis of Burkholderia contaminans FFH2055 Strain Reveals the Presence of Putative ß-Lactamases.

Burkholderia contaminans is a member of the Burkholderia cepacia complex (Bcc), a pathogen with increasing prevalence among cystic fibrosis (CF) patients and the cause of numerous outbreaks due to the use of contaminated commercial products. The antibiotic resistance determinants, particularly ß-lactamases, have been poorly studied in this species. In this work, we explored the whole genome sequence (WGS) of a B. contaminans isolate (FFH 2055) and detected four putative ß-lactamase-encoding genes. In general, these genes have more than 93% identity with ß-lactamase genes found in other Bcc species. Two ß-lactamases, a class A (Pen-like, suggested name PenO) and a class D (OXA-like), were further analyzed and characterized. Amino acid sequence comparison showed that Pen-like has 82% and 67% identity with B. multivorans PenA and B. pseudomallei PenI, respectively, while OXA-like displayed strong homology with class D enzymes within the Bcc, but only 22-44% identity with available structures from the OXA family. PCR reactions designed to study the presence of these two genes revealed a heterogeneous distribution among clinical and industrial B. contaminans isolates. Lastly, blaPenO gene was cloned and expressed into E. coli to investigate the antibiotic resistance profile and confers an extended-spectrum ß-lactamase (ESBL) phenotype. These results provide insight into the presence of ß-lactamases in B. contaminans, suggesting they play a role in antibiotic resistance of these bacteria.

Interspecies DNA acquisition by a naturally competent Acinetobacter baumannii strain.

The human pathogen Acinetobacter baumannii possesses high genetic plasticity and frequently acquires antimicrobial resistance genes. Here we investigated the role of natural transformation in these processes. Genomic DNA from different sources, including from carbapenem-resistant Klebsiella pneumoniae strains, was mixed with A. baumannii A118 cells. Selected transformants were analysed by whole-genome sequencing. In addition, bioinformatics analyses and in silico gene flow prediction were also performed to support the experimental results. Transformant strains included some that became resistant to carbapenems or changed their antimicrobial susceptibility profile. Foreign DNA acquisition was confirmed by whole-genome analysis. The acquired DNA most frequently identified corresponded to mobile genetic elements, antimicrobial resistance genes and operons involved in metabolism. Bioinformatics analyses and in silico gene flow prediction showed continued exchange of genetic material between A. baumannii and K. pneumoniae when they share the same habitat. Natural transformation plays an important role in the plasticity of A. baumannii and concomitantly in the emergence of multidrug-resistant strains.

Effect of Host Human Products on Natural Transformation in Acinetobacter baumannii.

Our previous data show that serum albumin can trigger natural transformation in Acinetobacter baumannii. However, extracellular matrix/basal membrane components, norepinephrine, and mucin did not have a significant effect on this process. Therefore, the effect of human products appears to be albumin specific, as both BSA and HSA have been shown to increase of natural transformation.

Human fluids alter DNA-acquisition in Acinetobacter baumannii.

Transformation is one of the mechanisms of acquisition of foreign genetic material leading to the emergence of multidrug resistant (MDR) bacteria. Recently, human serum albumin (HSA) was shown to specifically increase transformation frequency in the nosocomial pathogen Acinetobacter baumannii. To further assess the relevance of HSA as a possible modulator of A. baumannii transformation in host-pathogen interactions, in this work we examined the effect of different human fluids. We observed a significant increase in transformation frequencies in the presence of pleural fluid, whole blood cells and liquid ascites, and to a lesser extent with urine. The observed effects correlate with both HSA and bacterial content found in the assayed patient fluids. Taken together, these results are in agreement with our previous findings that highlight HSA as a possible host signal with the ability to trigger natural transformation in A. baumannii.

Genome sequence analysis of an extensively drug-resistant Acinetobacter baumannii indigo-pigmented strain depicts evidence of increase genome plasticity.

Acinetobacter baumannii is a multidrug resistant nosocomial pathogen that shows an outstanding ability to undergo genetic exchange, thereby acquiring different traits that contribute to its success. In this work, we identified genetic features of an indigo-pigmented A. baumannii strain (Ab33405) that belongs to the clonal complex CC113B/CC79P. Ab33405 possesses a high number of genes coding for antibiotic resistance and virulence factors that may contribute to its survival, not only in the human host, but also in the hospital environment. Thirteen genes conferring resistance to different antibiotic families (trimethoprim, florfenicol, ß-lactams, aminoglycosides and sulfonamide) as well as the adeIJK genes and the capsule locus (KL) and outer core locus (OCL) were identified. Ab33405 includes 250 unique genes and a significant number of elements associated with Horizontal Gene Transfer, such as insertion sequences and transposons, genomic islands and prophage sequences. Also, the indigo-pigmented uncommon phenotype that could be associated with the monooxygenase or dioxygenase enzyme coded for by the iacA gene within the iac cluster was probably conferred by insertion of a 18-kb DNA fragment into the iacG gene belonging to this cluster. The Ab33405 genome includes all type VI secretion system genes and killing assays showed the ability of Ab33045 to kill Escherichia coli. In addition, Ab33405 can modulate susceptibility antibiotics when exposed to blue light.

Human serum albumin alters specific genes that can play a role in survival and persistence in Acinetobacter baumannii.

In the past few decades Acinetobacter baumannii has emerged as a notorious nosocomial pathogen because of its ability to acquire genetic material and persist in extreme environments. Recently, human serum albumin (HSA) was shown to significantly increase natural transformation frequency in A. baumannii. This observation led us to perform transcriptomic analysis of strain A118 under HSA induction to identify genes that are altered by HSA. Our results revealed the statistically significant differential expression of 296 protein-coding genes, including those associated with motility, biofilm formation, metabolism, efflux pumps, capsule synthesis, and transcriptional regulation. Phenotypic analysis of these traits showed an increase in surface-associated motility, a decrease in biofilm formation, reduced activity of a citric acid cycle associated enzyme, and increased survival associated with zinc availability. Furthermore, the expression of genes known to play a role in pathogenicity and antibiotic resistance were altered. These genes included those associated with RND-type efflux pumps, the type VI secretion system, iron acquisition/metabolism, and ß-lactam resistance. Together, these results illustrate how human products, in particular HSA, may play a significant role in both survival and persistence of A. baumannii.