Roles of DJ41_1407 and DJ41_1408 in Acinetobacter baumannii ATCC19606 Virulence and Antibiotic Response.

Acinetobacter baumannii is a major cause of nosocomial infections, and its highly adaptive nature and broad range of antibiotic resistance enable it to persist in hospital environments. A. baumannii often employs two-component systems (TCSs) to regulate adaptive responses and virulence-related traits. This study describes a previously uncharacterized TCS in the A. baumannii ATCC19606 strain, consisting of a transcriptional sensor, DJ41_1407, and its regulator, DJ41_1408, located adjacent to GacA of the GacSA TCS. Markerless mutagenesis was performed to construct DJ41_1407 and DJ41_1408 single and double mutants. DJ41_1408 was found to upregulate 49 genes and downregulate 43 genes, most of which were associated with carbon metabolism and other metabolic pathways, such as benzoate degradation. MEME analysis revealed a putative binding box for DJ41_1408, 5'TGTAAATRATTAYCAWTWAT3'. Colony size, motility, biofilm-forming ability, virulence, and antibiotic resistance of DJ41_1407 and DJ41_1408 single and double mutant strains were assessed against wild type. DJ41_1407 was found to enhance motility, while DJ41_1408 was found to upregulate biofilm-forming ability, and may also modulate antibiotic response. Both DJ41_1407 and DJ41_1408 suppressed virulence, based on results from a G. mellonella infection assay. These results showcase a novel A. baumannii TCS involved in metabolism, with effects on motility, biofilm-forming ability, virulence, and antibiotic response.

Epidemiology, resistance genomics and susceptibility of Acinetobacter species: results from the 2020 Spanish nationwide surveillance study.

BackgroundAs increasing antibiotic resistance in Acinetobacter baumannii poses a global healthcare challenge, understanding its evolution is crucial for effective control strategies.AimWe aimed to evaluate the epidemiology, antimicrobial susceptibility and main resistance mechanisms of Acinetobacter spp. in Spain in 2020, and to explore temporal trends of A. baumannii.MethodsWe collected 199 single-patient Acinetobacter spp. clinical isolates in 2020 from 18 Spanish tertiary hospitals. Minimum inhibitory concentrations (MICs) for nine antimicrobials were determined. Short-read sequencing was performed for all isolates, and targeted long-read sequencing for A. baumannii. Resistance mechanisms, phylogenetics and clonality were assessed. Findings on resistance rates and infection types were compared with data from 2000 and 2010.ResultsCefiderocol and colistin exhibited the highest activity against A. baumannii, although colistin susceptibility has significantly declined over 2 decades. A. non-baumannii strains were highly susceptible to most tested antibiotics. Of the A. baumannii isolates, 47.5% (56/118) were multidrug-resistant (MDR). Phylogeny and clonal relationship analysis of A. baumannii revealed five prevalent international clones, notably IC2 (ST2, n = 52; ST745, n = 4) and IC1 (ST1, n = 14), and some episodes of clonal dissemination. Genes bla OXA-23, bla OXA-58 and bla OXA-24/40 were identified in 49 (41.5%), eight (6.8%) and one (0.8%) A. baumannii isolates, respectively. ISAba1 was found upstream of the gene (a bla OXA-51-like) in 10 isolates.ConclusionsThe emergence of OXA-23-producing ST1 and ST2, the predominant MDR lineages, shows a pivotal shift in carbapenem-resistant A. baumannii (CRAB) epidemiology in Spain. Coupled with increased colistin resistance, these changes underscore notable alterations in regional antimicrobial resistance dynamics.

Co-production of metallo-ß-lactamase and OXA-type ß-lactamases in carbapenem-resistant Acinetobacter baumannii clinical isolates in North East India.

Carbapenem-resistant Acinetobacter baumannii poses a significant threat to public health globally, especially due to its ability to produce multiple carbapenemases, leading to treatment challenges. This study aimed to investigate the antibiotic resistance pattern of carbapenem-resistant A. baumannii isolates collected from different clinical settings in North East India, focusing on their genotypic and phenotypic resistance profiles. A total of 172 multidrug-resistant A. baumannii isolates were collected and subjected to antibiotic susceptibility test using the Kirby-Bauer disk diffusion method. Various phenotypic tests were performed to detect extended-spectrum ß-lactamase (ESBL), metallo-ß-lactamase (MBL), class C AmpC ß-lactamase (AmpC), and carbapenem hydrolyzing class D ß-lactamase (CHDL) production among the isolates. Overexpression of carbapenemase and cephalosporinase genes was detected among the isolates through both phenotypic and genotypic investigation. The antibiotic resistance profile of the isolates revealed that all were multidrug-resistant; 25% were extensively drug-resistant, 9.30% were pan-drug-resistant, whereas 91.27% were resistant to carbapenems. In the genotypic investigation, 80.81% of isolates were reported harbouring at least one metallo-ß-lactamase encoding gene, with blaNDM being the most prevalent at 70.34%, followed by blaIMP at 51.16% of isolates. Regarding class D carbapenemases, blaOXA-51 and blaOXA-23 genes were detected in all the tested isolates, while blaOXA-24, blaOXA-48, and blaOXA-58 were found in 15.11%, 6.97%, and 1.74% isolates respectively. Further analysis showed that 31.97% of isolates co-harboured ESBL, MBL, AmpC, and CHDL genes, while 31.39% of isolates co-harboured ESBL, MBL, and CHDL genes with or without ISAba1 leading to extensively drug-resistant or pan drug-resistant phenotypes. This study highlights the complex genetic profile and antimicrobial-resistant pattern of the isolates circulating in North East India, emphasizing the urgent need for effective infection control measures and the development of alternative treatment strategies to combat these challenging pathogens.

Biodiversity of carbapenem-resistant bacteria in clinical samples from the Southwest Amazon region (Rondônia/Brazil).

Brazil is recognized for its biodiversity and the genetic variability of its organisms. This genetic variability becomes even more valuable when it is properly documented and accessible. Understanding bacterial diversity through molecular characterization is necessary as it can improve patient treatment, reduce the length of hospital stays and the selection of resistant bacteria, and generate data for health and epidemiological surveillance. In this sense, in this study, we aimed to understand the biodiversity and molecular epidemiology of carbapenem-resistant bacteria in clinical samples recovered in the state of Rondônia, located in the Southwest Amazon region. Retrospective data from the Central Public Health Laboratories (LACEN/RO) between 2018 and 2021 were analysed using the Laboratory Environment Manager Platform (GAL). Seventy-two species with carbapenem resistance profiles were identified, of which 25 species carried at least one gene encoding carbapenemases of classes A (blaKPC-like), B (blaNDM-like, blaSPM-like or blaVIM-like) and D (blaOXA-23-like, blaOXA-24-like, blaOXA-48-like, blaOXA-58-like or blaOXA-143-like), among which we will highlight Klebsiella pneumoniae, Pseudomonas aeruginosa, Acinetobacter baumannii, Serratia marcescens, and Providencia spp. With these results, we hope to contribute to the field by providing epidemiological molecular data for state surveillance on bacterial resistance and assisting in public policy decision-making.

[Characteristics of drug resistance and biofilm formation in carbapenem-resistant Acinetobacter baumannii in hospitalized children]. / 住院患儿耐碳青霉烯类鲍曼不动杆菌的耐药及生物膜特征分析.

OBJECTIVES: To study the distribution, drug resistance, and biofilm characteristics of carbapenem-resistant Acinetobacter baumannii (CRAB) isolated from hospitalized children, providing a reference for the prevention and treatment of CRAB infections in hospitalized children. METHODS: Forty-eight CRAB strains isolated from January 2019 to December 2022 were classified into epidemic and sporadic strains using repetitive extragenic palindromic sequence-based polymerase chain reaction. The drug resistance, biofilm phenotypes, and gene carriage of these two types of strains were compared. RESULTS: Both the 22 epidemic strains and the 26 sporadic strains were producers of Class D carbapenemases or extended-spectrum ß-lactamases with downregulated outer membrane porins, harboring the VIM, OXA-23, and OXA-51 genes. The biofilm formation capability of the sporadic strains was stronger than that of the epidemic strains (P<0.05). Genes related to biofilm formation, including Bap, bfs, OmpA, CsuE, and intI1, were detected in both epidemic and sporadic strains, with a higher detection rate of the intI1 gene in epidemic strains (P<0.05). CONCLUSIONS: CRAB strains are colonized in the hospital, with sporadic strains having a stronger ability to form biofilms, suggesting the potential for forming new clonal transmissions in the hospital. Continuous monitoring of the epidemic trends of CRAB and early warning of the distribution of epidemic strains are necessary to reduce the risk of CRAB infections in hospitalized children.

Structure of the K141 capsular polysaccharide produced by Acinetobacter baumannii isolate KZ1106 that carries KL141 at the chromosomal K locus.

The structure of the K141 type capsular polysaccharide (CPS) produced by Acinetobacter baumannii KZ1106, a clinical isolate recovered from Kazakhstan in 2016, was established by sugar analyses and one- and two-dimensional 1H and 13C NMR spectroscopy. The CPS was shown to consist of branched tetrasaccharide repeating units (K-units) with the following structure: This structure was found to be consistent with the genetic content of the KL141 CPS biosynthesis gene cluster at the chromosomal K locus in the KZ1106 whole genome sequence. Assignment of the encoded enzymes allowed the first sugar of the K unit to be identified, which revealed that the ß-d-GlcpNAc-(1→3)-d-GlcpNAc bond is the linkage between K-units formed by the WzyKL141 polymerase.

Three novel multiplex PCR assays for rapid detection of virulence, antimicrobial resistance, and toxin genes in Acinetobacter calcoaceticus-baumannii complex species.

The Acinetobacter calcoaceticus-baumannii (ACB) complex is an often-overlooked group of nosocomial pathogens with a significant environmental presence. Rapid molecular screening methods for virulence, antimicrobial resistance, and toxin (VAT) genes are required to investigate the potential pathogenicity of environmental isolates. This study aimed to develop and apply novel ACB complex-specific multiplex PCR (mPCR) primers and protocols for the rapid detection of eight VAT genes. We optimized three single-tube mPCR assays using reference DNA from ACB complex and other Acinetobacter species. These assays were then applied to detect VAT genes in cultured ACB complex isolates recovered from clinical and environmental sources. Widespread detection of VAT genes in environmental isolates confirmed the validity, functionality, and applicability of these novel assays. Overall, the three newly developed ACB complex species-specific mPCR assays are rapid and simple tools that can be adopted in diagnostic and clinical lab settings. The detection of VAT genes in environmental isolates suggests that environmental niches could serve as a reservoir for potentially pathogenic ACB complex and warrants further investigation. The newly developed mPCR assays are specific, sensitive, and efficient, making them well-suited for high-throughput screening in epidemiological studies and evaluating the potential pathogenicity of ACB complex recovered from various sources.

Isolation and genome-wide analysis of the novel Acinetobacter baumannii bacteriophage vB_AbaM_AB3P2.

A lytic Acinetobacter baumannii phage, isolate vB_AbaM_AB3P2, was isolated from a sewage treatment plant in China. A. baumannii phage vB_AbaM_AB3P2 has a dsDNA genome that is 44,824 bp in length with a G + C content of 37.75%. Ninety-six open reading frames were identified, and no genes for antibiotic resistance or virulence factors were found. Genomic and phylogenetic analysis of this phage revealed that it represents a new species in the genus Obolenskvirus. Phage vB_AbaM_AB3P2 has a short latent period (10 min) and high stability at 30-70°C and pH 2-10 and is potentially useful for controlling multi-drug-resistant A. baumannii.

Tracing clinically-relevant antimicrobial resistances in Acinetobacter baumannii-calcoaceticus complex across diverse environments: A study spanning clinical, livestock, and wastewater treatment settings.

Acinetobacter baumannii has become a prominent nosocomial pathogen, primarily owing to its remarkable ability to rapidly acquire resistance to a wide range of antimicrobial agents and its ability to persist in diverse environments. However, there is a lack of data on the molecular epidemiology and its potential implications for public health of A. baumannii strains exhibiting clinically significant resistances that originate from non-clinical environments. Therefore, the genetic characteristics and resistance mechanisms of 80 A. baumannii-calcoaceticus (ABC) complex isolates, sourced from environments associated with poultry and pig production, municipal wastewater treatment plants (WWTPs), and clinical settings, were investigated. In total, our study classified 54 isolates into 29 previously described sequence types (STs), while 26 isolates exhibited as-yet-unassigned STs. We identified a broad range of A. baumannii STs originating from poultry and pig production environments (e.g., ST10, ST238, ST240, ST267, ST345, ST370, ST372, ST1112 according to Pasteur scheme). These STs have also been documented in clinical settings worldwide, highlighting their clinical significance. These findings also raise concerns about the potential zoonotic transmission of certain STs associated with livestock environments. Furthermore, we observed that clinical isolates exhibited the highest diversity of antimicrobial resistance genes (ARGs). In contrast to non-clinical isolates, clinical isolates typically carried a significantly higher number of ARGs, ranging from 10 to 15. They were also the exclusive carriers of biocide resistance genes and acquired carbapenemases (blaOXA-23, blaOXA-58, blaOXA-72, blaGIM-1, blaNDM-1). Additionally, we observed that clinical strains displayed an increased capacity for carrying plasmids and undergoing genetic transformation. This heightened capability could be linked to the intense selective pressures commonly found within clinical settings. Our study provides comprehensive insights into essential aspects of ABC isolates originating from livestock-associated environments and clinical settings. We explored their resistance mechanisms and potential implications for public health, providing valuable knowledge for addressing these critical issues.

Sub-inhibitory concentrations of colistin and imipenem impact the expression of biofilm-associated genes in Acinetobacter baumannii.

Acinetobacter baumannii is an opportunistic pathogen that is responsible for nosocomial infections. Imipenem and colistin are drugs that are commonly used to treat severe infections caused by A. baumannii, such as sepsis, ventilator-associated pneumonia, and bacteremia. However, some strains of A. baumannii have become resistant to these drugs, which is a concern for public health. Biofilms produced by A. baumannii increase their resistance to antibiotics and the cells within the inner layers of biofilm are exposed to sub-inhibitory concentrations (sub-MICs) of antibiotics. There is limited information available regarding how the genes of A. baumannii are linked to biofilm formation when the bacteria are exposed to sub-MICs of imipenem and colistin. Thus, this study's objective was to explore this relationship by examining the genes involved in biofilm formation in A. baumannii when exposed to low levels of imipenem and colistin. The study found that exposing an isolate of A. baumannii to low levels of these drugs caused changes in their drug susceptibility pattern. The relative gene expression profiles of the biofilm-associated genes exhibited a change in their expression profile during short-term and long-term exposure. This study highlights the potential consequences of overuse and misuse of antibiotics, which can help bacteria become resistant to these drugs.

Host range expansion of Acinetobacter phage vB_Ab4_Hep4 driven by a spontaneous tail tubular mutation.

Bacteriophages (phages) represent promising alternative treatments against multidrug-resistant Acinetobacter baumannii (MDRAB) infections. The application of phages as antibacterial agents is limited by their generally narrow host ranges, so changing or expanding the host ranges of phages is beneficial for phage therapy. Multiple studies have identified that phage tail fiber protein mediates the recognition and binding to the host as receptor binding protein in phage infection. However, the tail tubular-dependent host specificity of phages has not been studied well. In this study, we isolated and characterized a novel lytic phage, vB_Ab4_Hep4, specifically infecting MDRAB strains. Meanwhile, we identified a spontaneous mutant of the phage, vB_Ab4_Hep4-M, which revealed an expanded host range compared to the wild-type phage. A single mutation of G to C was detected in the gene encoding the phage tail tubular protein B and thus resulted in an aspartate to histidine change. We further demonstrated that the host range expansion of the phage mutant is driven by the spontaneous mutation of guanine to cytosine using expressed tail tubular protein B. Moreover, we established that the bacterial capsule is the receptor for phage Abp4 and Abp4-M by identifying mutant genes in phage-resistant strains. In conclusion, our study provided a detailed description of phage vB_Ab4_Hep4 and revealed the tail tubular-dependent host specificity in A. baumannii phages, which may provide new insights into extending the host ranges of phages by gene-modifying tail tubular proteins.

Outbreak of Multidrug-resistant Acinetobacter baumannii in a Tertiary Health Center from Northwestern Nigeria.

Background: In spite of its global notoriety and WHO alarm, Acinetobacter baumannii is still an understudied critical-priority pathobiont in Nigeria. We characterized its antimicrobial susceptibility profile and resistance genes during an outbreak. Materials and Methods: This cross-sectional study involved collection of patients' urine samples and swabs from unit staff's hands and ward environments for the identification of A. baumannii strains using standard morphologic and biochemical methods. The disk diffusion method was used to assess the antimicrobial susceptibility profile of the isolates with the production of extended-spectrum beta-lactamases (ESBLs) confirmed by the combined disk test screening method. Characterization of the resistance genes of the ESBL producers was carried out using polymerase chain reaction polymerase chain reaction technique. Results: A.total of eight (six clinical and two nonclinical) A. baumannii isolates were identified. The overall isolate susceptibility and resistance rates to all the antimicrobial agents was 56.3% (27/48) and 35.4% (17/48), respectively. Similarly, all (8/8; 100.00%) isolates were susceptible to meropenem and 75.0% (6/8) to ampicillin-sulbactam while 62.5% (5/8) were resistant to trimethoprim-sulfamethoxazole and 50.0% (4/8) to each of ciprofloxacin and ceftazidime. In addition, 37.5% (3/8) of the isolates were multidrug resistant (MDR) with nonclinical isolates exhibiting more antimicrobial resistance than their clinical counterparts (9/12%-75.0% vs. 8/36%-22.2%). Phenotypic detection and molecular characterization revealed three ESBL-producing isolates that each harbored blaSHV and blaTEM genes with blaCTX-M gene being absent. Conclusion: MDR strains of A. baumannii harboring blaSHV and blaTEM genes were recovered from clinical and environmental sources during the outbreak, which was contained with preventive measures recommended.

Résumé En dépit des alertes faites par l'organisation mondiale de la Santé (OMS), Acinetobacter baumannii demeure un pathobiont sous-étudié et très peu priorisé au Nigeria. Nous avons procedé à sa caractérisation phénotypique et génotypique en dressant son profil de sensibilité aux antimicrobiens et ainsi que les gènes de résistance impliqués au cours d'une épidémie. Matériel et méthodes: Cette étude transversale a consisté à collecter des échantillons d'urine de patients et des écouvillons des mains du personnel des soins et de l'environnement hospitalier. L'identification des souches d' A. baumannii était faite par des méthodes bactériologiques standard. le profil de sensibilité aux antimicrobiens des isolats a été faite par la méthode de diffusion de disque , les bêta-lactamases à spectre étendu (BLSE) étaient recherchée par la méthode de dépistage combinée de disque ainsi que leur caractérisation moléculaire par la mise en évidence des gènes de résistance BLSE à l'aide d'une PCR (réaction en chaîne par polymérase). Résultats: Au total, huit isolats d'A. baumannii (6 cliniques et 2 de l'environnement) ont été identifiés. Les taux globaux de sensibilité et de résistance des isolats à tous les agents antimicrobiens étaient respectivement de 56,3 % (27/48) et de 35,4 % (17/48). De même, tous les isolats (8/8 ; 100,00 %) étaient sensibles au méropénème et 75,0 % (6/8) à l'ampicilline-sulbactam, tandis que 62,5 % (5/8) étaient résistants au triméthoprime-sulfaméthoxazole et 50,0 % (4/8) à la ciprofloxacine et à la ceftazidime. En outre, 37,5 % (3/8) des isolats étaient multirésistants (MDR), les isolats non cliniques présentant une plus grande résistance aux antimicrobiens que leurs homologues cliniques (9/12 %-75,0 % contre 8/36 %-22,2 %). La détection phénotypique et la caractérisation moléculaire ont révélé trois isolats producteurs de BLSE qui hébergeaient chacun les gènes blaSHV et blaTEM, le gène blaCTX-M étant absent. Conclusion: Des souches multirésistantes d'A. baumannii portant les gènes blaSHV et blaTEM ont été identifiées sur des prélevements cliniques et environnementaux au cours de l'épidémie, qui a été gerée grâce aux mesures préventives recommandées. Mots-clés: Surveillance de la résistance aux antimicrobiens, blaSHV carbapénème, pathogène ESKAPE, infections associées aux soins de santé, pratiques de prévention et de contrôle des infections, one health, uropathogènes.

Genomic Surveillance Uncovers a 10-Year Persistence of an OXA-24/40 Acinetobacter baumannii Clone in a Tertiary Hospital in Northern Spain.

Infections caused by carbapenem-resistant Acinetobacter baumannii are a global threat causing a high number of fatal infections. This microorganism can also easily acquire antibiotic resistance determinants, making the treatment of infections a big challenge, and has the ability to persist in the hospital environment under a wide range of conditions. The objective of this work was to study the molecular epidemiology and genetic characteristics of two blaOXA24/40Acinetobacter baumannii outbreaks (2009 and 2020-21) at a tertiary hospital in Northern Spain. Thirty-six isolates were investigated and genotypically screened by Whole Genome Sequencing to analyse the resistome and virulome. Isolates were resistant to carbapenems, aminoglycosides and fluoroquinolones. Multi-Locus Sequence Typing analysis identified that Outbreak 1 was mainly produced by isolates belonging to ST3Pas/ST106Oxf (IC3) containing blaOXA24/40, blaOXA71 and blaADC119. Outbreak 2 isolates were exclusively ST2Pas/ST801Oxf (IC2) blaOXA24/40, blaOXA66 and blaADC30, the same genotype seen in two isolates from 2009. Virulome analysis showed that IC2 isolates contained genes for capsular polysaccharide KL32 and lipooligosacharide OCL5. A 8.9 Kb plasmid encoding the blaOXA24/40 gene was common in all isolates. The persistance over time of a virulent IC2 clone highlights the need of active surveillance to control its spread.

Dynamics of blaOXA-23 gene transmission in Acinetobacter spp. from contaminated veterinary environmental surfaces: an emerging One Health threat?

BACKGROUND: Carbapenem-resistant Acinetobacter baumannii is a common pathogen associated with healthcare-acquired infections, and robust infection prevention and control protocols exist in human healthcare settings. In contrast, infection prevention and control (IPC) standards are limited in veterinary medicine, necessitating further investigation. AIM: Examine the possible transmission of carbapenem-resistant Acinetobacter spp. in a veterinary practice where a cat was diagnosed with an OXA-23-producing A. baumannii ST2 strain. METHODS: Environmental samples together with nasal and hand swabs from the veterinary personnel were collected. All swabs were screened for the presence of extended-spectrum-ß-lactamase- and carbapenemase-producing Enterobacterales, meticillin-resistant staphylococcus and multi-drug-resistant Acinetobacter spp. Whole-genome sequencing was performed for carbapenemase-producing strains. RESULTS: Of the veterinary staff, 60% carried meticillin-resistant Staphylococcus epidermidis. Environmental evaluation showed that 40% (N=6/15) of the surfaces analysed by contact plates and 40% (N=8/20) by swabs failed the hygiene criteria. Assessment of the surfaces revealed contamination with five OXA-23-producing Acinetobacter spp. strains: an OXA-23-producing Acinetobacter schindleri on the weight scale in the waiting room; and four OXA-23-producing Acinetobacter lwoffii strains, on different surfaces of the treatment room. The blaOXA-23 gene was located on the same plasmid-carrying Tn2008 across the different Acinetobacter spp. strains. These plasmids closely resemble a previously described OXA-23-encoding plasmid from a human Portuguese nosocomial Acinetobacter pittii isolate. Distinctly, the OXA-23-producing A. baumannii ST2 clinical strain had the resistant gene located on Tn2006, possibly inserted on the chromosome. CONCLUSION: The detection of an OXA-23-producing A. baumannii ST2 veterinary clinical strain is of concern for companion animal health and infection, prevention and control. This study established the dynamic of transmission of the plasmid-mediated blaOXA-23 gene on critical surfaces of a small animal veterinary practice. The genetic resemblance to a plasmid found in human nosocomial settings suggests a potential One Health link.

A 19-year longitudinal study to characterize carbapenem-nonsusceptible Acinetobacter isolated from patients with bloodstream infections and the contribution of conjugative plasmids to carbapenem resistance and virulence.

BACKGROUND: This study aimed to characterize carbapenem-nonsusceptible Acinetobacter (CNSA) isolated from patients with bacteremia from 1997 to 2015. METHODS: A total of 173 CNSA (12.3%) was recovered from 1403 Acinetobacter isolates. The presence of selected ß-lactamase genes in CNSA was determined by PCR amplification. The conjugation test was used to determine the transferability of metallo-ß-lactamase (MBL)-carrying plasmids. Whole genome sequencing in combination with phenotypic assays was carried out to characterize MBL-plasmids. RESULTS: In general, a trend of increasing numbers of CNSA was observed. Among the 173 CNSA, A. baumannii (54.9%) was the most common species, followed by A. nosocomialis (23.1%) and A. soli (12.1%). A total of 49 (28.3%) CNSA were extensively drug-resistant, and all were A. baumannii. The most common class D carbapenemase gene in 173 CNSA was blaOXA-24-like (32.4%), followed by ISAba1-blaOXA-51-like (20.8%), ISAba1-blaOXA-23 (20.2%), and IS1006/IS1008-blaOXA-58 (11.6%). MBL genes, blaVIM-11,blaIMP-1, and blaIMP-19 were detected in 9 (5.2%), 20 (11.6%), and 1 (0.6%) CNSA isolates, respectively. Transfer of MBL genes to AB218 and AN254 recipient cells was successful for 7 and 6 of the 30 MBL-plasmids, respectively. The seven AB218-derived transconjugants carrying MBL-plasmids produced less biofilm but showed higher virulence to larvae than recipient AB218. CONCLUSIONS: Our 19-year longitudinal study revealed a stable increase in CNSA during 2005-2015. blaOXA-24-like, ISAba1-blaOXA-51-like, and ISAba1-blaOXA-23 were the major determinants of Acinetobacter carbapenem resistance. MBL-carrying plasmids contribute not only to the carbapenem resistance but also to A. baumannii virulence.

The Acinetobacter baumannii K239 capsular polysaccharide includes heptasaccharide units that are structurally related to K86 but joined by different linkages formed by different Wzy polymerases.

The K239 type capsular polysaccharide (CPS) isolated from Acinetobacter baumannii isolate MAR19-4435 was studied by sugar analysis, one- and two-dimensional 1H and 13C NMR spectroscopy. K239 consists of branched heptasaccharide repeats (K-units) comprised of five residues of l-rhamnose (l-Rhap), and one residue each of d-glucuronic acid (d-GlcpA) and N-acetyl-d-glucosamine (d-GlcpNAc). The structure of K239 is closely related to that of the A. baumannii K86 CPS type, though the two differ in the 2,3-substitution patterns on the l-Rhap residue that is involved in the linkage between K-units in the CPS polymer. This structural difference was attributed to the presence of a gtr221 glycosyltransferase gene and a wzyKL239 polymerase gene in KL239 that replaces the gtr80 and wzyKL86 genes in the KL86 CPS biosynthesis gene cluster. Comparison of the two structures established the role of a novel WzyKL239 polymerase encoded by KL239 that forms the ß-d-GlcpNAc-(1→2)-l-Rhap linkage between K239 units. A. baumannii MAR19-4435 was found to be non-susceptible to infection by the APK86 bacteriophage, which encodes a depolymerase that specifically cleaves the linkage between K-units in the K86 CPS, indicating that the difference in 2,3-substitution of l-Rhap influences the susceptibility of this isolate to bacteriophage activity.

The Mechanism of Tigecycline Resistance in Acinetobacter baumannii under Sub-Minimal Inhibitory Concentrations of Tigecycline.

The presence of sub-minimal inhibitory concentration (sub-MIC) antibiotics in our environment is widespread, and their ability to induce antibiotic resistance is inevitable. Acinetobacter baumannii, a pathogen known for its strong ability to acquire antibiotic resistance, has recently shown clinical resistance to the last-line antibiotic tigecycline. To unravel the complex mechanism of A. baumannii drug resistance, we subjected tigecycline-susceptible, -intermediate, and -mildly-resistant strains to successive increases in sub-MIC tigecycline and ultimately obtained tigecycline-resistant strains. The proteome of both key intermediate and final strains during the selection process was analyzed using nanoLC-MS/MS. Among the more than 2600 proteins detected in all strains, we found that RND efflux pump AdeABC was associated with the adaptability of A. baumannii to tigecycline under sub-MIC pressure. qRT-PCR analysis also revealed higher expression of AdeAB in strains that can quickly acquire tigecycline resistance compared with strains that displayed lower adaptability. To validate our findings, we added an efflux pump inhibitor, carbonyl cyanide m-chlorophenyl hydrazine (CCCP), to the medium and observed its ability to inhibit tigecycline resistance in A. baumannii strains with quick adaptability. This study contributes to a better understanding of the mechanisms underlying tigecycline resistance in A. baumannii under sub-MIC pressure.

Characterization of the Zinc Uptake Repressor (Zur) from Acinetobacter baumannii.

Bacterial cells tightly regulate the intracellular concentrations of essential transition metal ions by deploying a panel of metal-regulated transcriptional repressors and activators that bind to operator-promoter regions upstream of regulated genes. Like other zinc uptake regulator (Zur) proteins, Acinetobacter baumannii Zur represses transcription of its regulon when ZnII is replete and binds more weakly to DNA when ZnII is limiting. Previous studies established that Zur proteins are homodimeric and harbor at least two metal sites per protomer or four per dimer. CdII X-ray absorption spectroscopy (XAS) of the Cd2Zn2 AbZur metalloderivative with CdII bound to the allosteric sites reveals a S(N/O)3 first coordination shell. Site-directed mutagenesis suggests that H89 and C100 from the N-terminal DNA binding domain and H107 and E122 from the C-terminal dimerization domain comprise the regulatory metal site. KZn for this allosteric site is 6.0 (±2.2) × 1012 M-1 with a functional "division of labor" among the four metal ligands. N-terminal domain ligands H89 and C100 contribute far more to KZn than H107 and E122, while C100S AbZur uniquely fails to bind to DNA tightly as measured by an in vitro transcription assay. The heterotropic allosteric coupling free energy, ΔGc, is negative, consistent with a higher KZn for the AbZur-DNA complex and defining a bioavailable ZnII set-point of ≈6 × 10-14 M. Small-angle X-ray scattering (SAXS) experiments reveal that only the wild-type Zn homodimer undergoes allosteric switching, while the C100S AbZur fails to switch. These data collectively suggest that switching to a high affinity DNA-binding conformation involves a rotation/translation of one protomer relative to the other in a way that is dependent on the integrity of C100. We place these findings in the context of other Zur proteins and Fur family repressors more broadly.

Novel mechanisms of diversity generation in Acinetobacter baumannii resistance islands driven by Tn7-like elements.

Mobile genetic elements play an important role in the acquisition of antibiotic and biocide resistance, especially through the formation of resistance islands in bacterial chromosomes. We analyzed the contribution of Tn7-like transposons to island formation and diversification in the nosocomial pathogen Acinetobacter baumannii and identified four separate families that recognize different integration sites. One integration site is within the comM gene and coincides with the previously described Tn6022 elements suggested to account for the AbaR resistance island. We established Tn6022 in a heterologous E. coli host and confirmed basic features of transposition into the comM attachment site and the use of a novel transposition protein. By analyzing population features within Tn6022 elements we identified two potential novel transposon-encoded diversification mechanisms with this dynamic genetic island. The activities of these diversification features were confirmed in E. coli. One was a novel natural gain-of-activity allele that could function to broaden transposition targeting. The second was a transposon-encoded hybrid dif-like site that parasitizes the host dimer chromosome resolution system to function with its own tyrosine recombinase. This work establishes a highly active Tn7-like transposon that harnesses novel features allowing the spread and diversification of genetic islands in pathogenic bacteria.