Carbapenem-resistant Acinetobacter baumannii (CRAB): metabolic adaptation and transcriptional response to human urine (HU).

Carbapenem-resistant Acinetobacter baumannii (CRAB) is a major human pathogen and a research priority for developing new antimicrobial agents. CRAB is a causative agent of a variety of infections in different body sites. One of the manifestations is catheter-associated urinary tract infection, which exposes the bacteria to the host's urine, creating a particular environment. Exposure of two CRAB clinical isolates, AB5075 and AMA40, to human urine (HU) resulted in the differential expression levels of 264 and 455 genes, respectively, of which 112 were common to both strains. Genes within this group play roles in metabolic pathways such as phenylacetic acid (PAA) catabolism, the Hut system, the tricarboxylic acid (TCA) cycle, and other processes like quorum sensing and biofilm formation. These results indicate that the presence of HU induces numerous adaptive changes in gene expression of the infecting bacteria. These modifications presumably help bacteria establish and thrive in the hostile conditions in the urinary tract. These analyses advance our understanding of CRAB's metabolic adaptations to human fluids, as well as expanding knowledge on bacterial responses to distinct human fluids containing different concentrations of human serum albumin (HSA).

Hetero-antagonism of avibactam and sulbactam with cefiderocol in carbapenem-resistant Acinetobacter spp.

The emergence of Gram-negative bacteria resistant to multiple antibiotics, particularly carbapenem-resistant (CR) Acinetobacter strains, poses a significant threat globally. Despite efforts to develop new antimicrobial therapies, limited progress has been made, with only two drugs-cefiderocol and sulbactam-durlobactam-showing promise for CR-Acinetobacter infections. Cefiderocol, a siderophore cephalosporin, demonstrates promising efficacy in the treatment of Gram-negative infections. However, resistance to cefiderocol has been reported in A. baumannii. Combination therapies, such as cefiderocol with avibactam or sulbactam, show reduced MICs against cefiderocol-non-susceptible strains with in vivo efficacy, although the outcomes can be complex and species-specific. In the present work, the molecular characterization of spontaneous cefiderocol-resistant variants, a CRAB strain displaying antagonism with sulbactam and an A. lwoffii strain showing antagonism with avibactam, were studied. The results reveal intriguing insights into the underlying mechanisms, including mutations affecting efflux pumps, transcriptional regulators, and iron homeostasis genes. Moreover, gene expression analysis reveals significant alterations in outer membrane proteins, iron homeostasis, and ß-lactamases, suggesting adaptive responses to selective pressure. Understanding these mechanisms is crucial for optimizing treatment strategies and preventing adverse clinical outcomes. This study highlights the importance of preemptively assessing drug synergies to navigate the challenges posed by antimicrobial resistance in CR-Acinetobacter infections.

Draft genome sequence of Lacticaseibacillus rhamnosus CRL 2244, a strain with strong killing effect on carbapenem-resistant Acinetobacter baumannii.

We report here a draft genome assembly of Lacticaseibacillus rhamnosus CRL 2244, recovered from wastewater in Argentina. The genome has a size of 2,898,100 bp, with G + C content of 46.73%. Comparative analysis reveals that its closest relative is L. rhamnosus 1.0320 (GCF_006151905.1), with an average nucleotide identity of 97.46%.

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 ß-lactam resistance. However, transcriptional analyses demonstrated significant modifications in levels of expression of genes associated with iron-uptake systems or ß-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 ß-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.

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.

Comparative genome analysis of the genus Shewanella unravels the association of key genetic traits with known and potential pathogenic lineages.

Shewanella spp. are Gram-negative rods widely disseminated in aquatic niches that can also be found in human-associated environments. In recent years, reports of infections caused by these bacteria have increased significantly. Mobilome and resistome analysis of a few species showed that they are versatile; however, comprehensive comparative studies in the genus are lacking. Here, we analyzed the genetic traits of 144 genomes from Shewanella spp. isolates focusing on the mobilome, resistome, and virulome to establish their evolutionary relationship and detect unique features based on their genome content and habitat. Shewanella spp. showed a great diversity of mobile genetic elements (MGEs), most of them associated with monophyletic lineages of clinical isolates. Furthermore, 79/144 genomes encoded at least one antimicrobial resistant gene with their highest occurrence in clinical-related lineages. CRISPR-Cas systems, which confer immunity against MGEs, were found in 41 genomes being I-E and I-F the more frequent ones. Virulome analysis showed that all Shewanella spp. encoded different virulence genes (motility, quorum sensing, biofilm, adherence, etc.) that may confer adaptive advantages for survival against hosts. Our data revealed that key accessory genes are frequently found in two major clinical-related groups, which encompass the opportunistic pathogens Shewanella algae and Shewanella xiamenensis together with several other species. This work highlights the evolutionary nature of Shewanella spp. genomes, capable of acquiring different key genetic traits that contribute to their adaptation to different niches and facilitate the emergence of more resistant and virulent isolates that impact directly on human and animal health.

Molecular and Phenotypic Characterization of a Multidrug-Resistant Escherichia coli Coproducing OXA-232 and MCR-1.1 in Argentina.

The spread of carbapenem-resistant Enterobacterales has raised concern in clinical settings due to the limited therapeutic options available. OXA-48-like enzymes are still sporadic in South America. The aim of this study was to characterize a multidrug-resistant Escherichia coli isolate from a hospitalized patient in Buenos Aires city. The isolate was characterized phenotypically by determination of its susceptibility pattern, synergistic and colorimetric tests, and molecularly, by PCR, whole genome sequencing, and plasmid analysis. It belonged to ST-744, phylogroup A, and serotype O162/O89: H9. It remained susceptible to ceftazidime, meropenem, aminoglycosides, trimethoprim/sulfamethoxazole, and tigecycline. The presence of blaOXA-232 harbored by a nonconjugative plasmid ColKp3, and blaCTX-M-14, mcr-1.1, and fosL1 in 2 conjugative plasmids, together with their genetic environment, was revealed. To the best of our knowledge, this is the first report of the coproduction of the enzyme OXA-232 and the mcr-1.1 gene in an E. coli clinical isolate in South America in a patient who had not received colistin therapy.

Infections due to Vagococcus spp. Microbiological and clinical aspects and literature review.

INTRODUCTION: An increase in recent years in the isolation of Vagococcus spp. is suggestive of emerging infection by this pathogen in our hospital. METHODS: Prospective, descriptive study. PERIOD: July 2014-January 2019. Phenotypic identification of 15 isolates of Vagococcus spp. was performed by conventional biochemical tests, automated methodology and mass spectrometry (MALDI-TOF MS). Molecular identification was achieved by sequencing the 16S rRNA gene. The Vitek™ 2C automated system was used to test antibiotic susceptibility. RESULTS: The molecular method identified 11 Vagococcus fluvialis, one Vagococcus lutrae and three Vagococcus spp. MALDI-TOF MS facilitated the rapid recognition of the genus. The most active antibiotics were ampicillin, trimethoprim/sulfamethoxazole, vancomycin, teicoplanin and linezolid. Most of the cases of isolation were associated with skin and soft tissue or osteoarticular infections in patients with diabetes. CONCLUSION: This article is the most extensive review of cases of Vagococcus spp. infection reported in the literature and highlights the microbiological and clinical aspects of this pathogen.

Infecciones por Vagococcus spp. Aspectos microbiológicos y clínicos y revisión de la literatura / Infections due to Vagococcus spp. Microbiological and clinical aspects and literature review

Introducción: En los últimos años un aumento en la frecuencia de aislamiento de Vagococcus spp. estaría indicando la urgencia de la infección por este patógeno en nuestra institución. Métodos: Estudio prospectivo y descriptivo que abarca el periodo de julio de 2014 a enero de 2019. La identificación fenotípica de 15 aislados de Vagococcus spp. se realizó por pruebas bioquímicas convencionales, por metodología automatizada y por espectrometría de masas (MALDI-TOF MS). La identificación molecular por secuenciación del gen ARNr 16S. La sensibilidad antibiótica fue realizada utilizando el sistema automatizado Vitek® 2C. Resultados: Se identificaron 11 Vagococcus fluvialis, un Vagococcus lutrae y tres Vagococcus spp. por metodología molecular. MALDI-TOF MS permitió el rápido reconocimiento de este género. Los antibióticos más activos fueron ampicilina, trimetoprima/sulfametoxazol, vancomicina, teicoplanina y linezolid. La mayoría de los aislamientos se asociaron con infecciones en la piel y partes blandas u osteoarticulares en pacientes diabéticos. Conclusión: Esta comunicación representa la mayor revisión de casos de infecciones por Vagococcus spp. reportados en la literatura, en la que se destacan los aspectos microbiológicos y clínicos de este patógeno.(AU)

Introduction: An increase in recent years in the isolation of Vagococcus spp. is suggestive of emerging infection by this pathogen in our hospital. Methods: Prospective, descriptive study. Period: July 2014-January 2019. Phenotypic identification of 15 isolates of Vagococcus spp. was performed by conventional biochemical tests, automated methodology and mass spectrometry (MALDI-TOF MS). Molecular identification was achieved by sequencing the 16S rRNA gene. The Vitek™ 2C automated system was used to test antibiotic susceptibility. Results: The molecular method identified 11 Vagococcus fluvialis, one Vagococcus lutrae and three Vagococcus spp. MALDI-TOF MS facilitated the rapid recognition of the genus. The most active antibiotics were ampicillin, trimethoprim/sulfamethoxazole, vancomycin, teicoplanin and linezolid. Most of the cases of isolation were associated with skin and soft tissue or osteoarticular infections in patients with diabetes. Conclusion: This article is the most extensive review of cases of Vagococcus spp. infection reported in the literature and highlights the microbiological and clinical aspects of this pathogen.(AU)

Interplay between Meropenem and Human Serum Albumin on Expression of Carbapenem Resistance Genes and Natural Competence in Acinetobacter baumannii.

Acinetobacter baumannii A118, a carbapenem-susceptible strain, and AB5075, carbapenem resistant, were cultured in lysogeny broth (LB) or LB with different supplements, such as 3.5% human serum albumin (HSA), human serum (HS), meropenem, or meropenem plus 3.5% HSA. Natural transformation levels were enhanced in A. baumannii A118 and AB5075 cultured in medium supplemented with 3.5% HSA. Addition of meropenem plus 3.5% HSA caused synergistic enhancement of natural transformation in A. baumannii A118. Medium containing 3.5% HSA or meropenem enhanced the expression levels of the competence and type IV pilus-associated genes. The combination meropenem plus 3.5% HSA produced a synergistic enhancement in the expression levels of many of these genes. The addition of HS, which has a high content of HSA, was also an inducer of these genes. Cultures in medium supplemented with HS or 3.5% HSA also affected resistance genes, which were expressed at higher or lower levels depending on the modification required to enhance resistance. The inducing or repressing activity of these modulators also occurred in three more carbapenem-resistant strains tested. An exception was the A. baumannii AMA16 blaNDM-1 gene, which was repressed in the presence of 3.5% HSA. In conclusion, HSA produces an enhancement of natural transformation and a modification in expression levels of competence genes and antibiotic resistance. Furthermore, when HSA is combined with carbapenems, which may increase the stress response, the expression of genes involved in natural competence is increased in A. baumannii. This process may favor the acquisition of foreign DNA and accelerate evolution.

Corynebacterium phoceense - a rare Corynebacterium species isolated from a urine sample.

Corynebacterium spp. are Gram-positive rods that are recognized to cause opportunistic diseases under certain predisposing clinical conditions. Some species have been described in urinary tract infections. In this report we document a new episode of urinary tract infection caused by Corynebacterium phoceense and describe the whole-genome sequencing, phenotypic characteristics and mass spectra obtained by matrix-assisted desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Based on genome identification and DNA-to-DNA hybridization, we can assume that our strain is the second isolate of C. phoceense to be described in a urine sample. No other infectious diseases have been reported to be associated with this species.

An Acinetobacter non-baumannii Population Study: Antimicrobial Resistance Genes (ARGs).

Acinetobacter non-baumannii species are becoming common etiologic agents of nosocomial infections. Furthermore, clinical isolates belonging to this group of bacteria are usually resistant to one or more antibiotics. The current information about antibiotic resistance genes in the different A. non-baumannii species has not yet been studied as a whole. Therefore, we did a comparative study of the resistomes of A. non-baumannii pathogens based on information available in published articles and genome sequences. We searched the available literature and sequences deposited in GenBank to identify the resistance gene content of A. calcoaceticus, A. lwoffii, A. junii, A. soli, A. ursingii, A. bereziniae, A. nosocomialis, A. portensis, A. guerrae, A. baylyi, A. calcoaceticus, A. disperses, A. johnsonii, A. junii, A. lwoffii, A. nosocomialis, A. oleivorans, A. oryzae, A. pittii, A. radioresistens, and A. venetianus. The most common genes were those coding for different ß-lactamases, including the carbapenemase genes bla NDM-1 and bla OXA-58. A. pittii was the species with the most ß-lactamase resistance genes reported. Other genes that were commonly found include those encoding some aminoglycoside modifying enzymes, the most common being aph(6)-Id, ant( 3 ″ )-IIa, and aph( 3 ″ )-Ib, and efflux pumps. All or part of the genes coding for the AdeABC, AdeFGH, and AdeIJK efflux pumps were the most commonly found. This article incorporates all the current information about A. non-baumannii resistance genes. The comparison of the different resistomes shows that there are similarities in the genes present, but there are also significant differences that could impact the efficiency of treatments depending on the etiologic agent. This article is a comprehensive resource about A. non-baumannii resistomes.

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.

Functional Analysis of the Acinetobacter baumannii XerC and XerD Site-Specific Recombinases: Potential Role in Dissemination of Resistance Genes.

Modules composed of a resistance gene flanked by Xer site-specific recombination sites, the vast majority of which were found in Acinetobacter baumannii, are thought to behave as elements that facilitate horizontal dissemination. The A. baumannii xerC and xerD genes were cloned, and the recombinant clones used to complement the cognate Escherichia coli mutants. The complemented strains supported the resolution of plasmid dimers, and, as is the case with E. coli and Klebsiella pneumoniae plasmids, the activity was enhanced when the cells were grown in a low osmolarity growth medium. Binding experiments showed that the partially purified A. baumannii XerC and XerD proteins (XerCAb and XerDAb) bound synthetic Xer site-specific recombination sites, some of them with a nucleotide sequence deduced from existing A. baumannii plasmids. Incubation with suicide substrates resulted in the covalent attachment of DNA to a recombinase, probably XerCAb, indicating that the first step in the recombination reaction took place. The results described show that XerCAb and XerDAb are functional proteins and support the hypothesis that they participate in horizontal dissemination of resistant genes among bacteria.

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.