Multifunctional Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9-Based Nanobomb against Carbapenem-Resistant Acinetobacter baumannii Infection through Cascade Reaction and Amplification Synergistic Effect.

Carbapenems have been considered to be the preferred antibiotics against Acinetobacter baumannii thus far. However, carbapenem-resistant Acinetobacter baumannii (CRAB) has gradually escalated worldwide, and it frequently causes respiratory and bloodstream infections. Its resistance may lead to high mortality. Thus, there is an urgent need to develop antibacterial drugs. In our research, the pH-sensitive sgRNA-I/L@ZS nanosystem delivered imipenem and better released it in infected tissues to synergistically damage bacteria with nanoparticles. Gene editing of the CRISPR-Cas9 nanosystem amplified the synergistic effect by reversing the drug-resistance of imipenem. Nitric oxide, which l-arginine reacted with ROS to produce in cascade reaction and bacterial infection sites, was beneficial to heal the infected tissues and induce bacteria death for further enhancing antibacterial effects. In addition, this nanocomposite influenced host-bacteria interactions and restrained and destroyed biofilms. The sgRNA-I/L@ZS nanosystem, similar to a nanobomb, was a high-efficiency bactericide against CRAB. Eventually, in acute pneumonia and peritonitis mouse models, the sgRNA-I/L@ZS nanosystem could combat bacteria and protect tissues from infection. It had marked suppressive effects on inflammation and promoted healing and proliferation of infected tissues. This multifunctional nanosystem is expected to be an effective antibacterial agent in the clinic based on good biocompatibility and no toxic side effects. Therefore, developing the nanocomposites will take a favorable step toward solving intractable public health issues.

The extensively antibiotic resistant ST111 Acinetobacter baumannii isolate RBH2 carries an extensive mobile element complement of plasmids, transposons and insertion sequences.

The complete genome of RBH2, a sporadic, carbapenem resistant ST111 Acinetobacter baumannii isolate from Brisbane, Australia was determined and analysed. RBH2 is extensively resistant and the chromosome includes two transposons carrying antibiotic resistance genes, AbaR4 (oxa23 in Tn2006) and Tn7::Tn2006 (dfrA1, sat2, aadA1, oxa23). The chromosome also includes two copies of Tn6175, a transposon carrying putative copper resistance genes, and 1-17 copies of six different insertion sequences. RBH2 has six plasmids ranging in size from 6 kb - 141 kb, four carrying antibiotic resistance genes. Plasmids pRBH2-1 (aadB) and pRBH2-2 (aphA6 in TnaphA6) were found to be essentially identical to known plasmids pRAY*-v1 and pS21-1, respectively. The largest plasmids, pRBH2-5 (oxa23 in AbaR4) and pRBH2-6 (oxa23 in AbaR4::ISAba11 and sul2, tet(B), strA and strB in Tn6172) have known transfer-proficient relatives. pRBH2-5, an RP-T1 (RepAci6) plasmid, also carries a different putative copper resistance transposon related to Tn6177 found in pS21-2. The backbone of pRBH2-5 is related to those of previously described RepAci6 plasmids pAb-G7-2 and pA85-3 but has some distinctive features. Three different RepAci6 backbone types were distinguished, Type 1 (pAb-G7-2), Type 2 (pA85-3) and Type 3 (pRBH2-5 and pS21-2). pRBH2-6 is closely related to pAB3 and their backbones differ by only 5 SNPs. Plasmids pRBH2-3 and pRBH2-4 do not carry antibiotic resistance genes. pRBH2-3 does not include an identifiable rep gene and is a novel plasmid type. pRBH2-4 is of the R3-T3 type and includes segments of the larger pABTJ2 that heads this group. Other ST111 genomes carry different plasmids.

Mobile genetic elements in Acinetobacter antibiotic-resistance acquisition and dissemination.

Pathogenic Acinetobacter species, most notably Acinetobacter baumannii, are a significant cause of healthcare-associated infections worldwide. Acinetobacter infections are of particular concern to global health due to the high rates of multidrug resistance and extensive drug resistance. Widespread genome sequencing and analysis has determined that bacterial antibiotic resistance is often acquired and disseminated through the movement of mobile genetic elements, including insertion sequences (IS), transposons, integrons, and conjugative plasmids. In Acinetobacter specifically, resistance to carbapenems and cephalosporins is highly correlated with IS, as many ISAba elements encode strong outwardly facing promoters that are required for sufficient expression of ß-lactamases to confer clinical resistance. Here, we review the role of mobile genetic elements in antibiotic resistance in Acinetobacter species through the framework of the mechanism of resistance acquisition and with a focus on experimentally validated mechanisms.

Evolutionarily stable gene clusters shed light on the common grounds of pathogenicity in the Acinetobacter calcoaceticus-baumannii complex.

Nosocomial pathogens of the Acinetobacter calcoaceticus-baumannii (ACB) complex are a cautionary example for the world-wide spread of multi- and pan-drug resistant bacteria. Aiding the urgent demand for novel therapeutic targets, comparative genomics studies between pathogens and their apathogenic relatives shed light on the genetic basis of human-pathogen interaction. Yet, existing studies are limited in taxonomic scope, sensing of the phylogenetic signal, and resolution by largely analyzing genes independent of their organization in functional gene clusters. Here, we explored more than 3,000 Acinetobacter genomes in a phylogenomic framework integrating orthology-based phylogenetic profiling and microsynteny conservation analyses. We delineate gene clusters in the type strain A. baumannii ATCC 19606 whose evolutionary conservation indicates a functional integration of the subsumed genes. These evolutionarily stable gene clusters (ESGCs) reveal metabolic pathways, transcriptional regulators residing next to their targets but also tie together sub-clusters with distinct functions to form higher-order functional modules. We shortlisted 150 ESGCs that either co-emerged with the pathogenic ACB clade or are preferentially found therein. They provide a high-resolution picture of genetic and functional changes that coincide with the manifestation of the pathogenic phenotype in the ACB clade. Key innovations are the remodeling of the regulatory-effector cascade connecting LuxR/LuxI quorum sensing via an intermediate messenger to biofilm formation, the extension of micronutrient scavenging systems, and the increase of metabolic flexibility by exploiting carbon sources that are provided by the human host. We could show experimentally that only members of the ACB clade use kynurenine as a sole carbon and energy source, a substance produced by humans to fine-tune the antimicrobial innate immune response. In summary, this study provides a rich and unbiased set of novel testable hypotheses on how pathogenic Acinetobacter interact with and ultimately infect their human host. It is a comprehensive resource for future research into novel therapeutic strategies.

Complete genome of the extensively antibiotic-resistant GC1 Acinetobacter baumannii isolate MRSN 56 reveals a novel route to fluoroquinolone resistance.

OBJECTIVES: To examine the causes of antibiotic resistance in the extensively resistant global clone 1 (GC1) Acinetobacter baumannii isolate MRSN 56 recovered at a US military treatment facility. METHODS: MRSN 56 was sequenced using MinION (Oxford Nanopore) and the reads combined with available Illumina MiSeq data using Unicycler. Acquired resistance genes were identified using ABRicate and their environment examined. ISAba1 and ISAba125 copies were located. RESULTS: MRSN 56 is ST1IP:ST231Ox:KL1:OCL1 and the complete genome includes four small plasmids, none of which carry resistance genes. The acquired resistance genes were found at four locations in the chromosome in addition to AbaR28 (aphA1, aacC1, aadA1, sul1) in comM. Tn2006 (oxa23, carbapenem resistance) was both in AbaR4 and alone elsewhere. Two copies of Tn7 (dfrA1, sat, aadA1) were identified. One was associated with a 22 852 bp adjacent segment [tetA(B), sul2] derived from the AbGRI1 island, and this novel configuration was designated Tn7+. Tn7+ was incorporated in the position preferred by Tn7, downstream of glmS, by transposition using a sequence in AbGRI1 resembling the Tn7 terminal inverted repeats. Tn7 was found at a secondary site. Fluoroquinolone resistance appears to involve a mutation in gyrA combined with inactivation by ISAba1 of the marR gene in the mar operon and constitutive expression of marA from the promoter internal to ISAba1. CONCLUSIONS: MRSN 56 represents a new sublineage of GC1 lineage 1 with novel features that had not been detected previously. The involvement of the mar operon in fluoroquinolone resistance has not been noted previously.

Scarless excision of an insertion sequence restores capsule production and virulence in Acinetobacter baumannii.

We identify a new mechanism mediating capsule production and virulence in the WHO and CDC priority ESKAPE pathogen Acinetobacter baumannii. Non-capsulated and avirulent bacteria can revert into a capsulated and virulent state upon scarless excision of an ISAba13 insertion sequence under stress conditions. Reversion events fully restore capsule production and in vivo virulence. This increases our knowledge about A. baumannii genome dynamics, and the regulation of capsule production, virulence and resistance.

Antiseptic 9-Meric Peptide with Potency against Carbapenem-Resistant Acinetobacter baumannii Infection.

Carbapenem-resistant A. baumannii (CRAB) infection can cause acute host reactions that lead to high-fatality sepsis, making it important to develop new therapeutic options. Previously, we developed a short 9-meric peptide, Pro9-3D, with significant antibacterial and cytotoxic effects. In this study, we attempted to produce safer peptide antibiotics against CRAB by reversing the parent sequence to generate R-Pro9-3 and R-Pro9-3D. Among the tested peptides, R-Pro9-3D had the most rapid and effective antibacterial activity against Gram-negative bacteria, particularly clinical CRAB isolates. Analyses of antimicrobial mechanisms based on lipopolysaccharide (LPS)-neutralization, LPS binding, and membrane depolarization, as well as SEM ultrastructural investigations, revealed that R-Pro9-3D binds strongly to LPS and impairs the membrane integrity of CRAB by effectively permeabilizing its outer membrane. R-Pro9-3D was also less cytotoxic and had better proteolytic stability than Pro9-3D and killed biofilm forming CRAB. As an LPS-neutralizing peptide, R-Pro9-3D effectively reduced LPS-induced pro-inflammatory cytokine levels in RAW 264.7 cells. The antiseptic abilities of R-Pro9-3D were also investigated using a mouse model of CRAB-induced sepsis, which revealed that R-Pro9-3D reduced multiple organ damage and attenuated systemic infection by acting as an antibacterial and immunosuppressive agent. Thus, R-Pro9-3D displays potential as a novel antiseptic peptide for treating Gram-negative CRAB infections.

Genome sequencing and molecular characterisation of XDR Acinetobacter baumannii reveal complexities in resistance: Novel combination of sulbactam-durlobactam holds promise for therapeutic intervention.

Emerging nosocomial strains of Acinetobacter baumannii are of recent concern as they are expressing extensive drug resistance (XDR). Using whole-genome sequencing and molecular characterisation analysis, the current study reveals the presence of carbapenemase genes in 92.86% of studied Indian isolates. These included blaOXA-51 , blaOXA-23 , blaOXA-58 , and blaNDM genes, with over a third expressing dual carbapenemase genes. As per the MLST scheme, IC2Oxf /CC2Pas was the predominant clone, with 57.14% isolates belonging to this lineage. The presence of these carbapenemase genes resulted in sulbactam (SUL) resistance (MIC: 16-256 µg/ml) in all of the studied isolates. The efficacy of durlobactam (DUR), a novel ß-lactamase inhibitor that also inhibits PBP2 was assessed through in silico intermolecular interaction analysis. Several nonsynonymous single nucleotide polymorphisms were identified in PBP2 (G264S, I108V, S259T) and PBP3 (A515V, T526S) sequences. Minimal variations were recorded in the protein backbone dynamics in active-site motifs of wild-type and mutants, which correlated with negligible binding energy fluctuations for the PBP3-SUL (-5.85 ± 0.04 kcal/mol) and PBP2-DUR (-5.16 ± 0.66 kcal/mol) complexes. Furthermore, higher binding affinities and low inhibition constants were noted in OXA23-DUR (-7.36 kcal/mol; 4.01 µM), OXA58-DUR (-6.44 kcal/mol; 19.07 µM), and NDM-DUR (-6.82 kcal/mol; 10.01 µM) complexes when compared with the conventional drugs avibactam and aztreonam. Stable interaction profiles of DUR with carbapenemases can possibly restore SUL activity against both PBP3WT and PBP3MTs . The study establishes the efficacy of the novel SUL-DUR combination as a successful treatment strategy in combating emerging XDR strains of A. baumannii.

Molecular and epidemiological investigation of a colistin-resistant OXA-23-/NDM-1-producing Acinetobacter baumannii outbreak in the Southwest Indian Ocean Area.

Dual resistance to colistin and carbapenems is a milestone reached by certain extensively-drug resistant (XDR) Gram-negative bacteria. This study describes the first outbreak of XDR colistin- and carbapenem-resistant OXA-23-/NDM-1-producing Acinetobacter baumannii (CCRAB) in the European overseas territory of Reunion Island (France, Indian Ocean). Between April 2019 and June 2020, 13 patients admitted to the University Hospital of Reunion Island were involved in the outbreak, of whom eight were infected and six died. The first case was traced to a medical evacuation from Mayotte Island (Comoros archipelago). An epidemiological link could be established for 11 patients. All of the collected CCRAB isolates showed the same resistance profile and co-produced intrinsic ß-lactamases OXA-69 and ADC-191, together with acquired carbapenem-hydrolysing ß-lactamases OXA-23 and NDM-1. A mutation likely involved in colistin resistance was detected in the two-component system PmrAB (D82N in PmrA). All of the isolates were found to belong to STPas1/STOx231 clonal complex and were phylogenetically indistinguishable. Their further characterization by whole-genome sequence analyses (whole-genome multi-locus sequence typing, single nucleotide polymorphisms) provided hints about the transmission pathways. This study pleads for strict application of control and prevention measures in institutions where the risk of imported XDR bacteria is high.

Acinetobacter baumannii up-regulates LncRNA-GAS5 and promotes the degradation of STX17 by blocking the activation of YY1.

Acinetobacter baumanniitriggers autophagy, affects the degradation of autophagy, and causes severe inflammatory injury. LncRNA growth arrest-specific transcript 5 (LncRNA-GAS5) and Yin and Yang 1 (YY1) are known to play an important role in the regulation of autophagy, however, the precise role of LncRNA-GAS5 and YY1 in the damage to autophagy caused by Acinetobacter baumanniiremains unclear. The aim of this study was to investigate the role of LncRNA-GAS5 and YY1 in the regulation of autophagy induced by Acinetobacter baumannii. We found that LncRNA-GAS5 was up-regulated following infection with Acinetobacter baumannii, thus resulting in the degradation of STX17, autophagy disorders, and the aggravated replication of Acinetobacter baumannii. We also analyzed the mechanism of interaction between LncRNA-GAS5 and YY1 and found that YY1 regulated its expression in a negative manner by binding to the promoter of LncRNA-GAS5. LncRNA-GAS5 and YY1 had opposite effects on the expression of STX17, this process maintained the stable expression of STX17. Following Acinetobacter baumannii infection, YY1 was down regulated and then separated from the binding region of LncRNA-GAS5, thus resulting in the activation of LncRNA-GAS5 transcription and reduction in STX17 protein expression. Finally, we infected LncRNA-GAS5 knockdown mice with Acinetobacter baumannii, the expression levels of IFN-ß in the lungs increased significantly, this alleviated lung injury. In conclusion, our work demonstrated the mechanism by which Acinetobacter baumannii infection can cause the degradation of STX17. We also demonstrated that LncRNA-GAS5 may be a potential therapeutic target for the treatment of lung injury induced by Acinetobacter baumannii.

Capsule carbohydrate structure determines virulence in Acinetobacter baumannii.

Acinetobacter baumannii is a highly antibiotic-resistant bacterial pathogen for which novel therapeutic approaches are needed. Unfortunately, the drivers of virulence in A. baumannii remain uncertain. By comparing genomes among a panel of A. baumannii strains we identified a specific gene variation in the capsule locus that correlated with altered virulence. While less virulent strains possessed the intact gene gtr6, a hypervirulent clinical isolate contained a spontaneous transposon insertion in the same gene, resulting in the loss of a branchpoint in capsular carbohydrate structure. By constructing isogenic gtr6 mutants, we confirmed that gtr6-disrupted strains were protected from phagocytosis in vitro and displayed higher bacterial burden and lethality in vivo. Gtr6+ strains were phagocytized more readily and caused lower bacterial burden and no clinical illness in vivo. We found that the CR3 receptor mediated phagocytosis of gtr6+, but not gtr6-, strains in a complement-dependent manner. Furthermore, hypovirulent gtr6+ strains demonstrated increased virulence in vivo when CR3 function was abrogated. In summary, loss-of-function in a single capsule assembly gene dramatically altered virulence by inhibiting complement deposition and recognition by phagocytes across multiple A. baumannii strains. Thus, capsular structure can determine virulence among A. baumannii strains by altering bacterial interactions with host complement-mediated opsonophagocytosis.

Colistin Dependence in Extensively Drug-Resistant Acinetobacter baumannii Strain Is Associated with ISAjo2 and ISAba13 Insertions and Multiple Cellular Responses.

The nosocomial opportunistic Gram-negative bacterial pathogen Acinetobacter baumannii is resistant to multiple antimicrobial agents and an emerging global health problem. The polymyxin antibiotic colistin, targeting the negatively charged lipid A component of the lipopolysaccharide on the bacterial cell surface, is often considered as the last-resort treatment, but resistance to colistin is unfortunately increasing worldwide. Notably, colistin-susceptible A. baumannii can also develop a colistin dependence after exposure to this drug in vitro. Colistin dependence might represent a stepping stone to resistance also in vivo. However, the mechanisms are far from clear. To address this issue, we combined proteogenomics, high-resolution microscopy, and lipid profiling to characterize and compare A. baumannii colistin-susceptible clinical isolate (Ab-S) of to its colistin-dependent subpopulation (Ab-D) obtained after subsequent passages in moderate colistin concentrations. Incidentally, in the colistin-dependent subpopulation the lpxA gene was disrupted by insertion of ISAjo2, the lipid A biosynthesis terminated, and Ab-D cells displayed a lipooligosaccharide (LOS)-deficient phenotype. Moreover, both mlaD and pldA genes were perturbed by insertions of ISAjo2 and ISAba13, and LOS-deficient bacteria displayed a capsule with decreased thickness as well as other surface imperfections. The major changes in relative protein abundance levels were detected in type 6 secretion system (T6SS) components, the resistance-nodulation-division (RND)-type efflux pumps, and in proteins involved in maintenance of outer membrane asymmetry. These findings suggest that colistin dependence in A. baumannii involves an ensemble of mechanisms seen in resistance development and accompanied by complex cellular events related to insertional sequences (ISs)-triggered LOS-deficiency. To our knowledge, this is the first study demonstrating the involvement of ISAjo2 and ISAba13 IS elements in the modulation of the lipid A biosynthesis and associated development of dependence on colistin.

Molecular Characterization of Carbapenem-Resistant Acinetobacter baumannii Blood Culture Isolates from Three Hospitals in Turkey.

We aimed to investigate the clonal relationships, common sequence types, and carbapenemase genes in 177 non-repetitive blood culture isolates of Acinetobacter baumannii collected from patients at three university hospitals in Turkey in 2016. Molecular epidemiological characteristics of the isolates were examined using pulsed-field gel electrophoresis (PFGE), and multilocus sequence typing (MLST) (Pasteur scheme-cpn60, fusA, gltA, pyrG, recA, rplB, and rpoB). Multiplex PCR was used to investigate the carbapenemase genes, including blaOXA-23-like, blaOXA-24-like, blaOXA-48-like, blaOXA-58-like, blaIMP, blaVIM, and blaNDM. PFGE genotyping yielded 92 pulsotypes with a clustering ratio of 69.7%. As per a ≥85% similarity coefficient, 159 (90.9%) isolates were found to be clonally related. The blaOXA-23-like and blaOXA-58-like genes were identified in 100% and 28.2% of the isolates, respectively. The blaNDM gene was identified in two isolates. The MLST analysis included 54 isolates with different pulsotypes, and 29 sequence types (STs). Most of the isolates (n = 36) belonged to the clonal complex (CC)2, one isolate belonged to CC1, and one isolate belonged to CC164. Sixteen new STs (ST1235-ST1250) were identified. Identifying both global ST2 and a large number of new STs, revealed high genetic diversity in A. baumannii isolates in the study population.

Outbreak of Multidrug-Resistant Acinetobacter baumannii ST208 Producing OXA-23-Like Carbapenemase in a Children's Hospital in Shanghai, China.

Aims: Acinetobacter baumannii is notorious for acquiring antibiotic resistance and causing nosocomial infections worldwide. This study aimed to investigate the prevalence and molecular characteristics of A. baumannii isolates obtained from inpatients and the intensive care unit (ICU) environment of a pediatric hospital in Shanghai, China. Methods: Between July 2017 and January 2018, a total of 88 A. baumannii isolates, including three obtained from ICU environmental specimens, were characterized by antibiotic susceptibility, multilocus sequence typing, and resistance genes. Results: Carbapenem-resistant A. baumannii (CRAB) isolates, which were resistant to all the antibiotics tested except colistin, accounted for 69.3% (61/88) of all isolates. Three sequence types (STs) were identified among the CRAB isolates, and the predominant clone was ST208 (93.4%, 57/61), which included three environmental isolates and 54 clinical isolates collected from ICU patients. Carbapenem-susceptible isolates, none of which was multidrug resistant (MDR), showed a more diverse genetic background with three known STs and 21 novel STs identified. Intrinsic blaOXA-51-like and blaAmpC were detected in all isolates, while blaOXA-23-like was only detected in all CRAB isolates. ISAba1-blaOXA-23-like, ISAba1-blaOXA-51-like, and ISAba1-blaAmpC were identified in 69.3% (all CRAB isolates), 0%, and 65.9% (58 CRAB isolates) of all isolates, respectively. Conclusions: A nosocomial outbreak of MDR A. baumannii ST208 producing OXA-23-like carbapenemase occurred, highlighting the necessity for strict infection control interventions in the ICU.

Analysis of Immune Responses in Acinetobacter baumannii-Infected Klotho Knockout Mice: A Mouse Model of Acinetobacter baumannii Infection in Aged Hosts.

Acinetobacter baumannii is an important opportunistic pathogen that primarily afflicts elderly people. To clarify the pathogenicity of A. baumannii in the elderly, we investigated immune responses to A. baumannii ATCC 19606 infection in klotho knockout (KO) mice, the mouse model of aging. Following intravenous inoculation, the mice seldom displayed severe symptoms. However, the survival rate was 56% at 7 days post-infection. Bacteria were detected in the lungs of klotho KO mice but not klotho wildtype (WT) mice at 7 days post-infection. Neutrophils, eosinophils, interstitial macrophages, and monocyte/dendritic cell subset in the lungs of klotho KO mice were transiently induced after infection with A. baumannii. The number of alveolar macrophages in klotho KO mice was lower than that in klotho WT mice, except for 1 day post-infection. CD11b expression on neutrophils and alveolar macrophages in the lungs of klotho KO mice was seldom upregulated by the infection. These results suggested that immune functions eliminating bacteria in the lungs of klotho KO mice were insufficient. CD11blow conventional DC cells hardly increased in klotho KO mice infected with A. baumannii. Additionally, the production of interleukin (IL)-10 in the sera of klotho KO mice was significantly higher than that in klotho WT mice, whereas that production of interferon-gamma was not detected in the sera of klotho KO mice. These results suggested that acquired immune responses were hardly induced in klotho KO mice. IL-1ß, CXCL1, CXCL2, and CCL2 expression was significantly higher in the lungs of klotho KO mice infected with A. baumannii than in those of klotho WT mice at 1 day post-infection. These results suggested that pulmonary inflammation was elicited in klotho KO mice during early infection. The expression levels of proinflammatory cytokines significantly correlated with TLR9 expression in the lungs of klotho KO mice. The collective results demonstrate an A. baumannii infection state in aged hosts and suggest that pulmonary inflammation and bacterial burden should be noted in aged hosts even in the absence of severe symptoms of A. baumannii infection.

Mechanisms Protecting Acinetobacter baumannii against Multiple Stresses Triggered by the Host Immune Response, Antibiotics and Outside-Host Environment.

Acinetobacter baumannii is considered one of the most persistent pathogens responsible for nosocomial infections. Due to the emergence of multidrug resistant strains, as well as high morbidity and mortality caused by this pathogen, A. baumannii was placed on the World Health Organization (WHO) drug-resistant bacteria and antimicrobial resistance research priority list. This review summarizes current studies on mechanisms that protect A. baumannii against multiple stresses caused by the host immune response, outside host environment, and antibiotic treatment. We particularly focus on the ability of A. baumannii to survive long-term desiccation on abiotic surfaces and the population heterogeneity in A. baumannii biofilms. Insight into these protective mechanisms may provide clues for the development of new strategies to fight multidrug resistant strains of A. baumannii.

Colistin heteroresistance in carbapenem-resistant Acinetobacter baumannii clinical isolates from a Thai university hospital.

Colistin is the last resort for the treatment of infections with carbapenem-resistant (CR) Gram-negative bacteria particularly Acinetobacter baumannii (CRAB). Currently, both colistin-resistant and -heteroresistant A. baumannii isolates have been reported globally. We therefore investigated the colistin heteroresistance rate in 75 non-duplicate colistin-susceptible CRAB clinical isolates from a Thai university collected in 2016. Minimum inhibitory concentrations (MICs) of colistin for all isolates were determined by broth microdilution method and carbapenemase genes were detected by PCR methods. All isolates were genotyped by ERIC-PCR method and screened for colistin heteroresistance by modified population analysis profile (PAP) method. The colistin MIC range for the 75 isolates was 0.5-2 µg/mL, with MIC50 and MIC90 of 1 and 2 µg/mL, respectively. Thirty-three isolates (44%) were considered colistin-heteroresistant with subpopulations growing at 3-8 µg/mL of colistin. After three daily passages of the subpopulations on antibiotic-free medium, their colistin MICs ranged from 4 to > 32 µg/mL, with MIC50 and MIC90 of 32 and > 32 µg/mL, respectively. Eight different ERIC-PCR profiles were obtained among the 33 isolates and all carried blaOXA-23-like. The high rate of colistin heteroresistance in the CRAB isolates highlights the possibility of treatment failure of CRAB infections by colistin due to the selection of colistin-resistant subpopulations.

Distribution of carbapenemase genes in clinical isolates of Acinetobacter baumannii & a comparison of MALDI-TOF mass spectrometry-based detection of carbapenemase production with other phenotypic methods.

Background & objectives: Carbapenemase-producing Acinetobacter baumannii (CRAB) poses a continuous threat to the current antimicrobial era with its alarming spread in critical care settings. The present study was conducted to evaluate the diagnostic potential of phenotypic methods for carbapenemase [carbapenem-hydrolyzing class D ß-lactamases (CHDLs) and metallo-ß-lactamases (MBLs)] production, by comparing with molecular detection of genes. Methods: One hundred and fifty clinical CRAB isolates collected between August 2013 and January 2014 were studied. Multiplex PCR was performed to identify the carbapenemases produced (class D blaOXA-51, blaOXA-23, blaOXA-48, blaOXA-58; class B blaVIM, blaNDM-1, blaIMP; class A blaKPC). Each isolate was evaluated for carbapenemase production by studying the pattern of imipenem hydrolysis using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). Results: The most commonly encountered carbapenemase genes were blaOXA-51(100%), blaOXA-23(98%), blaVIM(49.3%), blaNDM-1(18.7%) and blaOXA-58(2%). MALDI-TOF MS was able to detect 30.6 per cent carbapenemases within three hours (P=0.001 for MBL and P>0.05 for CHDL) and 65.3 per cent within six hours (P=0.001 for MBL and P>0.05 for CHDL). Interpretation & conclusions: MALDI-TOF MS reliably detected carbapenemase activity within a short span of time, thus helping in tailoring patient therapy. MALDI-TOF MS, once optimized, can prove to be a useful tool for timely detection of carbapenemase production by A. baumannii and consequently in directing appropriate antimicrobial therapy.

Distribution of Carbapenemases and Efflux Pump in Carbapenem-resistance Acinetobacter baumannii.

Acinetobacter baumannii has emerged as an important pathogen related to serious infections and nosocomial outbreaks around the world. In this study, 100 isolates of carbapenem-resistance in Acinetobacter baumannii (CRAB) were collected from clinical specimens. Agar dilution was conducted to determine the minimum inhibitory concentrations (MICs) for 15 kinds of antibiotics. Genes of carbapenemases and efflux pumps were amplified by PCR. The expression difference of pump genes was analyzed by real-time PCR between CRAB and carbapenems-sensitive Acinetobacter baumannii (CSAB). We found that most antibiotics, including aminoglycosides, fluoroquinolones, and cephalosporins showed high MIC values in CRAB. All isolates were sensitive to polymyxin B. Among the CRAB specimens, 54, 32 and 16 isolates were positive for SHV-12, PER-1 and TEM-1, respectively. 86 isolates were positive for OXA-23. 55 and 33 isolates carried adeB and adeJ genes, respectively. The expression level of adeB in CRAB was ten times higher than that in CSAB. We speculate that SHV-12, PER-1, TEM-1, OXA-23 and the AdeABC efflux pump may participate in high-level carbapenems resistance in Acinetobacter baumannii Moreover, adeE may be related to low-level resistance of carbapenems and quinolones in Acinetobacter baumannii.

Characteristics of invasive Acinetobacter infection: A multicenter investigation with molecular identification of causative organisms.

We examined microbiological and clinical characteristics of invasive Acinetobacter infection occurring in four hospitals located in the Minami-Ibaraki Area. Glucose-non-fermentative Gram-negative bacilli isolated from the blood and the cerebrospinal fluid in independent cases between 2001 and 2014 were consecutively collected and those possibly to be Acinetobacter species were re-identified using molecular methods. Of 158 strains identified as Acinetobacter species, 155 were classified into 16 officially designated species, including 42 Acinetobacter pittii and 40 Acinetobacter baumannii. Imipenem non-susceptibility was detected only in 4 strains, none of which demonstrated multidrug resistance. Retrospective analyses of 154 cases for which medical records were fully available showed that the most common cause of infection was primary bloodstream infection (134 cases), of which 128 were related to intravascular catheter use. The mortality on day 28 after the onset was independently associated with cerebrovascular disease, moderate to severe renal disease, the Pitt bacteremia score, and infection other than primary bloodstream infection but not with appropriate empiric antimicrobial therapy. Isolation of A. baumannii was significantly associated with septic shock but not with the 28-day mortality. These findings, obtained in a region where drug-resistant Acinetobacter strains were much less prevailing, indicated that non-baumannii Acinetobacter species were common pathogens, that the most predominant cause of invasive Acinetobacter infection was intravascular catheter-related infection, that virulence of A. baumannii might be higher than those of other species but its association with mortality was unclear, and that administration of broad-spectrum antibiotics targeting Acinetobacter species might be deferrable in a certain situation.