Comparison of available methods to evaluate cefiderocol susceptibility in Acinetobacter spp.

Recently, considerable uncertainty has arisen concerning the appropriate susceptibility testing for cefiderocol in gram-negative bacilli, particularly in the context of its application to Acinetobacter spp. The optimal method for assessing the susceptibility levels of Acinetobacter spp. to cefiderocol remains a subject of debate due to substantial disparities observed in the values obtained through various testing procedures. This study employed four minimum inhibitory concentration (MIC) methodologies and the disk diffusion to assess the susceptibility of twenty-seven carbapenem resistant (CR)-Acinetobacter strains to cefiderocol. The results from our study reveal significant variations in the minimum inhibitory concentration (MIC) values obtained with the different methods and in the level of agreement in interpretation categories between the different MIC methods and the disk diffusion test. Among the MIC methods, there was relatively more consistency in reporting the interpretation categories. For European Committee on Antimicrobial Susceptibility Testing (EUCAST) breakpoints, the categorical agreement (CA) for MIC methods ranged between 66.7 and 81.5%. On the other hand, the essential agreement (EA) values were as low as 18.5-29.6%. The CA between MIC methods and disk diffusion was 81.5%. These results emphasize the need for a reliable, accurate, and clinically validated methodology to effectively assess the susceptibility of Acinetobacter spp. to cefiderocol. The wide variability observed in our study highlights the importance of standardizing the susceptibility testing process for cefiderocol to ensure consistent and reliable results for clinical decision-making.

Genomic Comparative Analysis of Two Multi-Drug Resistance (MDR) Acinetobacter baumannii Clinical Strains Assigned to International Clonal Lineage II Recovered Pre- and Post-COVID-19 Pandemic.

BACKGROUND: After the emergence of COVID-19, numerous cases of A. baumannii/SARS-CoV-2 co-infection were reported. Whether the co-infecting A. baumannii strains have distinctive characteristics remains unknown. METHODS AND RESULTS: A. baumannii AMA_NO was isolated in 2021 from a patient with COVID-19. AMA166 was isolated from a mini-BAL used on a patient with pneumonia in 2016. Both genomes were similar, but they possessed 337 (AMA_NO) and 93 (AMA166) unique genes that were associated with biofilm formation, flagellar assembly, antibiotic resistance, secretion systems, and other functions. The antibiotic resistance genes were found within mobile genetic elements. While both strains harbored the carbapenemase-coding gene blaOXA-23, only the strain AMA_NO carried blaNDM-1. Representative functions coded for by virulence genes are the synthesis of the outer core of lipooligosaccharide (OCL5), biosynthesis and export of the capsular polysaccharide (KL2 cluster), high-efficiency iron uptake systems (acinetobactin and baumannoferrin), adherence, and quorum sensing. A comparative phylogenetic analysis including 239 additional sequence type (ST) 2 representative genomes showed high similarity to A. baumannii ABBL141. Since the degree of similarity that was observed between A. baumannii AMA_NO and AMA166 is higher than that found among other ST2 strains, we propose that they derive from a unique background based on core-genome phylogeny and comparative genome analysis. CONCLUSIONS: Acquisition or shedding of specific genes could increase the ability of A. baumannii to infect patients with COVID-19.

A New Twist: The Combination of Sulbactam/Avibactam Enhances Sulbactam Activity against Carbapenem-Resistant Acinetobacter baumannii (CRAB) Isolates.

An increasing number of untreatable infections are recorded every year. Many studies have focused their efforts on developing new ß-lactamase inhibitors to treat multi-drug resistant (MDR) isolates. In the present study, sulbactam/avibactam and sulbactam/relebactam combination were tested against 187 multi-drug resistant (MDR) Acinetobacter clinical isolates; both sulbactam/avibactam and sulbactam/relebactam restored sulbactam activity. A decrease ≥2 dilutions in sulbactam MICs was observed in 89% of the isolates when tested in combination with avibactam. Sulbactam/relebactam was able to restore sulbactam susceptibility in 40% of the isolates. In addition, the susceptibility testing using twenty-three A. baumannii AB5075 knockout strains revealed potential sulbactam and/or sulbactam/avibactam target genes. We observed that diazabicyclooctanes (DBOs) ß-lactamase inhibitors combined with sulbactam restore sulbactam susceptibility against carbapenem-resistant Acinetobacter clinical isolates. However, relebactam was not as effective as avibactam when combined with sulbactam. Exploring novel combinations may offer new options to treat Acinetobacter spp. infections, especially for widespread oxacillinases and metallo-ß-lactamases (MBLs) producers.

Bridged Nucleic Acids Reloaded.

Oligonucleotides are key compounds widely used for research, diagnostics, and therapeutics. The rapid increase in oligonucleotide-based applications, together with the progress in nucleic acids research, has led to the design of nucleotide analogs that, when part of these oligomers, enhance their efficiency, bioavailability, or stability. One of the most useful nucleotide analogs is the first-generation bridged nucleic acids (BNA), also known as locked nucleic acids (LNA), which were used in combination with ribonucleotides, deoxyribonucleotides, or other analogs to construct oligomers with diverse applications. However, there is still room to improve their efficiency, bioavailability, stability, and, importantly, toxicity. A second-generation BNA, BNANC (2'-O,4'-aminoethylene bridged nucleic acid), has been recently made available. Oligomers containing these analogs not only showed less toxicity when compared to LNA-containing compounds but, in some cases, also exhibited higher specificity. Although there are still few applications where BNANC-containing compounds have been researched, the promising results warrant more effort in incorporating these analogs for other applications. Furthermore, newer BNA compounds will be introduced in the near future, offering great hope to oligonucleotide-based fields of research and applications.

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.

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.

The Genetic Analysis of an Acinetobacter johnsonii Clinical Strain Evidenced the Presence of Horizontal Genetic Transfer.

Acinetobacter johnsonii rarely causes human infections. While most A. johnsonii isolates are susceptible to virtually all antibiotics, strains harboring a variety of ß-lactamases have recently been described. An A. johnsonii Aj2199 clinical strain recovered from a hospital in Buenos Aires produces PER-2 and OXA-58. We decided to delve into its genome by obtaining the whole genome sequence of the Aj2199 strain. Genome comparison studies on Aj2199 revealed 240 unique genes and a close relation to strain WJ10621, isolated from the urine of a patient in China. Genomic analysis showed evidence of horizontal genetic transfer (HGT) events. Forty-five insertion sequences and two intact prophages were found in addition to several resistance determinants such as blaPER-2, blaOXA-58, blaTEM-1, strA, strB, ereA, sul1, aacC2 and a new variant of blaOXA-211, called blaOXA-498. In particular, blaPER-2 and blaTEM-1 are present within the typical contexts previously described in the Enterobacteriaceae family. These results suggest that A. johnsonii actively acquires exogenous DNA from other bacterial species and concomitantly becomes a reservoir of resistance genes.

External guide sequence technology: a path to development of novel antimicrobial therapeutics.

RNase P is a ribozyme originally identified for its role in maturation of tRNAs by cleavage of precursor tRNAs (pre-tRNAs) at the 5'-end termini. RNase P is a ribonucleoprotein consisting of a catalytic RNA molecule and, depending on the organism, one or more cofactor proteins. The site of cleavage of a pre-tRNA is identified by its tertiary structure; and any RNA molecule can be cleaved by RNase P as long as the RNA forms a duplex that resembles the regional structure in the pre-tRNA. When the antisense sequence that forms the duplex with the strand that is subsequently cleaved by RNase P is in a separate molecule, it is called an external guide sequence (EGS). These fundamental observations are the basis for EGS technology, which consists of inhibiting gene expression by utilizing an EGS that elicits RNase P-mediated cleavage of a target mRNA molecule. EGS technology has been used to inhibit expression of a wide variety of genes, and may help development of novel treatments of diseases, including multidrug-resistant bacterial and viral infections.

Draft Genome Sequence of Empedobacter (Formerly Wautersiella) falsenii comb. nov. Wf282, a Strain Isolated from a Cervical Neck Abscess.

Empedobacter (formerly Wautersiella) falsenii comb. nov. strain Wf282 was isolated from a cervical neck abscess sample from an 18-year-old female patient. The isolate was resistant to many antibiotics, including meropenem and colistin. The total DNA from the multidrug-resistant E. falsenii comb. nov. Wf282 clinical isolate was sequenced.

Whole-genome sequence analysis of the naturally competent Acinetobacter baumannii clinical isolate A118.

Recent studies have demonstrated a high genomic plasticity in Acinetobacter baumannii, which may explain its high capacity to acquire multiple antibiotic resistance determinants and to survive in the hospital environment. Acinetobacter baumannii strain A118 (Ab A118) was isolated in the year 1995 from a blood culture of an intensive care unit patient. As this particular strain showed some peculiar characteristic such as being naturally competent and susceptible to numerous antibiotics, we performed whole-genome comparison (WGC) studies to gain insights into the nature and extent of the genomic differences. The Ab A118 genome is approximately 3,824 kb long with a 38.4% GC content and contains 3,520 coding sequences. WGC studies showed that the Ab A118 genome has 98% average nucleotide identity with that of A. baumannii ATCC 17978, and 96% average nucleotide identity with that of strains AYE and ACICU. At least 12 inversions, 275 insertions, and 626 deletions were identified when the Ab A118 genome was compared with those of strains ATCC 17978, AYE, and ACICU using MAUVE WGC. Multiple gene order arrangements were observed among the analyzed strains. MAUVE WGC analysis identified 19 conserved segments, known as locally colinear blocks. The number of single nucleotide polymorphisms found when comparing the Ab A118 genome with that of strains ATCC 17978, AYE, and ACICU was 43,784 (1.1496%), 44,130 (1.158%), and 43,914 (1.153%), respectively. Genes comEA, pilQ, pilD, pilF, comL, pilA, comEC, pilI, pilH, pilO, pilN, pilY1(comC), pilE, pilR, and comM, potentially involved in natural competence were found in the Ab A118 genome. In particular, unlike in most strains where comM is interrupted by an insertion of a resistance island (AbaR), in strain Ab A118 it is uninterrupted.

Genome Sequences of Two Klebsiella pneumoniae Isolates from Different Geographical Regions, Argentina (Strain JHCK1) and the United States (Strain VA360).

We report the sequences of two Klebsiella pneumoniae clinical isolates, strains JHCK1 and VA360, from a newborn with meningitis in Buenos Aires, Argentina, and from a tertiary care medical center in Cleveland, OH, respectively. Both isolates contain one chromosome and at least five plasmids; isolate VA360 contains the Klebsiella pneumoniae carbapenemase (KPC) gene.

Differential distribution of plasmid-mediated quinolone resistance genes in clinical enterobacteria with unusual phenotypes of quinolone susceptibility from Argentina.

We studied a collection of 105 clinical enterobacteria with unusual phenotypes of quinolone susceptibility to analyze the occurrence of plasmid-mediated quinolone resistance (PMQR) and oqx genes and their implications for quinolone susceptibility. The oqxA and oqxB genes were found in 31/34 (91%) Klebsiella pneumoniae and 1/3 Klebsiella oxytoca isolates. However, the oqxA- and oqxB-harboring isolates lacking other known quinolone resistance determinants showed wide ranges of susceptibility to nalidixic acid and ciprofloxacin. Sixty of the 105 isolates (57%) harbored at least one PMQR gene [qnrB19, qnrB10, qnrB2, qnrB1, qnrS1, or aac(6')-Ib-cr)], belong to 8 enterobacterial species, and were disseminated throughout the country, and most of them were categorized as susceptible by the current clinical quinolone susceptibility breakpoints. We developed a disk diffusion-based method to improve the phenotypic detection of aac(6')-Ib-cr. The most common PMQR genes in our collection [qnrB19, qnrB10, and aac(6')-Ib-cr] were differentially distributed among enterobacterial species, and two different epidemiological settings were evident. First, the species associated with community-acquired infections (Salmonella spp. and Escherichia coli) mainly harbored qnrB19 (a unique PMQR gene) located in small ColE1-type plasmids that might constitute its natural reservoirs. qnrB19 was not associated with an extended-spectrum ß-lactamase phenotype. Second, the species associated with hospital-acquired infections (Enterobacter spp., Klebsiella spp., and Serratia marcescens) mainly harbored qnrB10 in ISCR1-containing class 1 integrons that may also have aac(6')-Ib-cr as a cassette within the variable region. These two PMQR genes were strongly associated with an extended-spectrum ß-lactamase phenotype. Therefore, this differential distribution of PMQR genes is strongly influenced by their linkage or lack of linkage to integrons.

Inhibition of cell division induced by external guide sequences (EGS Technology) targeting ftsZ.

EGS (external guide sequence) technology is a promising approach to designing new antibiotics. EGSs are short antisense oligoribonucleotides that induce RNase P-mediated cleavage of a target RNA by forming a precursor tRNA-like complex. The ftsZ mRNA secondary structure was modeled and EGSs complementary to two regions with high probability of being suitable targets were designed. In vitro reactions showed that EGSs targeting these regions bound ftsZ mRNA and elicited RNase P-mediated cleavage of ftsZ mRNA. A recombinant plasmid, pEGSb1, coding for an EGS that targets region "b" under the control of the T7 promoter was generated. Upon introduction of this plasmid into Escherichia coli BL21(DE3)(pLysS) the transformant strain formed filaments when expression of the EGS was induced. Concomitantly, E. coli harboring pEGSb1 showed a modest but significant inhibition of growth when synthesis of the EGSb1 was induced. Our results indicate that EGS technology could be a viable strategy to generate new antimicrobials targeting ftsZ.

Small plasmids harboring qnrB19: a model for plasmid evolution mediated by site-specific recombination at oriT and Xer sites.

Plasmids pPAB19-1, pPAB19-2, pPAB19-3, and pPAB19-4, isolated from Salmonella and Escherichia coli clinical strains from hospitals in Argentina, were completely sequenced. These plasmids include the qnrB19 gene and are 2,699, 3,082, 2,989, and 2,702 nucleotides long, respectively, and they share extensive homology among themselves and with other previously described small qnrB19-harboring plasmids. The genetic environment of qnrB19 in all four plasmids is identical to that in these other plasmids and in transposons such as Tn2012, Tn5387, and Tn5387-like. Nucleotide sequence comparisons among these and previously described plasmids showed a variable region characterized by being flanked by an oriT locus and a Xer recombination site. We propose that this arrangement could play a role in the evolution of plasmids and present a model for DNA swapping between plasmid molecules mediated by site-specific recombination events at oriT and a Xer target site.

Internalization of Locked Nucleic Acids/DNA Hybrid Oligomers into Escherichia coli.

Delivery inside the cells is essential for practical application of antisense technologies. The hybrid locked nucleic acid (LNA)/DNA CAAGTACTGTTCCACCA (LNA residues are underlined) was labeled by conjugation to Alexa Fluor 488 (fLNA/DNA) and tested to determine its ability to penetrate Escherichia coli cells and reach the cytoplasm. Flow cytometry analysis showed that the fLNA/DNA was associated with 14% of cells from a stationary phase culture, while association with a labeled isosequential oligodeoxynucleotide was negligible. Laser scanning confocal microscopy confirmed that the fLNA/DNA was located inside the cytoplasm.

Inhibition of aac(6')-Ib-mediated amikacin resistance by nuclease-resistant external guide sequences in bacteria.

Inhibition of bacterial gene expression by RNase P-directed cleavage is a promising strategy for the development of antibiotics and pharmacological agents that prevent expression of antibiotic resistance. The rise in multiresistant bacteria harboring AAC(6')-Ib has seriously limited the effectiveness of amikacin and other aminoglycosides. We have recently shown that recombinant plasmids coding for external guide sequences (EGS), short antisense oligoribonucleotides (ORN) that elicit RNase P-mediated cleavage of a target mRNA, induce inhibition of expression of aac(6')-Ib and concomitantly induce a significant decrease in the levels of resistance to amikacin. However, since ORN are rapidly degraded by nucleases, development of a viable RNase P-based antisense technology requires the design of nuclease-resistant RNA analog EGSs. We have assayed a variety of ORN analogs of which selected LNA/DNA co-oligomers elicited RNase P-mediated cleavage of mRNA in vitro. Although we found an ideal configuration of LNA/DNA residues, there seems not to be a correlation between number of LNA substitutions and level of activity. Exogenous administration of as low as 50 nM of an LNA/DNA co-oligomer to the hyperpermeable E. coli AS19 harboring the aac(6')-Ib inhibited growth in the presence of amikacin. Our experiments strongly suggest an RNase P-mediated mechanism in the observed antisense effect.

Complex class 1 integrons with diverse variable regions, including aac(6')-Ib-cr, and a novel allele, qnrB10, associated with ISCR1 in clinical enterobacterial isolates from Argentina.

Transferable quinolone resistance has not previously been reported in Argentina. Here we describe three complex class 1 integrons harboring the novel allele qnrB10 in a unique region downstream of orf513, one of them also containing aac(6')-Ib-cr within the variable region of integrons. The three arrays differed from bla(CTX-M-2)-bearing integrons, which are broadly distributed in Argentina.

Evidence for the active role of a novel nuclease from Helicobacter pylori in the horizontal transfer of genetic information.

Helicobacter pylori is a gram-negative bacterium that colonizes the human stomach, causes gastritis, and is associated with ulcers and gastric cancer. H. pylori is naturally competent for transformation. Natural genetic transformation is believed to be essential for the genetic plasticity observed in this species. While the relevance of horizontal gene transfer in H. pylori adaptiveness and antibiotic resistance is well documented, the DNA transformation machinery components are barely known. No enzymatic activity associated with the transformation process has been determined experimentally and described. We isolated, microsequenced, and cloned a major DNA nuclease from H. pylori. This protein, encoded by the open reading frame hp0323, was expressed in Escherichia coli. The purified protein, NucT, has a cation-independent thermostable nuclease activity that preferentially cleaves single-stranded DNA. NucT is associated with the membrane. NucT-deficient H. pylori strains are one or more orders of magnitude less efficient than the parental strain for transformation with either chromosomal or self-replicating plasmid DNA. To the best of our knowledge, NucT is the first nuclease identified in a gram-negative natural transformation system, and its existence suggests that there is a mechanism of DNA processing and uptake similar to the mechanisms in well-studied gram-positive systems.